HPTLC - HIGH PERFORMANCE THIN-LAYER CHROMATOGRAPHY, Volume 9 (Journal of Chromatography Library)

Journal of Chromatography Library - Volume 9

HP"LC HIGH PERFORMANCE THIN-LAYER CHROMATOGRAPHY

Journal of Chromatography Library - Volume 9

HP"LC HIGH PERFORMANCE THIN-LAYER CHROMATOGRAPHY

JOURNAL OF CHROMATOGRAPHY LIBRARY Volume 1 Chromatography of Antibiotics by G. H. Wagman and M. J. Weinstein Volume 2 Extraction Chromatography edited by T. Braun and G. Ghersini Volume 3 Liquid Column Chromatography. A Survey of Modem Techniques and Applications edited by Z. Deyl, K. Macek and J. Janhk Volume 4 Detectors in Gas Chromatography by J. SevEik Volume 5 Instrumental Liquid Chromatography: A Practical Manual on High-Performance Liquid Chromatographic Methods by N. A. Pams Volume 6 Isotachophoresis. Theory, Instrumentation and Applications by F. M. Everaerts, J. L. Beckers and Th P. E. M. Verheggen Volume 7 Chemical Derivatization in Liquid Chromatography by J. F. Lawrence and R. W. Frei Volume 8 Chromatography of Steroids by E. Heftmann Volume 9 HPTLC: High Performance Thin-Layer Chromatography edited by A. Zlatkis and R. E. Kaiser

Journal of Chromatography Library - Volume 9

HPTLC HIGH PERF'ORMANCE THIN-LAYER CHROMATOGRAPHY Editors

A. Zlatkis Universityof Houston, Houston, Texas

R E. Kaiser Institute of Chromatography, Bad Durkheim

ELSEVIER SCIENTIFIC PUBLISHING COMPANY AMSTERDAM - OXFORD - NEW YORK

INSTITUTE OF CHROMATOGRAPHY BAD DURKHEIM 1977

ELSEVIER SCIENTIFIC PUBLISHING COMPANY 335 Jan van Galenstraat P.O. Box 211, Amsterdam, The Netherlands Distributors for the United States and Canada:

ELSEVIER/NORTH-HOLLAND INC. 52, Vanderbilt Avenue New York, N.Y. 10017

ISBN 0 4 4 1 5 2 5 . 4 Copyright 0 1977 by Institute of Chromatography, Bad Durkheim All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the Institute of Chromatography, P.O. Box 1308, D-6702 Bad Durkheim-1, G.F.R. Printed by Graphische Kunstanstalt, D-6702 Bad Durkheim.

Contributors to this volume: J. Blome Lerchenweg 7, D-7919 Au/Iller

H. Halpaap E. Merck, P.O.B. 41 19, D-6100 Darmstadt U. B. Hezel Carl Zeiss, P.O.B. 35/36, D-7082 Oberkochen

D. Jaenchen CAMAG AG, Homburgerstr. 24, CH-4132 Muttenz R. E. Kaiser Institute of Chromatography, P.O.B.1308, D-6702 Bad Duerkheim-1

J. Ripphrrhn E. Merck, P.O.B. 4119, D-6100 Darmstadt

Table of Contents Preface and Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

.

Simplified theory of TLC (R.E Kaiser) . . . . . . . . . . . . . . . . . Rf-value and k. Real Rf-values . . . . . . . . . . . . . . . . . . . . . . . Flow function in HPTLC. Rf-value in HPTLC . . . . . . . . . . . . . . . Correlation between circular HPTLC Rfand linear Rf-values . . . . . . . . Separation capability and separation power in HPTLC . . . . . . . . . . . Plate height and separation number in TLC. . . . . . . . . . . . . . . . . Resolution, selectivity. separation capability . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 The separation number in linear and circular TLC (J.Blome) . . . . . . .

Calculation of the separation number in linear TLC . . . . . . . . . . . . The space diffusion model . . . . . . . . . . . . . . . . . . . . . . . . . Determination of the separation number in circular TLC . . . . . . . . . . Critical questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

9 14 15 18 18 26 28 31 34 38 39

40 42

44 49

3 Advantages. limits and disadvantagesof the ring developing technique

51 (J.Blome) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prerequisits for the ring developing technique . . . . . . . . . . . . . . . 52 Working technique in circular chromatography . . . . . . . . . . . . . . . 53 ComparisonofthemethodslinearTLCandcircularTLC . . . . . . . . . 61 Ratio of Rf to RRf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Gradient effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Reduction of vapor pressure . . . . . . . . . . . . . . . . . . . . . . . . 65 Flowing around effect . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 Dynamic area. Trace analysis . . . . . . . . . . . . . . . . . . . . . . . . 67 Effect of solvent atmosphere . . . . . . . . . . . . . . . . . . . . . . . . 68 Material required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Space required. handling ability. miscellaneous . . . . . . . . . . . . . . . 71

4 The U-chamber (R.E.Kaiser)

...................... 73 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Disadvantages of the normal U-chamber system. Conclusions . . . . . . . 77 Comparison of migration speed . . . . . . . . . . . . . . . . . . . . . . . 78 81 Principle of wet dosage in HPTLC . . . . . . . . . . . . . . . . . . . . . Continuous HPTLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Coupling with another separation or detection systems . . . . . . . . . . . 83 Plate adjustment and dosage . . . . . . . . . . . . . . . . . . . . . . . . 84

5 Dosage techniques in HPTLC (R.E.Kaiser)

. . . . . . . . . . . . . . . 85 Primary optimized dosage . . . . . . . . . . . . . . . . . . . . . . . . . 86 87 Dosage volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Instrumentation for nano dosage. Self loading sample capillary . . . . . . . 88 88 Glass-metal capillary . . . . . . . . . . . . . . . . . . . . . . . . . . . . Constant dosage capillary . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Sample application on the wet layer . . . . . . . . . . . . . . . . . . . . 92 Thermal and chemical focusing . . . . . . . . . . . . . . . . . . . . . . 93

6

High performance thin-layer chromatography: development, data and results (H.Halpaap. J.Ripphahn) Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Development of the pre-coated HPTLC plate . . . . . . . . . . . . . . . . 96 Chromatographc performance of the HPTLC plate . . . . . . . . . . . . 102 Chromatographic characteristics . . . . . . . . . . . . . . . . . . . . . . 109 Influences of the type of chamber and of sorbent and solvent activity . . . . 109 Influence oE temperature . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Differences between linear andcircular chromatography . . . . . . . . . . 119 Advantages of HFTLC . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Chromatographic and physical parameters of solvents. elutropic series 126 breakdown related to silicagel . . . . . . . . . . . . . . . . . . . . . . .

7 Consideration on the reproducibility of TLC separations (D.Jaenchen) . . 129

Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Modification of the stationary phase by substances other than components 134 of the developing solvent . . . . . . . . . . . . . . . . . . . . . . . . . . Influence of layer preconditioning with components of the developing 136 solvent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The role of solvent pre-adsorbed by the dry layer . . . . . . . . . . . . . . 139 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 Transfer TLC-HPLC: Compatible experimental approach; is equilibration via the gas phase comparable to liquid phase equilibration? . . . . . . . . . . . . . . . . . . 141 144 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8 Potential and experience in quantitative HPTLC (U.B.Hezel)

. . . . . . 147

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Application techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . Photometric procedures for HFTLC evaluation . . . . . . . . . . . . .

147 150

. . 154

Absorption measurements in reflectance and transmission mode . . . . . . Smoothing of baselines by simultaneous reflectance and transmission measured . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Absorption measurements in UV and fluorescence “quenching” . . . . . . Fluorescence measurements . . . . . . . . . . . . . . . . . . . . . . . . Reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Qualitative initial examinations . . . . . . . . . . . . . . . . . . . . . . . Practical examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Requirements of the instrument for the photometric evaluation of HFTLC chromatograms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

154 157 158 163 166 171 173 180

9

Application of a new high-performancelayer in quantitative TLC (J.Ripphahn, H.Halpaap) . . . . . . . . . . . . . . . . . . . . . . . . . Advantages of HPTLC precoated plates . . . . . . . . . . . . . . . . . Applicator for TLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Apparatus for nanoliter application . . . . . . . . . . . . . . . . . . . . . Peak area and baseline determination . . . . . . . . . . . . . . . . . . . . Evaluation procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . Wavelength optimization . . . . . . . . . . . . . . . . . . . . . . . . . . Comparison of the detection at right-angles to solvent flow and in flow direction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Limit of detection, Detection of phenylanaline in serum . . . . . . . . . Regression line and correlation coefficient . . . . . . . . . . . . . . . . Aflatoxine measurements . . . . . . . . . . . . . . . . . . . . . . . . . Coloured figures Principle of simultaneous three phase circular separation in a IfC-U-Chamber . . . . . . . . . . . . . . . . . . . . . . . .

......

189

. 190 191 192 209 212 212

. .

215 216 218 221

193

SimultaneousmultiphasecircularHPTLCofdyemixture . . . . . . 194. 195 Detection of trace level actions . . . . . . . . . . . . . . . . . . . . . . . 197 Power of two dimensional HPTLC on 50 x 50 mm plates . . . . . . . . . . 198 Separation power and Rf data precision of HPTLC layers . . . . . . . . . . 201 40 samples simultaneously separated in one run . . . . . . . . . . . . . . 203 50 x 50 mm circular HPTLC for precise qualitative and quantitative 208 analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix Conversion table Rf to k and k to Rf values.

. . . . . . . . . . . . . . . . 225

Pocketcomputerprogramforlinearregressioncalculation . . . . . . . . . 226 Index .

...................................

227

Preface and Introduction The publication of this book appears at a time when many colleagues who are actively involved in analytical research, consider thin-layer chromatography as one of the less important analytical tools. Another considerable group of scientists have concentrated their efforts on the problems of HPLC, a method which is called high pressure liquid chromatography but should be high performance liquid chromatography. The separation columns - rarely operated under optimum conditions hardly exceed lo00 theoretical plates. Irreversible adsorptionof samplecomponents on the column material, often limits their general application. Most of the shortcomings of HPLC can be avoided by using TLC methods. This book is not intended to discredit HPLC. Indeed, it is meant for those who use HPLC. An elegant, fast, inexpensive and sufficiently precise preliminary technique for HPLC can now be provided which may be the method of choice for certain analytical problems. The technique has been named HPTLC, high performance thin-layer chromatography and the editors are fully aware of the implication of this notation. The following list of characteristics demonstrates the potential of HPTLC. Separation capability: up to 40 different substances, completely separated in one single run. Separation power:

separation of more than 5 components per minute (time averaged).

Analysis capacity:

- HPTLC as a preliminary technique for HPLC: analysis of one sample or a group of substances. - HPTLC as a routine technique: analysisofup to 12 samples in one single run using the micro-circular technique; analysisof up to 8 sampleswith the macro-circular technique and up to 40 samples in the linear technique. Using the continuous flow method, more than 10 samples can be analyzed successively.

Qualitative analysis: Near a Rf-value range of 0.5, real Rf-values with relative standard deviations of f 1% can be obtained when the circular technique (Uchamber-CAMAG) is applied. Quantitative analysis: Relative standard deviationsof+ 2.3%or better are obtained with the linear technique in the nanogram range. The circular technique on 100 x 100 mm plates has relative standard deviations of 1.5 to 1% or better.

*

9

Rf-value range in qualitative analysis:

In circular TLC: from Rf,eal = 0.01 to Rfreal = 1.00, U-chamber, controlled flow of the mobile phase and known composition of the gas phase.

Rf-value range in quantitative analysis: From Rf 0.5 to Rf 0.7, ratios of 1 :10,OOO. The method has the potential of operating at the femtogram level. Sample dosage:

Precision in the 100 nanoliter range: f 1%of the applied volume, when dosed on the wet layer; no memory effects or system losses.

Transfer of data:

From HPTLC to HPLC; transfer also possible from the circular continuous flow method when a multiple component mobile phase is used.

Time:

As low as 120 seconds per analysis.

HPTLC is defined as the combined action of several variables which include: 1. an optimized coating material with a separation power superior to the best HPLC separation material. 2. a new method of feeding the mobile phase 3. a novel procedure for layer conditioning 4. a considerably improved dosage method 5. a competent data acquisition and processing system.

Thus a complete system and procedure is discussed here. This should be understood as a stepwise improvement of an analytical method, which has been a powerful tool since the pioneering work of E. Stahl. The results thus far obtained, as well as the promising aspects of the new method are encouraging enough to refer to the technique as the second generation of thin-layer chromatography. The final judgement however, will be left to those who use this new methodology. This book is not intended for theoreticians, however, we would like to correct some biased opinions with the aid of some new methods of presentation. We would like to assert that real Rf-values can be obtained with very high precision on a 5 x 5 cm plate in 150 sec., when the phase conditions are optimized. It will be shown’that the real Rf-value can be transformed directly into the chromatographically more useful capacity factor k = $/h. This can be effected even if we deal with adsorption chromatography, where elution is generally done with mixtures of organic solvents of different polarity. It will also be demonstrated that in TLC it is now meaningful to refer to plate height. It is possible and useful

10

to measure precisely basic chromatographic data such as the phase ratio, which has not been readily accessible. The application of HPTLC with respect to quantitative analysis is described in detail. HPTLC has been developed in close cooperation with Ute Hezel, J. Blome, D. Jaenchen, H. Halpaap, J. Ripphahn and R. E. Kaiser. The research, done in the Institute for Chromatography, was initiated by J. Blome, who recognized the principal advantage of circular TLC, L.V. Andreev, who pointed out the efficiency of extremely thin TLC coatings, F. Eisenbeiss, who prepared a 5 p-plate without binder which could separate 10 dyestuffs in 13 seconds (l), D. Jaenchen who combined the prototypes ofthe new chambers, dosage systems and accessories with the CAMAG dosage system and thus created a technically useful tool, and H. Halpaap who prepared a general applicable HPTLC plate, which was exclusively used by the authors of this book. At this writing, there were no plates available which could have been used for other than adsorption based separations. J. Blome begins his article “Advantages, Limits and Disadvantages of the Ring Development Technique” with a review of chromatography on thin layers during the last 150 years, stating that in the earlier period, the superiority of the circular technique over the linear technique was already recognized. It appears to be the fate of this chromatographic method, that knowledge previously acquired must sink into oblivion before being revived and acknowledged. The short analysis times, which can be accomplished today are not a novel achievement, except for the work by F. Eisenbeiss which will be discussed later. It is also quite likely that controlled dosage of a continuously flowing mobile phase in TLC has been used by others. Reliable, fast and inexpensive analytical data are needed. Problems will be more readily solved, if the data are more accurate, or if they provide a basis from which we can operate. The final source of errors will be mistakes in the interpretation of the data. J. Goldman and R. R. Goodall have recently reported TLC separations within 60 seconds at migration distances of 20 mm (2-8, 1969-1973). In 1967, E. Stahl discussed the reduction of analysis time as a function of particle size of the coating material (9). H. Halpaap was one of the first to examine the influence of various particle sizes on running times, Rf-values and plate heights in his article “Erzielung von reproduzierbaren Trennungen durch Verwendung standartisierter Sorbentien in definierten Systemen” (10, 1973). L. V. Andreev drew considerable attention to HPTLC, as we know it today when he described his rapid separations by micro thin-layer chromatography. However, the data which can be found in the current literature are not as impressive as those presented during the 1973 meeting in Novaky CSSR. The results were indeed remarkable for the times (1 1). 11

The potential and scope of HPTLC can be seen from different points of view, and various aspects can be emphasized in the application of the new, optimized plates. Continuation of the conventionaltechnique will lead to disappointing results since regular dosing would neutralize the tremendous separation power of the new coatingmaterials.The new circular separationchambers which have been especially developed for the HPTLC plates will give results which will be impressive: accurate data can be obtained within seconds, the size of the TLC plate can be reduced by a%, only 1/1OOO of the mobile phase is necessary and the analysis time can be reduced by a factor of 10. The precision can be improved ten-fold, compared with conventional TLC. Basically this is what is meant by the concept of HPTLC, and the way it should serve those now using TLC or who are new in the field. For those who are engaged in the problems of HPLC, W L C offerspossiblealternatives. HPTLC does not require expensive instrumentation, when only modest precision and reproducibility are necessary. Under such circumstances HPTLC can be carried out almost anywhere. All the necessary tools, including chemicals can be conveniently carried. In the first chapter of this book, a brief introduction into the theory of TLC is presented. Then, J. Blome describes the theory and practice of the circular development technique. In the next chapter the U-chamber will be discussed. Besides the precise determination of real Rf-values, qualitative HPTLC can be used for routine analysis and as a rapid preliminary technique for HPLC. The continuous flow micro TLC is another stronghold of the method. The last section of the first part of this book is dedicated to standard dosage techniques in HPTLC. Since they are the basis for precise, quantitative data, they will also be mentioned by the authors in the following chapters. A very structured article by Ute Hezel describes the “Potential and Experience in Quantitative HPTLC”. Her excellent work in circular micro TLC is indicated by the quality of the quantitative data and the practicality of these methods. In his article “Considerations on the Reproducibility of TLC”, D. Jaenchen emphasizes the latest findings and those items which although important, are generally not published, e.g. the problem of relative humidity in adsorption TLC, and similar effects in distribution TLC. The next article “Application of a New High Performance Layer in Quantitative TLC” was written by J. Ripphahn and H. Halpaap. The authors screened a multitude of materials while developing an optimized TLC plate, the most important accessory in HPTLC. Used improperly, e.g. spots of 1 mm in size - the plates perform like conventional materials. The adjustment and control of the physical and physicalchemical conditions of the gas phase, liquid phase and solid 12

phase in TLC can be readily achieved by miniaturization and other techniques. If the conditions are chosen correctly, very interesting results can be obtained. The book concludes with a set of tables, which contain new data of H. Halpaap. This particular set of data represents a sequence of silica gel polarities for different mobile phases (listed incorrectly in previous literature). The authors of this volume have demonstrated that HPTLC, as a new competitive analytical method, is able to provide solutions for complex separation problems. The editors trust that the readers will be benefited by these studies.

November 1,1976

13

REFERENCES (1) F. Eisenbeiss-Darmstadf private communication; very narrow silica gel 60, approximately 5 vm, layer without binder, mechanically unstable, but highly permeable. Public experiment during a HPLC-seminar at the Institute for Chromatography, Bad Diirkheim, 1973. Separation number higher than 20, migration distance 20 mm, migration time 13 second, 10 components, completely separated. Layer permeability strongly increased after two years of storage, stable sharpness of separation. (2) J. Goldman and R. R. Goodall, J. Chromatog. 32 (1968) 24 (3) J. Goldman and R. R. Goodall, J. Chromatog. 40 (1969) 345 (4) J. Goldman and R. R. Goodall, J. Chromatog. 47 (1970) 386 (5) J. Goldman and R. R. Goodall, J. Chromatog. 71 (1972) 297 (6) J. Goldman and R. R. Goodall, J. Chromatog. 73 (1972) 161 (7) J. Goldman and R. R. Goodall, J. Chromatog. 78 (1973) 7 (8) J. Goldman and R. R. Goodall, J. Chromatog. 78 (1973) 153 (9) E. Stahl,Z. f. Analytische Chemie 236 (1968) 294-310 (10) H. Halpaap, J. Chromatog. 78 (1973) 77-78 (1 1) L. V. Andreev, ref. in Chemical Abstr. 81 (1974) 9487u L. V. Andreev, ref. in Chemical Abstr. 78 (1973) 4069k Discussions in a conference during the Bratislava-Chromatography meeting, fall, 1973, in Novaky.

NOMENCLATURE In order to aid the reader through the chapters where varying nomenclature has been used, the following explanations are given: HPTLC - high performance thin-layer chromatography Rf might also be written as Rf or RF Data labeled with RRf = Rfc are obtained by circular TLC Laminar TLC and linear TLC are synonymous and should be distinguished from circular TLC. The symbols used in this book were adopted from Geiss "Die Parameter der DC" (The Parameters of TLC). Net retention time $ and dead time are used to emphasize the principal theoretical relationship with the elution methods. Considering these time values, one should keep in mind that they represent migration distances, clearly, the relative migration distance. All time related chromatographic data are expressed as relative values. 14

Chapter 1

Simplified theory of TLC R. E. Kaiser

This section demonstrates the practical use of some basic formulae as an aid in the optimization of routine HPTLCand data transfer from HPTLC to HFLC. Although it goes beyond the scope of the work of Geiss (1)it does not represent a complete theoretical treatment of TLC. 'Ibe Practical Experiment and its Interpretation Ihe Rf-value and k The Rf-value is defined as the ratio of the migration distances. Zsubstance =Rf h o b i l e phase System errors influence this fundamental qualitative TLC-value, when the exact position of the solvent front (Zmobile phase) cannot be located. The loss of mobile phase or the "piling up" of mobile phase components already present in the layer will also affect the Rf-values.In the first case the Rf-values become systematically too large and in the second case too small. These Rf-values cannot be used to calculate k. However, real U-values can be obtained when the following conditions are met: - no gradients along the separation path - no loss of mobile phase - the correct position of the front can be calculated or measured without errors - by excluding any influences resulting from prevaporization The experimental approach to these conditions is a technical question and is not relevant to this section. The small k, the capacity factor of a substance is defined as "the ratio of the retention time in the stationary phase to the retention time in the mobile phase".

15

This is the simplestand most fundamental formulation of the qualitative-chromatographic behavior of a substance. In TLC everything takes place during the period:

In elution chromatography measurements can be taken only after the period of 1 x tm. A substance with a capacity factor of k will be eluted after the time:

This simple observation leads us to the basic relationship between Rf and k.

Suppose a substance has a Rf-value of 0.1, it would take a time of 9 x tm until this substance had reached the position of the original solvent front, i.e. if we would have used continuous flow TLC, which is essentially the same as column elution chromatography. Thus we can formulate:

Rf=

ts tm

Since k = -we can conclude:

tm ~

tmS

~

1 k+l

(3)

tm

= Rf = ___

tm+ts

where tms = tm

+ ts

the gross retention time or the total time, which the substance is retained in the stationary phase and mobile phase. In other words, it is the same retention time which passes from dosage to exit of the substance in column elution chromatography. A relation of absolute time values is clear and unambiguous and therefore it is of advantage to use the k values. However, this does not guarantee correct values, because ,$, and ts respond independently to similar or different chromatographic conditions. In case the temperature changes, and perhaps tm will change, but most probably not in the same direction or magnitude. To a great extent k is a function of the chemical state of the phases. HPTLC data can be transferred to HPLC systems only if the phases are the same. Closed (adsorption) columns must be conditioned to an extent where the chemical state of the chromatographicallyactive surface is stabilized. The same equilibrium conditions are required for systems with an exposed layer. Abrupt changes in the chemical state of this layer, which is in equilibrium with the surrounding gas phase are initiated, when the flow of mobile phase stops even fora short time. The mobile phase consists of solvents of different volatility or polarity. It is therefore very important to execute a dynamic continuous flow TLC, even during dosage of the 16

sample,This requirement is more stringent in TLC than in LC, where the stationary phase is embedded in a column. The flow through an equilibrated column can be stopped for a few minutes without any effect. Thus the data transfer from HPTLC to HPLC must be a dynamic process. The experimental approach will be described later. How are the data obtained? (a) by photography where the substances can be seen on a photographic plate, hseconds after the start of the sample. (b) by stopping the flow of the mobile phase, calculating the location of the extrapolated front, measuring the positions of the stained substance and computing the Rf-value. Dynamic continuous flow TLC can be achieved in a circular mode, and is easy to control because of the flow function. This function as well as the conversion from circular to linear Rf-values will be described later in connection with data transfer. This will be followed by a discussion of the recovery of the real values.

If a substance does not migrate, i.e. not even a trace of the substance can be detected in the mobile phase, then its elution would last 00 x h;the Rf-value is zero. A substance which migrates half of the separation length (Rf = 0.5) would require a time of h + h for its elution. Its retention time in the mobile phase would be the Same as the retention time in the stationary phase, and therefore:

A substance which migrates with the front is not retained by the stationary phase. Its Rf-value is 1 and:

k =&t

= 0/1

=O

From these few observations we are able to obtain the table Rfvs. k. Intermediate values can be found at the end of the book; they were calculated from the equation: k=-- 1 - R f

Rf

(4)

under the condition of real Rf-values. 17

k

Rf

00

0

9 4 2

0.1 0.2

1 0.5

0.5

0

1.0

0.333 0.666

Real Rf-values

In order to obtain real Rf-values it is importar! to preven any loss of mobile phase by evaporation or any increase, caused by condensation from the gas phase. Consequently a HFTLC chamber for the obtention of real Rf-values has almost no gas phase, does not exhibit a temperature gradient, the layer is in a horizontal position and is fully symmetrical. This is the only effective way of eliminating any disturbance of the phase ratios. In other approaches, complex equipment is used to compensate and regulate the many variables which influence the Rf-value. A symmetrical, small, flat chamber, which has a controlled constant mobile phase flow is primitive in a positive sense and therefore simple and functional. The constant phase flow is measured directly: 0 prior to the layer, the flow is directed on an analytical balance, or it is measured by volume integration, or a precision syringe is used which causes a constant flow by a constant piston movement. This is easily accomplished by the constant rotation of an electronically controlled stepping motor, which has the flexibility of a stepwise adjustable constant frequency. Conclusion: The correct position of the point is obtained by calculation or calibration, and is not distorted by a preloading. It is understood that the layer is homogeneous in all three dimensions, i.e. it displays high performance quality. Before the position of the front can be calculated the flow function in circular TLC must be understood. It is generally known that the migration velocity in regular, linear TLC is dependent on many factors, such as the specific, flow controlling properties of the layer, the surface tension, the wettability, the viscosity of the mobile phase, the temperature, etc. All these factors lose significance when the phase flow is controlled. The flow is fed up to the adsorbing capacity of the layer. ' h e flow function in HPTLC

In conventional TLC, the feed of the mobile phase is a potential source of errors. It is influenced by many factors which are difficult to control. A more detailed discussion of these factors can be found in "Die Parameter der DC" by Geiss. 18

The flow function can be expressed as: (Zf)2 = t x K: IC = velocity constant in cm2/s zf = migration distance of the front in cm after t seconds. t = time in seconds. The mobile phase is not aware of its flow direction, therefore the flow function can be applied for linear as well as circular TLC: (Zf)2 = t x K The circular area, covered by the mobile phase = TT (Zf)’. Consequently this area is a linear function o f t x IC,which means that the phase flow is constant. In linear TLC, the phase flow decreases quadratically as a function of time. The flow is further reduced by unavoidable evaporation of the mobile phase. Since the evaporation rate is proportional to the area, the expansion rate of the wetted area decreased quadratically. A controlled flow in circular TLC can easily be maintained. If high precision is not needed, the mobile phase is directed onto the plate by gravitational flow using a capillary tube which is connected to a reservoir. A controlled flow in linear TLC, can only be accomplished with electronically regulated pumps. Here two pumps can be used for gradient techniques. The controlled flow of the mobile phase in circular TLC is relatively independent of the chemical and physical nature of the mobile phase. Controlled flow can be applied in the range of 0.4 to 2 pl/s, for almost every mobile phase, except water and materials which have a viscosity similar to n-butanol. A flow of 1 pl/s on HPTLC plates is a realistic value for most of the phases. Why is this possible? The flow of a liquid along a flow resistance is described by Poiseuille’s equation:

d = effective width of the flow resistance L = length of the flow resistance p = pressure drop (in TLC: capillary force) q = viscosity of the mobile phase b = factor to balance the different dimensions If the mobile phase is pumped onto the plate, the layer will adsorb a considerable amount until that point is reached where the flow resistance builds up. The larger 19

the area, by which the mobile phase enters the plate - or more correct - the wider the circumference of the “entry circle” on the adsorbing layer - i.e. a larger “d” in Poiseuille’s equation - the higher is the flow in the layer. Since the amount of mobile phase, which can be adsorbed per millimeter entry band within a certain time (at a given flow) is determined by the flow function, the length of the entry band (i.e. the diameter of the drop) is related to the location of the point of entry for the mobile phase. The plate must be mounted in a horizontal, vibration free position which excludes any “draft” or similar effects. The size of the drop can be used to adjust the flow to the optimum adsorption capacity of the layer. Homogeneous layers, consisting of the same material, require the same amount of mobile phase for optimum flow conditions, provided that the chemical nature of the mobile phase and temperature are the same. The Rf value of 1.00, i.e. the exact location of the front after t seconds, (after the last drop of the pumping step is used up) can be determined as follows: A. Drill a completely circular area into the layer of the TLC plate (a 1.5 mm borer is placed in a hole of a dosage template, 10mm away from the center of the plate).

B. Measure the total volume of this disc: Vt =Dn? r,D = radius and thickness of the disc (can be measured with magnifying glass and micrometer). C. Apply the mobile phase on the center of the disc. Use a reasonable flow of approximately 1 pl/s. (i) maintain the flow until the periphery of the disc is reached (“effective”). (ii) continue the flow until the disc is completely saturated (“full”).

Observations here will determine whether the mobile phase evaporates and at what rate. In this case it is necessary to readjust the flow, so that the plate always contains a tiny, excess drop of mobile phase (ths can be observed through the glass plate).

D. Either the time, or the flow multiplied by the time - the dosed volume to fill the disc - is a measure for Rf = 1.00 with Zf = r. A complete calibration curve can now be obtained for any value r = Zf as a function o f t or V dosage. The loss of mobile phase by evaporation is included in this calibration. 20

T on the disc after saturation is:

vs = Vt-V,

(7)

Vs = volume of solid phase = stationary phase Vm = volume of mobile phase Vt = total layer volume The phase ratio is given by:

In the following calculation, the non-saturated phase volume Vm effective is more important, therefore the value for As/Am in the Martin relation will change slightly. log

I -Rf = log Rf

As Am

--

+-Lip RT

This equation, which is of significance, provides a relation between structure and chromatographic behavior. A basic factor in the determination of real Rf values, is the comparison of the visual position and theoretical position of the front. It will reveal whether losses or increases (due to humidity conditioning) need to be considered.

F = flow, i.e. F . t As/Am

= phase

=

dosed volume

ratio, measured as “effective”

Zf(nomina1) = nominal value after t seconds (after the drop is used up) Zf(nominal) represents the valid value of the solvent front (in circular TLC) Example: HPTLC - silica gel plate, Merck, Dec. 1974. Mobile phase: toluene, 20 ppm (w/w) HzO, temperature: 200 C, relative humidity: 40%

Surveying the plate: Total Thickness (glass + layer)

Thickness of glass only

1.435 mm 1.430 mm 1.435 mm 1.433 mm 1.432 mm 1.425 mm

-

1.242 mm 1.250 mm 1.240 mm 1.240 mm 1.240 mm 1.240mm

1.432 mm average value 1.242 mm 33.0038 mm standard deviation .+M.004 mm +

+

+

From these data we can calculate the layer thickness D = 0.190 mm kO.004 mm. By repeated measurements with a micrometer, a standard deviation of f 4 microns can be obtained. The precision of the micrometer is k2.5 microns, however, the main error arises from the difficulty in stabilizing the contact pressure on the layer. Using the “dosage template” for the U-chamber (CAMAG) a circle was scratched in four plates. The plates (50 x 50 mm) are retained in the U-chamber plate holder. The radius of the circle was measured by three different methods. tadius ‘circle

=

10.58 k0.03 mm

Knowing D and r we can calculate the total volume Vt of the disc layer: Vt = 0.190 TT (10.58)2 Vt = 66.75 mm3, with a 2%experimental error. Toluene, n-butanol, water and methanol were pumped on the layer at a flow of 1 pl/s, until the solvent reached the periphery of the disc (“effective”). Pumping was continued to the point of saturation (“full”). In order to recognize the solvent front more clearly when it reached the “effective”state, the flow was maintained for a very short time. However, these values represent phase consumptions between “effective”and “full”. Results in mm3 after 50 s the brim of the disc was reached “effective” toluene rrbutanol water methanol

48 48 48 47

average value

47.9 k0.4 mm3

22

48 48

48

‘Ydl” 51 50 53 55 52.3

53 51

evaporation rate 53

3mm3/min 1

-

* 1.7 mm3

6

Therefore: Vm = 47.9 mm3 with 1%uncertainty for four different mobile phases. There is a difference of 8.4%between “effective” loading and “full” loading of the layer. This value is, however, not very realistic because HPTLC never operates under “full” conditions and should not be considered. Since: Vt - Vm = Vs eff. Vs eff = 66.75 - 47.9 = 18.9 mm3 ASIAm = 18.9f47.9 = 0.395

With these data we are able to compute Zf (nominal)

J=.

21- (nominal)

47.9 (0.335 + 1) = 10.6 mm 3.142 x 0.190

This value has an uncertainty of 2%. The main errors result from inaccurate measurements of the layer thickness, which is assumed to be constant throughout the entire plate. The correctness of this assumption can be examined in an experiment where we measure the rate of increase of the wetted area as a function of time. Provided the flow of mobile phase is constant we are able to draw conclusions regarding the constancy and linearity of the increase. A suitable mobile phase is applied on a conditioned, equilibrated plate and the time and wet area are recorded by repeated photography. This arrangement allows the measurements of Zf as f (t) to a precision of 0.02 mm.

Here the problem arises, that within a short time a “drop” is formed which is not yet added to the dosed volume. Linear regression will account for the volume, or the equivalent of its volume in the wet layer, which is determined by the specific properties of mobile and stationary phase. These properties, combined with the given or adjusted permeability of the layer make up the flow resistance.

If all the previous statements are true, a linear relationship between time and wetted area should be found. Deviations would indicate system errors in Rf-values, which, for example, are time dependent. Simultaneously, Rf-values should be meusured. They must be independent of the migration distance, otherwise system errors must again be considered. 23

Measured values:

Time (seconds)

Measured, wetted area (mm2).

area and A calculated by regression

8 23 39 54 68 83 113 143 173 203 233 292 353 413 473

69.4 134.8 188.7 254.5 314.2 373.3 490.9 611.4 735.4 855 962 1200 1452 1669 1885

77.2 136.2 199.1 258.2 313.3 372.3 490.3 608.4 726.4 844.5 963 1195 1435 1671 1907

- 7.8 - 1.4 - 10.4 - 3.7

+ 0.9 + 1.0 + 0.6 + 3.0 + 9.0

+10.5 1.0 + 5.0 +17.0 - 2.0 -22.0

-

measured and calculated migration distance Zf

4.7 6.6 7.8 9.0 10.0 10.9 12.5 14.0 15.3 16.5 17.5 19.5 21.5 23.0 24.5

5.0 6.6 8.0 9.1 10.0 10.9 12.5 13.9 15.2 16.4 17.5 19.5 21.4 23.1 24.6

After 8 seconds, the area is 7 mm2short. This is caused by the volume of the drop or the delay in the expansion of the wetted area. The regression coefficient over all values is = 0.9998 i.e. there is a strong linear relation between time and wetted area. Zf measuredkalculated differs only in the limits of the measuring error (0.1 mm), therefore we can conclude a strong lineariry between time and wetted area. The area in question is circular to the extent of M S % . This means, the HP coating was homogeneous within these limits. Consequently the migration distance of the front zf can be calculated from the area with an accuracy of 0.1 mm, and, as we will see later, it also can be measured with the same accuracy. Since the calculated and time correlated value for Zfas well as the measured Zfare identical, any loss or increase of the phase is insignificant; even errors resulting from prevaporization are unimportant. (Toluene, 20' C, silica gel, Merck, HPTLC plate, no prevaporization, CAMAG U-chamber). The position of a hypothetical substance with an Rf = 1.00 can be measured or calculated with an accuracy of 1%, at migration distances as small as 10 mm. Assuming a sample contains a substance which is soluble in the mobile phase and is not retained by the stationary phase, the Rf-value of this substance Rf = 1.00 and this will be a real Rf-value.

24

Since Zf (limit) can be obtained accurately, all the other Rf-values have to be real values, q.e.d. The controlled flow of mobile phase on a fully homogeneous layer, which is wetted completely symmetrical, with almost no gas phase above it, as well as a precise dosing in terms of position and quantity, introduces a new quality in 7ZC. The combined action of the optimized coating and this specific development produce real and therefore transferrable data which allows the use of qualitative analysis to its full potential, such as structure determination by means of chromatographic data. HPTLC data should not be discussed in terms of conventional TLC. To prevent any misunderstanding, it should be emphasized that our findings are not based on one experiment or the use of toluene as mobile phase, however, only one HPTLC plate material was used since no competitive product was commercially available in 1976. Mobile phases which are more volatile require an adjustment of the development conditions in the U-chamber. These include the control of the temperature and determination of the evaporation rate. The method was described earlier. Whereas accuracy requires some effort, it indeed merits the additional work.

Rf-value as a function of the migration distance in circular HFTLC

The measurement and results, mentioned at the end of this section must now be confirmed in practice. The Rf-value ofa substance was photographedand measured during its development. The results are interpreted as follows: 1. The reproducibility of the Rf-values impose high requirements on the method: no dosage errors no plate irregularities and no phase errors.

2. Factors which influence the flow of the mobile phase and the position of Rf = 1.00, i.e. the front, have to be considered in the result as a trend. 3. If a trend is apparent, it is necessary to examine by means of statistical, mathematical tests whether the trend is significant or can be neglected. Results

Coating material: HPTLC silica gel Dec. 1974 Mobile phase: benzene, containing 15 ppm H 2 0 (w/w) Flow: 1 pl/s at 200 C 40% relative humidity, equilibrium conditions, red dye, lo-’ gram, U-chamber, circular TLC 25

Time (s)

Rfcircular

Rfiinear

68 113 173 233 292 353 413 473

0.236 0.237 0.234 0.225 0.227 0.230 0.230 0.234

0.0557 0.0562 0.0548 0.0506 0.0515 0.0529 0.0529 0.0548

Rfiinear Calculated from (Rfc)* after zf = 10.0 mm 12.5 15.3 17.5 19.5 21.5 23.0 24.5

Evaluation

Average value Rfc = 0.232 kO.004 Average value Rflin = 0.0537 +0.002 n = 8 different chromatograms Trend: no trend noticed The question here is not the reproducibility of given Rf-values, by measuring the position of the ring maximum and front line, but the repeated measurements of changing chromatograms. Therefore, any erroneous position of Rf = 1.00 can be detected if it is 3 times larger than the standard deviation, i.e. kO.015. In this case, a trend would be assured. However, no influence of this kind was located in the above set of measurements.

In circular HPTLC, Rf-values in the range of 0.2 are reproducible to +2% of this volume. (at Rf = 0.5 : s = 1% rel). Transformations to linear Rfiralues, which are the only basis fork determinations or other data, are therefore reliable and this will be shown.

+

Correlation between circular HPTLC Rf-values and real linear Rf-values

According to Geiss (1):

This relationship is sometimes considered controversial. HPTLC would be a suitable technique for resolving the problem because of its highly accurate data. However, a linear chamber, comparable to the extremely accurate circular chamber - the L-chamber of the Institute for Chromatography is available only as a prototype (1976). Consequently the following data are non-homogeneous. 26

Linear Rfvalues, obtained from a micro-chamber of the N-type

Even though the cost of using a micro chamber of the N-type is considerably higher than that of the classical N-chamber, the speed and accuracy of the results merit this investment. Circular Rflvalues obtained from a U-chamber

Comparison: (R, B, G , V: sample components) HPTLC layer Dec. 1974, toluene Start of the sample: linear at Rf

R B GI G2 V1 V2

0.12 0.20 0.36 0.44 0.53 0.76

=0

0.12 0.20 0.35 0.45 0.52 0.76

Circular: a) Rf = (Rfc)’ application exactly at the entry point of the mobile phase

b) eccentric application

0.12 0.20 0.36

0.06 0.13 0.26 0.31 0.40 0.66

0.12 0.20 0.35

0.44

0.44

0.53 0.78

0.53 0.79

0.04 0.10 0.22 0.32 0.39 0.71

Conclusion Rflinear is completely similar to (Rfcircular)2, when the starting point is located exactly in the center of the circular chromatogram. If the substance is applied more distant from the center point, the chromatogram will resemble a run in linear TLC. The data also indicate that the Rf value in regular TLC may be erroneous because of a change in the Rf = 1.00 position. The discrepancy between RflinV2 = 0.76 and RfcircV2 = 0.78 is not accidental. Probably the value 0.785 will be correct. However this is of secondary importance, since we wanted to confirm the equation by means of the HPTLC circular technique

The starting point of the sample, and the entry point of the mobile phase must be the same. This imposes certain requirements on the technique. It is well known that a substance, surrounded by an excess of mobile phase, does not migrate but diffuses in all directions. The drop formation, as a consequence of the flow resistance, causes a “blind” spot where the mobile phase is fed.

27

It is therefore necessary to:

1. Start the HPTLC analysis with the lowest possible flow, when circular TLC is applied or 2. Start or stop the flow, alternately, until the essential sample components have left the "blind" area of the drop. This can easily be automated, and would provide a routine aid in preventing starting errors.

This is only of interest when real Rf-values are needed for data transfer, structural information, TLC in combination with other analytical techniques or Rf-value determination in the range of 0.1 or smaller, where circular TLC is superior to linear TLC.

If micro circular TLC is applied as a routine technique, constant correction factors may be used which are dependent on the diameter of the starting circle for Rf = 1.00 and which can be calculated for each Rfc value. In this case, dosage is done exactly in a circular line, which can be easily accomplished with the dosage template of the CAMAG U-chamber or a circular Evachrom applicator (Chapter 4). The drop of the mobile phase no longer has any influence since its diameter and the dosing circle will be adjusted to each other, to avoid any overlapping. It is obvious that high precision data are only useful if combined with a maximum separation power of the layer, and the smallest possible starting spots or circles for the sample. One of the practical aspects of qualitative and quantitative TLC, is the direct influence of the starting point width on the final result and separation power. It is also of practical interest to know the lower limit of differentiation between two Rf-values, i.e. the point where the system error becomes noticeable. The migration distance, in relation to the quadratically increasing analysis time but only linear increasing separation capability, is another practical and maybe even economic aspect. A very simple and compact theory will now be presented, which can answer these questions by means of a minimum amount of measurements and calculations.

Separation capability and separation power in HPTLC

In other areas of chromatography, the separation number is an established concept, which offers definite advantages: measurable with sufficient accuracy, simple, relatively independent of the chromatographic technique, easy to handle and suitable for comparison and optimization. 28

Separation number

Definition: the separation number defines the number of substances, which are completely separated between Rf = 0 and Rf = 1, provided that the separation conditions are isocratic (restriction: in quantitative chromatography, the separation number is defined as the highest possible number of completely separated substances, between k = 0 and k = 10. In terms of Rf-values, this means the number of completely separated substances between Rf = 1.00 and Rf = 0.091).

Two substances are completely separated, when the distance between the two adjacent peak maxima and the sum of both peak widths at half peak height are the same (according to the linear regression line). Start plot the widths b0.5 above rhe Z values in an extended scale. Extrapolarefor 61 and bo

h-

Determination of the separation number:

Separation number: SN =

~

Zf -1 b+bl

zf

=

migration distance of the front

bo

=

extrapolated width of the starting spot at half-height of the concentration curve

bl

=

extrapolated width of the spot with Rf way as above).

* this

is

=

1 (extrapolation done the same

an approximation. A morr sophisticdted formula leads to sligthly higher numbers. See chapter4

29

Even if the sample to be separated does not contain a component which either remains at the starting point or migrates to Rf = 1, the separation number still can easily be measured. Linear interpolation is done by plotting or with the aid of a pocket calculator, which can carry linear regression. A computer program is listed in the appendix of the book. Measurement and calculation of the separation number (valid for linear and circular TLC). Substance

Width of photometrically measured spots at half peak height “b”

migration distance (same scale as b)

1 2 3 4 5 6

(4.8 mm) 4.8 5.6 6.4 7.2 bl

19 mm 30.5 47.6 70.5 101 127 (= Zf, front)

Linear regression shows, that “b” of substance 1 does not correlate with the other values. An examination of the chromatogram reveals that the layer might have been overloaded by substance 1. Consequently the peak width at half-height is not the result of the size of the starting point or time dependent diffusion caused by exchange processes during the chromatographic separation. Additional effects (in addition to overloading) need to be considered, such as nonlinearity in the signal transfer from the photometrically measured gram/mm2 on the plate, to the recorder or integrator signal of the data processing unit. Conclusion on the source of the error can be drawn only from a set of data and not from one single chromatogram. Linear regression therefore uses only correct values. Plotting of the data will immediately show which value does not correlate with the others. Results

Substance ‘0’ 1 2 3 4 5

Substance “1” Separation number

30

=

b measured

b calculated by linear regression

bo = ? (4.8) 4.8 5.6 6.4 7.2 bl = ?

=

127 3.9 + 8.2

3.9

4.5 4.9 5.5 regression coefficient = 0.98

6.3 7.3 8.2

- 1 = 9.5

Certainly not an excellent result, considering separation numbers of20 to40, which can be obtained by HPTLC, but up to 18 substances could be detected simultaneously on this plate. The accuracy of the Rf-value determination depends on SN

A Rf

=

Rf differences between two adjacent, but completely separated spots. ARf=-

1

SN+ 1

Applied to the above data:

A Rf difference of 0.05 is the lowest value for two substances to be separated. Important: SN determinations require isocratic, i.e. gradient-free TLC. Changes in the mobile phase flow must be homogeneous.

Plate height and separation number in TLC Recording plate height or plate numbers in TLC, has been reported as meaningless. The question remains however, whether it is useful to apply the concept of plate height. We believe it to be worthwhile, especially when comparing coatings and separation systems to be optimized. We would like to correct a common opinion in chromatography, that the use of theoretical and effective plate height is of value. One cannot assume that a mathematically elegant solution of some problem is necessarily of practical value. The plate height or number of plates is dependent on diffusion processes which result in a spreading of the sample spot. This is true for all chromatographic techniques, and is demonstrated in the following figure under the condition of isothermal and isocratic chromatography.

b

31

The ratio t/b is the basis for the following expressions: number of effective plates: n = (ts/I~().5)~ x 5.54 number of theoretical plates: N = (tms/b().5)*x 5.54 effective plate height: h =L/n theoretical plate height: H = L/N L N n H h

= length of the

separation path in mm number of theoretical plates, dimensionless = number of effective plates, dimensionless = theoretical plate height in mm = effective plate height in mm hs= gross retention time in mm 6 = net retention time (6+ h= hs), which is different from the gross retention time by the dead time h,the time in which TLC separation takes place. b.5 = peak width at half peak height, bo 5-values of TLC spots or rings are obtained only after recording the regression line. Dimensions: in mm, which is the same scale as the time values or the equivalent migration distances. =

Imporrant: The time values are recorded by fast, linear scanning of the TLC

chromatogram along the migration distance. It is obvious from the above figure that the t/b ratio is dependent on the t value. This is the precise point: since the theoreticians are not concerned with the practical aspect that each chromatographic analysis requires time and material, they neglected a very simple fact, which will be presented in the following figure. The relationship between t and b is given by:

but never by: bib,

32

= k/bc

The expression accounts for the fact that:

1. the dosage requires space and time 2. the t/b ratio is a measure for separation power and separation capability of a given system, independent of the actual chromatographic position (t) of a substance. The distinction between “theoretical” and “effective” values now becomes unnecessary; thus we can formulate the number of real plates:

and the real plate height

as substance independent expressions. Nred values will be determined by means of linear regression or regression coefficients. In order to characterize a chromatographic system we need not rely on incorrect values and calculated linearizations, whose basis might not even be linear, Since this new concept (whch was proposed by the author in 1959 (2) has been applied in comparison studies, using different techniques in gas, liquid and thin-layer chromatography, we feel that all theoretical “interpretation problems” have been eliminated. It was stated earlier: SN+l=-

Zf bo + bl

and

where N1 redis the number of real plates for the substance at Rf = 1.00 from the expressions above we obtain:

33

Since: H r d =L m r d and in TLC: H1 red = zfm1real the separation number can be expressed:

The following conclusions can be drawn from these expressions. The determination of the number of plates is meaningfa for the substance with Rf = 1.00, i.e. the substance which migrated the longest possible distance. The corresponding b-values (bl - bo) are obtained by regression or plotting, with a high degree of accuracy.

Resolution, selectivity,separation capability The width of two adjacent peaks determines the selectivityof a separation system, which is necessary for a complete separation. We define the selectivity of a system as the ratio of the Rf-values:

Selectivity for two substances p, a =

!9 > 1 Rfa

The peak width depends on the migration distance

where

Z,is the migration distance of substance a

21- is the migration distance of the mobile phase, i.e. the front The “chromatographic” distance of two substances a and p can be expressed by:

34

This distance can also be represented as resolution R times an appropriate peak width. hence we can formulate:

The plate height can be calculated under the same conditions. The minimum migration distance, necessary to obtain a separation (according to Rf-values)can be calculated when SN is known The separation number - the result of a combined action between mobile phase and stationary phase - is determined by the plate height value, i.e. the coating material and by bo, which itself is a function of the dosage technique. The experimental parameters are fully integrated. (HPTLC, of course, would allow a fast, preliminary experiment, but calculationis less expensive, more elegant, more relevant and more efficient). The factor:

~

b1-bg becomes very practical. bl+b

Its value is not influenced by concrete data, but is dependent on the dosage technique and is therefore an important measure for this quality. Subsequently, an analysis should start with the smallest bo and end with the lowest bl, a logical and well known requirement. The following expressions are presented without comment: The number of real plates for Rf = 1.00

Real plate height of the layer

The minimum migration distance Zf, which is necessary to obtain a certain separation number SN, at a given layer quality, expressed in plate height Hi real:

Optimized chromatography utilizes the full potential of the most effective parameter: selectivity. The conditions for a separation problem, on a given coating material can be optimized by the right choice of mobile phase, i.e. its chemical composition, which means satisfactory separation in the shortest possible time. Since:

and Nu

=

( ")

ba - bo

2

x5.54

we obtain

The real plate number however, is independent of the concrete value for Z, therefore Na = N1 real and also

hence:

36

From this expression we obtain the basic equation of TLC:

or

In words: A satisfactoryresolution requires a minimum migration distance Zf,i.e. a minimum analysis time and a good coating material (low Hi red). The best results are obtained, when the system has a high selectivity for the two substances a and P to be separated. Or: a sufficiently high separation number and an optimum dosage technique combined with a maximum selectivity, guarantees an excellent separation.

Of practical interest, is the quantitative effect of the dosage quality.

is l%, 5%, lo%, up to 40% of bl, and the width of the spot Let us assume at the start, is of the same relative size as the spot of the substance with Rf = 1.00 at the end of the analysis (naturally determined by means of the regression lines). The relationship between bo and dosage quality is given below (The QD-VdUe always has a negative effect on the separation). bo values, relative to

bl

=

QD =

0.7%

1.3%

2.5%

5%

10%

20%

40%

0.99

0.97

0.95

0.91

0.82

0.61

0.43 37

Conclusions:

Slight inadequaciesof the dosage technique considerably reduce the quality of the separation, even if the coating material is excellent, because the factor QD has a very strong influence on the final result. The effects of a spread starting spot of a substance with a high Rf value are less pronounced than those of a substance with a low Rf value. This is a known fact, especially in circular TLC. It should be emphasized that bo, even for such substances with an insufficient HFTLC dosage system, can reach 80%of bl i.e. an effective QD ofO.11. The significanceof different phases has not been discussed. The reader is referred to the book of F. Geiss (l), who presents basic and important concepts in TLC. The basic data were obtained from dyestuffs on silica gel plates - although test substances with a different chemical structure should behave in a similar manner. The theory presented here is applicable to the A1203 - HFTLC plates which may be available soon, and plates suitable for stable reversed phase chromatography. We have used time-values, such as k = &/h, which require a linear interpretation. For a better understanding, one ought to keep in mind that these values are derived from the migration distance. The flow function should be viewed within this perspective. The expressions, which have been derived in this chapter emphasizing the time concept can be applied to all other chromatographic techniques. This versatility is indeed helpful.

Literature (1) F. Geiss, “Die Parameter der Dunnschicht Chromatographie, eine moderne Einfuhrung in Grundlagen und Praxis”, published by Friedr. Vieweg + Sohn Braunschweig,now Wiesbaden, Germany (1972), 282 pages. (2) R. E. Kaiser: Gas-Chromatographie, Akademische Verlagsgesetlschaft, GeestBEPortig KG,Leipzig(1960)23,BibliographischesInstitutAG,Mannheim (1960) 41.

38

Chapter 2

The separation number in linear and circular TLC J. BIome

1. Width of the Starting Peak b()

This section deals primarily with a mathematical model of the high separation power in circular TLC. According to R. E. Kaiser (Chapter 1,p. 29) peak broadening in TLC is, in the first approximation, a function of the migration distance (in contrast to LC, where the migration distances are the same for all substances). Since all substances have the same diffusion time (in contrast to LC, where different substances have different diffusion times, i.e. the peak broadening by diffusion is proportional to time), the effects of diffusion (B term in the van Deemter equation) are the same for all substances. Therefore diffusion and starting peak width are factors with a constant numerical value contributing to the migration dependent peak broadening. The difference in spot width at Rf = 0 (measured or calculated by extrapolation) before and aftera chromatographic run, is a measure for the “rest diffusion”. In the following, the term bo, the starting peak width, is understood as the sum of starting spot width and rest diffusion. It is important that both factors remain small. They are dependent on the migration time (long analysis times result in poor separation) and on the molecular mobility of the substance with respect to the stationary phase. We limit ourselves to the evaluation of average values, because fine differences will be levelled by individual substance parameters, such as different concentrations, etc. However temperature is an important factor (m/2 x $), its negative effect on TLC is known and usually avoided. The same is true for LC, except for ion exchange chromatography. Starting peak width and time dependent molecular diffusion are additive terms within a geometric progression, which describes the necessary space for the substances, distributed between start and front. Assuming a resolution of 4.7 sigma for these substances, i.e. the Rf-value differences of all successive substances (Rf‘m+l - Rfm) is equal to the sum of their peak widths at half peak height (Rfm+l - Rfm = bm+l + bm), we can express the separation number - the maximum possible number of substances, completely separated in the range of Rf = 0 to Rf = 1 under isocratic conditions - as the number of terms in a geometric progression.

39

2. Calculation of the Separation Number in Linear TLC After a linear TLC separation is completed, the peak width of a substance near the origin and the front, are extrapolated to Rf = 0 respectively Rf = 1. The corresponding peak widths at half peak height, bo and bl, are plotted as ordinates (Fig. 1) above the extrapolated abscissa values Rfo and Rf1. If the straight line through bl and bo is elongated to the point of intersection with the abscissa and the migration distance between Rfo and Rf1 is set equal to 1, we can express the elongated, straight line (v) from the point of intersection to Rfi as: bl

v=-

bl

-bo

The term v can be utilized to calculate the width (2 bn) of the last peak Rfn preceeding Rf1 :

As for the measuring technique, it is more reasonable to use the width of the last peak as starting term of the geometric progression. The number of terms represents the separation number (SN) and the sum of the terms represents the separation length (= 1). The quotient of the progression can be calculated as: q

40

v - 2bn = y

(3)

The terms bo and bl are inserted into the equation and the expression is converted into logarithms. Substituting into the equation for the sum of a progression

S=a

q" - 1 q-1

we obtain the separation number in linear TLC:

log

bo

-

bl l-bl+bg log l+bl-bg

SN = -

(4)

The results obtained from this expression (4) differ from those calculated by the equation l/(bo bl) - 1 according to R. E. Kaiser. The deviations between both expressions increase with the difference bl - bo. The following table represents both separation numbers with respect to bo and bl - bo (the values in parentheses are calculated by the equation of R. E. Kaiser).

+

bl

b

-bo

0.0 1 0.02 0.03 0.05 0.08

0.02

0.03

0.05

0.07

0.1

27.5 (24.0) 17.3 (15.6) 12.8 (11.5) 8.4 ( 7.3) 5.6 ( 4.6)

23.1 (19.0) 13.3(13.3) 11.6(10.1) 7.8( 6.7) 5.3 ( 4.3)

17.9 (13.3) 12.5(10.1) 9.8( 8.1) 6.9( 5.7) 4.9 ( 3.8)

14.8 (10.1) 10.7( 8.1) 8.6( 6.7) 6.2 ( 4.9) 4.5 ( 3.3)

11.9(7.3) 8.9(6.1) 7.3(5.3) 5.5 (4.0) 4.0 (2.9)

I I

The table provides information regarding the basic parameters, controlling the separation number in linear TLC: the plate quality (bl - bo) expresses the peak broadening as a function of the separation length and the starting peak width. Decreasing bo values result in higher separation numbers. Starting peak widths (the sum of starting spot width and time dependent diffusion) which are lower than 1-2% of the separation length are rarely obtained. The framed values in the above table are more realistic, especially if no specific techniques or materials are applied, e.g. the thinfim plate according to E. Cremer. 41

3. The Space Diffusion Model

The earlier derived separation number (4), which accounts for the geometric progressionof the peak widths, is based on the assumption that the broadeningofthe starting peak is proportional to the migration length. A three dimensional diffusion model (sphere), where the expansion of the space dimension is limited by the thickness of the TLC-layer, seems to offer more advantages. Instead of a spheric diffusional space, an equivolume cylinder is formed, whose height is defined by the thickness D of the TLC layer, and whose radius is the peak width, measured at half peak height. The starting peak width bo corresponds to the radius do of the spherical diffusion space, and the front peak width bl corresponds to the radius dl. Assuming a linear increase from do to dl in analogy to bo and bl, we are able to calculate the corresponding, corrected values for the peak widths (b’ values). Except for b’o and b’l which are identical to bo and bl, the b’ values form a curve, which appears below the b line (figure 2). The values for do and d l can be calculated according to:

42

subsequently:

and

the b’ curve is obtained from:

A regression calculation (calculation of the partial migration distance Rf1 to Rfo 5 and Rfo 5 to Rfo from b’ at Rfo 5) reveals the deviation of the separation number (SN’) with respect to the values obtained from equation (4). The parameters for this deviation are the starting - and front peak width, not the layer thickness D, which can be seen from the following table.

dl b.5

0.02 0.12 0.025 0.0196 0.0646 0.0631

0.02 0.12 0.001 0.0067 0.0221 0.0631

0.01 0.1 1 0.005 0.0072 0.0357 0.0515

0.01 0.11 0.025 0.0123 0.0610 0.0513

0.02 0.07 0.005 0.0115 0.0264 0.0425

0.02 0.07 0.025 0.0196 0.0451 0.0425

0.01 0.06 0.005 0.0072 0.0238 0.0354

SN SN’

8.93 9.45

8.93 9.43

11.94 13.11

11.94 13.11

12.52 12.9

12.52 12.9

17.9 19.0

bo

bl D

do

Since the b curve is located below the b line, the SN’values are somewhat higher (approximately 1% of the bl/bo relation). This correction is significant only if a small starting peak width is obtained on a low efficiency plate (an unlikely assumption). However, the verified applicability of the space diffusional model is important, because it agrees with the diffusion laws of vant’ Hoff, Fick and Nernst. They understand diffusion primarily as a space filling effect, where the shape of the space is not actually defined. Diffusion occurs into the macroscopic shape of a space which is pre-shaped by the convective stream of the medium. 43

4. Determination of the Separation Number in Circular TLC

By means of this concept, the separation numbers in circular TLC (SNc) can be calculated in a relatively simple manner. The basic values bo and bl are obtained as described earlier. They allow the evaluation of dosage quality and time proportional molecular diffusion, as well as the separation quality of a TLC layer (bl - bo = f(H) ), etc. The actual calculation of SNc can be carried out in different ways. (i) All consecutive peaks are assumed to have a constant surface relation. SNc is calculated as exponent, similar to (4)via the sum equation of a geometric progression. (ii) Assuming a two dimensional peak broadening from bo2 to b12, which is proportional to the migration distance, the surfaces are equated to the corresponding surfaces of the ring zones. Their radii are the Rfc values and the ring width corresponds to the second, the area dimension. (iii) The theoretically more correct approach is the relation to the distance proportional increase of the corresponding spherical diffusion radius. The calculation is straightforward as in the other two cases; the results are independent of the layer thickness. 4.1 The formulation of the quotient (Rq) between two successive rings requires that the b values at half peak height for a 4.7 sigma separation, must be doubled (2b = B). The radii, which define the ring zones are labelled ‘1, r2 and ‘3, starting from Rfcl. The ratio of the surfaces of the two outer ring zones (after cancelling n) can be expressed as:

where:

44

from this, Rq is obtained:

This quotient and the first term B12 are applied to the sum equation of the geometric progression and the sum itself is set equal to 1. After converting into logarithms and substituting bvalues:

4.2 If SNc is calculated via the second route, the first step in circular TLC is important. The separation begins with the starting spot in the center of the plate, until a ring with a radial distance of 4.7 sigma is reached. At this point its b value is doubled and its area is quadrupled. If 2b is selected as radius in the equation for the area, the ring zone widths are obtained as 2 P values.

[ 2 ki) + Rfc x 2 x (bl - bg)]

xn

= 2 Rfc

x2 x pxn

after elimination of 2 n:

P=

[ bo + Rfc x (bl - boll2 RfC

(8)

Fig. 3 shows peak width curves (ordinate) for some models of average chromatographic conditions. The ordinate is extended with respect to the abscissa (migration distance) by a factor of 20. The plots reveal a strong decrease of the peak width at the start. The lowest P values appear around Rfc values of 0.5, and do not increase noticably near the front. This seems unusual and appears to be in contradiction to the theory of chromatographic processes. However the praxis confirms this effect. It can be explained with the earlier mentioned concept (Section 3), that diffusion is not restricted to a defined shape of a space, but can be influenced by a convective stream of the milieu. Additionally the figure demonstrates the extremely small peak widths which may be obtained by focusing, even at a low dosage - and plate quality. The peak widths in linear TLC are represented in curve 111 - linear. The chromatographic conditions are the same as for curve 111, however the differences are extreme.

45

/

1.0

*r

I

0.5

-.-•

I

2F 0

I

I

I

I

0.5

I

I

I

d

I

I ?F 1

figure 3: Peak width over RRFforsome models of average chromatographic conditions.

46

Furthermore, the figure showsthat the assumption ofaconstant quotient of kvalues for two consecutive peaks (prerequisite for equation (7) ) leads only to a rough approximation. The elementary calculation of SNc uses the sum of the pvalues between e.g. Rfcl and Rfq)2. The number and the necessary space of these values - which are measured in 0.1 Rfc intervals - are recalculated to 80% of the migration distance (in this special case) and supplemented by the possible number of peaks between Rfq.2 and Rfq. Sufficientprecision is obtained by this method. The curves are plotted with values from the following table. Model

I

I1

I11

Iv

b

0.02 0.12 (8)

0.05 0.15 (8)

0.03 0.06 (8)

0.03 0.10 (8)

~~

bl equation 213

Q’ = bo2/3 dl’ = bl Rf-C

1.o 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1

(9a) 0.0737 0.2433

2 p 103

(9a) 0.1337 0.2823

(94 0.0966 0.1533

2 p 103

28103

(9a) 0.0966 0.2154

2p103

b

28.8 26.8 25.0 23.2 21.4 19.6 18.0 16.6 16.0 18.0 40.0

28.8 25.8 22.9 20.3 18.0 13.9 14.2 12.9 12.5 14.9 40.0

45.0 43.6 42.2 41.2 40.4 40.0 40.3 42.6 49.0 72.0 100.0

43.0 42.6 40.3 38.7 37.3 36.3 36.7 38.7 45.0 68.0 100.0

7.2 7.2 7.3 7.4 7.7 8.1 8.8 10.1 12.9 21.8 60.0

7.2 7.1 7.1 7.2 7.4 7.8 8.3 9.8 12.6 21.4 60.0

20.0 19.2 18.4 17.8 17.2 17.0 16.8 17.4 19.4 27.4 60.0

20.0 18.7 17.6 16.6 15.8 15.2 15.0 15.4 17.4 25.5 60.0

SNc SNc (7) SNlin (4)

43.7 36.6 8.9

45.9

21.1 17.9 5.5

22.3

98.6 96.0 11.5

101.2

48.2 42.5 8.6

52.1

The table contains (from top to bottom):

- a reference to the chromatographicmodels (correspondingto those in the curves) - bo and b values of these models

- d’ = b2/1 values, calculated from the b data 47

- Klc-values, downwards from 1 to the start, in 0.1 intervals. The start is not exactly located at Rfco, but in front of it, by a factor of bo. In addition to the Rfc-values, 2 P values are listed, which are calculated according to equations (8) and (9). The circular TLC separation numbers which are obtained from (8) and (9) are next. They are followed by SNc, determined via equation (7) imd SNlin, obtained from equation (4). Linear TLC was carried out under the same conditions as circular TLC. The small difference between the separation numbers calculated according to equation (8) and (9) was expected, especially after the earlier observations (Section 3). Because of the explanation given in Section 3.2, the results from equation (7) are also not surprising. Comparison of the separation numbers reveals that SNc are four to five times higher than SNlin under average chromatographic conditions.

4.3 The previously mentioned third route for the calculation of the separation number which considersa three-dimensionaldiffusion model is the most reasonable approach. The radius Q i.e. d l at the diffusion sphere is obtained from bo and b l through equation (5). During the distance-proportionalincrease from Q to dl, the diffusion sphere, which is equivolume to the zone-ring space, expands in cubic proportions. The Rfc - dependent peak width is determined in a similar manner to the previous calculation as: (Q

P=

+ Rfc.[dl - $I3)

4

RfC

X3D

(9)

Since the layer thickness (D) does not influence the P values it is not necessary to measure D. Therefore the calculation can be simplified, if d in equation (5) is expressed by D:

Using equation (9), we obtain:

or

P=

( b2I3+ Rfc *[bl2l3- b2I3] 1 Rfc

Using the peak width the separation numbers can be determined according to the previously mentioned routes (Section 4.2.1). 48

5. Critical Questions

Attempts to formulate experimental observations by means of mathematical equations have been made. The basis for these calculations is that diffusion is a space effective phenomenon, and that the space can be deformed by a flow, which in turn causes a diversion of the diffusion. The peak widths therefore decrease, similar to focusing. This effect is much more.than a trend. The macroscopic space, in which the flow occurs, is large compared to the diffusional motion of the molecules which migrate in this flow, and therefore a diffusion out of this flow is unlikely. This concept may even be used as a suitable criterion for differentiating between the three systems of liquid chromatography. All three systems, column chromatography, linear TLC and circular TLC, have one unhindered longitudinal diffusion, however the first system has two hindered transverse diffusion. Linear TLC has one hindered and one unhindered transverse diffusion, while circular TLC has one hindered and one enhanced transverse diffusion, because the ring circumferences behave as their doubled radii. Questions, concerning the credibility of separation numbers in the range of 100 are justified (see previous table). Here we must consider the fact that this separation number (Model iii) was obtained at peak widths of bo = 0.03 and bl = 0.06. The value of bl - bo = 0.03 corresponds to a plate number Neff = 1500 for this particular system. However other requirements must also be met: the starting spot width (e.g. after focusing, as described in Chapter 3 and 5) and the molecular diffusion together should not exceed 3% of the separation length, temperature and separation time, e.g. viscosity should have low values. The value bo = 0.03 is a difficult requirement. Is there any practical value for such high separation numbers? It is usually emphasized that the efficiency in chromatography of the liquid phase is achieved rather by a good selectivity than by high separation numbers. According to L. Rohrschneider (Innsbruck, May 1975) this is not true. Selectivity is aimed at separating two substances, and can be optimized for these two substances. However optimization means consideration of the mobile phase and this can be a very tedious process. In addition who is lucky enough to have only two components in his analytical sample? A third component may interfere with the above mentioned optimization. A high separation number i.e. the number of available spaces is the best guarantee for good selectivity.

49

This Page Intentionally Left Blank

Chapter 3

Advantages, limits and disadvantages of the ring developing technique J. Blome

1. In Retrospect

It would be interesting, not just from the historical aspect, to trace back the numerous experiments involved in the conception of separation techniques, which in a liquid phase, allow a mixture of substances to be separated into its component parts due to their differing physical properties, without a material change taking place. What directions these efforts, always independent, took over one and a half centuries, what ideas influenced their direction, what problems this work produced or how it lent speed to the developing art of experimenting, is not certain. What is certain, however, is the fact that in this discipline of chromatography it seems to be fate that discoveries first had to be forgotten before they could be re-discovered and recognized: Over two centuries passed before Tswett’s column chromatography was re-discovered by Kuhn and Lederer and again twice that time before its further introduction into the modem high pressure liquid chromatography. A similar period of time elapsed between the first successful experiments in thin-layer chromatography by Izmailov and Shraiber in 1938 and the real breakthrough by Egon Stahl. Paper chromatography had to wait even longer for full recognition. Its beginning dates back to before Goppelsroeder and Schoenbein, even before Runge, whose “Paragons for Friends of the Beautiful” and “Independently Grown’’ enthusiastically illustrate a “Formative Power of the Substances” and who, at the same time, was successful in associating capillary diffusion with chemical reaction and thus gave the impulse to the spot reaction. The new method remained linked with capillary analysis (Runge, Diss. 1822) for a long time although a similar process was in fact common amongst dyers even earlier. In 50 years Goppelsroeder produced exceedingly comprehensive and versatile capillary analysis material, which received a firm place in phytochemical chemistry and in the official Homoeopathischen Arznei Handbuch. The first experiments to decouple capillary analysis from paper chromatography as we know it today originate from him, when he tried to separate substances loaded on paper with pure solvent. Once more, decades passed before paper chromatography became a recognized technique; through the amino acid analysis of Consden, Gordon, Martin and Synge in 1944. 51

It is interesting to note that at the beginning of both capillary analysis, paper chromatography, as well as at the beginning of thin-layer chromatography, the ring developing technique was used. The question whether the return of this concept of ring development was coincidental or based on developing possibilities and associations appears to be justified.

2. Prerequisites for the ring developing technique

If the advantages and disadvantages of thin-layer chromatographic techniques are to be compared, then the following must be undertaken. Firstly, the criteria by which the results obtainable by these techniques are to be judged must be described and secondly the working procedure of the ring or circular chromatography (CTLC) - with which a similar performance would be obtained - must be described. This working technique has, apart from technical perfection, as is now available in the "U-Chamber", the minimum of prerequisites. These are listed as follows and should be taken into consideration when varying the technique. (1) The most important aim of the following working technique described is the maintaining of, as far as possible, an exact radial symmetry. Prerequisites for this are: (2) Qualitatively good, i.e., pure, TLC plate material, whose layer is as isotropic as possible; in other words, one that does not produce streaking.

(3) Elimination of an unsymmetrical gravitational effect through good horizontal positioning of the TLC plate. (4) Uniform flow of the developing solvent over the plate center, which has not

been damaged (e.g., by application of the substance) and which affords a radially symmetric and uniform flow into the plate layer. (5) The atmosphere in the sorption layer region should be stabilizablebya chamber, uniformly tightly closed all around, and also be as reproducible as possible. Heat transfer by contact or radiation leads to a disturbed or unstable atmosphere and should be eliminated by taking the appropriate precautions.

(6) If, due to substance application, starting pre-run, preliminary separation or the use of a warm air dryer, a non-homogeneous water or solvent loading occurs, then it is advisable to re-equilibriate the layer before running the plate.

52

3. Working technique in circular chromatography Two possibilities are available: Type 1. The TLC plate is placed coated side down and the developing solvent supply proceeds ascending against the layer (3.1). Type 2. The developing solvent supply proceeds from above onto the TLC plate, coated side up (3.5). The first method has many advantages through simple and safe execution also as a routine method, as the following description indicates, and is therefore to be chosen in practice. The second method is advantageous when working with gradient development and when using aluminium TLC sheets instead of the usual glass plates, or when working without chamber saturation. 3. I Developing Solvent Supplyfrom Below

If, as according to type 1, the coated side is facing downwards and the developing solvent supply also proceeds from underneath, then the important saturation of the atmosphere with solvent vapors (for the stabilization of the chromatographic separation) is easily attained by placing the TLC plate on a petri dish containing some of the developing solvent. The usual 9 cm diameter size is suitable for the most commonly used 10 x 10 cm plates. The dish should have a planar rim so that a tight fitting onto the TLC plate is possible. If this is not the case, then the rim should be ground flat to the plate with water and corundum (about 10-15 minutes), or with a glass blowing apparatus (if one has more time). The distance between layer surface and developing solvent is small and can be reduced even more by increasing the amount of development solvent, which is normally 5 to 10 ml. Moreover, the distance is uniform, so long as the horizontal position (see above 2.3) is maintained. Advantages result from this technique, in contrast to the attempts to attain a uniform chamber saturation in the usual TLC chamber, which are only too apparent to the practical chemist; advantages that are rewarded subsequently with, amongst others, reproducible RRf values. The developing solvent is transported onto the plate layer by capillary action. This means a system stabilizing and therefore advantageous self-regulating liquid flow, because the TLC layer only takes that amount of solvent from the wick tip that can be transported by the layer by capillary action. The probability of an over wet transmitting point occurring (“puddle formation”) is therefore reduced. Puddle formation is particularly detrimetal in central application, as the transport of the substance in those regions is disturbed.

53

figure 1.

Here a teflon tube f i e d with cotton, which is a few mm shorter than the inner height of the petri dish, is recommended (Fig. 1). A thin piece of stainless steel wire (diameter 0.3-0.5 mm) is wrapped around the tube to enable it to be firmly fixed in the dish. The free end of the wire is bent after 2 cm, a further 3 cm and a further 3 cm to form a triangular base; the triangle being an equilatorial triangle with sides 3 cm long. In the middle of this triangle the cotton tilled teflon tube is held in a vertical position by the wire. The teflon tube should be placed in the petri dish roughly in line with the sides of the dish or so that the wadding extends behond the dish sides by about 1 mm. The elasticity of this device allows a good contact to be established between plate and developing solvent.

Figure 2. Petri dish

If doubts aboutusing a stainlesssteel wire witha particular developingsolventshould arise, (reaction with halides, organic oxyacids, complexones, etc), then the stainless steel wire can be replaced by a Pt wire (length 12 cm, diameter 0.5 mm equivalent to 0.5 g Pt) or PVIr ligand. The choice of the inner diameter of the teflon tube determines the travelling speed of the solvent system, whose maximum depends on the particle size or particle size distribution of the TLC layer and the viscosity of the developing solvent. A distinct retarding effect is achieved by reducing the inner diameter of the teflon tube from the usual 1-1.6 mm to 0.7-0.8 mm. This can be advantageous in extreme cases, for example improved separation, particularly on coarse particle layer (H/urelation!). In this case it is most important that the chamber is tightly closed. When working with indium oxide treated layers then the upper limit is a diameter of 0.7 mm and, accordingto our still very limited experience, the optimum diameter is 0.5 mm. The best utilization of the indium plates is probably first possible in a Uchamber with automaticallycontrolled,variable developing solventtravelling speeds. The silica gel plate industry also offers a long awaited "HP" material, where the silica gel has a firmerand narrower particle distribution.First experiments with sample plates showed an improved separationalso by non-retarded developing solvent times, i.e., with a normal teflon tube, inner diameter of ca. 1.6 mm. 54

3.2 Application of the Substance Here we must distinguish between application of a single substance (e.g., in the search for an optimal solvent system) or application of more solutions, whose components are to be compared singly and against each other. In the simplest case, where only one substance is to be applied, when the solvent and solvent system are the same and the sample volume is small in comparison to the wick volume, then the sample can be applied directly onto the dry wick. The wick is then replaced into thb solvent system in the petri dish and the TLC plates placed on top. In this case, exact centering is not necessary. The wick is easily sucked dry by the application of a filter paper on to the wick. The single application of heterogeneous solvents, in particular polar solvents or highly diluted samples, should take place centrally in the middle of the plate. The center of the plate can be ascertained using an applicator platform (Figure 3) or by drawing in the diagonals faintly. The center can be seen against the light on the reverse side and can be marked with a felt-tip pen.

J 0 0 0

0.0 000

Figure 3.

When more than one sample is to be applied (usually 4 or 8 samples) then these are applied in the middle of either a 900 or a 4 5 O angle. The angles are established with a platform or pencil cross. The best possible uniform distance from the center is achieved with an application volume of 4-10 ml, i.e., so large that the spots do not overlap on application. A small overlapping ofsolvent zones is not necessarily disadvantageous, so long as no substance is carried out to the periphery. That is dependent on the polarity of the solvent chosen for the sample. Amongst the applicatorsavailable,the simple applicationcapillariesor the Barollier pipettes are preferable. They guarantee the best reproducible application volume, the smallest chance of damaging the coat, no metallic contact with the substance, no leakage problems, and are memory-effect free and quick to clean. The 55

“disadvantage”that no variation in dosing is possible is no true disadvantage, then it is far better to be able to use a scaled dilution for quantitative determination, where the dosage is correct and exact, by preparing standards of differing concentrations but using the same application volume. This is especially true where the dose volume is important in the geometrical formation of the applied spot.

3.3 The Reliminary Run An important improvement in the separation performance and the quantitative evaluation is achieved by first developing the applied substance spots into a smaller circular zone, from which the chromatographic separation of the components is started. With normal silica gel plates, this takes place with a solvent that is sufficiently polar to transport all the components present or, when a reverse phase system is being used, sufficiently apolar. The pre-run development solvent is applied onto the center of the plate in approximately 10 microliter portions, coated side up, using a Barollier pipette or a capillary. The pre-run is allowed to run about 1-2 mm over the applied spots. If all the components do not run with the solvent font - and this is often the case - then the procedure is repeated several times, taking care that the solvent front always runs to the same position. Figs. 3a-e illustrate the model pre-run of a start ring formation for a plate with several applied spots.

Figure3a-e

3 a

b

C

d

e

Subsequently the pre-run solvent system should be dried off as carefully and thoroughly as possible so that the developing solvent for the chromatographic separation is not interfered with by the polar pre-run solvent. Before chromatographic development takes place, the pre-run ring position is marked with a pencil to facilitate the evaluation of Rf values. This pre-run technique can, however, be recommended in the laminar TLC technique, in place of the streak application. On the one hand it permits the best quantitatively reproducible streak application and on the other hand a good reproducible streak length. The greater the applied volume and the greater the polarity 56

of the sample solvent, then the greater the length of the streak. If that is not sufficient, then one can apply several spots at defined intervals, which unite into a streak in the pre-run.

3.4 7he Development of the Ring

Before using the transport wick described above (3.1) or when changing the solvent system, it should be cleaned as follows: by placing the bottom of the wick onto filter paper it is first sucked dry. The wick is then placed in the petri dish containing the required solvent and by putting a filter paper on top of the wick, solvent is drawn through to clean the wick. It may at first appear difficult to set the TLC plate on the petri dish, with the center exactly on the teflon tube wick. For this reason the center is marked on the reverse side of the plate with a felt tip pen (see 3.2). When setting the plate onto the wick, one notes the position of the wick and, holding the plate horizontally between both hands, moves it over the wick without “forgetting” its position. The plate is then placed upon the petri dish and the movement of the solvent system noted so that, if necessary, the plate may be raised a little to correct any errors in development. Even when the plate must be moved two or more times for correction purposes, this does not necessarily affect the success of the run, as in the central region (due to the pre-run) there are usually no analysis substances present to be affected. After correcting with respect to the plate center and teflon capillary, the plate is, if necessary, slid across the petri dish (this time without raising the plate!) to ensure a tight fitting between the plate and the dish on all sides. The wick and stand move with the plate without changing their respective position. After a little practice, one is capable of achieving a placing accuracy of within 1-2 mm and often correction is not required at all.

3.5 Solvent @stem Supplyfrom Above In a simple apparatus (Fig. 5 ) the solvent system is allowed to flow through a correspondingly long teflon capillary (inner diameter 0.5-0.7 mm) onto the plate, coated side up. The teflon tube passes through a hole in a glass plate and is thereby held in place. Distance from the plate and sealing between the two plates is performed by a 2-3 mm thick, silicon rubber plate, with a circle 90 mm in diameter cut out, stuck onto the glass plate. The other end of the teflon tube is connected to or dips into a reservoir containing the solvent system. The solvent system level must be adjusted during free flow and is dependent to a certain degree on length and inner diameter of capillary, solvent system viscosity and flow rate required.

57

figures

The capillary must be, and remain (!) air-free. Gas bubbles increase the flow resistance in a capillary to a great degree and can stop the flow entirely. A constant flow supply by means of a pump avoids difficulties of this type. Figure 6 shows another possibility. An inversely placed fdter funnel on the TLC coating forms a chamber. A bulb tube is fixed by means of a silicon tube in the narrow end of the filter. Cotton is placed in the bulb tube tip and the cotton can be so packed as to achieve the required flow rate (ca. 0.03-0.1 ml/minute). The distance of the bulb tube tip to the TLC coat should be about 0.3 mm so that damage to the TLC coat as well as drop formation are avoided. Real advantages can be reaped in two cases in the solvent system supply from above: when using aluminum TLC sheets or other opaque TLC material and when using a solvent system gradient (3.6).What is missing in this technique, however, is the formation of a similarly stable solvent vapor atmosphere above the plate layer. This flows out from the moistened center over the solvent front to the periphery.The radial symmetry of this system is therefore substantially endangered so that the tightness, horizontal position, isolation against heat transfer and good reequilibriation after the pre-run must be particularly well executed. As the solvent system atmosphere is first formed from solvent that has evaporated from the layer, and as the solventsystem usually contains differentcomponents with differingvapor pressures and requiring differing times to reach their partial pressure, then the 58

composition of the developing solvent usually remains inconstant until the end of the run. This fault is unimportant ifa change of the solvent system composition during the development is envisaged (3.6).

3.6 The Solvent System Gradient Good possibilities are offered by the ring development technique with regard to a simple and reproducible development using a solvent system gradient (See also 4.6). A simple apparatus is shown in Figure 7. Two bulbs of ca. 3 ml capacity each are joined together by a short capillary tube. The lower bulb contains a small magnetic stirrer bar and a vertically turning magnetic stirrer is placed at the same height. The lower bulb is also fitted with a drain tube, running into a teflon tube. This runs to the center of a TLC plate according to Fig. 5. Before gradient development is started, solvent system I (i.e., for silica gel plates usually the less polar solvent) is drawn through the teflon tube, the lower bulb and out through

Figure 7.

Magnetic stirrer

the upper bulb, thereby cleaning away any remaining solvent from a previous run. Finally, after removing the sucking tube, the solvent is allowed to run out through the teflon tube until it comes to a standstill in the capillary between the two bulbs. This is achieved by raising the teflon outlet tube to this height. Solvent system I1 is then filled through the upper opening into the upper bulb, the magnetic stirrer switched on and the teflon capillary placed on the TLC plate center. The solvent supply from above has the advantage that its position can be directly observed from above and “puddle formation” for example can then be avoided by varying the hydrostatic pressure. The safest solution here is also compulsory flow by means of a pump, whereby the solvent system I1 is forced directly, i.e., without the upper 59

bulb, into the lower tube filed with solvent system I. This possibility should ideally be realized with the aid of a "U-ctiamber" system. The resulting gradient is not linear but follows the e-function (11) = 1-l/en, where Il is the concentration of solvent I1 and in the ratio of the solvent volume that has flown to the mixing bulb volume. The smaller the volume of the mixing bulb is, the steeper the gradient runs. It should be quoted when reporting results. Figure 8 indicates the curve. The bulb recommended above with a volume of 3 ml minus ca. 1 ml for the stirring bar, i.e., ca. 2 ml liquid, is large enough for the development of a 10 x 10 cm plate (circular area of diameter 90-95 mm); in fact, only 1/3 to 1/2 of this volume is required. In this way, solvent system I1 concentrationsof approximately4O-50% are reached in the final stages. Accordingly one is working approximately in the straight line region of the graph. What polarity 100

figure 8.

this final stage corresponds to is dependent on the chosen polarity of both of the solvent systems. The formula, or its graphic representation in Figure 8, reproduces the concentration in YO of solvent system I1 against n, which is, however, not identical with the increase in polarity! For this reason it seems unnecessary to pose the question, whether an exactly straight line gradient would be better. A straight line gradient does not result in a straight line polarity rise. In addition, it is still very expensive. The advantage of this simple gradient formation, which 4 years previously was suggested and tested in a pressure resistant form for high pressure liquid chromatography and which was demonstrated to and discussed by the IfC in September 1974, lies in its simple and accurate reproducibility. 60

4. Comparison of the methods linear TLC and circular TLC (CTLC) The question of the criteria by which the performances of both methods were to be measured, has been mentioned previously (2.). Is CTLC a “HPTLC” and if so, why? The following points were considered and summarized in 7 groups. 4.1 Number of plates, separation number, separation numbedtime = (separation performance) 4.2 Resolution, selectivity, use of the separating region (theor. reasons) 4.3 Separating capacity, dynamic extent, trace analysis 4.4 Reproducibility of the Rf or RRf values 4.5 Detection, quantitative evaluation 4.6 Solvent system gradient 4.7 Time - material space requirements, handling technique, miscellaneous

4.1 Number of plates, Separation Number, Separation Numbernime (=separation pe~ormance)

The superiority of circular TLC over the usual linear TLC is indeed known and easy to demonstrate. By using an advanced and improved technique (U-chamber, see Chapter 3) it can be even several orders of magnitude higher. This compares with the separation performance of capillary gas chromatography. To avoid unattainable expectations the nomenclature must be defined carefully and the question, which of these three similar terms belongs to the practical evaluation function, clarified. Whether the term “number of plates” in the circular TLC can be upheld and whether in this connection a plausible explanation can be found for the astounding performance of the circular TLC, which we have observed for many years. The terms that suggests economics, “separation performance” (= separation numbedtime) must certainly take an economic blow when only a relatively viscous solvent system is used to produce the required selectivity in a specific separating problem, i.e., when a good separation can only be achieved by using a poor separation performance. This rather paradoxical sounding formulation indicates why the recommended nomenclature should be observed. The remaining term, “separation number,” as the “number of substances completely separated from one another within the development region, start to solvent front” has the advantage that it is based on practice. An important fact here is that it is even separable from the classical number of plates, which, as will be shown, cannot be used in CTLC. The relationship to the theory of chromatography justifies the treatment of the question of a plausible explanation of the CTLC in connection with the following terms: 61

4.2 Resolution, Selectivi& Use of the Separating Region The resolution of two neighboring substances, i.e., the ratio of the distance between peaks to the average peak width R = ARf/(Wm + wn> is, according to the well known formulation (see L. R. Snyder 1968) a proportional function of the selectivity S = kn/km -1, which is primarily a function of the solvent system. The resolution is, however, also a function of the separation number or the square root of the number of plates. This group is therefore related to the previous group.

Ratio of Rf to RRf Uses of the Separating Region The optimum range in linear TLC is, as is known, the Rf region from 0.3-0.6 or also 0.2-0.7; that is, 30 or at the most 50%of the separation length. The reason is evident: in the range below 0.3 or 0.2 a sufficient separation is not available and above 0.6 or 0.7 the peak width, which rises with Rf, has usually increased and therefore the substance concentration decreases, so that the detection is correspondingly difficult. The Rf values in circular TLC (here denoted as RRf value) signify in linear TLC the ratio of the distance travelled by the component of the substance to the distance from the start to the solvent front. Both radii correspond however to the square of their solvent system quantities, so that according to Geiss:

(RW2 = Rf This leads to the important conclusion: Due to this square function, the flat, wide peaks of the higher Rf regions are squashed together and the narrow steep peaks of the lower Rf region are, in circular TLC, spread out. This means an optimization not only of the ”use of the separatingregion,” in which a “more just” place distribution is achieved. It also means that the peak widening and concentration diminution proportional to the distance run, which is applicable (theoreticallyand practically) inlinear TLC, is fundamentallyinapplicablein circular TLC. It means more over that, as indicated in another section of this book (Chapter 2: J. Blome, The separation Number in Linear and Circular TLC), that the band widening of the peaks is only a fraction of that in linear TLC, which leads to an increase in the separation number. Finally, it also means that the terms number of plates and plate height are no longer applicable and the calculation of astronomical number of plates from peak widths at high RRf values is therefore not admissable.

62

Figure 9 presents an informative picture of the changed place ratios. In crossing from the Rf(1inear) to the RRf(circular) values, the Rf region between 0.0 and 0.1 is spread over 3 times that area on the RRf side, while the upper half of the RRf is reduced to about 30% of that of the Rf. The Rf values used here correspond to a maximum separation number. The illustration corresponds to the conditions of a start peak width - measured at half height - of 2% of the separation length as well as a peak widening of around 7% of the separation length.

Rf 1

RRf

,

Figure 9.

If the start ring pre-run (as recommended above, 3.3) is used the RRf positions are shifted by a certain amount independent of the radius of the pre-run ring (v). When calculating the pertinent RRf positions, one must choose whether these are defined as from the center or - what appears to be more sensible - from the start ring perimeter. In the latter case, the ring area for the substance with the radius (S + v) and for the front (1 + v) should each be reduced by the start ring pre-run area:

-

( S + $2 * n - v2 * n (1 v)L * TT - VL . n

+

=

Rf

or RRfv

= [Rf. (1

+ 2v) + v

~ ] -~v ' ~ 63

Figure 10 illustrates the changes, using the same example, once for a pre-run of 20%,a pre-run of 10%and the separation run.

figure 10.

Gradient Effect If the comparison between liquid chromatography and TLC is made (and this should be done) then it is apparent that LC operates, in contrast to TLC and CTLC, in the region above (retention time in the mobile phase). Taking this into consideration, the considerably poorer performance (measured with respect to separation number) compared not only to CTLC but also to common TLC is amazing. In this connection one only has to think of the efforts to obtain a very tine, narrow class of sorbent, even spherical particles to pack the column as tight as possible, that are required to make the present HPLC performance possible.

To clarify this situation, the remark made earlier (IfC course September 1974) may be of use. This was that practically every TLC system is a gradient system, as far as one sees “pure” adsorption chromatography, in which “only” the solid adsorbent competes for the substance molecule with a “pure” solvent. Pure adsorption chromatography is a borderline theoretical case. Just a trace of water in the adsorbent (and it is almost impossible to keep silica gel anhydrous until it is placed in the developing chamber!) or a small amount of another polar component (not necessarily water) deactivates the plate from the start and results in a gradient formation. Thus, a front is formed whose more polar component 64

concentration decreases in the direction of the front (solvent gradient). This is certainly true for all dry plates used. The question, how much this gradient effect determines the difference in performance between TLC and LC can be clarified now that it is possible, using the “U-chamber”, to dose the wet, equilibriated plate, i.e., during the run. In this case - in a strong analogy to liquid chromatography a gradient can be suppressed and the effect of gradient on separation quality can be investigated.

Reduction of Vapor Pressure Another hypothesis is presented here. This, like the previous one, should help to clarify the question of the difference in performance between TLC, CTLC and LC. It is based upon an observation of an earlier colleague, F. Jordan/Briihl, made 10 years ago and which remained unsolved fora long time. We had attempted to separate and quantitatively evaluate a group of substances, with nearly all of which we were (qualitatively) familiar, using the streak application technique, at that time on chromatographic paper. On another streak we applied the substances in defined quantities and developed both streaks, a distance of 1-2 cm apart, using the ascending technique. Despite numerous modified experiments (different types of paper, different or same direction of manufacture, in line or lateral to the direction of manufacture, development in cylindrical vessel so that both stripes ran under the same spatially symmetric conditions, without or later with a tempering jacket) the same effect was observed. Even when both fronts had run satisfactorily in a horizontal direction, a “bleeding” of the substance streak occurred and each in the opposing direction. If the front streak bent upwards to the left and downwards to the right then the lower streak bent downwards to the left and upwards to the right. It was, however, not possible to influence the direction of the strips in the streak. The streaks crossed fairly accurately in the middle, i.e., their average Rf value coincided. This effect, which naturally occurs when two TLC plates are placed together with the coated sides facing, could be stopped by spatially separating theatmosphere into two individual atmospheres, by the insertion of a glass plate. Obviously the effect of the substances to be separated on the solvent system is underestimated. Even when only gamma quantities are involved, the quantity of solvent in which the substance component is dissolved, is also very small. If50 x g of substance component are present in an area of 25 mm2,that would be correspondingly covered by 2.5 microliters of solvent, even then a 2%(!) solution is formed. For a distribution system saturated with a component (e.g., Partridge-Mixture) this already means a de-mixing, with more simple solvent systems, a reduced vapor pressure compared to that of the pure solvent system. Due to the close proximity of the second 65

strip, which transported the same substance at the same height, a competing effect was established due to reduced vapor pressures at both places. This upset the equilibrium state and resulted in “bending” away from the horizontal position.

In liquid chromatographythis state of equilibrium is not reached. As far as simplified explanations are acceptable, the following is offered: In TLC (regarded as “liquid chromatography against a gas-phase formed from the solvent”), the lengthwise diffusion (“peak widening”) is hindered by the fact that the individual substance components are accompanied by a front of reduced vapor pressure with which they maintain an equilibrium through a mutual reciprocal action. This equilibrium is preferentially formed with that component of the solvent system in which the corresponding substance component is dissolved. It is obvious that a wall of reduced vapor pressure regions is more effective on a streak application than on a spot and agrees with our experience of better separation performance by streak application as opposed to spot application. As one would expect, the “circular wall” formed in CTLC is more effective, simply because there are no edge effects present. The “healing effect” (R. E. Kaiser, May 1975) of a fault should be immediately comprehendible.

Flowing Around Effect

The many different types of spot formation (tailing in front or behind the spot, half-moon shapes, mushroom shapes, etc.) are often observed but usually disregarded by the TLC practitioner. Most of these deformations (apart from tailing backwards to the start, which is usually due to activity remaining in the adsorbent) originate from differing flow or current effects. The substances influence on the solvent (!) (see also 4.2) is the reason why the solvent flows around the substance and doesn’tsimplytake up and later release the substance completelyandvoluntarily from the adsorbent (both are endogemic processes and require time). The streak application is better because it lengthens the solvent detour. However, the circular chromatogram is best of all as there are no detours! 4.3 Separating CapaciQ, Dynamic Area, Trace Analysis

The consequence of the above paragraph is that a considerable increase in the amount of substance that can be separated is attained. Under the same conditions, the quantity applied in CTLC can be up to 10 times that applied in TLC. In this connection it is understandable why a round paper chromatogram (!) was so valued in the days of preparative isolation by paper chromatography. The ring was cut out and eluted. 66

Dynamic Area It is nearly always possible to create conditions in which a proportionally small substance component is forced to remain in the start region; under normal conditions this is easy for more polar components and in reverse phase chromatography, easy for less polar components. If this is possible it means a noticeable raising of the dynamic area, for in the peripheral region the space grows proportionally to the RRf value and also to the peak widening, which is however consierably smaller than by linear TLC, which hardly exceeds a doubling of its smallest state.

If the substances cannot be placed as wished (see above), i.e., the minor component near the start and the major component(s) ina high Rfregion, then liquid chromatography should be chosen, if possible, as this has an incomparable advantage due to: k=- 1 - ~ f- ~-(RR$ Rf (RW2

In other words, the substances (with high Rf values) that “hurry out in front” are registered first, and therefore very sensitively, as pointed peaks.

h c e Analysis

In problems concerning trace analysis, two different cases occur - low quantities or low concentrations are available. If one assumes that in both cases classical methods of separation have been exhausted, then for the first case, the larger dynamic region of linear TLC would be advantageous. In the second case, CTLC is preferable because, as shown above, a 10 x substance loading factor is possible and therefore the substance identification is increased by this factor. The pre-run technique previously mentioned, also similarly applicable in linear TLC, can be used when the ratio of components is very great. In this case, a suitable solvent is used in the pre-run, to transport first the major component(s) away from the application area, before the trace substance are chromatographically separated from the substances left behind. The removability of this “auxiliary solvent system” is, compared to liquid chromatography, as similarly advantageous as the start ring centralization.

67

4.4 Reproducibility of the Rf or RRf Values Efmt of the Plate Quality

Two main criteria affect the reproducibility of Rf or RRf values. The operator has normally little influence on one of these. This is the differing quality between (as well as within) the individual batches of ready-made plates produced by the manufacturer. These differences are mainly those of average particle size and distribution, and quantity and type of the various impurities in the coating material. They also consist of differing pH’s and average pore sizes, differing activities, differing inner surface areas and technical faults in coating and drying; cloudy, stripey or non-uniformly coated layers. The alternative is to prepare the plates personally; this requires experience and specialized knowledge and the economics of such a procedure should be calculated on the throughput of plates. Sometimes cleaning, by dipping or “bathing” the plate in the proposed solvent, helps. Beforehand, the plate can be “prewashed”with a strongly polar mixture (e.g., methanol/dichloromethane, 1 : 1, if necessary adding 3%ammonia [25% aq. solution] or acetic acid depending on the solvent to be used or the substances to be separated.) Special care should be taken to avoid renewed contamination by the laboratory air during drying; this is best avoided by using a vacuum desiccator. The “everyday user” of ready-made plates can improve the reproducibility of his Rf or RRf values by making sure that an extremely uniform loading of the layer is made alter (!) complete removal of all application, prerun and other solvents. (If it is considered important and the substances are able to withstand it, the plate can be activated before putting the “dry” cooled plate into the “dry” development chamber) (see also 3.3).

Effect of Solvent Atmosphere A second possibility of influencing the reproducibility of the Rf or RRf values is by making sure that the stabilizationof the chamber atmosphere is reproducible. This is difficult to achieve in the normal TLC trough, as most operators will appreciate (those who have tried in vain to correct non-linear solvent fronts and “transverse relation” of Rf values of substances that have been applied precisely in a line, by lining the trough with filter paper or by tilting the plates in this way or that.) Edge effect?

Stabilization in circular TLC is easy to achieve, if the aforementioned technique (3.1) is observed. The degree of success may be gauged by measuring the deviation of a substance ring from a circle. A maximum deviation of 2% should be reached easily. Radial symmetry!

68

4.5 Detection, Quantitative Evaluation Detection by subjection to vapors is better than spraying because the reaction takes place without drop formation, i.e., finepored and cloudless. This is important for in situ quantitative evaluation. This detection form also has disadvantages. The popular and universally used iodine vapor is often less stable, because the iodine deposited is in equilibrium with its own atmosphere. Spots are then paler towards the edge of the plate and the evaluation is dubious. In circular TLC there are no edges, it is radially symmetric. A high separation quality can also be understood as a high concentration gradient with respect to the separation length, i.e., as an obstacle to diffusion in the direction of development. This means that the local concentration of an applied substance must lie correspondingly high at a high separation quality (see 4.2) and that therefore the quantitative evaluation is better due to a raising of the signal: noise mtio. It has been possible to confirm this. In order to measure the arc of a circular TLC, we had made a rotating disc, driven by a synchronized geared motor (6 or 2 r.p.m.) which could be used on a Zeiss chromatographic spectrophotometer. By positioning the beam (made extremely narrow at the entrance aperture) at a determined radial distance from the center of the plate, all substances (from a multiple application) with this RRf-value could be read off in 1 revolution, i.e., photometrically detected and integrated and compared in a familiar fashion.

We would like to thank Peter Stollnberger/Zeiss-Munich for his construction of a pilot model. The accuracy of the quantitative evaluation by the method described here is limited by the variations in circles due to poor chromatographic conditions. It is therefore essential to note the prerequisites described in 2.1-2.7. The method whereby the measurement is taken from the center of the plate to the periphery is indeed possible, but disadvantageous in so far as it is not so informative and as far as measuring technique is concerned incorrect. Finally, one disadvantage is that an appropriate instrument is not on the market.

4.6 Application of Solvent Gradient

When judging a TLC plate the “small amount of impurity” remaining at the start is not usually given a lot of significance, often because the diameter of such a start spot is small. However, it is then that caution should be exercized; the spot remained so small because its solvent (whose dispersal on application was really 69

greater) was not polar enough to take this remaining start substance with it. This means that this “small start spot” could be the tip of an iceberg, which can first be fully seen by using a considerably more polar solvent. This often results in a separation into several components. The use of a solvent gradient can be especially recommended here because then it is possible to detect all substances. Because of the simple working technique (described under 3.6) the accurate reproducibility and the fact that the time-consuming re-equilibration is not necessary (as opposed to LC), the circular TLC is almost predestined for the gradient development technique. This certainly outweighs the big disadvantage of CTLC against linear TLC, which must, however, not be overlooked: Two dimensional development is not possible in CTLC! 4.7 Time Required

The short development runs in CTLC result in short development times, especially as the solvent flows horizontally and not against gravity. For a 10 x 10 cm plate (11 cm vertical development length) 1/3 to 114 of the time is required. This is useful in routine analysis and a great advantage in methodical work, e.g., in the search for or optimization of a new solvent system when results from experiments must be awaited before subsequent experiments can be planned. Solvent changing is simple and quick. Emptying, cleaning out and drying of the dish is done while the wick is being cleaned (3.4.1) in 1-2 minutes. Another minute is required to wash the new solvent through the wick.

Material Required The usual 20 x 20 cm TLC plates should only be interesting in special cases, e.g., in quantitative analysis with very extreme dynamic conditions and only so long as no round TLC evaluator is available. The 10 x 10 cm format is usually sufficient for four or even eight substance applications, a considerable material saving justifying the use of a long awaited improved HP material. The amount of solvent required for a 9 cm diameter chamber is approximately 10 ml. If the distance plate to solvent is reduced (thus reducing the developing time some what), then the solvent volume can be doubled or tripled; alternatively it can be reduced.

Less solvent is used in the gradient development (3.6) and when the solvent is supplied from above (3.5) because, apart from that used for cleaning the wick, only the solvent to cover the development area is required, i.e., a maximum of ca. 5 ml. 70

Space Required, Handling Ability, Miscellaneous

The small apparatus and plate size results in a correspondingly small space requirement. The working top should be level (3.3). The handling technique is also very easy to learn (see description of working technique) and this is also true in practice. However, the very optimistic “rentability viewpoint” up till now cannot be allowed to cloud the usual disadvantages that all new methods bring with them. This requires time, a lot of time . . . , introductory time (e.g., to read this book), discussion time (whether it is all really worth it?), starting time (and who could do it?). Time for experiments (the first ones go wrong anyway.) If it does finally work (quicker than one thinks) then the discussion about which results are correct arise, the old ones or the new ones? Would it perhaps not be better (to be on the safe side!) to continue the old method until . . . the newer one has been verified. And what should we do with the old not inexpensive TLC chambers? (they are in fact ideal for plate washing, see 4.4) . . . All this belongs to the passive part of “rentability calculations”.

71

This Page Intentionally Left Blank

Chapter 4

The U-Chamber* R. E. Kaiser

The HPTLC layer, which is superior to a HPLC column, in terms of separation ability and separation power, displays its full potential only on short separation distances. This is true if the analysis is not carried out at a plate height optimum, determined in a conventional manner. Separation power means the result of a separation as a function of time. Since time is an important factor in an analysis, sampling and work-up procedures, consuming several hours cannot be tolerated. If a separation takes only two minutes, it can be repeated easily. Certainly, in such a case we might be confronted with the problem,of choosing the “right” value, but these are statistical-mathematicalquestions, which will not be discussed here. Multiple measurements and multiple calibrations are the basis for obtaining meaningful results, and it is important for the analysis to be completed in seconds rather than minutes. One must be aware of each millimeter of separation length which is wasted; the smallest migration distance necessary to achieve an acceptable separation is the best. Solutions to analytical problems are as good as the methods which provide the data. It has been often said that linear TLC is faster than circular TLC. This is true, if the two systems are compared in terms of migration distance only. However a few millimeters are of little consequence when we consider the higher separation sharpness and therefore higher separation power in circular HPTLC and its superior precision of quantitative (2-3 times better than in linear TLC) and qualitative data. Let us compare those values which can be measured directly: the migration distance in linear HPTLC and the ring diameter in circular HPTLC. In circular HPTLC, a diameter, 8 , of 40 mm is reached in 188 seconds, in linear HPTLC a distance of 40 mm is reached in 320 seconds. 40 mm has been selected as a reference, because it is the maximum migration distance that is technically possible in the normal CAMAG U-chamber (an optimized limit), and because the plate height optimum in linear HPTLC is usually measured at a migration distance of 40 mm. For special analysis and for simultaneous multiphase development a MACRO-U-chamber CAMAG is in preparation.

*

CAMAG U-chamber system, available from C A M A G Inc., 2855 South 163rd St. New Berlin, Wisconsin 53151

73

Advantages of the U-chamber system in TLC: 1. The mobile phase is isolated from the surrounding atmosphere. Chromatographically pure solvents, the basis for real Rf-values, remain pure. Each troughchamber, eveh those in which sandwich plates are inserted, cause contamination of pure solvents within seconds or minutes. This is especially critical in adsorption-TLC, where polar contaminations, even in trace amounts must be excluded.

2. The flow of the mobile phase is electronicallycontrolled, which allows adjusting the H/u optimum. The standardization of operation instructions imposes no problem. Comparison of the results is possible because the controlled flow conditions of the mobile phase can be reproduced. The exact flow rate is measured with an analytical balance. 3. Gradient techniques can be applied. 4. The gas phase is controlled and can be adjusted in counter flow or parallel flow

with respect to the mobile phase. This excludes any influences of the gas phase, usually one of the main interferences with linear TLC. Thus the Rf values are completely stabilized. The data pair technique, which consumes time (50%) and sample capacity (50%) and is still necessary in linear HPTLC can be eliminated. 5. The chamber is constructed in a fully symmetrical manner, allowing development of the plate in a horizontal position. Thermal effects can be excluded, such as the formation of a micro climate in the through chamber (with or without sandwich adapters).

6. The dosage position of the sample and the entry point of the mobile phase are defined with an accuracy of 0.1 mm, without any adjustment. This obviates system errors of this nature.

7. In the central dosage technique the separation starts without delay. In linear TLC there is some loss in time and migration length. The latter can be minimized if a precisely positioned and exactly loaded mini trough-chamber is available. 8. The plates in the plate-holder can be labeled providing means for an automatic evaluation. The sample, or 6-12 samples, in case a ring dosage technique outside of the centerpoint is applied, could be marked with a code which is read by mechanical, optical or electromagnetic methods. The procedure can be 74

optimized with respect to wave length, etc. After the separation, the plate is dried and placed in a scanning device, ready for evaluation.

9. Material consumption: Plate material: 1/16 compared to convenional TLC 1/4 compared to linear HPTLC Mobile phase: extremely low; only 0.15 ml of mobile phase is necessary for one analysis. It has been estimated that the entire system can be amortized in 14 days, because of the low solvent consumption. 10. Time of analysis: a maximum of 180 seconds per 1

-6-

12 separations.

11. The system can be used in combination with HPLC and GC, including capillary column GC. The plate is positioned very precisely under transfer capillaries or transfer spraying devices. A mechanical accuracy of 0.1 mm is easily accomplished, (height adjustment with a lab jack). The time controlled transfer is achieved by rotating the plate. 12. Single as well as multiple eccentric dosage is improved by precisely adjustable, chemical focusing (starting ring widths of 0.1 mm are possible). 13. The technical difficulties of continuous flow HPTLC have been overcome (figure page 76)

14. The system is compact. 15. Operation demands only moderate skill.

75

HPTLC - continuousflow technique in the I/-chamber, Orlita type AE-10 pump and heated metal ring (see alsofigure 4.5).

76

Disadvantages of the normal U-chamber system in TLC* The number of samples is limited to 12, and the maximum migration distance measured from the centerpoinf does not exceed 20 mm. Conventional TLC plates cannot be used in the U-chamber. These restrictions confine the U-chamber technique to certain applications. However, the scope is still wide enough for most qualitative und quantitative separations where speed is essential. A macro U-chamber version offers a capacity of 60 sample spots and 40 mm migration. Its disadvantage relatively to the normal U-chamber is the larger time consumption: 10-16 minutes for one run with up to 60 samples. The U-chamber is round. Flat chambers, such as the F-chamber(IfC-prototype) combine the advantages of a controlled mobile phase flow, although the electronic equipment is more complex, because the flow is time programmed or more than one pump has to be used and controlled in a close loop. Advantages of the linear technique, such as two-or multidimensional TLC and a large number of samples compared to the U-chamber technique are available. The Rf-values are better than those obtained from trough-chambers, or chambers with sandwich adaptors. A detailed description of the U- and F-chambers can be found in the operation manuals of the manufacturers of these chambers. This discussion will be restricted to the principle of the U-chamber technique with some basic examples of applications.

Conclusions: The final measured values were reached after 3 min in the U-chamber and after 5 min in the linear technique. The earlier mentioned differences between the two techniques need to be considered. Our future work in instrumental micro TLC is concerned with the application of the wet dosage technique (see Chapter 7) in linear TLC, a method used only in circular TLC up to now. A new technique for direct optical evaluation of the plate needs to be designed. The 100 x 100 mm F-chamber may also be equipped for circular operation, for carrying out complex, qualitative separations but a macro U-chamber type offers more precision. This is carried out at the expense of an increase in analysis time.

* In the german version of this book the author wrote "disadvantage No. 1: Two dimensional separations cannot be carried out in the U-chamber. This is a basic drawback of the circular technique." Since M A Y 1976 this statement is obsolete. I1 is a powerful technique. to do multiphase - multidimensional analysis simultaneously by CTLC. Unexpected precise data concerning phase mixtures, whose composition can be read expressed as an angle, can begained. A macro U-chamber version was developped for this highly informative technique by IfC as a prototype. See page 77

z

(mm) Migration distance (linear HPTLC) i.e. diameter (circular HPTLC)

limitlin:5.3 min

Figure 4.1 Comparison of migration speeds of the linear and circular techniques. Mobile phase: toluene, temperature 2P C,relative humidity: 40%, HFTLC silica gel precoated plate. Flow function applied to the linear technique

(z? = 0.201 x t z in millimeters t in seconds

Flowfunction applied to the circular technique at aflow rate of 1 p l h

(Z?

78

= 0.195 x t

Z

= diameter

t

= in seconds

in millimeters

Figure 4.2 Plate holder for the 5 x 5 em HP7ZC plate. 7he loaded holder is placed in a precisely horizontal position on the U-chamber dosage turret, scanner or coupling devicefor GC and HPLC. Bayonet lock: the plate can be loaded and consequently adjusted within 5 seconds. If the plate is not cut accurately it can be made to fit by breaking ofl the edges.

79

figure 4.3 Cross sectional view of the U-chamber made bv CAMAG. S: Dosage syringe to maintain the flow of the mobile phase, operated by a stepping motor (e.g. glass teflon syringe with a volume of 250 microliters or 1 microliter). g: hlet or outlet for parallel - or counter current gas flow, to remove vaporized mobile phase, to dry or moisten (impregnate) the plate.

The mobile phase is fed through a platinum-iridium capillary. It is a necessary accessory if the sample is to be applied centrally, with or without the wet plate applicator. Conventional capillaries show memory effects and have a significant dead volume. The capillary has a flat ground point and has to be positioned on the plate with a precision of 0.01 mm, otherwise the flow of sample and mobile phase is disturbed. In such a case, incorrect Rf-values are obtained, the separation time increases and the capillary may become clogged.

80

A k m -

per Soder P

figure 4.4 Principle of "wet plate dosage" on a 5 x 5 em HmZCplate. The separation is carried out with a power syringe (CAMAG dosage unit) or with a pump ORUTA type A E 10 (seefigure 4.5). Afer the layer has been impregnated with mobile phase, the sample is applied at a mobile phase flow of 0.4 pl/s. 7heflow is then a4usted to optimum speed. m: solvent inlet p : pump with aflow of 1 pl/s s: power syringe wd: wet dosimeter, 10-1OOO nl

81

Figure 4.5 ContinuousHPTLC as a preliminary techniquefor HPLCand for separationproblems, which are best solved by continuous development. gas inlet, N2, dry orpassed through a micro washbottlefilled with a conditioning solvent m: mobile phase; any suitable mixture p: pump Orlita Type AE 10, excellent performance over a long testing period. Can be operated at afrow of 1 pl/s to 10 mlhnin in HP’lZCand HPLC nd: nano dosimeter (see Chapter 5) h: heated metal ring, aluminum, outer diameter = 47 mm, inner diameter = 37 mm g:

At a ring diameter of 36 mm a toluene flow of 0.9 mg/s (measured with an analytical balance) can be completely evaporated. The heated ring (15-20 watts) reaches a temperature of 75O C measured by thermochromism. At the above mentioned flow, is approximately 280 seconds. This value allows the extrapolation of all data for substances with k = up to 10 x tm, as well as the measuring time in the isocratic technique using the 37 mm 8 ring. Smaller rings (tm becomes smaller). 82

11-I

Q b)

Figure 4.7 a and b: a. A TLCplate, anchored in a I/-chamber holder is coupled with the outlet of another separation system, e.g. gas chromatography or HPLC with a micro sprayer, prior to the coupling. b. Automatic adjustment for in situ optical evaluation. m e plate in the centered holder rests on a perpendicular mounted metal block which is operated by a steppingmotorand rotates3603 61.5, or 60min. memetalfacilitatesa temperature exchange. m e exact distance with an accuracy of 0.1 mm can be adjusted by means of a simple mechanical system. (Labjack).

The HPTLC micro plate remains in the holder and is placed on the optical scanning system which has a mechanical tolerance of only 0.1 mm. Consequently, adjustment of the optical center in circular TLC is automatic. In conventional TLC this operation is usually very time consuming. Rotational scanning, where data are processed with respect to the angle of rotation, will become a very rapid evaluation technique, if faster integrators become available and if the need is apparent.

83

m

Plate adjustment on the chamber

Eccentric dosage on the plate by means of the above dosing aid

figure 4.6 The CAMAG plate holder allows a precise positioning of dosage and plate.

The precision is better than f0.2 mm, because everything is axially symmetrical. Eccentric dosage is facilitated by a series of bores in various distances from the center. There are of course other techniques available, for dosage at precise positions. One of those uses a rotating device and carries the plate in its holder. An EVAChrom applicator is used together with the IfC-fixed nanoliter sampling device for HPTLC, see chapter 8, rocker dosage. a4

Chapter 5

Dosage techniques in HPTLC R . E. Kaiser

The influence of the spot size at the start of an analysis was discussed in the theoretical section Wide spots ruin the high separation power of HPTLC. The dosage quality (QD) is always a factor which affects the chromatographic separation power. The expression bi - bn

tells us how to approach this problem. The broadening of a peak B = bl - bo during the separation is unavoidable. But if bo becomes as small as possible, QDapproaches 1, which means conservation of the separation power of the system. The HPTLC separation can be carried out on a “wet or dry” layer. After dosage, the solvent is removed from the layer and the plate can be developed with a different mobile phase. This diminishes the negative influence of wide starting spots, a common dosage error. J. Blome describes this method in his article “Advantages, Limits and Disadvantages of the Ring Development Technique”. However, dosage errors cannot be corrected when the sample is applied on a “wet” layer. Here it is important to keep bo as small as possible - unless focusing is done.

Primary optimized dosage

Certain techniques and an especially selected mobile phase can counterbalance the effects of an inadequate dosage. Since this method is time consuming and has other disadvantages, a primary optimized dosage technique is important. Itsgoal: bo=O.I mm This can be achieved by: minimum dosage volume - highest possible sample concentration, most suitable solvent for the sample and efficient instrumentation. 85

The dosage volume We assume that the sample has been dissolved in an appropriate solvent, which yields the smallest Rf-values for most of the sample components on a given TLC stationary phase. However, this choice is often limited because we are not always dealing with a model mixture. Furthermore, we assume that the permeability is the same throughout the entire layer. The dosage volume then spreads out symmetrically on the layer, and assumes the shape of a hemisphere, according to the TLC flow function. A rough calculation demonstrates that a hemisphere cannot be obtained, because it is technically impossible to apply such a small sample volume. Any volume higher than 2 nanoliters will penetrate to the glass plate. Thus the solvent in which the sample is dissolved will assume the shape of a disc, with the thickness D of the layer (in our case the thickness of the HPTLC layer is: D = 0.19 mm). Further, the volume of the dosage solution is increased according to: Vdos = Vsol (1 + As/Am)

=

vs01x 1.395

(see theoretical section)

Subsequently the values for the dosage volume, spot-diameter and bo can be obtained.

Dosage volume Vs01 dSpotcalculated in mm dSpot measured* in nanoliters (at Rf = 1 for sample in mm 1 nl = 0.001 pl and solvent)

bo in mm

lo00 nl 500 200 100 50 20 10 5 __

1.8 1.3 0.8 0.6 0.4 0.3 0.2 0.1 0.1

2

3.06 2.16 1.37 0.97 0.68 0.43 0.31 0.22 0.14 ~

* measured values: red dyestuff in toluene on silica gel

86

3.1 2.2

1.1

-

0.6

Conclusions:

Only dosage volumes below 10 nl = 0.01 pl (Hamilton 7101 syringe)are compatible with HPTLC, unless a sample solvent can be selected in which even the least polar component has an Rf-value of less than 0.1. In this case the dosage volume may be increased to 100 nl in order to obtain a bo of approximately 0.1 mm. If a dosage of 0.1 microliter = 100 nanoliters is repeated 10 times, large amounts of sample can be applied, or traces can be enriched on the plate; however in this case, even a “Rf = 0.1 - mm - solvent” cannot correct dosage errors.

The value Vdosage = 2 nanoliters at bo = 0.1 mm was calculated without taking into consideration the dosage system. The needle of the Hamilton syringe 7101 has a “discharge area” of 0.45 mm. If metal is wetted by the solution, the spot will increase to a starting point width of 0.5 mm and bo becomes 5 times as large as it should be. Some of the data in the above table, e.g. dspot = 0.6 mm versus dspot cal. = 0.43 mm, are already critical. Starting spots < 0.3 mm at a dosage volume of 10 nl are only obtained by tricks, provided that a “Rf = 0.1 solvent” is used (e.g. evaporation of the solvent within milliseconds). The surface of the HPTLC plate, Merck, silica gel, is very hard. If the dosage system, which is in mechanical contact with the plate, is operated carefully, the surface of the layer is only ruptured to a small extent. This is unavoidable and always causes a decrease in separation power.

The following recommendationsfor aprimaty optimized dosage, without any corrective after-treatments, are based on the above discussion, and other facts not mentioned here.

1. Choose the highest possible sample concentration. 2. Select a sample solvent with the properties of a mobile phase in which all sample components have a Rf-value below 0.1. 3. The area covered by the dosage needle should be less than 0.5 mm in diameter; 0.3 mm is still technically feasible. 4. The needle rests on the layer with a pressure lower than 100 g/mm2. This corresponds to a “pressure” of 5 grams, if the needle has an 0.d. of 0.3 mm and an i.d. of 0.15 mm. Training is required to carry out an accurate 0.1 mm dosage without rupturing the layer.

a7

Instrumentation for nano dosage Nanoliter dosage by means of the Hamilton syringe and micrometer is described in Chapter 6. An important improvement is the rocker-dosage described in Chapter 8. The rocker combines the following:

- highly accurate dosage optimum contact pressure which can be regulated similar to the pressure of a sound pickup on a record player - easy, manual operation.

-

In order to achievegood drainage of the capillary it is necessary to apply a minimum contact pressure between capillary and layer. This should be enough to slightly deform the upper 1/1OOO mm of the layer. The nanoliter dosage was considerably improved when the glass capillaries were replaced by ones made of platinumiridium. Chemically inert and very smooth on the inside, they hardly show any memory effects. The capillaries can be flamed, are durable, if handled correctly and are an ideal contact material for the hard HPTLC layer. figure 5.1 (see page 90) Self-loading, plaritrutn-iridiumsample capillary

Advantages: The dosage volume of different samples is reproducible to 3-5% of the applied volume. If the same sample is applied, the reproducibility is f0.7%of the dosage volume. (Standard deviation of 10 individual measurements). The strong discrepancy between similar and different samples is caused by the amount of solution retained in the capillary, which is dependent on the polarity, viscosity and wettability of the sample solution. Maximum dosage volume: 22 nanoliters per 1 mm capillary. The capillary is treatable by a flame. Disadvantage: No disposable parts. Figure 5.2 (see page 90) Glass-metal capillaiy with window, to view loadingprocas (very useful in combination with the Merck applicator type: Nr. 10226 in conventional EC).

The disposable capillaries, which are used in this applicator, have a macro-volume of 750 nl, a volume much too large for HPTLC. Advantage: total costs are less than the Pt-Ir capillary (figure 5.1) and the reproducibility is approximately the same. Memory effects can be washed out; rinsing three times with the new solution is usually sufficient. Dosages of 50 and 100 nl are possible. Disadvantages: the capillary is very small and cannot be flamed. The capillary can only be exchanged with the use of tools. 88

Figure 5.3 (seepage 90) Combination of R-Ir needle and metering capillary for the macro application, which can be used in the dosage system described in Chapter 4 or in the above mentioned applicator.

The capillary is loaded with excess of solution and drained to the desired dosage volume. Dosage itself is carried out with a "rocker" holder or with a calm hand. A contact pressure of approximately 5 grams should be applied. This can be developed in a relatively simple manner: Place a 5 x 5 cm used TLC plate on a balance and zero the scale. Apply pressure until 5 grams are reached on the scale. Advantage: wide scope of application, suitable also for qualitative TLC (micro separations) and line dosage. Disadvantage: in some cases the dosage volume can only be measured with an accuracy of k 100 nl. Excellent results in routine application are now possible by using the 100 or 200 nl fixed volume Pt-Ir sampler Fig. 5.4 (constant dosing capillary). Used instead of system 5.1 in the so called rocker dosage mode described in chapter 8. A magnetic holder offers rapid selfpositioning. Spot positioning is better than k 0.1 mm even when repeated dosage into the same spot is necessary in order to samp1e"absolutely" (avoiding loss of substance by using a wash solvent), see figure 5.4. Figure 5.4 (see page 90) Constant dosugc cupillaiy as shown in Figure 5.1. This has the advantage of being easily cleaned; macro dosages can be carried out.

89

0,3mm +I-/

v

=

50nl

/

3

1

mm

I

1

fig. 5.1

/ L)

4

v = 90nl

O,IS mm

fig. 5.2

3

fig. 5.3

/ V = 5000 -15000 nl .

v

= 35nl

\

fig,5.1 - 5.4

90

a

figure 5.5 Comparison of some manual dosage systems

Figure 5.6 Dosage unit for application on the wet layer. 10-250.000 nanoliters in the CAMAG U-Chamber. 91

Sample application on the wet layer The principle is quite simple: A T-piece is inserted between the mobile phase reservoir and the layer. The sample (20 to 1000 nl) is introduced directly into the running system after the plate is oxygen or water free, or after it is equilibrated by different polar phases. The following requirements must be met: Dead volume: in the nanoliter range Memory effects: none Thus it is desirable to introduce a dosage system for TLC, - which allows a precise and real dosage in the 20-1000 nanoliter range without memory effects or losses and without spreading of the sample (unavoidable in other dosage units). - which makes continuous flow TLC meaningful, because the dosage can be carried out without disturbance of the sensitive phase equilibrium. The starting point width bo is dependent on instrumental factors and is usually larger when the application is done on the wet layer instead of on the dry layer. Larger bo values mean a loss in separation power. However this decrease is much lower than the effects of a regular 200 nl dosage. The characteristicsof the dosage on the wet layer can be summarized as follows:

- dosage volumes of 10 nl and larger - relative standard deviation for repeated dosage: *0.6%

regular: 1-2%, even at an absolute volume of 20 nl (provided that dosage systems are used, such as templates. “reprojector Shandon” or the electronic dosage system CAMAG) - no phase loss by evaporation - no enrichment - no memory effects - durable - expensive The “Nan?‘-system for dosage on the wet layer consists of a 1-microliter Hamilton syringe (type 7101), whose needle is inserted into a 0.5 mm platinum-iridium tube. This connection, which does not have any dead volume is sealed by a 0.5 mm conical teflon gasket which operates in a manner similar to the separation casset gaskets of the Institute of Chromatography. The diameter of the 0.5 mm Pt-Ir tube is gradually decreased to 0.2 mm and the tube is positioned onto the layer of the HPTLC plate. The entire system is accommodated in the CAMAG U-chamber. 92

Figure 5.5 illustratesthe dosage units,with the exception of the systemin Figure 5.2. Because of the extremely small dimensions, the dosage system for wet application is capable of bo values of approximately 0.4 mm, at a dosage volume of 50 nl and a mobile phase flow of 0.4 pl/s (HPTLC plate, silica gel, toluene). At larger sample volumes, bo values of 0.3 mm are only obtained by focusing. This can be achieved principally in two differentways: 1. Thermal focusing in circular HPTLC: the mobile phase is continuously fed at a flow of 0.4 PUS. The sample is applied on the wet layer. Gas enters centrally through the gas dosage ring of the Uchamber. The heat., which is necessary to evaporate completely the mobile phase flow, is supplied through the glass plate by a metal ring similar to the ring kiln technique by Weiss. bo values of 0.1 mm or smaller can be obtained. 2. Chemical focusing was described by J. Blome.

Sample dosage on the wet layer opens new perspectives in the circular technique. 'Materials which were not previously accessible to TLC, can now be chromatographed (light, oxygen, water vapor, sometimes interfered with the separation, or the sample applied on a dry layer, might have undergone a chemical change). The continwushw circular TLC with sample application on the wet layer, is the best technique for the data transfer from TLC to HPLC. Figure 5.6 demonstrates the dosage sharpness of a wet layer application, which can be expected, if one of the earlier mentioned focusing techniques is applied. Dosage volume: 500 nl of a lkomponent mixture of dyestuffs in toluene, applied on a wet HFTLC silica gel plate. The maximum migration length is reached at the outer ring, where the mobile phase is completely evaporated

93

Figure 5.7 Sample application on the wet layer, volume: SO0 nl, focusing.

94

Chapter 6

High performance thindayer chromatography: development, data and results H. Halpaap J. Ripphahn E. Merck, Darmstadt, Germany

1. Introduction

Basic parameters in connection with precoated TLC preparations have been described in two earlier publications, “Charakterisierung von Kieselgelen nach Porensystem und Aktivitiit” (1) (“Characterization of silica gels on the basis of pore systemsand activity”)and “Erzielungvon reproduzierbaren Trennungen durch Verwendung von standardisiertenSorbentienindefinierten Systemen”(2) (“Obtaining reproducible separations by using standardized sorbents in defined systems”). The chromatographic process taking place within the pore system of a sorbent on the surface of its walls has been discussed. Mean pore size, pore size distribution, total length of available pores as well as the type of atom grouping at the walls are characteristic parameters of a sorbent determining its specific chromatographic properties. As in high pressure liquid chromatography, the plate height in chromatography using layers is to a large extent influenced by the mean particle size, the particle size distribution of the sorbent, and by the quality of the layer. However, it is somewhat more difficult to obtain optimum separations with chromatographic techniques using layers in as much as the migration time cannot be variably adjusted via system pressure changes, but depends solely on the capillary forces of the layer, i.e., on secondary parameters of the sorbent The manufacture of high quality sorbents for thin-layer chromatography requires a production plant and equipment set up according to the latest technological advances as well as continuous, multiplecheck supervision. Carefully selected standardized pre-coated TLC preparations in well-defined chromatographic systems are no doubt the best guarantee for reproducible results both in qualitative and quantitative terms. In summary, the following should be stated: The activity of a silica gel is determined by its pore system, the surface texture of the skeleton substance, and the degree of loading of the silanol groups with water. Standardized silica gels with 95

different pore widths and pore distributions,but with definedpore volumes, surfaces and water adsorption isotherms can be associated with specific chromatographic properties. Besides these primary parameters of a sorbent, i.e., pore system and activity, the particle size and particle size distribution are of equal importance. These parameters determine via the quality of the layer the solvent flow in the intermediate space between the individual particles and thus within the pores, too. These secondary parameters have an important influence on separation times, Rf-values and plate heights. Thus, standardization of a few selected silica gels of different types, with MITOW pore and particle size distribution ranges is a precondition to reproducibility of a chromatographic separation process.

2. Development of the prwoated HPTLC plate

Further important relationships between primary and secondary characteristics of sorbent layers and their chromatographic properties have been found during the past three years. A first account of this work was given during the IfC courses at Bad Durkheim in June 1975.The resultsare being published here for the first time. Fig. 1 can be considered as a preliminary result of our work. It shows a circular chromatogram with about 50 micro points of 20 nl each in an eccentric application pattern. The chromatogram was completed within 10 minutes, accurate dose application and actual chromatography included. A very high degree of precision was obtained thanks to the quality of the layer, which was confrmed by means of screen electron microscopic photographs of an extremely thick packing of particles of practically equal size and a smooth, homogeneous surface. Fig. 2 compares the surface homogeneity of the conventional precoated silica gel TLC plate with that of its high performance counterpart. The surface patterns were obtained from photometric measurements with the layers being inclined at an angle of 100 and microscopic surface photographs. In a very broad study, series sorbent layers consisting of silica gels of different particle size and different layer thickness were applied, with otherwise equal conditions, e.g., in terms of silicagel type, binderand indicatoradditions,suspension and coating procedure. Chromatography was then performed, the same chamber type, presaturationof layer, temperature and coating procedure beingused, whereas the migration distancesof the solvents were differentand three differentapplication amounts, and ten migration comes, yielded a total of 3240 individual data. The reflectance measurements were camed out on a Zeiss chromatogram spectrophotometer PMQ 11, the analog output being linked to a process control computer IBM 1800.

96

figure I : Circulare chromatogram, seepage 203, (C 11.).

Figure 2 Photometric su face measurements and microscopic suNacephotographs ofpre-coated TLC plates Top:pre-coated TLCplatesilica gel 60 F254; bottom:precoated HPlZCplatesilica gel 60 F254fornano-lLC;l@t:slit 2.5mmx1.5mm, magnifcation I :24, angleofincidence I@'; right: layer treated with ceres violet, magnifcation I :200, incident light.

97

Parameters determined: tf tfo t zf zfo

s

s mm mm zx mm K mm2/s hRf wx mm H pm RS

Migration time of solvent from starting line to front Migration time of solvent from immersion level to starting time Development time of chromatogram Migration distance of solvent from starting line to front Migration distance of solvent from immersion level to starting line Migration distance of substance Velocity coefficient Percentage of substance in mobile phase Base width of substance peak Plate height Resolution

Parameters determined occasionally:

k a ” N’/s

Partition factor Relative retentiori, selectivity Effective plate number Effective plate number/sec

Figs. 3 and 4 show H-zf curves. The migration distances zf in mm are plotted on the abscissa, the plate heights H in pm are plotted on the ordinate. It has been proved of value to relate the individual plate heights to a mean hRf value of 50. In this way all substance spots within an hRf range from 20 to 80 are covered. In Fig. 3 the four horizontal rows are related to different mean particle sizes of silica gel, with the coarser grades above and the finer grades below. The three vertical rows are related to different layer thicknesses covering the range from about 300 to 100 pm, with the larger numbers to the left and the smaller numbers to the right. Carefully classified silica gels were used in each case. The three curves of each individual diagram are related, from above to below, to three different application volumes, i.e., 2 pl, 0.75 pl and 0.1 pl, corresponding to 2000 ng, 750 ng and 100 ng of individual substance. Within the horizontal rows of different particle sizes an overall decrease of plate heights from row 4 to row 3 and from row 3 to row 2 can be recognized, whereas a strong increase is noted from row 2 to row 1. Within the vertical rows of different layer thicknesses, row I with the smallest layer thickness always shows larger, i.e., more unfavorable, plate heights as comared to those of rows I1 and 111, which show hardly any difference. The optimum plate height values can be read from the course of the individual H-zf curves. 98

8oM L

I

I

8u LO

LO

4

L 2b

I

1

1

80

1

20

I

1

figure 3

H - zf curves of precoated TLC plates (mended experimental studies). Abscissa = migration distance zf (10 - 100 mm); ordinate = plate height H (pm); horizontal rows 4 - 1 =particle sizesfrom coarser tofiner; vertical rows III - I = layer thicknesses from larger to smaller application amounts: upper curve 2 p l = 2000 ng eachoflipophilicdyes, mediumcurve0.75pl= 7SOngeach,lowercurveO.11 = lOOng each. 99

Y

0 0

100-

100%

W TLC

8 0-

60-

LO-

20

1OO nI /

-

HPTLC

80

60-

CO-

L

20.

I

I

I

I

20

LO

60

80

Zf

Figure 4

H - zfcurves of pre-coated iTCplates (pmduction setting) Left: pie-coated Z C plate silica gel 60 F254; right: precoated HPlZCplate silica gel 60 F254 for nano-lZC; abscissa = migration distance zf(10 - 100 mm); ordinate = plate height H (pm); application amounts of lipophilic dyes: I@ 2.0 p l (2000 ng each), 0.75 pI (750 ng each), 0.1 p l (100 ng each); right 0.75 p I (750 ng each), 0.1 pI (100 ng each), 0.02 pl(20 ng each).

The course of a curve is influenced by the particle size range of the silica gel used. In the uppermost horizontal row 4 the optimum plate height value has not yet been reached at a migration distance of 100 mm. In row 3 the optimum value is found at about 100 mm, whereas in row 2 it is found at a distinctly lower level, at about 50 mm; it is to be noted here that the curves rise only very gradually as the migration distances become larger. In row 4, associated with a silica gel of very fine particle size, the optimum value is fount at stdl lower migration distances. It should be noted that in this case there are some very sharp rises with the larger migration distances. From these patterns it can be concluded that each particle size range of a sorbent can be associated with an optimum migration distance and thus with an optimum plate height. Diagrams 3/11 and 2/11 are grossly related to the particle size ranges of conventional pre-coated TLC plates and of the new HPTLC pre-coated plates.

While the results obtained in the coating studies discussed so far were derived from extended experimental studies, the results shown in Fig. 4 are derived from a production setting.The results obtained in this latter case are clearly superior to the extended experimental ones. This can be explained by the superior coating procedures achieving greater packing thightnes. With the pre-coated TLC plate (left diagram) optimum plate heights ofabout 30pm areobtained. With the HPTLC precoated plate (right diagram) plate heights of 12 pm are obtained with smaller application volumes. The application of minute amounts down to 5 nl is very easy to perform if a Hamilton syringe (1 p1) is used in connection with a micrometer. In this way about ten spots can be applied per minute with a high degree of dosage accuracy.

Fig. 5 compares the different volumes of the substance solutions applied on the one hand with the thickness. and surface of the sorbent layer on the other hand. For reasons of simplification, neither spherical spreading of spots nor additional adsorption effects were considered. Whereas conventional thin-layer chromatography called for application volumes of a few microliters, the volumes used in high performance thin-layer chromatography must be distinctly lower in the nanoliter range. A volume of 100 nl corresponds to a cube of 464 pm edge length. Thus, volumes of 20, 10 and 5 nl mean edge lengths of 271, 215, and 171 pm, respectively. The table shows the increase in spot spreading with declining layer thickness. With a supposed layer thickness of 10 pm a volume of only 10 nl would spread over a surface of I mm2,which means an extremely unfavorable initial diffusion at the starting point. According to the table, the layer thicknesses of about 200 pm which have so far been used in TLC are to be considered as optimal for HPTLC, as well. 101

1 = la, m x

1.0

loo w

x

loo w

5 nl

-

171 pm x 171 Urn x 171 pm

1

ul=

1om l w x l m

20 nl = 271 pm x 271 pm x 271 pm

1

nl-l~pmxl@3pmxlCOpm

50 n l = 368 pm x 368 pm x 368 pm

1

ml=

1 o w x

l w x

10 n l = 215 pm x 215 pm x 215 WI

1 o w x

100 n l = 464 pm x 464 Urn x 464 pm

layer thickness 250

m

layer surface 10 n l

5 nl

20 nl

141 wn x 141 pm

2mplnx

2mpm

283 pm x

283 pm

200 pm

158 pm x 158

wn

224 pm x

224 pm

316 pm x

316 UUI

150 pm

183 WI x 183

wn

258 pm x

258 pm

365 p m x

365 pm

1aJ pm

224 pm x 224 pm

316 pm x

316 pm

447 pm x

447 pm

50 WI

316 pm x 316 pm

447 pm x

447 pm

632 pm x

632 wn

25 pm

447 pul x 447 pm

632 p m x

632 pm

894 pm x

894 pm

10 pm

707

wn x 707

pm

l r n p m x l r n p m

1 414 ion x 1 414 pm

&we 5 Spatial spreading of dflerent volumes of substance solutions, related to layer thickness and layer suface.

3. Chromatographicperformance of the HPTLC plate Fig. 6 shows the number of available separation steps with different migration distances zf as a function of the plate height H, based on a maximum hRf value of 80, which prevails in chromatography using a normal chamber (Nchamber). Plate heights between 10 and 15 pm, which can be obtained in HPTLC, give several thousands of separations steps, depending on the migration distances. Fig. 7 gives a tabulated survey of the number of complete separations of neighbored substance pairs, again based on a maximum hRf value of 80, with different migration distancesq a n d differentplate heights H,andalso based on40 separations, corresponding to a resolution R, = 1. The large number of complete separations obtained with short migration distances within the plate helght range from 10 to 15 m, i.e., the range of interest in this connection, is remarkable. Since the resolution R, is inversely proportional to 2 V H, a decline in plate height from the conventional precoated TLC plate (about 30 pm) to the new precoated HPTLC plate (about 12 pm) means an improvement in resolution of about 60Y0,i.e., a remarkable gain in performance. The relationships between plate height and resolution are shown in Fig. 8. 102

plate height H (hRf 50) !Jm

different migration distances zf 25 nm 50 w 75 mn = 20 nmQlRf40) 40 nm(hR+-80) 60 w(hRf80)

la, w 80 mn(hRf80)

50

400

800

1 200

1 600

40

500

1000

1 500

2000

30

667

1 333

2000

2 667

25

800

1600

2400

3 200

20

1000

2000

3000

4000

15

1 333

2 667

4000

5 333

10

2000

4000

6aa3

8000

5

4000

8000

12 OOO

16 000

figure 6 Number of Hectiveseparation steps "depending on theplate height and themigration distance Basis: maximum hRf value = 80

different migration distances zf 75 nm 50 w 25 nm = 60 nm(hRy80) 40 w(hRf=eO) 20 nm(hR(80)

100 nm i

80 nm(hRf=sO)

50

6.3

8.9

10.8

12.6

40

7.1

10.0

12.2

14.1

30

8.2

11.5

14.1

16.3

25

8.9

12.6

15.5

17.9

20

10.0

14.1

17.3

20.0

15

11.5

16.3

20.0

23.1

10

14.1

20.0

24.5

28.3

5

20.0

28.3

34.6

40.0

Figure 7 Number of complete separations of neighbored substance pairs (4 a separations corresponding to Rs = I), depending on plate height and migration distance Basis: maximum hRf value = 80

103

H

-

100

141

173

200

224

245

265

283

XI0

316

71

100

123

141

158

173 165 158 152

187

200

212

224

14 15

58

82

100

116

129

146 141

153

163

173

183

20

50

71

87

100

112

123

132

141

150

158

25

45

63

78

89

100

110

118

126

134

141

5 pm 10 11 12 13

~~

30

41

58

71

82

91

100

108

115

123

129

35

38

53

66

76

85

93

100

107

113

120

40

35

50

61

71

79

87

94

100 106

112

45

33

47

58

67

75

82

88

100

105

94

Figure 8 Relations between resolutions with d@erent plate heights

Fig. 9 shows the changes in the hRfvalues, plate height H and velocity coefficient K foramigrationdistancezfwithlineardevelopmentinaN-chamberwithsaturation of the atmosphere. As the migration distance increases, the hRf values of the three dyes violet 0,green (G) and blue (B) decline, due to an increasing uptake of solvent via the vapor phase. For the same reason the velocity coefficient K is increased because less solvent is required via the capillary flow for pore filling with larger migration distances zfand correspondingly longer rest times. The diagrams in Figs. 3 and 4 have already shown a decline in plate height H with increasing migration distances zfin the 10-50 mm range. 104

figure 9 Chromatographic characteristics (hRf values, H values and K values) depending on the migration distance N-chamber/saturation; benzene; 20 nl = 20 ng each of lipophilic dyes; 8 parameters each.

The eluotropic series of commonly used solvents - related to medium pore silica gels in adsorption chromatography and to hydrophilic stationary phase partition chromatography - as well as their important chromatographic and physical characteristics are given in Table I. The values for the velocity coefficient K with different migration distances and, partly, different temperatures apply to precoated TLC plates silica gel 60 (Merck). The corresponding values for the precoated HPTLC plates silica gel 60 (Merck) are somewhat lower. As the numerical K value increases, the separation time declines. The polarity series begins with lipophilic solvents and ends with hydrophilic solvents. The use of the more lipophilic solvents as a rule results in a decline of Rf-values whereas an increase is obtained from the more hydrophilic solvents. 105

velocity coefficient 2

PIP

/sec

different migration distwces 25

m

50 m

75

m

t (sec)

2

7.5

25.2

53.3

91.9

5

3.0

10.1

21.3

36.8

10

1.5

5.1

10.7

18.4

15

1.o

3.4

7.1

12.3

20

0.8

2.5

5.3

9.2

25

0.6

2.0

4.3

7.4

30

0.5

1.7

3.6

6.1

-(Zf

+

5)

2

figure 10 Development times t (min) of chromatograms depending on the velocity coefficient and the migration distance

plate height

H WfW

w

different migration distances 75 mn 60 mn (hRf40) = 20 mn (hRf=80)

100 mn

25 mn

c

80

mn (hRf80)

50

44

26

19

15

40

56

33

23

18

30

74

44

31

24

25

89

53

38

29

20

111

66

47

36

15

148

88

63

48

10

222

132

94

73

5

444

264

188

145

figure 11 Separation eciency per chromatogram. aprmsed as @hive stzparation step number Nhec, depending on theplate height and the migration distance Basis: maximum hRf value = 80

106

Since, in contrast to column chromatography, in thin-layer chromatography such factors as UV transmittance and refractive index of a solvent need not be considered (the solvents are removed from the layer before evaluation), the large number of solvents with their molecular group specific parameters can be incorporated into the chromatographicprocess. From this, it follows that the selectivity range is considerably larger in thin-layer chromatography. In selecting solvents for routine work, solvents with higher K values, i.e., shorter migration times, will be preferred. Butanol and propanol, too, may be used to advantage for special TLC separations in spite of their unfavorably low K values. Fig. 10 contains a tabulated presentation of linear ascending chromatograms with different K values of the solvents used and different migration distances zf, allowance having been made for a 5 mm distance zf between the immersion 0 level of the solvent and the starting line. Since the law of flow for TLC requires the square of the migration distance, the development times are prolonged considerably with larger migration distances, particularly in the case of solvents with low K values. Thus, the separating efficiency,expressed as effective separation step number N’/sec, is significantly higher with small migration distances zf and with layers of low plate height than with larger migration distancesand more unfavorable plate heights. These relationships are shown in tabulated form in Fig. 11, based on a maximum hRf value of 80, a K value of the solvent of 10 mm2/s as well as parallel development of ten chromatograms per plate. Indeed, the revolution and separation number are increased with a larger migration distance, in spite of a more unfavorable separating efficiency expressed as effective separation step number N’/sec. sphere diameter

sphere nmber per u1 (m3)

hole ncrober between spheres per u1 (m?l

relations of hole nmbers

(vn) 1

1.2

lo9

3.6

lo9

125

law,

2

1.5 x 108

4.5

lo9

16

125

3

4.5

lo7

1 . 3 x 10’

5

31

4

1.9

lo7

5 . 6 x 10’

2

16

5

1.0

lo7

2.9

1

8

1 /8

1

lo7

3.6 x lo6

10

1.2 x lo6

25

7.1

lo4

2.3

lo5

1/125

1/16

50

1.0

lo4

2.9

lo4

l/law,

1/125

figure 12 Relationship betweenflow resistance and particle size in a sphere packing Statistical spacefilling = 63%. 107

The relationship between particle size and flow resistance can be studied on a sphere packing with a statisticallydense space filling. The solvent contained in the layer moves within the intermediate spaces between the solid particles, on the basis of capillary forces. The smaller the intermediate spaces and the larger their number, the higher is the flow resistance, but also the sooner wiU the exchange of substance within the pores of the adjacent sorbent particles proceed. Fig. 12 shows that, comparing a packing with spheres of 10 pm diameter with 5 pm diameter spheres, the number of holes is eight times larger.

20 Lo hrio - KS- chamber

60

m%

figure 13 Chromatographiccharacteristics (hRf values, K values)for various types of chamber (N-chamberhaturation, narrow chamber) and with dlCfeent relative humidities (VarioKS-chamber) zf = 50 mm: benzene: 30 nl = 30 ng each of lipophilic dyes 108

4. Chromatographic characteristics 4.1 Influences of type of chamber and of sorbent and solvent activity

Figs. 13 and 14 show that chromatographic characteristics are influenced by the type of chamber used as well as by the activity of the adsorbent. The conditions prevailing in a normal chamber with saturation of the atmosphere as well as in a narrow chamber (S-chamber) (1 mm spaced) and the conditions prevailing in a Vario-KS-chamber with different relative humidities of the sorbent (between 0% and 80%)are plotted on the left and right sections of the abscissae. The hRf values and the K values (Fig. 13) and the values for plate heights and resolutions of neighbored substance pairs (Fig. 14) are plotted on the associated ordinates.

5- -25 &-

-- 20

3--

-- 15

2--

-- lo

1

-- -- 5 a -

----

H 50 Rs

L Vano KS - chamber

figure 14 Chromatographic characteristics (H values, Rs values) for various rypes of chamber (N-chamberhaturation, narrow chamber) and with different relative humidities (VarioKS-chamber) zf = SO mm; benzene; 30 nl = 30 ng each of lipophilic dyes

109

As compared to the Nchamber with a saturated atmosphere, in the S-chamber the hRf values are increased considerably, particularly in the higher range, due to the fact that there was hardly any presaturation of the layer via the vapor phase. For the same reason the K values are lower, i.e., the migration times are somewhat prolonged.

The increase in Rf-values with higher humidity figures in the Vario-KS-chamber is produced by the selectivelyhigher inactivation of the sorbent The slight increase in plate height in the narrow chamber as compared to the normal chamber with saturated atmosphere cannot be explained satisfactorily. There might be a connection between this phenomenon and the somewhat longer migration time. The strong increase in plate height with increasing relative humidity in the Vario-KSchamber is explained by the increasing deactivationof the sorbent (hydrationof the silanol groups). The improvement in substance pair resolutions in the narrow chamber as compared to the saturated normal chamber is attributed primarily to the larger differences in hRf values. On the other hand, the deterioration in resolution with sorbents with higher humidities in the Vario-KS chamber is attributable to the increase in plate height Fig. 15 gives a graphic representation of the velocity coefficient value K and its being influenced by the migration distances of five selected solvents, n-hexane, carbon tetrachloride, benzene, 1-propanoland acetone, in three different chamber types, N-chamber with saturation, S-chamber, and open Nchamber. The open normal chamber need not be considered further; due to the differences in evaporation effects, the K values are considerably reduced, which means distinctly prolonged migration times. In the narrow chamber the K values ought to be constant irrespective of the migration distance. This pattern has been obtained for benzen and 1-propanol. The strong increase of the K value in the case of acetone, up to 100 mm, would suggest a pre-saturation of the adsorbent via the vapor phase due to the high vapor pressure of acetone, taking place even in the MITOW chamber of 1 mm spacing. The decline in K values for hexane above 70 mm and for carbon tetrachlorideabove 60 mm can only be explained by evaporation effects in this range. It is possible to calculate from the K value differences between the saturated N-chamber and the 1 mm S-chamber the percentage pre-loading of pores via the vapor phase, depending on the migration distance. This has been shown in the lower right-hand section of Fig. 15. It can be recognized, for instance, that in the saturated Nchamber in the case of benzene with a migration distance of 100 mm the pores on the layer are loaded with solvent to more than 30%on an average. 110

KI

KI

K

20-

20-

20-

-

2: n-Hexane

6: Carbontetrachloride

10: Benzene

l~-~z.loto

* - - - - _

-

. \

1

1

1

t

I

1

M

Lo

figure 1.5 K - zf curves of different solvents for various types of chamber (diagrams 1 - 5) Abscissa = migration distance zf (10 - 100 mm); ordinate = velocity coflcient K (mdlsec);full line = N-chamberhaturation; broken line = narrow chamber 1 mm spaced; dotted line = N-chamber open; diagram 1 = n-hexane (polarity score 2). diagram 2 =carbon tetrachloride(6),diagram 3 = benzene(lO),diagram4 = I-propanol (21), diagram 5 = acetone (23) Percentage pore loading with development using a number of solvents in a saturated N-chamber, depending on the migration distance (diagram 6) n-htxane (2), carbon tetrachloride(6), benzene (10). acetone (23) 111

1

25

5-

100.

2 0.

4-

80.

15'

3-

D-

10.

2-

O-

5s

1-

20.

I

'

figure 16 Chromatographic characteristics (hRf values, H values, a values) depending on the solvent used N-chamberhaturation; zf = 50 mm; 50 nl = 50 ng each of lipophilic dyes. 112

Findings like this lead to the conclusion that further characteristicssuch as specific gravity, boiling point, vapor pressure, and evaporation number should be considered in selecting a solvent or solvent mixture (Table I). In the case of solvent mixtures such considerations are even more important. According to our own findings, the surface tension of a solvent does not play any role in chromatography. In the presence of a sorbent the value y is obviously altered to a large extent. However, the viscosity of a solvent is a very important factor which has a distinct influence on the K value and thus on the migration time. The decline in viscosity at higher temperatures has a favorable influence on the K values.

654-

h Rf

3-

B 182-233

2G 40.0-475 1-

V I 58.5-697 I

I

10

I

I

2 0 3 0

o:

A

-

Zf(mm)

Figure I7 Standard deviations of hRf values depending on the migration distance Abscissa = migration distance y(mrn); ordinate = standard deviation s (%); N-chamberhaturation: benzene: 20 nl = 20 ng each of lipophilic dyes; eight applications at 10 mm intervals.

113

None of the different polarity parameters given in Table I, such as dielectric constant, dipole moment, solvent polarity parameters and I" values according to L. R Snyder, are completelycomparablewith one another; nor are they in harmony with our own polarity sequence measurements. The product of the velocity coefficient K and the viscosity q is surprisingly consistent with almost all solvents (exception: n-pentane, tert-butanol, diethyl ether, dioxane, pyridine). This means that the viscosity is the factor which determines the velocity.

Relations

Figure 18 Relations between peak areas (mvsec) of three diflerent substances depending on the migration distance Abscissa = migration distance zf(mm); ordinate = relations ofpeak areas, related to the dye G (100) with medium Rfvalues; N-chamber/saturation;benzene: 20 nl = 20 ng of lipophilic dyes ceres violet BRN (V), ceres green (G), solvent blue (B); reflectance measurements at 586 nm; eight measuring points each.

114

Fig. 16 shows the relationships between a number of chromatographic characteristics and the solvent used in each case. In accordance with the polarity sequence (Table I), the adsorption of the substance to the sorbent declines from xylene to diisopropyl ether, which leads to an increase in the Rf-values. In the case of diisopropyl ether, separation of the three dyes violet 0,green (G), and blue (B), hardly yields any noticeable adsorption differences. This is also evidenced by the distinct decline in selectivity values a from xylene to diisopropyl ether. The decline in the adsorption tendency from the more lipopilic to the less lipophilic solvents is also shown by an increased diffusion of spots, which in turn results in an increase in plate height H and a deterioration of the separation quality.

In ordertoobtainconsistent hRfvaluesoverthe entirewidthofthelayer,aminimum substance migration distance is required in spite of a high degree of accuracy with regard to constant chromatography conditions in terms of development chamber, pre-saturation and temperature (Fig. 17). Thus, a standard deviation of hRf values below 1% is obtained for the dye V I with the highest Rf value between 0.6 and 0.7 at a migration distance zf of the solvent of about 35 mm, which means a migration distance zx of the substance of about 20 mrn. The two other dyes, G and B, with smaller absolute migration distances, show correspondingly more unfavorable standard deviations in their hRfvalues. Certain errors occurring during application of the substances at the same height (not dosing errors) or during initial wetting of the layer are corrected gradually during the process of solvent flow.

The relations between the peak areas of the individual substances, too, are subject to change as the migration distances zf change (Fig. 18). As a consequence, absolutely consistent Rf-values must be ensured for the entire plate if an internal standard is used for quantitative determination. The good harmony of Rf-values is shown in Figs. 19 and 20. In this case amounts of 30 nl of solution each were spotted by means of a Hamilton syrings and 1 pl of solution was applied in a line measuring 80 mm by means of a Hamilton syringe and an automatic applicator. Development was camed out in a normal chamber with saturation of the atmosphere under standard conditions.

Figure 19, see page 199, (C 7.) figure 20, seepage 201, (C 9.)

115

x n

o hRf lipophilic

3

rn hRf hydrophilic o ).c lipo-

K hydro-

16-

80.

PL /

// 12,

60-

8

40.

4

20,

I

20

46

66

S b O C

figure 21 Chromatographic characteristics(hRf values, K values)depending on the temperature, using a normal chamber Lipophilic system: 20 nl = 20 ng each of lipophilic dyes: toluene; hydrophilic system: 50 nl = 10 ng each of amino acids: propanol-water80 - 20: ninhydrin. 116

4.2 Influence of temperature In the diagrams that follow the relationships between a number of chromatographic characteristics on the one hand and temperature on the other hand are demonstrated. The data are based on a lipophilic and a hydrophilic chromatographic system in a saturated Nchamber and in an S-chamber. In Fig. 21 a very strong increase of the K value in a lipophilic chromatographicsystem withina temperature range of 22-60'' C as well as a distinct increase of the K value in a hydrophilic system in the same temperature range are seen. This increase is explained by a decline in viscosity of the' solvent used and the increase of its vapor pressure. The increase applies to the normal chamber, where the pre-saturation of the sorbent via the vapor phase increases very strongly as temperatures rise. This is also manifested by a distinct fall in the Rf values as temperatures rise (Fig. 21). In Fig. 22 the considerable decline in resolution of neighbored substance pairs with rising temperature, particularly in a lipophilic chromatographicsystem,can be recognized. Due to the strong pre-loading of the sorbent via the vapor phase, only the remainder of non-loaded pore volume is available for the actual chromatographic process.

Rs Rs lipophilic Rs hydrophilic

figure 22 Chromatographic characteristics (Rsvalues) depending on the temperature using a normal chamber Lipophilic system: 20 nl = 20 ng each of lipophilic dyes: toluene; hydmphiiic Jystem: 50 nl = 10 ng each of amino acids;pmpanol-water 80 - 20: ninhydrin. 117

The data obtained from the narrow chamber are considerably more favorable. Here, both in the lipophilic and hydrophilic systems no clear reductions of the Rf-values are noticed up to a temperature of 60" C, while the migration time is shortened especially in the hydrophilic system (Fig. 23). The distinct decline in resolution at higher temperatures found in the lipophilic system must be due to greater diffusion. In contrast, the resolution is only slightly impaired at higher temperatures in the hydrophilic system (Fig. 24). These data allow the general conslusion that high temperatures should be used in chromatography only if solvents with low vapor pressure are employed. Under conditions like this, shorter migration times are achieved although we have not been able to establish improved separations.

o hRf lipophilic

hRf hydmphilic

100-

BO-

16-

o

K

lipo-

0

K

hydro-

VI

12-

60-

8-

LO-

4-

20-

o

n

I

I

I

20

LO

60

eb .c

figure 23 Chromatographiccharacteristics(hRf values, K values) depending on the temperature, using a namw chamber Lipophilic system: 20 nl = 20 ng each of lipophilic dyes; toluene: hydrophilic system: 50 nl = 10 ng of amino acids; propanol-water 80 - 20; ninhydrin. 118

4 3 Differences between linear and circular chromatography

In the diagrams that follow, linear chromatography and circular chromatography are compared for a number of chromatographic characteristics on the basis of different migration distances zf of the solvent. Development is carried out in the ascending way in a saturated Nchamber or in a Petri dish with solvent transfer to the inverted layer via a felt wick of 2 mm diameter. In Fig. 25 the resolutions Rs of two dye pairs each in a lipophilic chromatographic system with linear and circular chromatographywith centric application are compared. In both chromatographic systems the migration distance zf is measured from the contact point of the solvent depot with the sorbent to the solvent front.

Rs

7

9 6

5 GIB VIIG

4 *hydrophilic

3.

2-

1 figure 24 Chromatographic characteristics (R, values) depending on the temperature, using a narrow chamber Lipophilic system: 20 nl = 20 ng each of lipophilic dyes: toluene; hydrophilic system: SO nl = 10 ng each of amino acids: propanol-water 80 - 20; ninhydtin.

119

In the case of the separation of the substance pair with higher Rf-values, i.e., the violet and green dye, the resolutions Rs between 20 and 50 mm show a very strong increase for linear chromatography,while a moderate increase is shown for circular chromatography. Only at a migration distance of 20 mm an equivalent separation eficiency is obtained for the two methods. At a 50 mm migration distance, the resolution ratio is 5.1 :3.4 in favor of linear chromatography, which means that separation is improved by 50% in linear chromatography. Things are somewhat different when comparing the separation efficiency of substance pairs with lower Rf-values, i.e., the green and blue dyes. Here a strong increase in resolution with increasing migration distance is found in both chromatographic systems. With a migration distance of 20 mm, the resolution is by 24% more favorable in circular chromatographythan in linear chromatography,but the figure is reduced to 3% with a migration distance of 50 mm. These results confirm the well-known fact that circular chromatography favors the separation of substances with low Rf-values.

Rs

-

zn

2. €2

2

1

A

I

1 0 3 O 4 0 5 0

2 0 3 O U ) Y )

linear chromatography

circular centric chr.

Zf (Jt-d

figure 25 Comparison of chromatogmphiccharacteristics (R,values)between linear andcircular chromatography (centric application), depending on the migration distance Abscissa = migration distance r f (10 - 50 mm); ordinate = resolution R,; linear chromatography:N-chamberhaturation,20 nl = 20 ng each of lipophilicdyes,benzene; circular chromatography: Petri dish, centric application, 1.5 pI = 1.5 pg mch of lipophilic dyes, benzene; evaluation slit width = 2 mm. 120

In Figs. 26 and 27 further chromatographic parameters are compared for the two procedures. Eccentric application in circular chromatography made it possible to usethe sameapplicationvolumesas in linearchromatography.Whencomparingthe hRf values, the relatively slight decline with higher migration distances found in linear chromatography and the very strong decline found in circular chromatography are noticed. This is caused solely by the system employed. In the case of circular chromatography in the Petri dish, the space between the solvent and the sorbent layer is small. There is, therefore, a very high degree of pre-saturation of the sorbent via the vapor phase. For the same reason the K values increase with increasing migration distance. The very much lower K value for circular chromatography as compared with linear chromatography is remarkable. It amounts to no more than about one-third, which means that in circular chromatography development times must be three times longer than in linear chromatography when the migration distance is the same (Fig. 26). Fig. 27 shows that the plate heights, too, are more favorable in linear chromatography. When comparing resolutions it becomes evident here, too, that the substance pair with higher Rf-values, W/G, is separated better by linear chromatography, whereas practically equivalent separations are obtained by the two methods in the case of the substance pairs with lower Rf-values, GIB.

5. Advantages of HPTLC

Fig. 28 shows the base widths of substance peaks wx depending on the migration distance in the 20 to 70 mm range. In the three diagrams from left to right, 750, 100 and 20 ng of lipophilic dyes were applied. Spreading of peaks is not consistent with the various substances. It is always larger with the green dye G with a medium Rf-value than with the blue dye B with a low Rf-value, whereas the violet dye V with a high Rf-value shows slight diffusion with a small migration distance but increased spreading as the migration distance is prolonged. The smaller the application amounts, the more favorableare the base widths of the substance peaks. The relationships are summarized in Fig. 29, which also includes a comparison with column chromatography on an order of size level. Calculated on the basis of the maximum peak base width, a sorbent volume between 3 and 42 mm 3 is required for an application amount of 20 ng,depending on the migration distance of solvent in the 10 to 70 mm range. Converted to columns of corresponding lengths, this means inner column diameters between0.6 and09 mm. It is a question whether columns of such minute limina can ever be used in high-pressure liquid chromatography.It remains to be stated: Normal separationonaprecoated HFTLC plate is achieved by means of surprisingly small, optimally packed volumes of adsorbent between 10 and 30 pl. 121

71

25

T’OO L-

-20

1-

-6

\ ,’A

05’ 2-

-10

-

I-

1 10

figure 26

15

10

----

/

-5

15

K

-hRf I = linear chromatography

c = circular chr., eccentric

Rs

-H 50

figure 27

figure 26 Comparison of chromatographiccharacteristics(hRf values, K values) between linear and circular chromatography (eccentric application) depending on the migration distance Abscissa = migration distance zf(5 - 20 mm); ordinate = hRf values and K values (m&/sec); linear chromatography:N-chamberhaturarion,25 nl = 25 ng of lipophilic dyes, benzene; circular chromatography:Petri dish, eccentric application,25 nl = 25 ng lipophilic dyes, zf = 15 mm, benzene.

figure 27 Comparison of chromatographiccharacteristics (H values, R, values) between linear and circular chromatography (eccentric application), depending on the migration distance Abscissa = migration distance zf (5 - 20 mm): ordinate = H values and Rs values; linear Chromatography:N-chamber/saturation,25 nl = 25 ng each of lipophilic dyes, benzene: circular chromatography: Petri dish, eccentric application, 25 nl = 25 ng each of lipophilic dyes, zf = 15 mm, benzene. 0

122

-s

x-

E E

u

0

N

c

Y

Figure 28 Base widths of substancepeaks depending on the migration distance Abscissa = migration distancezf(mm); ordinate = base width wx (mm); N-chamber/ saturation: application amounts: ldt, 750 nl = 750 ng each of lipophilic dyes: center, 100 nl = 100 ng each; right, 20 nl = 20 ng each.

123

application ammt: 750 ng migration distance

of solvent

=f IllD

peak width W lpax

mn

wlme of sorbent required per chrurmtogram calculated per coltmn length diameter m3

n

m

mn

application amunt: 100 ng peak width W lpax lllp

wlu~ of sorbent required per chnmutogram calculated per colunn length d i e t e r 3 nm mn mn

10

application munt: 20 ng peak width wnrax

volupe of sorbent required per chmmtogram calculated per c o l m length diameter

lllp

1.33

2.7

10

0.582

20

2.59

10.4

20

0.812

1.97

7.9

20

0.7021

1.69

6.8

20

0.656

30

2.98

17.9

30

0.817

2.28

13.7

30

0.762

2 .oo

12.0

30

0.714

40

3.34

26.7

40

0.922

2.57

20.6

40

0.809

2.30

18.4

40

0.765

50

3.67

36.7

50

0.967

2.84

28.4

50

0.850

2.56

25.6

50

0.807

60

3.96

47.5

60

1.004

3.07

3.8

60

0.884

2.79

33.5

60

0.843

70

4.20

58.8

m

1.0%

3.41

47.7

70

0.931

2.98

41.7

70

0.871

figure 29 Sorbent volumes required per chromatogram, depending on the migration distance and application amount N-chamberhaturation; benzene

The pre-coated HPTLC plate with its outstanding quality ensures that the user can work with an optimally standardized layer in terms of type of adsorbent, particle size range and packing tightness. This plate offers a pre-set solvent flow pattern and separation efficiency provided that substance application is performed adequatelyand certain chromatographicconditions are fulfilled. The easy handling of substance application and chromatographic development, the multiplicity of chromatographic systems that can be employed without giving rise to problems and of detection possibilities,as well as the highly sensitivemethods of direct evaluation of substances from the sorbent free from solvent are among the general advantages of chromatographic separation procedures employing layers. They apply in particular to high performance thin-layer chromatography.

'H. Halpaap, J. Chromatogr.78 (1973) 63. *H. Halpaap, J. Chromatogr. 78 (1973) 77.

Pre-coated HPTLC plates for nano-TLC have been used exclusively in all experiments covered by Figs. 1,9 and 13 to 29. Types of plate: Silica gel 60 F254

10 cm x 10 cm 25 plates (Cat. No. 5628) 10 cm x 10 cm 100 plates (Cat.No. 5629) 10 cm x 10 cm 50 plates (Cat. No. 5642)

Silica gel 60

10 cm x 10 cm 25 plates (Cat. No. 5631) 10 cm x 10 cm 100 plates (Cat. No. 5633) 10 cm x 10 cm 50 plates (Cat. No. 5641)

E. Merck, Postfach 41 19, D-6100 Darmstadf Germany EM Laboratories, Inc., 500 Executive Boulevard, Elmsford, N.Y. 10523 U.S.A. BDH Chemicals Ltd., Poole/Dorset, BH12 4NN, England 125

Table 1 Chromatographic and physical parameters of solvents, eluotropic series breakdown related to silica gel (re.to H.H.lp.lp)

A Solvenu

B

C

Formula

b4ol.w

D K

E K

50 mm '5 mn 2P

-

22"

F K 100 m s 2P

9.2

0.6

11.4

2 n-Hexanc 1n-Pentane 4 Cyclohexane 5 Carbon disullide 6 Carbon tetrachloride 7 Triclomethylene 8 Xylene

86.18 12.5 72.15 10.6 84.16 5.4 76.14 13.4 53.82 6.1 31.39 8. I 06.17 6.7

1.9 2.6 6.3 5.7 6.7 9.6 7.6

14.6 13.9 6.7 17.7 7.0 10.6 8.2

9 Toluene

92.14

8.1

9.8

11.0

10 Benzene l l Chloroform 12 Dichlommethane 13 di-iso-Pmpylether 14 ten-Butanol IS Diethyl ether 16 iso-Butanol 17 Acetonitrile 18 iw-Butyl methyl ketont 19 2-Pmpaml (- iso-pmpyl alcohol) 20 Ethyl acetate 21 I-Propanol (= n - h p y l alcohol)

78.11 19.38 84.91 02.18 74.12 74.12 74.12 41.05 00.16 60.10

8.6 9.0 10.1

10.4 11.6 13.2 11.2

12.6 7.0 2. I

9.8 0.5 1.8 1.4 1.1 1.3 1.5 4.0 8.2 2.3

15.3 1.6 15.4 9.1 2.5

88.10 60.10

9.2 2.3

0.9 2.6

12.1 2.9

22 Ethyl methyl ketone (- 2-butanone) 23 Acetone 24 Ethanol 25 Dionane

72.11

11.1

2.8

13.9

saon 88.11

12.7 3.4 5.2

4.7 3.9 6.0

16.2 4.2 6.5

72. I I 32.04 79.10

10.9 5.6 6.3

1.9 6.5 7.2

12.6 7.1 8.0

A B C D E

F G H I J K

I

J

K

K I00mm

U\

50mm Dl60181

75 mm 0160181 4016018(

-

00.2 I

26 Tetrahydmfuran 27 Methanol 28 Pyridine

H

K

11.0

1.0 11.0 1.3

1.8 5.8

4.3 7.6

126

-

cut Ofl

14.6 19.2

195

1.388

195 Mo

1.372 1.358 1.426

!10 180

1.460 1.481 !% 1.495-I 1.505 !05 1.499

!65

0.4 6.0 7.2

1.9 7.2

12.9 18.1

!85 !45

!M

1.501 1.447 1.424 1.368

1.1

'10

1.354 1.198

90 '05

65

1.9 5.5 9.9

4.3 6.0 1.1

4.5 6.3

1.3% 1.378 1.372 1.186 1.379

135 05

7.5 1.6 7.4

8.2 2.5

blarily sequence from lipophilicto hydmphilic Formula Molecular weight Velocity coellicient K (mm21see), prc-cmlcd TLC plate silica gel 60 F254 Merck, N-chamberl saturation, 2 P C. solvent mimation distance zf 50 mm same as D. bul if 75 mm same as D.but q 100 rnm same as D. bu1WC. 600 C, 800 C same as E. bu1WC. 600 C. KP C same as F, but W C , 60°C W C UV cutafipoints in nm. laver lhicknesscm. reference = water. transmittance 20/. Refractive index n@

--

K nDB

nm -

I n-Heptane

46.07

G

8.5 13.2

'IS

1.359 1.383 1.422

M 05 05

1.311 1.509

A Solvents

L d4m

N

M

hil.1 Vap.pr OC toil

0 R P Q E ~ PncP n c l Y nb. 2 P 40" d W c n

-

-

S

T

MAC ppm

D.c

U

z

Y

V

W

X

D.N D

ET

P' K . r

(0) m

(D -

I n-Heptanc

0.684 98.4

2 n-Hexane 3 n-Pcntane 4 Cyclohexsne 5 Carbon disulfidc 6 Carbon tetrachloride 7 Triclomethylcnc 8 Xylene

0.659 0.626 0.779 1.26) 1.594 1.462 0.860

9 Toluene

0.872

68.8 36.1 80.8 46.3 76.8 86.9 37/ 40 10.6

10 Benzene

80. I

1 I Chloroform 12 Dichlommethane 13 di-iso-Pmpylether 14 tert-Butanol

61.3 39.7 68.0 82.6 34.6 m.7 82.0 15.9 82.4

0.879 1.480 1.325 0.725 0.786 IS Dicthyl ether 0.714 16 iso-Butanol 0.803 17 Acelonilrile 0.782 18 iso-Butyl methyl ketone 0.801 19 2-Pmpanol 0.785

0.40 0.33 20.4

120

1.4

500

77 298 91 58 517

1.8 4 3

0.31 0.22 0.94 0.36 0.94 0.57

3.5

0.68 0.54

21

6. I

D.57 0.47

75 160 356 135 31 449 9

3 2.5

xrcin.

3.5

0.26 18.4 16.0 0.71 0.32 0.74 27.0 0.48

100

0.901 0.W

77.2 97.2

1.9

0

Im,

m 10 10

50

20 0 0 0 26 22 0.28 0 3.4 0.89

m 28.1

ml

4.56

81

30.9 OS 4.53 3.06 31.2 0.C 6.30 32.6 1.( 6.37 32.5 1.J 6.58 6.04 2.4 1.58

82 55 1 I4 I IS I I9

24 0.38 0.4 33.9 2.3

109 101

5.27

I I4

I5 32

0 0

0.63 0.49 28.9 0.56 0.47 27.1 3.43 0.36 26.5 3.35 32.0 2.82 1.79 3.24 0.20 17.0 3.71 2.12 1.39 29.3 3.59 22.7 2.27 1.35 21.7

77 14

2.9 6

1.44 0.36 23.9 1.09 1.40 23.8

400

6.0 I .9 18. I 0. I 3.10 1.7 w.7

1.3 i.32 1.9 i.06

96 I10

1.8

1.6 1 1

4

40

23 4.7 8.9 3.9 2.2 4.2 8.2 7.5

5 400

a3

50 ml 500 100

400 100

(= ioo-propylalcohol)

20 Ethyl acetate 21 I-Pmpanol

Relat mv. K.n

0.13 0 34.5 1.27 1.1 39.1 1.69 I.! 41.1 I.! 34.0 I .i 13.9 I .40 I,2 34.6 3.12 3.5 16.0

3.c 5.55 4.4 i.50 3.4 1.68 2.2 1.62 3.4 1.10 2.9 1.67

I I9 118

103 84 56 66 108 109 97 103

5.94

6.2 5.01 i.37 1.7 18.6 1.3 i.68

(- n-Propyl alcohol)

0.805

79.6

72

2.8

1.43

24.6

200

as

1.5 i.98

108

23 Acetone 24 Ethanol 25 Dioxane

0.791 0.791 1.034

56.2 78.3 11.3

180 44

1.32 0.27 1.14 0.82 1.21 0.92

13.7 12.8 13.7

m,

0.7 1.09 2.J 12.2 5.4 i.18 4.3 1.04 1.7 51.9 5.2 1.79 22 1.40 1.4 16.0 1.8 1.87

94

aa,

30

2. I 8.3 7.3

143

26 Tetrahydrofuran 27 Melhanol 28 Pyidine

0.887 0.792 0.982

16

131 96 IS

1.3 13 2.7

1.47 0.38 1.52 0.45 L2.6 1.92 0.73

1.4 1.7 17.4 1.2 i.92 26 1.93 1.7 5.5 5.6 1.69 2.3 L.28 2.2 10.2 i.3 '3 i.52

107 67 133 100

22 Ethyl methyl ketone (- 2-butanone)

54.7 15.3

100

m

m 5

2.7 11.3

m

L Specific gravity ~ 2 0 M Boiling point at 760 torr. i n T N Vapor pressure at 28 C. in torr 0 Evaporation number (diclhyl ether = I ) P Viscosity r( (cP) at 2 P C Q Viscosityq(cP)stWC R Surface tension y (dynlcm) at 200 C S M A C values i n ppm 1975 (maximum allowed concenlration) T Dielectric constants acc. l o Wollmann, Phanazie 29 (1974). 708 U Dipole moments. calculation acc. to Onsager (acc. lo Wollmann, see T) V Dipole moments, calculation acc. to Debye, measured i n benzene, acc. lo Reifhamil, MsungsmittelElfekte i n der organishen Chemie. Verlag Chemie GmbH. Weinheim 1969 W Solvent polarity parameter ET at 2S°C. in kcal/molc, measurementsacc. 10 Dimroth el al. (acc. lo Reichardl, see V) X P values, separatecharacterizationaccording lo Solvent polarityand selstivity,acc. 10 L. R. Snyder, derived from Rohrschneider data. J. Chromalogr. 92 (1974). 223

Y K100mm(F).n2PC(P) Z Relation to mean value K . q = 5.52 (100)

127

This Page Intentionally Left Blank

Chapter 7

Considerations on the reproducibility of thin-layer chromatographic separations D.Janchen, CAMAG, Muttenz, Switzerland

Two chromatographic methods are available for the separation of non-volatile substances: flat-bed chromatography and column chromatography of which thinlayer chromatography (TLC)and high performance liquid chromatography (HPLC) are the most versatile and powerful variations. TLC being a rapid and on the surface, simple and straightforward method, has gained acceptance and importance in most analytical laboratories in the early sixties. As HPLC became known many researchers expected that this method would replace TLC in many applications. Today we know that both chromatographic methods will continue to exist, each in its own right, and that they are well suited to complement each other. Table 1 Comparison of benefits and limitations of HPLC and TLC HPLC

TLC

Changing the stationary phase Changing the mobile phase Qualitative identification of separated compounds

very difficult difficult

easy very easy

difficult

possible

Costs per analysis

medium

low

Detection limit, easily accessible

10-12

10-9. . .10-l1*

* with high performance TLC (HPTLC)

From Table 1 it can clearly be seen where each method is preferable: HPLC for routine separations employed over a long period of time TLC in all those cases where flexibility and easy optimization are advantages

129

There are some criteria precluding HPLC: Generally speaking these involve cases in which the stationary phase changes under the influence of the mobile phase. Examples are: permanent deterioration of the stationary phase after only a few chromatographiccycles; or, the necessity of using such elution gradients that cause regeneration periods of long duration. Table 1 furthermore indicates that TLC would make an excellent pilot technique for HPLC (routine) separations provided valid and practicable transfer rules could be found. Column chromatographers often consider TLC to be a non-scientific, non-reproducible method that is only looked at if all else fails. To a certain extent this resentment is understandable. Most thin-layer chromatographers indirectly admit that they do not expect reproducibility of their method: In quantitative TLC analysis with in-situ scanning it is taken for granted that at least one, better several calibration standards have to be chromatographed side by side with the unknowns on the Same plate. This means, however, that one does not expect to be able to develop, on a second plate, the same quantity of the same substance to an identical separation zone. The main reason for poor reproducibility in TLC lies in the developing process. The difference between column chromatographyand TLC is the introduction of a third phase, i.e., the gas phase, in TLC. The most common single source of error, even though it is confined to adsorption chromatography, is negligence of the influence of environmental factors on the layer prior to chromatography, e.g., humidity. The common TLC developing procedure comprizes the following steps: The TLC plate (or sheet) is taken out of the storage container, packing, etc. During sample application, lasting at least several minutes, the layer is allowed to interact with the laboratory atmosphere. It is known that the half-time value for the equilibration between a silica gel layer and humidity in the atmosphere is approximately 90 seconds (1). After sample application the plate is placed in the developing tank which contains the developing solvent, so that the tank atmosphere is more or less saturated with the solventvapors. The developingprocess starts with the immersion of the layer in the solvent. The speed of solvent migration follows the chromatographic flow law (2), i.e., it decreases by the square of time (= distance). Equilibration between the dry layer and the solvent vapors starts with insertion of the plate in the tank and ends when the solvent front reaches the respective area. Equilibration i.e., the time for solvent pre-adsorption, is short at the "lower" area of the chromatogram and long at the "higher". If the solvent consists of more than one component preferential pre-adsorption will occur. From a critical point

130

of view it must be stated: In this type TLC a chromatographic system is used consisting of a non-defined stationary phase and an illdefined mobile phase, both of which change in an unknown manner during chromatography.

Attempts to gain control of the experimental parameters in TLC have been made as long as the method has existed: Complete saturation of the tank atmosphere with solvent vapors (3); developing devices of extremely small internal volume (4); saturation of such a “Sandwichchamber” with solvent vapors (5); control of relative humidity (1); systematic investigation of the influence of layer preloading (6,7), and many more. Very little has changed in the TLC developing routine. It is often pointed out that prime interest lies in “good separations”rather than in reliable Rfvalues. It will be shown that reproducible Rf-values are a prerequisite for correct TLC analyses.

Complete control of all parameters of the developing process, i.e., all phases, stationary, mobile and gas phase is essential for the following reasons: 1. Only reproducible chromatograms give reliable, quantitative and qualitative, analytical correctness. An extra advantageof reproducibility is a reduction in the number of calibration standards required. 2. Intentional control of all three phases permits optimization of the chromatographic resolution. 3. With experimentally compatible techniques, TLC can produce reliable information suitable for transfer to HPLC techniques. A few basic considerations may help in the understanding of certain conclusions derived from experimental data.

The Rf-value =

migration distance of the substance migration distance of the solvent front

only describes a completed chromatogram. The true or “corrected” Rf-value R’f from which retention data are accessible can differ considerably from the observed Rf.An accepted relation is

131

R’f the true Rf-value, i.e., the migration result achieved by the mobile phase actually passed through the system, Vm the apparent volume of the mobile phase, Vv that part of the apparent mobile phase that was preadsorbed by the dry layer, i.e., by-passed chromatographic action; (Vm-Vv) is the volume of the actual mobile phase,

1.1 an empirical (average) factor compensating for the gradual decrease of solvent concentration from 100% saturation to 0% near the solvent front. This factor becomes 1.0 if the solvent migration is stopped by a score line (recommended). The factor by which Rf has to be multiplied to give R fis called “xi-value.” As all meaningful information can only be derived from R’f, and Vv is difficult to measure, chromatographic conditions should be chosen to give xi-values as close to 1.0 as possible. The relation between R f and the chromatographic partition coefficient, k, also called “capacity” factor is 1 R’f= whereby k k+l’

ts tm

= -;

k is, for a given substance, the ratio of its residence time, in the stationary and the mobile phase. The chromatographic resolution Rs is defined in TLC as

1 kl Rs = -. (- 4 k2

1)VG.

(1 - R’f)

or, after substitution or R’f by k . . .

term 1 (N = number of theoretical plates)

132

term 2

term 3

Term 1 defines the selectivity of the system. If there is no difference between k l and k2 no separation is possible. Term 2 specifies the “quality” of the stationary phase. Term 3 takes into account the position of the two substances in the chromatogram, i.e., the numerical values of kl and k2.

Figure 1 shows the curve of Rs versus mean Rf (R;.) for a given selectivity (term I 0

0.2

0.4

0.6

0.8

= const.).

1.0

It can be seen that (in a layer without a gradient) the maximum resolution will occur at R’f = 0.3. The Rf range within which no less than 75% of the maximum achievable resolution can be expected is from R’f = 0.1 to 0.6. If there is a gradient in the layer the range of maximal Rs may be shifted slightly, but maximum Rs is reduced.

Experimental

Precoated plates were used for all experiments: CAMAG aluminium oxide DSF-B, 20 x 20 cm, MERCK silica gel 60 F-254,20 x 20 cm, MERCK HPTLC silica gel 60 F-254, for nano-TLC, 50 x 50 mm, MACHEREY, NAGEL silica gel for nano-TLC, 50 x 50 mm. Various types of developing devices served for chromatographic development: CAMAG VARIO - KS chamber (8); stationary layer preconditioning by liquids contained in the respective conditioning trays with subdivisions; development after insertion of the sandwich slide. In all cases where preloading conditions were varied across the plate, the layer was subdivided in tracks by score lines. The solvent migration was always limited by a score line 100 mm from the sample application, and development of all tracks was carried to that distance. 133

CAMAG twin trough chamber (22255) for 20 x 20 cm plates, (22155) for HPTLC plates; one of the troughs contained preconditioningliquid and a saturation wick; plate inserted in the other (empty) trough with a layter facing the wick; development started by adding solvent. Some of the chromatograms developed in the “sandwich” configuration, i.e., with a cover glass plate. CAMAG U-chamber system (9,10), comprising U-chamber mounted on levelling table, dosage unit for elution solvent, sample injection valve; preconditioning of the layer, when applicable, by feeding air at a rate of approximately 50 cc/min through the chamber after passing the stream through a wash bottle containing the conditioning liquid; feed rate of developing solvent 1.4 sec/Vl if not stated otherwise. Sample application on macro plates with capillary pipettes or, in the form of bands, with Chromatocharger (CAMAG 27500); application of dry HPTLC plates with platinum-iridium pointed capillary (CAMAG 28701) or, into the solvent wet layer, with sample injection valve (28710). Dyes were used in most of the experiments since colored substances allow the location of separatedzones at allstagesof the procedure. The argument occasionally heard by the author, that surprises of the kind described below only happen with dyes, not with ”real chromatograms” must be classified as wishful thinking.

Modincation of the stationary phase by substances other than components of the developing solvent “Activity” of the stationary phase is an important parameter in chromatographic systems. “Activity“ in this connection stands for “availability of free surface.” In adsorption chromatography activity is mainly determined by the degree of presaturationofthe adsorptive sitesby water. Suchlayersadsorb waterfromtheambient atmosphere within minutes. Figure 2 shows the separation of a multi-component dye mixture (8) on alumina at different activities. VARIO-KS-chamber; preconditioning with sulfuric acidwater mixtures for 40 minutes; developed with benzene. Apart from the general increase of Rfs with relative humidity, note several (unexpected) inversions, i.e., changes in the sequence of the substances. 134

72X 65% 47% 42% 32% 18%14% 9%

figure 2

Conclusion: In adsorption chromatography it is strongly recommended that each chromatographic system be checked for its sensitivity towards a gradient of relative humidity at right angles to the direction of chromatography. Relative humidity usually cannot be controlled in routine TLC procedures. If Rfs change drastically with relative humidity, substances may migrate in an unsuitable Rf range under adverse climatic conditions. Should an inversion occur, however, either relative humidity must be kept under control, or the chromatographic system must be changed. Under conditions where the substances change their sequence they cannot be separated at all. In partition chromatography relative humidity is not critical, except that at very low relative humidities the stationary phase (water) may dry out so that the capacity of the stationary phase breaks down. On the other hand, the stationary liquid phase in partition chromatography may be modified by preconditioning via the g a s phase. Figure 3 shows the separation of dansyl amino acids on silica (MN) in the U-chamber, with developing solvent chloroform-ethyl acetate-methanol-water 45 :45 : 10 : 1; a, b 100 pl, c-f 150 pl. Samples applied (clockwise): DANS-val, -leu, -gly, -ala, -mixture, and Sudan-I1 marking Rf = 1. Dynamic preconditioning via the gas phase with: a) none, b) 2’ 50% acetic acid, c) 2’ 5% NH3, d) 5’ DMF, e) 5’ 20% formic acid, f) conc. acetic acid. The effect of preconditioning with acetic acid as well as with ammonia is rather insignificant, however, a significant influence can be seen with formic acid as well as with DMF.

135

a

c

e

b

d

f

Figure 3 a-f

Influence of layer preconditioning with components of the developing solvent Preferential adsorption of individual components of the developing solvent must always be expected in adsorption chromatography using tank development. The degree of preferential adsorption vanes with the chromatogram area. For a somewhat systematic investigation of this phenomenon the system silica with benzenemethanol 99 : 1 and a sample mixture of acetorange, indophenol, butter yellow was chosen and first checked for any critical behavior upon changes in the relative humidity.

figure 4

Figure 4 shows a VARIO-KS chromatogram with a gradient of relative humidity from 72%(left) to 14% (right) with 30 minutes preconditioning.There is a significant influence of the activity on the Rfs, but no inversion that could have made

136

further results ambiguous. All further chromatograms of this system were preconditioned at 42% relative humidity, i.e., corresponding to track No. 5 from the left.

Figure 5

Figure 5 shows a chromatogram of the same system, also run in the VARIO-KS chamber. The three tracks at the left side had been exposed 10’ to the vapors of the developing solvent, the three tracks at the right side were left unsaturated. The center two tracks were presaturated 10’ with pure benzene. The result is absolutely striking. By exposure of the dry layer to the vapors of the developing solvent the sequence of indophenol and acetorange is inverted. No such inversion occurs when the layer is exposed to pure benzene, i.e., the component that forms 99% of the developing solvent. This finding simply means that, within this chromatographic system, the result of “normal” tank development is entirely haphazard. This is demonstrated by Figure 6: Same chromatographic system as before; plate preconditioned at 42% relative humidity outside the chamber, then placed in twin trough chamber and developed immediately. Indophenol and acetorange are not separated. Had the time of exposure of the dry layer to the solvent vapors been longer, the separation had become similar to the left part of the chromatogram in Figure 5, had it been shorter, it had more likened the right part. The time of exposure in a tank is a function of the solvent level with respect to the sample origin.

figure 6

These findings were confirmed by a number of chromatograms, basically using the same chromatographic systems, but on HFTLC silica gel plates 50 x 50 mm. 137

After sample application the plates were placed in a twin-trough chamber saturated with vapors of benzene-methanol 99 : 1. Development was started by adding developing solvent to the trough in which the plates had been inserted. Variations of the conditions are listed in Table 11.

Table I1 Layer exposed to benzene-MeOH 99 : 1 a) prior to

no

10'

no

lo'

b) during development

no*

Yes

Yes

Yes

developing solvent

benzeneMeOH99:l

benzeneMeOH99:l

benzeneMeOH99:l

benzene

Rf butter yellow

0.56

0.55

0.59

0.51

Rf indophenol

0.08

0.40

0.17

0.32

Rf acetorange * sandwich cover plate ** resolution incomplete

0.18

0.29

0.21**

0.25

Conclusions:

If one of the components of a developing solvent is significantly more strongly absorbed than the other(s), development in a large tank is unsafe, as results become haphazard. Since it is generally recommended (1 1) that an adsorption chromatographic developing solvent be composed of a large portion of a comparatively non-polar solvent and of a small portion of a polar one, preferential adsorption must be expected in most cases. Reproducible conditions could be established either by complete presaturation of the layer or by leaving it completely unsaturated Consideration of the effect of high xi-values (see above) makes the unsaturated layer, i.e., the sandwich configuration more attractive. This holds true for adsorption chromatography. In partition chromatography pre-equilibration of the stationary liquid phase with the mobile phase appears advantageous, 138

The role of Vv, i.e., the solvent pre-adsorbed by the dry layer Sudan-I1 migrates on silica gel with ethyl acetate at Rf = 1. Therefore, it should be suitable for marking the true front of that solvent. Figure 7 shows a silica gel plate with Sudan41 applied in form ofa band and developed with ethyl acetate in the

figure 7

VARIO-KS chamber after pre-equilibrating the various tracks for 15 minutes with the vapors of different solvents. These were (from the left): first 3 tracks ethyl acetate, chloroform, benzene-methanol 98 :2, benzene, cyclohexane, no preequilibration (3 tracks). In the unsaturated tracks Sudan-I1 in fact migrates at Rf There are two possible explanations:

=

1, in all other at Rf< 1.

a) By pre adsorption of solvents more or less polar than ethyl acetate, the stationary phase is sufficiently modified to increase ts/tm. b) In all cases the true Rf, i.e., R’f is 1.0, which would mean that the solvent adsorbed by the dry later is pushed ahead by the migrating solvent front; the true solvent front only reaches the positions marked by the dye. The chromatogram depicted in Figure 8 precludes a).

Rgure 8

139

Silica gel layer, Sudan-I1 samples, pre-equilibration for 15 minutes and development in the VARIO-KS chamber. he-equilibration in the two tracks on the left: ethyl acetate-benzene 9 : 1, ethyl acetate-methanol 9 : 1; no pre-equilibration in the remaining 3 tracks. Track 1 and 5 developed with ethyl acetate-benzene 9 : 1, tracks 2 and 4 with ethyl acetate-methanol 9 : 1, center track with ethyl acetate. No matter whether the developing solvent is made more polar or less polar than ethyl acetate there is no difference between pre-saturation and unsaturation from this experiment to the results depicted in Figure 7.

figure 9

Explanation b) can be confirmed by Figure 9. On a silica gel phate Sudan-I1 samples were applied at the positions marked (x). The left side of the plate was presaturated for 15 minutes with ethyl acetate, the right side was left unsaturated. Then the plate was developed with ethyl acetate containing 0.115% Sudan-11. Development was allowed to proceed until the liquid at both sides had reached the score line. The true solvent front is recognizable by the limit of the red tinted area (marked - - - in the illustration). On the left side the true solvent front only reaches Rf = 0.65 (R’f = Rf 0.65). Ethyl acetate that got into the stationary phase via the gas phase is pushed ahead of the front of the Sudan labelled ethyl acetate migrating in the layer. The, Sudan-I1 samples applied to the layer are transported at Rf = 0.65, i.e., R’f = 1.0 by the non-labelled ethyl acetate. At the right side, the labelled developing solvent (as well as all samples) reach Rf = 1.0. 1

Conclusion:

Apart from partition chromatography (liquid-liquid chromatography) presaturation of the layer with the vapors of the developing solvent is of dis-advantage, since the available separation distance decreases, in certain cases drastically (Vv can reach 50% of Vm). Accordingly, separation zones beyond Rf = 5 can already be that close to R’f = 1 that resolution collapses. In that case the chromatogram

140

becomes unreliable with respect to qualitative as well as quantitative information. The unsaturated sandwich configuration in typical adsorption chromatography offers the best separation conditions. As we are aware of the fact that most TLC separations on silica gel are based on transition mechanisms between adsorption and partition chromatography the preference of either complete presaturation or complete unsaturation will depend on which separation mechanisms are expected to predominate. Intermediate saturationconditions, particularlythose which change during the developing process, should be avoided.

Transfer TLC-HPLC:Compatible experimental approach;is equilibration via the gas phase comparable to liquid phase equilibration? Development in the unsaturated sandwich configuration is obviously not suitable for TLC-HPLC transfer. In HPLC the sample is only introduced into the separation system after stationary and mobile phase have reached equilibrium. The CAMAG U-Chamber system offers a compatible experimental approach. When equipped with the sample injection valve (28710), it also permits sample introduction into the running system. The questions that had to be answered by experiment: a) What quantity of mobile phase has to be passed through the layer before equilibrium with the stationary phase has been reached? b) Can gas phase pre-equilibration substitute for equilibration by liquid phase? If so. to what extent?

In the first series of experiments Sudan-I1 (marking the R'f = 1 position) was chromatographed with ethyl acetate on H n L C silica gel MERCK 50 x 50 mm plates. After application of the first sample to the center of the plate mobile phase was added via the center capillary at a flow rate of 1.4 sec/pl. After each 30 pl the flow of mobile phase was slowed down, another small quantity (0.3 pl) of sample injected into the solvent capillary and the flow rate of the mobile phase set back to 1.4 sec/pl. After a total of 120 p1 the process was terminated, the chromatogram photographed and the radii of the concentric rings measured with a precision of k 0.15 mm (on the blown up print). The squares of mean radii measured in various directions from the center are recorded in Table 111 and plotted against pl of mobile phase in illustration 10. Table 111 also shows the various conditions of dynamic layer pre-equilibration. 141

Table 111 Experiment No:

61 “.”

62 “x”

63 “xn

Ring corresponding to volume of mobile phase

without pre-equilibr.

pre-equilibr. 2 minutes w. ethyl acetate

pre-equilibr. 2 minutes w. ethyl acetate -MeOH 9 : 1

473 408 252 112.4

564 4 10 256 115.6

546 410 256 115.6

~

~~

120 p1

90 60 30

~

?=

The position of the first sample of experiment No. 61, i.e., without layer preequilibration is off linearity, all others are on a straght line (with excellent precision). The straight line (calculated from ? by linear regression) intercepts x at 7 pl. Result: In this chromatographic system equilibrium is reached by 2 minutes dynamic gas phase conditioning or by passage of 30 p1 mobile phase respectively.

Figure I0

142

In the second series of experiments substances migrating with comparatively non-polar solvents at (linear) RfS 0.6 were investigated. A mixture of Ariabel red and butter yellow was chromatographed on the same type of silica gel layer under conditions described for the previous series. The intervals at which sample was 50 pl in this case. In experiments No. 73 and 76 the run was started with the sample introduction, in experiments No. 74 and 75 50 pl of mobile phase was added first. Further experimental data are listed in Table IV together with data on Ariabel red migration. These data are also plotted in Figure 11.

Table IV

Experiment No:

73

74

75

76

(Ariabel red ring corresponding to volume of mobile phase) Mobile phase

without pre-equilibr.

without pre-equilibr.

without pre-equilibr.

toluene

toluene MeOH 95 :5

toluene MeOH 95 :5

pre-equilibr. 5’ with toluene-MeOH 95 : 5 toluene MeOH 95 :5

-

-

-

-

600 484 289

1332 84 1 388

250pl 200 150 100 50

?=

306 234 174 114.5 50.4

676 676 529 309

.

All ? of experiments No. 73 and 76 are on straight lines, however, for different reasons. In case of the non-polar pure toluene, where no preferential adsorption can occur, the system is equilibrated as soon as the stationary phase is wetted by the solvent. In experiment No. 76 the intensive dynamic pre-equilibration with vapors of the developing solvent is suitable to equilibrate the system. Equilibration by the mobile phase itself requires the passage of more than 150 PI. At the first glance it might appear surprising that gas phase equilibration is more efficient This can be explained by the comparatively large methanol content of the gas phase and its access to all areas of the layer. 143

1200

-

Imm21

800

I

50

749,

150

250

figure 11.

Result: In the case of pronounced preferential adsorption of a solvent component present in a small proportion, gas phase pre-equilibrationis preferable. Equilibration via the mobile phase is also possible, but can require volumes of mobile phase that large that the solvent front may reach the edge of the layer before equilibrium is established. This situation can be coped with by continuous development. Again, all curves (calculated by regression analysis) intercept x at a value distinctly above 0, i.e., 8 pl. The conclusions drawn from these few experiments should in no way be considered as rules that can be generalized beyond the conditions investigated. It was the purpose of this article to suggest that chromatographers give more consideration to desired, and undesired, but inevitable interactions between all three phases in TLC - stationary, mobile and gas phase. 144

References 1. F. Geiss, H. Schlitt, A. Klose, Z. anal. Chem. 213,331 (1965) 2. C. L. de Ligny, E. C. Kok, J. Chromatog. 38,224 (1968) 3. E. Stahl, Chemiker Ztg. 82,323 (1958) 4. M. Brenner, A. Niederwieser, Experientia 17,237 (1961) 5. D. Janchen, J. Chromatog. 14,261 (1964) 6. F. Geiss, H. Schlitt, Chromatographia 1,387 (1968) 7. F. Geiss, “Die Parameter der Dunnschichtchromatographie,” Vieweg (1972). (Illustration 1 was reproduced from this book) 8. CAMAG, product bulletin 251-400 “The Vario-KS-chamber” (1969) 9. R. Kaiser, Z. anal. Chem. in print (Euroanalysis issue 11, 1976) 10. CAMAG, Firmendruckschrift 281-600 (1975) 11. e.g., D. L. Massart, J. Chromatog. 79, 157 (1973)

145

This Page Intentionally Left Blank

Chapter 8

Potential and experience in quantitative “high performance thin-layer chromatography” HPTLC Ute B. Hael Carl Zeiss, Oberkochen, Germany

Compared with conventional TLC, HPTLC is considerably faster and can be standardized much better. The various spot application techniques and procedures for the direct photometric evaluation of HFTLC plates will be discussed in detail (reflectance,transmission, simultaneousreflectanceand transmission, fluorescence “quenching”, fluorescence). The standard deviation of this method is better than 2.5%. The eficiency of HFTLC is illustrated by practical examples. The most important requirements of a photometer employed for the quantitative evaluation of HFTLC plates will be dealt with.

1. Introduction

Since about 1968 it has been possible to perform quantitative evaluation of thinlayer chromatograms by direct photometric scanning with commercialinstruments. After a great deal of scientificinvestigationthis technique has becomean invaluable tool for the daily routine in the analytical laboratories of the pharmaceutical industry, foodstuffs chemistry and clinical chemistry. In gas chromatography (GC) it is necessary to convert the samples into a gaseous state by heating them before analysis. Many of the organic samples - in particular those of higher molecular weight - decompose under this thermal load. A number of substances therefore has to be transformed into easily vaporizable derivatives before the GC analysis can take place. Latest estimates indicate that owing to these difficulties 90% of all organic substances could best be analyzed by liquid chromatography - i.e. column or thin-layer chromatography (LC or TLC). For many problems LC and TLC are based on the same separationprinciple; they differ only in how their mobile phase is moved (pressurekapillary action) and in the arrangement of their stationary phase (columdthin layer).

147

While in LC the chromatographic part is rigidly connected to the detector, thinlayer chromatography is performed in a chamber independent of the detector. For this reason TLC is more flexible for rapidly changing chromatographic separation problems and the development of new separations - a TLC photometer detector is generally not dependent on the separation systems used. While liquid chromatography can be rationally used in a laboratory always dealing with the same problem, thin-layer chromatography with subsequent direct photometric evaluation will be employed in laboratories dealing with many different separation problems. For the quantitative evaluation of such chromatograms only photometric procedures are really suited as even with experience the lowest error threshold reached in visual appraisal is still 10%. One possibility of quantitative evaluation is to scrape the substance spot with the silica gel from the chromatogram, then to elute the substance from the silica gel and finally to determine quantitatively the absorption or fluorescence of the solution with a photometer('). The disadvantages of this method are: a) Time requirement (about 2 hrs for 10 spots): Seen from a commercial point of view the large time requirement may be important, but it is also very critical for light-sensitive, oxygen sensitive or other decomposable substances which can change irreproducibly during the scraping and the elution. b) Incomplete elution of the substance from the silica gel and nonspecific apparent background absorbance by silica gel contained as acolloid in the elution solution: As a rule, after chromatography the substances have been firmly adsorbed to the silica gel and can be eluted from the silica gel only with accordingly large solvent quantities. Only in very rare cases is a substance eluted fully. Besides, a certain portion of the silica gel always enters into the solution during the elution process. The measuring light is scattered at this silica gel. That is why less light strikes the photomultiplier and this causes the apparent increase in absorbance. These two error sources can be eliminated only in part by determining blank values, samples and standards under identical conditions on the same chromatogram.

c) Large substance requirement: For an elution as complete as possible at least 5 ml of solvent are needed. To achieve asuff?cientmeasuringsensitivity,aboutlOto100pgofsubstance/spot and thus per 5 ml of solution should be available for absorption measurements. of course, smaller substance quantities per spot would do if the solution was concentrated after elution. But this would mean a further time requirement 148

and creating another source of error. - These large substance quantitieshpot resulted in an overload of the adsorbent layer and in tailing TLC spots with the previously used thin-layer chromatograms in the 20 cm x 20 cm size (in the followingcalled “macro-TLC”).The new HPTLC pre-fabricated plates described below are totally overloaded with substance quantities larger than 10 pg/spot and do not provide any separation then - they are therefore no longer suited for the elution method. In comparison with the latter, for the direct photometric measurement on the chromatogram, in macro-TLC only about a tenth of the substancehpot is required, in HPTLC only a hundredth (see Table l), to obtain the same peak height. In summary it can be said that a quantitative determination of HPTLC spots by the elution method is not practical. - The possibilities of photometric evaluation of HPTLC spots direct on the chromatogram are described in sections 3-6. Disadvantages of the macro-TLC employed up to now are the long chromatography duration and the unfavorable relation between the number of samples and the number of standards as only a maximum of 12 tracks can be used on one chromatogram. In this field the new “High Performance Thin-Layer Chromatography” = (HPTLC or nano-TLC)(” has brought about quite considerable progress. The HPTLC prefabricated plates* used in the investigations described below exhibita better chromatographic resolution, which isdueto the carefullyselected silica gel. Provided the diameter of the spots applied does not exceed 1.5 mm the optimum of the chromatographic resolution is reached at a 3-5 cm migration of the solvent. As in most cases the same solvent systems are used in HPTLC as in macro-TLC, in comparison with the latter the separation times can be reduced by a factor of 5 to 10. This way the HPTLC separation times are only about 4 to 20 minutes. Owing to the short separation distances and times the spot diffusion at right angles to the solvent flow is very small so that the diameter of the separated spots exceeds 4.5 mm only in very rare cases. This permitsa trackspacingof5mm;thisway 18trackscan beusedona10cmx lOcm plate or even 36 tracks on a 10 cm x 20 cm plate (Fig. 1). This creates substantial advantages for the standardization of the method in routine measurements (see section: Reproducibility).

* Supplier: E. Merck, Darmstadf Germany Available: HPTLC prefabricated plates for nano-TLC, silica gel 60 F254 or without fluorescent indicator, size 10 cm x 10 cm or 10 cm x 20 cm.

149

2. Application techniques The microliter syringes and glass microcapillaries used in macro-TLC yield spots with too large a diameter for HPTLC. In no case must the diameters exceed 2 mm in HPTLC. J. Ripphahn and H. Halpaap(’) use a 1 pl Hamilton syringe* in combination with an Agla micrometer screw**. Similarly, H. Jork uses a 10 pl Hamilton syringe* with a Desaga micrometer screw***. The advantage of this system is that - if the micrometer screw is turned slowly enough - volume quantities of the solutions of 500 to 2000 nl can be applied. This is of importance if sample solutions of very low concentration are to be examined. However, manipulation of such systems when one wants to exchange the sample solution for the reference solution is somewhat cumbersome. For this reason the tests described in this work were performed with a platinumiridium capillarym* designed by R. E. Kaiser (Fig. 2). The capillary has an internal diameter of about 0.15 mm. The volume is 0.23 p1 and was photometrically determined witha dye solution. (For this the capillarywas filled several times and emptied on a filter paper. After elution from the paper the dye solution volume aspirated by the capillary was determined with a photometer). The capillary is fixed with a platinum wire to a glass rod. During the first tests the capillary was held by hand. After spotting, the capillary should be kept in methanol, for example. It must on no account hit the bottom of the container as in this case it can be bent inwards. If this should happen the capillary can be straightened by inserting a platinum wire and by rotating the latter. When application is done by hand the capillary tends to be clogged with silica gel. It can be cleaned with a thin platinum wire. The clogging of the capillary with silica gel of the adsorbent layer can be avoided almost completely if the capillary is built into the EVA-Chrom-TLC-Applicator’) (Fig. 3). With the aid of a rocker arm the capillary can be let down smoothly on to the adsorbent layer in a vertical position. This system permits a standard deviation for 10 spots of better than 2.5% to be attained in routine (section 4: Reproducibility). The same capillary is used for all solutions; rinsing is done twice either with the new sample or reference solution. For Sudan red in benzene solution a carry-over error of 4% was found, i.e. the carry-over error is absolutely * Supplier: Hamilton Co., Reno, Nevada, USA ** Supplier: Hormuth + Vetter, Heidelberg, Germany *** Supplier: Dasaga, Heidelberg, Germany **** Supplier: Antech, D-6702 Bad Diirkheim, Germany I)

Also obtainable from Carl Zeiss, New York, N.Y.

150

negligible if rinsing is done twice with the solution to be applied. All sample and reference solutions are applied in identical volumes. Different substance quantitieshpot are caused by different concentrations in the dispensing solutions.

Fig. 1:

Comparison between macro-TLC and HPTLC for multicomponent dyestuff mixtures. Macro-TLCplate (top) with glass microcapillary in holder and H m C p l a t e (bottom) with platinum-iridium capillaryfor spotting. Both sides of the HP7ZCplates can be used. 151

2a

26

Fig. 2 a: Platinum-indium capillaryf u e d with a platinum wire to a glass rod (prototypefrom R. E Kaiser).

fig. 2 b: From prototype tofinal product: HPTLC sampler (platinum-indium capillary) according tofigure 5.4 on page 90;volume 100 nl, 200 nl, 500 nl (ANTECH GmbH, P. 0.B. 1308,04702 Bad Diirkheim-I, Germany) 152

fig. 3:

EVA-Chrom-TLC-Applicator with platinum-iridium capillaty for the application of HPIZC spots.

153

3. Photometric procedures(4d) for HPTLC evaluatiod2) Absorption measurements in reflectance and transmission modes:

In photometric measurements the TLC spot is scanned by a slit whose width can be adapted to the diameter of the spot. Following the solution photometry, an attempt was fust made to quantitate TLC spots with the aid of a transmission measurement via the Beer-Lambert law (E = E c * d). An essential prerequisite of this law is, however, that the sample does not scatter the measuring light. This condition is not met exactly as the light is strongly scattered at the adsorbent layer. For this reason there is no linear relationship between absorbance and substance quantity/spot for TLC plates (Fig. 4, curve 1). It is however this diffuse scattering (= reflectance) of the light at the adsorbent layer of TLC plates that permits quantitation of the spots: a spot-free background diffusely reflects all the light whereas the spot absorbs a portion of the incident light so that the spot diffusely reflects less light. This reflectance diminution is the larger the more light is absorbed; hence, it depends on the substance quantity/spot and on the measuring wavelength. It is true that, compared with reflectance measurement, transmission measurement provides a larger peak height (Fig. 5). But as in transmission mode measurement is made through the adsorbent layer; layer thickness fluctuations distinctly affect the transmittance. This results in fairly serious baseline fluctuation in transmission measurements. In practice the interest is after all in the ratio of peak height to background fluctuation and this is always better for reflectance (Fig. 5). That is why - compared with transmission measurement - reflectance measurements provide better detection limits, measuring reproducibilities and more favorable working ranges for quantitative measurements. Transmission measurements are also limited in wavelength by the UV absorption of the carrier material (glass in general) and of the silica gel. This means that transmission measurements below 325 nm are not possible unless fluorescence “quenching” is employed. Disadvantages of this system are described below. If you consider that more than 80%of all the substances separable by TLC absorb only in UV it proves a disadvantage in practice that transmission measurements cannot be employed in the UV range. The staining by spraying the plate, often used for this reason, results in a deterioration of the reproducibility and is also an additional time requirement. - Contrary to this, reflectance measurements can be easily performed over the entire spectral range from 196 nm to 2500 nm, i.e. above all in the UV range, which is so important in practice. 154

[rel integr.-units]

!(1-R)

[rel. integr.-units] 022

2

1 200

500

TIME 13 bb 102 126 150 186

LOO

300

215 237 262 266 342 368

100

200

dy ID

1 P I 1 5 0 0 SS 1 8L 6 0 TP 9999 U A AREA 231821 215888 190109 15117A 91391 58099

236698 211811 191080 150627 98045 57159

1oc

c [ng /spot]

d 10 20 LO o 80 100 fig. 4:

Comparison between reflectance and transmission measurements, 10-100 ng Sudan red, A = 500 nm, data-pair technique, calibration curves and printout of computer integrator.

flog 1/T dy ID

1 P I 20 ss 1 BL 6 0 TP 1000 M A AREA 5117 4a91 4424 3636 2417 1488

5460 5130 4601 3754 2399 1620

ng 100 I D 8 0 10 6 0 ID 4 0 10 2 0 10 1 0 ID

1 0 0 I0 80 I 0 60 I D 4 0 ID 20 I D 10 I D

If, in reflectance measurements, the area between the reflectance-location curve and the baseline is plotted vs. the substance quantity/spot, slightly curved calibration graphs are obtained which pass through the origin (Fig.4, curve 2). Small substances yield straight calibration lines through the origin (Fig. 6).

scale division = Skt

Fig. 5:

Comparison of the values of reflectanceand transmission measurements, 20 ng Sudan red, h = 500 nm.

156

'

F [rel. integr.-units]

4,000 273,000. 2,000

151,000* 1,000 68 ,000.

4,000 281,000* 2,000 145,000.

1,000 65,000.

c [ng / spot]

1

2

i4

R

0,998

N

6,000

B

2,000 69,357

A

Fig. 6: Rejlectance measurements of very small substance quantities, I-4 ng Sudan red, h = 500 nm, data-pair technique, calibration line and printout of desk computer for the calculation of the regression lines and the correlation co#ficient R.

Smoothing of baselines by simultaneous reflectanceand transmission

) '

The detection limit of photometric measurements of absorption is - with appropriate electronics - not determined by the instrument noise but by the inhomogeneities within the adsorbent layer. In the long-wave visible spectral range the measuring radiation is not as strongly scattered in the UV range and therefore penetrates into the layer. For this reason layer thickness differences make thernselves felt in the form of baseline fluctuations in the long-wave visible range. 157

Much larger baseline fluctuation can be observed with macro-TLC plates than with HPTLC plates. The cause for this is that the layer thicknesses of HPTLC plates are more constant and what owing to the greater light scattering at the adsorbent layer less light penetrates into the layer. Although the baseline drift of HPTLC plates is considerably lower, even here the exactly complementary run of the baselines in transmission and reflectance can be observed (Fig. 7). Where the layer is thicker, more light is diffusely reflected and less light transmitted. For this reason reflectance and transmission values are added according to a corresponding weighting. With the aid of this simultaneous reflectance and transmission measurement (German patent 2, 047, 952, GB patent 1, 328, 734, US patent 3, 746, 869) at the same time, at the Same place and at the same wavelength the baseline can be smoothed (Fig. 7); this improves the ratio of peak height to baseline and this way the detection limits.

Absorption measurements in

UV andfluorescence “quenching”

The reflectance method can also be applied to UV-absorbing TLC spots. Frequently fluorescence “quenching” is recommended here. This method is wellproven for visualizing UV-absorbing TLC spots. But if this method is employed for quantitative photometric evaluation of TLC plates it is accompanied by some disadvantages as compared with reflectance measurement: a fluorescent indicator is added to the adsorbent as homogeneously as possible; this indicator fluoresces mostly green if excited at short wavelengths (Fig. 8). If such a chromatogram with fluorescent indicator is exposed to light of short wavelength the UV-absorbing spots appear dark against the brightly fluorescing background as they act as a fdter, as it were, which absorbs a portion of the fluorescence excitation radiation; that is why at the place of the spots less fluorescence is produced. Consequently, in terms of physics, this is not a quenching effect but an absorption measurement at the wavelength of the fluorescence excitation. That is why this method should more appropriately be called fluorescence diminution(’). Fig. 9 11, IV shows that the reflectance measurement at 254 nm and the fluorescence diminution measurement with excitation at 254 nm (Hg-line) yield identical peak heights. But compared withreflectance measurement fluorescence diminution measurement exhibits severe background fluctuations which are a result of the inhomogeneous distribution of the fluorescent indicator in the adsorbent layer. For this reason - despite identical peak height - the ratio of peak height to background fluctuation is better for the reflectance measurement than for the fluorescence diminution measurement. But even with reflectance measurements on HPTLC plates with fluorescent indicator there is a background fluctuation 158

159

which is clearly larger than the electronic noise of the photometer. This residual background fluctuation can be explained in part by the intrinsic absorption of the indicator in UV. It can be reduced if H€“LC plates without fluorescent indicator are used for reflectance measurements in UV. In principle, fluorescence “quenching” also permits a photometric determination of UV-absorbing TLC spots in transmission as the long-wave fluorescence radiation can penetrate through the glass plate and the adsorbent layer. As shown in Fig. 9 111, however, this results in serious drawbacks for the peak heightlbackground fluctuation ratio. The fluorescence diminution method is used with fdter photometers having only one mercury burner with the 254 nm line for excitation (perhaps also 281 nm and 313 nm). For the UV range, so important for organic substances, there are therefore only few lines available above 254 nm for absorption measurements. Hence, fluorescence diminution measurement loses in specificity and sensitivity for UVabsorbing substances as normally measurement cannot be made at the absorption maximum of the substance. How the peak height can be optimized by varying the wavelength in a spectrophotometer is shown by Fig. 9 I, 11. Fluorescence diminution measurements are frequently used with monochromator instruments as this allows transmission measurements in W via the long-wave emission of the fluorescent indicator (Fig. 9, 111). Compared with reflectance

- ---- excitation radiation, 254 nm Hg green emission radiation

adsorbent + indicator carrier gias

Fig. 8: Schematic illustration of thefluorescence “quenching” method.

160

I

Fig. 9:

Comparison between reflectance and fluorescence diminution measurement, drug mixture.

measurement, this method is inferior as regards background fluctuation (Fig. 9, 11, 111). Furthermore, with the fluorescence diminution method only those substances can be detected whose absorption is above 240 nm. The excitation maximum of the fluorescent indicator is about 280 nm, below, 240 nm hardly any excitation can be observed. That is why substances only absorbing in the range below 240 nm do not act as a filter for the fluorescent indicator and cannot be detected in fluorescence diminution measurement. Such substances are, for example, benzoic acid (K = 226 nm), digitalisglucosides(A = 225 nm), phenobarbital (A = 215 nm) or atropine sulfate (A = 1% nm). In summary it can be said that for direct quantitative photometric evaluation, reflectance measurement - in comparison with transmission and fluorescence diminution measurements - yields the best results and optimum selectability of the measuring wavelength. Chromatograms with fluorescent indicator are also measured best in reflectance.

4 F [rel.integr.-units]

aoo600400-

200c [ngI spot] 1

20

4-0

60

80

100

fig. 10:

Fluorescencemeasur~enrsoflargesubstancequantities.calibrationcurvef o r b 1OOng rhodamine B, hat, = 546 nm Hg Aem. = 585 nm filter fi 56)

162

Fluorescence measurements

Compared with the fluorescence diminution measurement, the measurement of the intrisic fluorescence of TLC spots offers the following substantial advantages: a) Enhanced selectivity Non-fluorescing substances are not measured; the variation of the excitation and emission conditions increase the spectral selectivityover that of absorption measurement.

b) Enhanced measuring sensitivity This is improved by a factor of 10 to loo0 over absorption measurements.

F [rel. integr.-units]

2000 X(I), 0,010 34,000. 0,020 65,000.

0,050 166.000. 0,100 407,000'

0,200 797,000*

IAO fig. 11:

0,500 1905,000*

c [ng / spot] R

1,000

N

6,000

Fluorescence measurements of small substance quantities. B -0,633 0.01-0.5 ng rhodamine B, A 3838,750 A a C . = 546 nm Hg, Aem. = 585 nm filter E5 56) calibration line and printout of desk computerfor the calculation of the regression lines and correlation co&cient R.

163

c) Increased linearity Following the solution photometry there is an exponentialcorrelation between the fluorescence intensity IFI and the substance quantity c/spot:

where Ioh = light intensity at excitation wavelength This formula, which can be derived from the Beer-Lambert law, is confirmed by the example of rhodamine B (Fig. 10). To small products EA . c - i.e. for small substance quantities c - the linear approximation applies

this is known as the basic formula in fluorometry. Thus, for 0.01 - 0.5 ng rhodamine B a good straight calibration line through the origin is obtained (Fig. 11). To achieve measuring sensitivities as high as possible the proportionality factor Ioh EA should be as large as possible. If for excitation the xenon lamp (quasi continuum emitter) is used, EA is generally optimized. But practice has shown that owing to the high radiation intensity of the mediumpressure mercury burner the product Ioh * EA is better at the Hg-lines than that which is obtained with a xenon lamp for the wavelengths at the excitation maximum of the substances.

-

d) Independence of the result of the spot shape('') The fluorescence intensity is measured at the photomultiplier. The value obtained is a linear measure of the fluorescing substance quantity - no additional mathematical conversion is needed as is the case with absorption measurements. For this reason it is of no consequence how the fluorescent substance is distributed within the spot provided one makes sure that the total spot is scanned. As a measure of concentration the area between the fluorescence-location curve is taken. The independence of the result of the spot shape is ofgreat importance for the investigationsof extracts (comparisons between pure substance and substance in extract) and for two-dimensional chromatography (comparisons between one and two-dimensionally separated substances).

164

196

118 136 156 175

T I .3E 21 PO 60 01 99

815

10

48035 485Ub 40019 48211 48132 40116 48161 48316 40427 40394

AMEA

1 BL 60 TP ?939 MA

315 SS

I Ph

st.dev.

mean

1,9

483.7

,

484.0 485.0 484 0 482.0 481,O 488,O 482.0 483.0 484,O 484.0

4. Reproducibility

The total reproducibility of the direct photometric evaluation of HPTLC plates depends on: a) The spotting reproducibility: The results given in the following refer to the platinum-iridium capillary in the EVA-Chrom-TLC-Applicatoras described in section 2. A direct determination of the spotting reproducibility has not been possible yet. b) The reproducibility of the photometric system employed (mechanical characteristics of the scanning stage, the photometer and the computer integrator): Fig. 12 shows the reproducibility for 80 ng caffeine (measured with the Zeiss KM 3 chromatogram spectrophotometer). For this, one and the same HPTLC spot was scanned 10 times. Between the measurements the spot was moved out of the measuring area and then moved back with the aid of the full deflection of the recorder. It should be mentioned that the entire adjustment - very important for reproducibility - took less than 2 seconds per spot. The standard deviation* of the photometric system is far better than 1%. c) The reproducibility of the Rf-value within one HPTLC $ate: The Rf-values are, in the first place, affected by the layer thickness fluctuations of the adsorbent of the plate. Previous observations show that these are considerably less on HPTLC than on macro-TLC plates. On the other hand, the Rf-values can vary with a sagging solvent front. - These effects of the chromatographic parameters on the result in macro-TLC can be corrected by the datapair technique(") of Professor Frei (Fig. 13). For this, a double analysis of Standard and sample is performed on the chromatogram. Pairs are formed one member of which is in the one half of the plate and the second member in the other half. This method permits a reliable average to be taken of the varying chromatographic parameters in a plate. This effect is not achieved in the frequently used double analysis of adjacent tracks. - We successfully applied the data-pair technique also to HPTLC plates (Fig. 13). With its greater number of trackdplate this method is particularly suited for HPTLC: it is generally recommended to employ 3 standards for quantitations. On a macroTLC plate, for example, 10 tracks can be applied, i.e. 5 samples and standards

* This article always gives the standard deviation of the individual value which is calculated as follows: n-1

166

in double analysis. This permits only (5-3) = 2 samples to be determined on a macro-TLC plate when the data-pair technique is employed. A 10 cm x 10 cm HPTLC plate permits the use of 18 tracks, therefore 0.5 x 18 - 3 = 6 samples can be determined. For 10 cm x 20 cm HPTLC plates with approx. 36 tracks the number of samples/plate is still better: 0.5 x 36 - 3 = 15 samples. This is quite an essential point as it is necessary to determine the calibration curves for each plate with the aid of the standards. In summary, the following can be said: with the data-pair technique the number of samples per plate can be increased by a factor of 3 to 7.5 by the use of HPTLC plates as compared with macro-TLC plates. If you also consider that separation times are reduced by a factor of 5 to 10, then this means that with HPTLC - in comparison with macro-TLC - the chromatographic analysis time is reduced by a factor of 15 to 75. Figs. 14, 15, 16 show some examples of the reproducibility of photometric evaluation on HPTLC plates. For this, 10 spots each of the same sample were evaluated on one plate. The standard deviation in routine measurements is better than 2.5% and can be further improved by the data-pair technique. I

I @

II ;. .

I

0

.

.

. .

.

. . I . . I -

4

.

*

*

.

. .

I I

Xr

Fig. 13:

XPX3

s,

X4%

s2

4% 3 x 3 s3

s<

x,

x4xs s2

s3

Data-pair technique on a 10 cm x I0 cm HPTLCplate, standards: Si - S3, samples Xi - X6

167

a 4

ID

1 PI 500 ss 1 eL 6 0 TP

9 9 9 9 HA

, ,

2727 0 2724 0 2750.0 2715,O 2758 ,0 2731,O 2746 ,0 2738,O 2763.0 2702 0

hHEA

T 1HE

20 62 95 133 164 195 228 260 296 33 1

272608 272390 275034 271499 275779 273099 274589 273831 276336 270209

,

mean st.dev.

2735,4

,

2729 0 2735 ,0 2744 ,0 2739 ,O 2730.0

mean st.dev.

2735.4 6,3

data-pair technique

19,3

Fig. 14: Reproducibilityfor 10 spots each of 60 ng Sudan red, simultaneous rejlestance and transmission measurement,A = 5 0 nm, absorption-location curves, printout of the computer integrator, printout of the desk computerfor the calculation of the standard deviations without and with data-pair technique.

N,

a, Q) Q)

a

f

m c

--

+,

r

1

ai ur

i

o m w I-

U Y ~ ~ N O U Y N D O ~ U Y O C

o

o (D v)

v)

- -

169

e

4 0

I0 1 PW 250 SS

682,O 696.0 706.0 695.0 708.0 696 ,0 697 0 701.0 692,O 689.0

1 BL 60 Tp 9 9 9 9 nA TIME

AH€&

12 42 7A 106 135 171 20 I 232 264 289

681638 696009 705030 694869 706083 695645 697171 701230 692254 689425

,

mean st.dev.

696.2

689.0 696.5 703.5 693 ,5 698,5

mean st.dev.

696,2 5.4

data-pair-technique

7.7

fig. 16:

Reproducibilityfor I0 spots each of 20 ng thiabendazole. fluorescence measurement, Amc. = 313 nm Hg,Am. = 35s nm filter M365),fluorescence-locationcurves,printout of the computer integrator,printout of thedesk computer for the calculation of the standard deviations without and with data-pair technique.

5. Qualitative initial examinations For substance identification it is recommended that the reflectance or fluorescence-location curves be recorded. This, of course, can be done also for substances absorbing only in UV. The Rf-values can be determined directly from these recordings (Fig. 17). Of the substances under test, absorption spectra in

scale division = Skt I

I

I

-1

I

I

I

I 1 I

I

I

I I

I

I

I

I

I

I 1 1: I 1 I 1 I 1

I

I

I

I 1 1

11 I 1 I.

I I I

I 1 I I 1

I I-

I I

I

I 1 I

'

I -

I

I I I

1

I I I

I

I

I

I I

I

I

I I

1

I

I

9

I

--I

I

I

I

I I

1 I

I

I

I

I

I

I

I I

I

I

I I

I 1 I

I

I I

1

.I

I

I

I

I

.I I1

I I

I I

-1 I1

I

,

I

,

I

I

-

I

I

I

I1 I

I

I

I

I1 :I

I

I

I1 I1

I

I

~~~

I I I

I I I I

11

I

I

I I I

I I I

I

I I

II

I

I

I

II I

-'

I

I

i

I I

I I

I I I

1

'

I 1

I

I

1 I

=solverit =front 1

I

I

I I

I

I

1 1 1

I

I I

I

I

I

1

1

.I r

I I

-

I

I I

I :pp~i?EIE Pot

Fig. 17:

Example of the determination of the Rf-values from the reflectance-location curve. 17 1

reflectance, fluorescence spectra, excitation or emission spectra can be plotted directly from the chromatogram (Fig. 18). As the spectra of the chromatogram and of the solutions can differ, in critical cases the substance should always be examined in the sample as well as in the chromatographically separated pure state.

I I

-

I I

I I

-I I

I

I

I I I

I

I

I

I

1

1

I

I I

1

I

I

I I

'

I

I

I I

I

I

I

I

I

1

1

I

I

I

I

I

I I

I I I

I

I

I

I I I

fig. 18:

Absorption spectrum in rdectance mode, plotted relative to plate background, 80 ng caffeine h = 195 - 300 nm.

172

1 I I I

1 I I I I

I I

I

I

I

1 I

I

I

I I

I

I

1

I I

6. Practical examples

a) Caffeine and phenacetin (Fig. 19) : benzene/ether/acetic acid (60/30/10) Solvent Migration : 3 cm Separation time : approx. 5 min Measurement : reflectance, Acaffeine = 275 nm Aphenacetin = 250 b) Sorbic acid in wine (Fig. 20) : toluene/ethyl acetate/chloroform/formic acid (25/13/10/2)('*) Solvent Migration : 4 cm Separation time : approx. 5 min Measurement : reflectance, A = 262 nm

100

50

I

20

I

LO

I

60

I

80

I

100

-

fig. 19:

Quantitative determination of caffeine and phenacetin, reflectancemeasurement, hcafleine = 275 nm, hphenacetin = 250 nm. 173

For analysis the white or red wine is applied to the chromatogram without any preparation. Incompletely separated benzoic acid does not have any prejudicial effect as it hardly absorbs at 262 nm. : saccharose and glucose (Fig. 21) : dichloroethane/acetic acid/methanol/water

c) Sugar Solvent

(S0/2S/15/10)3) Migration : 2 x 6 cm Separation time : 2 x 20 min = 40 min : the plate is dipped into a reagent consisting of 2 ml aniline, Staining 10 ml of 85% phosphoric acid, 2 g diphenylamine ad 10 ml methanol. Of course, the plate can also be sprayed with the reagent but in this case background fluctuation becomes larger. HPTLC plates are especially suited for this immersion method as owing to the shorter migration distances a small volume of immersion solution is required.

1

F F [rel. [rel. integr.-units] integr.-units]

100

50

c [ng / spot]

20

LO

fig. 20:

Quantitative determination of sorbic acid, reflectance measurement, h = 262 nm. 174

60

80

Measurement : reflectance or simultaneous reflectance and transmission, h = 625 nm d) Quinine in bitter mineral water (Fig. 22)

Solvent

: benzene/diethyl ether/dichloromethane/diethylamine

(20/20/20/8)('3) Migration :4cm Separation time : approx. 5 min. Staining : immersion in 95 ml diethyl ether and 5 ml conc. sulfuric acid Measurement : fluorescence hexcitation = 365 nm Hg hemission = 460 nm (filter FL 43)

Fig. 21:

Quantitative determination of glucose and saccharose, simultaneous rejlectance and transmission measurement, h = 625 nm. 175

e) Thiabendazole in citrus fruit (Fig. 23) Solvent : ethyl acetatelmethyl ethyl ketone/formic acid/water (50/30/10/10)10) Migration :4cm Separation time : approx. 5 min Measurement : fluorescence hexcitation = 313 Hg = 355 IIIII (filter M 365) hemission The substance is known to be extremely light-sensitive(''. 14). But with the analytical conditions used no photochemical decomposition whatsoever of the sample has been observed("). - The spot shapes of the pure substance and of the sample differ markedly. For this reason the results are adulterated in reflectance. In fluorescence measurements no effect of the spot shape on the result can be observed(").

. i

0

16

fig. 22:

Quantitative determination of quinine sulfate, fluorescence measurement, hexc. = 365 nm Hg hem. = 460 nm filter I% 43).

176

24

f) Aflatoxins in peanut extract (Fig. 25)

The one-dimensional separation of aflatoxins on HPTLC plates has already been described(*). Especially in peanut extracts there are many fluorescing impurities which can be separated from the aflatoxins only in two-dimensional chromatography. Solvent

: first dimension: diethyl ether/methanol/water (96/4.5/ 1.5)

second dimension: chloroform/acetone (90/10)(15) Migration :2x8cm Separation time : 2 x 20 min = 40 min Measurement : fluorescence Hg hexcitation = 365 hemission B1, B2 = 428 nm (filter FL 43) hemission G1, (32 = 450 Nn (filter FL 43)

A

F [rel. integr.-units]

400 300 200 100 c [ng / spot] Y

I

5

I

10

I

I

I

15

20

25

m

Fig. 23:

Quantitative determination of thiabendazole, fluorescence measurement, ABC. = 313 nm Hg hem. = 355 nm filter M 365).

177

In most cases the aflatoxins are determined in the sample on the same plate with the aid of the one-dimensionally separated standards Fig. (24). But there are differences of up to 15% in the slope of the calibration lines for the two dimensions. This is due to the fact that the different solvents for the two dimensions affect the fluorescence quantum yield. The so-called anti-diagonal technique(”) of Beljaars prevents this by using the same solvent conditions for standard and sample and measuring the latter on the same plate. As shown in the sketch of Fig. 25, one sample and two standards are applied in an anti-diagonal in the left corner. For identification two additional standards are applied to the one-dimensional tracks (see sketch). With the solvent systems selected the impurities are in the upper “dirt zone” (Fig. 25). For quantitation the tracks must be marked by dots behind and in front of the aflatoxins under a UV lamp. These dots help to adjust the tracks on the scanning stage of the photometer. The quantitative results are shown in Fig. 25.

AC B D

s3 s2 S1

P Fig. 24: Sketch of a two-dimensionalchromatogram, P = sample S1- S3 = standard mixtures A - D = components.

178

I0

Ell

s, 0

2 PI 2500 S S 5 EL

0000

6 0 TP

5000

0 0

p = sample S,, S2= standard

U

TlUE

El0 27 33

NA

AREA

8

I0

46

113009 89444 179402 108482

El0 111 117 122 129

247404 165493 391504 226794

El0 315 322 328 336

194781 144552 212329 118303

38

2 2

2 3

16 I0

2 2 2 3

10

2 2 2 3

fig. 25: Quantitative determination of ajlatoxins in peanut extract with the aid of the antidiagonal technique, fluorescence measurement, hat. = 365 nm Hg, hem., BI,B2 = 428 nm hem., GI, G2 = 450 nm filter 43) sketch of the chromatogram,printout of the computer integrator,fluorescence-location CtlNes.

7. Requirements of the instrument for the photometric evaluation of HPTLC chromatograms

The requirements of quantitative HPTLC analysis can be combined under the following two formulas: optimum signal - depends on: the photometric method the amplification reserve of the photometer the electronic noise of the photometer the chromatography incl. staining (if needed) optimum reproducibility - depends on: the photometric method the precision of the mechanical system used for scanning the electronic noise of the photometer the spotting the chromatography incl. staining (if needed)

In view of the above a photometer for HFTLC should meet the following requirements: 1. It should be rapidly adaptable to a variety of problems by being capable of working in reflectance, simultaneous reflectance and transmission, and in fluorescence.

2. It should have a monochromator so that the user can choose the wavelength best suited for his task and plot spectra direct from the plate. 3. It should have a precise mechanical system for the scanning of the samples to ensure good reproducibility. 4. An optical imaging system carefully adjusted to the tasks and low-noise electronics should permit a large expansion of the measuring signal.

5. Operation should be as easy as possible, the time required for measurement as short as possible.

180

Fig. 26: Zeiss KM 3 chromatogramspectrophotometerfor routinemeasurements of reflectance, transmission, simultaneous rdectance and transmission, andfluorescence. (From right: lamp power supply, on base plate: housingfor tungsten, deuterium and mercury source, monochromator, intermediate optics, optical head with scanning stage, indicator unit, computer integrator and recorder).

All the measurements mentioned in this work were performed with the Zeiss KM 3 chromatogram spectrophotometeP (Fig. 26) which meets all these requirements:

Re 1: A simple switchover allows the instrument to be set to the three types of measurement without conversion ofthe instrument. The light ofa tungsten or deuterium lamp is spectrally dispersed by a monochromator and imaged through a slit on to the sample. The diffusely reflected light is measured at an angle of 45' by a photomultiplier.

* Supplier: Carl Zeiss, Oberkochen, Germany. 181

Fig. 27: Scanning stage of the Zeiss chromatogram spectrophotometer with Hp7zC plate.

For simultaneousreflectance and transmission measurement a second photomultiplier is fixed on the underside of the chromatogram. The signals of the reflectance and of the transmission photomultiplierare electronically added. For fluorescence measurement in general a mercury burner is used for excitation. With the monochromator the Hg-lines from 254 nm upwards are selected. Between sample and photomultiplier a cut-off or wide-band fiter is inserted which eliminates the excitation radiation. Apart from this basic design of the instrument chiefly used in routine analysis, further measuring set-ups for special research problems can be created by reversing the beam path. Or a xenon lamp may be used for fluorescence excitation. The possible setups are given in Table 2.

182

Re 2: A quartz prism monochromator is employed (A = 185-2500 nm) which is particularly efficient in UV. A special hydrogen source with Suprasil windows permits measurements below 200 nm without nitrogen flushing and yet without straylight. Wavelength reproducibility in the visible range is better than 0.5 nm and in UV better than 0.1 nm. Re 3: The scanning stage (Fig. 27) for chromatograms of up to max. 20 cm x 20 cm runs on ball bearings. It is movable in x and y direction by hand, in y direction it can be driven by a synchronous motor (1-300 mmlmin). Setting reproducibility is 0.1 mm in both directions. This precision is of particular importance for HPTLC evaluation. The scanning stage is easily accessible from all sides so that the individual tracks can be rapidly set. This feature also facilitates the adjustment of the TLC plates for two-dimensional chromatography. The scales for the x and y directions permit the rapid and reliable evaluation of two-dimensional chromatograms. Re 4: The entrance slit is imaged on the chromatogram. The imaging is achromatic, i.e. the size of the measuring area is independent of the measuring wavelength. The length of the slit-shaped measuring area (track width) can be set in steps from 3.5 to 14 mm. The theoretical local resolution can be continuously adjusted from 0.01-2 mm. For HPTLC plates a track width of 3.5 or 6 mm was used. The new K M 3 chromatogram spectrophotometer is equipped with fast response, low-noise transistor electronics so that the instrument despite the open scanning stage - is not sensitive to extraneous light The amplification reserve is larger than the factor lo4.The measuring sensitivity is so good that generally the first amplification step is used for measurement. For this reason the measuring signalscan be usefully expanded on the recorder up to a factor of 20. The analog output supplies 1 V for full deflection and is normally virtually undamped. Apart from a chart recorder a computer integrator* can be connected direct.

Re 5 : The few controls are clearly arranged on the indicator unit (Fig. 28). An automatic amplification unit sets the plate background to 100%or any other feedable value before measurement. The control accuracy is 0.1%. This automatic amplification unit also facilitates the plotting of reflectance spectra (Fig. 18). Quantitative evaluation can be performed either with an overlay

* Obtainable from Carl Zeiss, Oberkochen, Germany

chart thus determining height x half width of the peak or with a disc integrator in the recorder. However, evaluation of the peak areas is faster, easier and more reliable with a computer integrator*. This integrator available for the instrument has variable integration intervals and therefore provides an excellent reproducibility even at rapid scanning speeds (in HPTLC generally 30-120 mm/min). Drifting baselines and overlapping peaks are corrected. The computer integrator is easily programmable. Measurements can be also performed with the predecessor of the KM 3 - the chromatogram spectrophotometer with PMQ I1 electronics(2).The instrument provides the same results - but with considerably less operating comfort. My thanks are due to Mr. Scharf for having performed the tests. I am especially indebted to Dr. W. Tausch for the detailed discussions which greatly contributed to this article.

184

Fig. 28: Indicator unit of the Zeiss KM 3 chromatogram spectrophotometer Vrom top l@t to bottom right) - damping switch for galvanometer and analog output (normal position 1 = 0.7 sec), galvanometerfor fine adjustment of the chromatogram tracks and for setting the factor fR :fT for simultaneous reflectance and transmission measurement, selector switch for reflectance, transmission, simultanaus reflectance and transmission measurement, and fluorescencemeasurement, potentiometer for setting the reference point of the automatic amplification unit (in general 100%), potentiometerforfactorfR :fr switch and potentiometerfor data expansion. 185

Table 1 Macro-TLC

HPTLC

Sue:

20 cm x 20 cm

10 cm x 10 cm 10 cm x 20 cm

Adsorbent:

silica gel

silica gel

Plate height:

30 mm

12 mm

Dispensing volume/spot with capillaries:

1-5 pl

approx. 0.1-0.2 p1

Diameter of the spots:

approx. 3-6 mm

approx. 1-1.5 mm

Diameter of the separated spots:

approx. 6-15 mm

approx. 2-5 mm

Migration:

10-15 cm

3-6 cm

Separation time :

approx. 30-200 min

approx. 3-20 min

Absorption:

approx. 0.05-5 pg

approx. 0.005-0.5pg

Fluorescence:

approx. 1-1ooO ng

approx. 0.1-100 ng

Absorption:

0.005-0.05 pg

0.00054.005 pg

Fluorescence:

0.1-1 ng

0.014.1 ng

Tracks/plate

approx. 10

approx 18 or 36

Number of samples on one plate in double analysis if three standards are used:

0.5 x 10 - 3 =2

0.5 x 18 - 3 =_6 Or 0.5 x 36 - 3

Substance quantity/spot

Detection limits

186

=a

L81

0

0

@--+€lo

lJJu 008-581

uu

m-szr

R @erences :

1) H. Jork, Z. anal. Chem. 236,310 (1968) 2) J. Ripphahn, H. Halpaap, J. Chromatog. 112,81 (1975) 3) H. Jork, GDCh-Kurs Saarbriicken, Okt. 1975 4) W. Tausch, MeStechnik 2,38 (1972) 5) U. Hezel, Angew. Chem. 85,334 (1973) Inti Edit. 12,298 (1973) 6) S. Mazzola, U. Hezel, Boll. Chem. Farm. 113,379 (1974) 7) L. Treiber et al., J. Chromatog. 63,211 (1973) 8) H. Jork, J. Chromatog. 82,85 (1973) 9) W. Tausch, Chimia 23, 17 (1969) 10) H. Otteneder, U. Hezel, J. Chromatog. 109,181 (1975) 11) H. Bethke, W. Santi, R. W. Frei, J. Chromatog. Sci. 12,392 (1974) 12) V. M. Nos, Z. Lebensmittel-Unt und Forsch. 147,331 (1972) 13) K. Roder, E. Eich, E. Mutschler, Pharm. Ztg.115,1430 (1970) 14) S. Ebel, G. Herold, Deut. Lebens.-Rundsch. 70,133 (1974) 15) P. R. Beljaars, Maastricht, personal information

188

Chapter 9

Application of a new high-performance layer in quantitative thinlayer chromatography J. Ripphuhn and H. Hulpaup E. Merck, Darmstadf Germany

The “HPTLC pre-coated plate silica gel 60 F254 for nano-TLC@”*is based on silica gel 60A.Under the usual chromatographic conditions it has been possible to achieve a plate height (according to the classical definition) of 0.012 mm. The application and evaluation techniques were matched to the reduced scale and higher performance of this Merck “HPTLC plate”*, with the result that substances adsorbing in the visible and UV spectral ranges could be quantitatively determined in amounts between 10 pg and 100 ng The approximate standard deviations for the individual values were between 1% and 10%rel. depending on the concentration, and the regression lines mass/signal definitely passed through the origin with correlation coefficients between r = 0.997 and 0.9999. Finally, it was established that for resolutions Rs greater than 1.5 measurement of the chromatograms at right-angles to the solvent flow offers considerable advantages such as increased sensitivity detection by optimization of the wavelength, shorter measuring time and more measurement data and higher statistical significance of the results.

Quantitative determination of chemical substances on thin-layer plates has been practiced for more than 10 years (1-3). Optimization of techniques for application of the substances, chromatographic development of the TLC plate and optical evaluation has resulted in quantitation of substances with average adsorptivities in the region of amounts of 100 ng and in the case of fluorescing substances of about 10 ng, at an approximate standard deviation of srel = 2 to 10% (4-7).

* supplier: E. Merck, Darrnstadt, Germany and EM Laboratories, Inc., Elrnsford, New York.

189

Some inadequacies of the techmque have been improved by adopting special procedures: variations in the thickness of the layer on the plate, for example, markedly affect the quantitative result, particularly with transmittance measurements (4, 8, 9). Of the two procedures available for measurements in the visible range, i.e., transmittance and reflectance, it is reflectance which is preferred, or a combination of the two, Simultaneous Measurement (10-15). Further possibilities for reducing the error due to variations in layer thickness are afforded by the double-beam process at two loci and the double wavelength process at one locus. It can be shown, however, that irregularities in the layer thickness on a good plate only need to be considered over distances in excess of several centimeters. Reducing the scale of the TLC should therefore lead to further improvement. Optimum separation is achieved at varyingrunning distances of the solvent system according to the particle size range of the adsorbent: for smaller particles at shorter running distances and for larger particles at longer running distances. A normal good quality pre-coated plate Silica gel 60 exhibits the optimal plate height of 0.040 mm at a running distance of zf equal to or greater than 100 mm. When the same amounts of substance are applied, the optimal plate height for the “HPTLC pre-coated plate” of 0.012 mm is to be found at a running distance ofz =4omm. f

The advantages of the “HPTLC pre-coated plate” with reduction of the scale of the TLC are: - the three-fold number of theoretical plates or a

1.6 times better resolution

- shorter running times - smaller spot diameters and - lower limits of detection.

hence

The reflectance of the layer is increased as a result of the smaller particle size, and the measurement of the absorption by the substance meets the conditions of the Kubelka-Munk function to a greater extent (16). The measuring area can be reduced; the signal-to-noise ratio is thereby improved, particularly the noise component contributed by the thin layer. The plate height, and hence the resolution or the Separation Number, are greatly influenced by the amount of solution of the substance applied: the smaller the amount applied, the better the resolution. 190

fig. 9.1 Applicatorf o r TLC

The application of volumes of solution down to 100 nl can still be made with a simple applicator having appropriate microcapillary tubes of glass or metal or a combination thereof. A device which we have found to be best for application of amounts down to about 5 nl is a commercially available syringe (1~1)in conjunction with a micrometer such as is shown in Fig. 9.2. Methods for the automatic, rapid and economical application of such small amounts-for routine analysis are currently in the course of development, e.g., on the basis of commercial automatic samplers for gas chromatography. Application of such small amounts of solution of the substance gives spots which exhibit a diameter of bo less than 1 mm before chromatographic development and a diameter of 1-3 mm after development depending on the running distance. In order to evaluate the quality of the layer it was first necessary to optimize the very time consuming and inexact determination of the plate height and other values of interest. The analog output of a chromatogram spectrophotometer, in this case a Zeiss PMQ 11, was linked to a process control computer IBM/7, which is capable of recording the photometer signal directly or via a TE converter and correcting this as required by applying the Kubelka-Munk function. 191

fig. 9.2

Apparatus for Application of Volumes in the Nanoliter Range

Original signal, 1st and 2nd derivative. Xi is the point at which the first derivative crosses the x-axis and corresponds to the location of the peak maximum. X 2 and X'2' are the points at which the 2nd derivative crosses the x-axis and corresponds to the temporal location of the points of inflection. Y'2 and Y'2' correspond to the gradient of the tangents through the points of inflection. The first and second derivatives for each point on the signal curve are calculated by approximation. The points at which the second derivative crosses the x-axis indicate the points of inflection of the sections of curve on either side of the peak, whereas the value of the first derivative and the signal height show the positions of the tangents through the points of inflection. Continuation of t a t seepage 209 192

la

Ib

lc

Id

Figure C I Principle of simultaneous three phase (three dimensional) circular separation in a larger IjC'Wchamber 1 a: Sample as circle line applied 1 b: Three d@Eerentphases fed with constant and equalflow 1 c: composition of mobilephase versus angle on plate I d: Readyfor quantitative and qualitative evaluation

193

Figure C 2 Simultaneous multiphase circular HFTLC chromatography dye mixture Phase A = Toluene, B = TolueneDi-isopropyl ether 50.50 vh,C = Chlorofonn

194

Figure C 3 Same dye mixture as infigure C 2 left side, but Phase A = Toluene, B = Toluenemi-isopropyl ether 5050 vh,C isopropyl ether 9020 vh and all mixtures between these three phases

=

Toluenemi-

(K) 195

figure C 4

Simultaneous multiphase circular HPTLC chromatography Phase A , B, C a l l equal = Toluene

196

figure C 5 Interesting details at trace level of polar compounds (1% ethanol in Chlorofotm) rapid changes of Rf values in ranges

(UC) 197

Figure C 6 Power of two dimensional separations: time consumption 2 x 4 minutes plate: 50 x 50 mm Phase A = Toluene, B = Chloroform

198

figure C 7 Linear chromatogram Spotting of 30 nl - 30 ng each of lipophilic dyes using a I p l Hamilton syringe in connection with a micrometer; zy-= 30 mm

10 x 10 cmplates can be used twice: 30 to 40 mm migration distance ( . = 30 - 40 mm) is suflcient for an optimization of separation in time (Halpaap)

199

Rgure C 8 Examplefor loadability of l o x lOcm HPECplates, dyespots, Toluene, 40%humidity (K) 200

figure C 9 Linear chromatogram Application of I p l = 1 pg each of lipophilic dyes in a line measuring 80 mm, using a I pl Hamilton syringe in connection with an automatic applicator. Separation power and

Rf data precision of HPT layers; dye, toluene, 40% humidity

201

Circular chromatographyis able to improve thepoor starting conditions at thesampling spot. The samples had been applied in the form of circles. TheJigure shows clearly how overloading disturbs separation power but how circular chromatography can improve the limitations of overloaded sampling (?fC) 202

figure C 11 Circular chromatogram Eccentric application of 20 nl = 20 ng each of lipophilic dyes using a I pl Hamilton syringe in connection with a micrometer;zf = 15 mm; zf = 20 mm. 0

Ideal use of separation space: 40 samples simultaneous& separated on a I0 x 10 cm plate by precise circular instrumental Hp7zC (Halpaap)

203

figure C 12 Circular chromatography on precoated H p 7 z C plates f o r nano 7zC Silica Gel 60 F254. Lipophilic dyes, mobile phase: Hexane/ChlorofomLVH~70/30, zf = 30 mm

204

Figure C I3 Circular chromatogram of Dansyl amino acids. Plate material asfigure 12. Mobile phase: Dioxanemater 97/3, zf = 30 mm, detection: UV366 nm. (Halpaap) 205

Figure C 14 Separation of cholesteryl stearate, chlormadinone acetate, cholesterone, epitestosterone, pregnandiol, corticosterone, g each. Mobile phase: chlorofotm/methanol 9713, zf = 20 mm, detection perchloric acid/methanol5/95, dipping technique 1200 C, UV366 nm. (Halpaap, E. Merck) 206

figure C 1.5 Plates as figure 14, separation of steroids as figure 14, punctiform spotting of 500, 50,30, l o x 10-9g each. Mobile phase and detection asfigure 14 (Halpaap, E. Merck)

207

Egure C 16

50 x 50 mm Hp7zC plates in addressable holder, CAMAG U-chamberfor precise qualitative 1 % at 0.5 Rf) and quantitative 2% and better reproducibility at S) analysis

(+

(+

(K) 208

Continuation of t a tfrom page 192:

The position of the peak maximum with respect to time, i.e., the migration distance zx of the spot x, is determined by the point at which the first derivative crosses

the x-axis. The base-line is determined according to the customary chromatographic criteria. The peak width w is given by the difference with respect to time between the two points of intersection with the base-line of the tangents through the points of inflection. The number of theoretical plates of the plate height, and the resolution between two neighboring substances, are calculated for each substance on the chromatogram from the values for the peak width w and the migration distance zx, and printed out by the computer as record of the analysis. It therefore became possible to determine the plate height for practically any number of spots on a chromatogram in less than 2 min. The approximate standard deviation for this determination of plate height for 10 bands on one plate is better than S = _+ 1.5% The corresponding manual evaluation would take many hours and yield a standard deviation of greater than k 10%of the values. An example of a computer print out is given on page 21 1.

209

210

T1 =

10.000 SEC

T2 =

210.000

SEC

220.000

SEC

time

LAUFZ E I T

T =

l a y e r t r a n s p o r t a t i o n speed migration d i s t a n c e t o t a l distance

TRANSPORT-GESCHWINDIGKEIT

U =

8.125

LAUFSTRECKE

L =

38.663

MM

GESAMTSTRECKE

S =

35.663

MM

s o l v e n t constant

FLIESSKONSTANTE

KAPPA =

5.781

PEAK NR.:

hFtf data peak base width number o f p l a t e s p l a t e number per s e c p l a t e height i n micrometer p l a t e height i n micrometer resolution

HRF-WERTE PEAK-BASISWEITE

I N MM

TRENNSTUFENZAHL TRENNSTUFENZAHLISEC

MMISEC

MM;:x2/SEC

3

2

HRF =

11.526

16.375

w =

1.738

1.681

N =

573.74

871.09

4

5

6

35.294

43.799

61.685

2.059 1252.06

2.135 1444.46

2.167 1971.86

NS =

2.060

3.959

5.691

6.565

8.963

TRENNSTUFENHOEHE I N YM

H =

53.444

35.281

24.498

21.228

15.558

TRENNSTUFENHOEHE IN YM

H50 =

12.320

AUFLOESUNG

Computerprint-out

R =

11.528 0.869

17.287 3.181

18.596 1.243

19.160 2.537

The method described made it possible to include the determination of the separating efficiency in the form of plate height and other parameters such as resolution R, Rf-value, k values and velocity coefficients K in the assessment of the quality of separated substances and also in the quality control of pre-coated plates in general.

Evaluation procedures The methods of measuring absorption and reflectance in the visible and ultraviolet spectral ranges and fluorescence, together with their optimization and limitations will be illustrated using several classes of compounds as examples. Wavelength Optimization It is customary, and in elution chromatography methods such as GC and LC in particular even essential, to evaluate substances quantitatively in the order of the k values. In TLC it is always advisable to evaluate in the direction of the solvent flow when closely neighboring or overlapping substances also exhibit slmilar absorption or fluorescence maxima. If however, the resolution between the separated substances is sufficiently large, i.e., of the order of R> 1.5, then measurement at right-angles to the solvent flow has some important advantages.

In order to demonstrate a possible optimization of the wavelength, a mixture of 7 lipophilic dyestuffs was separated on an HPTLC.

I I

1

fig. 9.3

212

L

2

3

4

i

5 6

7

Mixture of 7 lipophilic dyestuffs, 10 ng each, mobile phase benzene, and measurement at 420 nm (lower curve), 500 nm (middle curve) and 580 nm (upper curve). 1 = Ceres violet BRN 4 = Fat yellow 3G 6 = Ceres red G

2 = Ceres black G 5 = Blue VIF Organ01 7 = Ceres brown BRN

In many instances a substance exhibits its absorption maximum at a particular wavelength at which the others do not absorb. For measurements in the direction of solvent flow it is therefore always necessary to seek a compromise, since the wavelength cannot be changed during measurement with certain equipment. In the case of measurements at right-angles to the solvent flow, the optimum wavelength is set for each substance, and maximum sensitivity thus achieved. The second advantage is the possibility of referring to the background rejlectance Ro in the immediate vicinity of the spot at one point that is guaranteed to be free from substance since it lay by the side of the chromatography band. This is very important for accurate determination of the base line. The only other comparable method available for solving this problem is the doublebeam procedure in the direction of solvent flow, but without optimization of the wavelength, and that means loss of sensitivity. The third advantage is that of speed of measurement: up to 35 bands can be measured on a 10 cm wide plate with a single equipment setting (see Fig. 9.4).

Fig. 9.4

213

Section of a measurement of the blue dyestuff at right-angles to the solvent flow across 70 bands on a 20 cm wide plate. Distance separating bands: 2.5 mm Measuring conditions: A = 420 nm; slit = 1.8/0.7 mm Measuring rate: v = 30 mm/min Another example:

4I 20

a13

250 I D 2 PW 2 0 0 0 ss

5 BL 6 0 TP 1 SP TIME

25

r =0,98

(r 4 , 9 9 8 1

35 46 61 91

Hy dr oc o r t is on

1 G6 121 136 145 148

AREA

34436 41770 42270 40491 3G607 17GS8 18725 17969 1UBOY 3188 2 21419 3 2933497

fig. 9.5

Chromatogram recorded at right-angles to the solvent flow of 20 ng and 50 ng of hydrocortisone. Integrator print-out and regression lines of the latter with and without statistically significant runaways. 214

Measuring conditions: h = 242 nm; slit 2.0/0.7 mm Measuring rate: 30 mm/min Spectra-Physics Autolab Minigrator as integrator. Hydrocortisone was determined alongside estrogens. The optimum wavelength of hydrocortisone is at 242 nm, that of the estrogens at 225 nm and 280 nm. The limit of detection of 8 x lo-'' (0.8 ng) was to be found at 1.3 x lo-'' g on measurement at right-angles to the direction of solvent flow, i.e., lower by a factor of 6. The corresponding difference can be seen clearly with the naked eye. The reason for this improvement is undoubtedly the lower background noise (see Fig. 6.6)

c

a:

4

100

)

b:

c

so

10%

20

Fig. 9.6

Comparison of the detection of 10 ng hydrocortisone (a) at right-angles to solvent flow (b) in the direction of solvent flow. 215

The fimir of detection was calculated from the equation proposed by H. Kaiser (19).

I U I-U so

= signal height =

signal height of the blank value at the same site of measurement

= limit of

detection = standard deviation of the blank values at the site of measurement

Amino acids as such are normally not detectable photometrically, and must therefore be converted chemically to absorbing or fluorescing substances before or after chromatography. Ninhydrin was used to convert amino acids into substances that absorb in the visible spectral range, and Fluescin, that is o-phthalaldehyde and mercaptoethanol in buffer solution* (17, 18) to convert such acids into substances that can be excited to produce fluorescence in the UV region. Both conversion products show similar limits of detection at about 0.1 ng, and satisfactory measurement from 1 ng upwards. Screening for phenylalanine is possible by direct application of dilute serum or plasma with a 200 nl capillary tube. In addition to the sera, phenylalanine was applied several times within pathological limits with respect to phenylketonuria, and a high, easily visible concentration attained which served to facilitate adjustment of the plate (see Fig. 9.7).

\ I

\ I Serum

PHA

fig. 9.7

Detection of phenylalnine in serum 216

Serum

PHA

Serum

Rhodamine B, a substance which can be excited to produce intense fluorescence, was applied in concentrations between 10 ng and 20 pg (1 pg = 1 x lo-'* g) in order to ascertain whether the signal/concentration correction curve is still linear at higher amounts of sample. About 6% of all values were significant runaways which were clearly attributable to errors in application. These values were eliminated (as prescribed by statistical methods). See Figs. 9.8, 9.9,9.10 and 1.12 for the results, which are based on the calibration line passing through the origin, and exhibit a linearity with a convincing regression coefficient of 0.9997 which speaks for itself.

ID 2 pn 80 s 5 5 bL 6 0 TP

I SP 1IW F

AIIEL

14

Ill34

25 33

22560

3f105

41

hO44

5: 62

22671 45628

1.

Y48U 2 23653 3

bl 92

44 IY;

9091 2345u

I U I

IIZ I22 I32

Fig. 9.8 Rhodamine B

05/40

10531 ?A593 41330

142

152 I l U

I 16

534 2 5871 3 3832601

1

A

I PW 50 SS

I a 0

. I

[of

-Ah?'

p&

sr

ID ID I P1 50 SS 5 aL 6 0 TP

I SP Tlhr

AREA

23

123U

34 43

3418 6831

5J

Ill? 2582 6461 1381 2d21

GJ

13 61 94

I03 llJ 123 133

Fig. 9.9

Rhodamine B

143

IS3 163

I83

6455

1043 2Y99 620b 1213 2934 6092

12G 1 533357 '

*I

h

I,

h 217

57

1503 2 19b 3

59 67 72 7b

6a5

131 142

-0 0

l5u 2

91 95 106 112

117

31 2

--

b59_ 3 3RG 2

___

lAg& 3

681

127 - 6 1 9 133 137 I 1U 8 5 151

153 157 I L7

56 2 216-3 . 2 48 2 123 2

195 3

- ?

116

161

fig. 9.10 Rhodamine B

-

z -T i

~

e

i

20 10

5 2 1

.. I

#

.A

fig. 9.11

Regression line and correlation coefficient for the values from Figs. 9.8-9.10 (see Table 1)

218

Table 1 Rhodamine B

Approximate recurring standard deviation measured in dirction of flow in O/o

2 ng

1 10 ng

5 ng

Approximate standard deviation measured at right angles to flow in O/o Rhodamine B Approximate standard deviation measured at right angles to flow in OIo

r a b

1 0 0 pg 50 PR 2 0

b'

n r

a

b

4,383

+0,07

6 0,997

4,386

+0,07

0,9998

4,502

+0,29

6 0,998

4,507

+0,27

16 0 , 9 9 8 8

4,183

-0,91

6 0,985

4,027

-0,18

pg

+3,3% +5,0%+15,8%

= linear correlation coefficient = gradient of

a'

0,9999

n' r ' -

regression line = coordinate section of regression line

(at 1.OOO there is a theoretical calibration line which in practice does not exist).

n' gives the number of individual measurements for one evaluation, and includes all values on the plate. n includes the neighboring values or two values in each case per concentration. At 2 ng and 20 pg the dosage was 20 nl, and the approximate standard deviation is therefore comparatively large.

It can be seen from Table 1 that there is no difference in reproducibility and accuracy of the quantitative results of the analysis between the values in and at right-angles to the direction of flow, apart from the considerably lowered limit of detection. There is, however, a considerable saving in time at rightangles to the direction of flow, since it is only necessary to adjust the band measured once, instead of 14 times as in the other case. For Rhodamine B a limit of detection N =6 x g was found. The aflatoxins B1, B2, G1 and G2 were separated on an HPTLC plate by twofold development with chloroform-acetone 9O:lO. Amounts of 200 pg, 500 pg and lo00 pg were applied to determine the calibration line. Twenty-four bands could be accommodated on a 10 cm wide plate, which made it possible to perform an eightfold determination for each concentration. Application of the substances, chromatography, measurement and evaluations took altogether about 1 hour working time. Three of the 24 bands are represented in Fig. 9.12. The limit of detection for the aflatoxins is at 10 pg. The regression line passes through the origin, and its correlation coefficients are in all cases better than r = 0.9987. In this range therefore, there is a linear relationship between fluorescence signal and amount of substance.

220

200 pg

500 P9

fig.9.12

Section from evaluation of a plate accommodating a total .of 24 bands aflatoxins. Excitation wavelength is 366 nm Measurement wavelength is 460 nm Slit dimension = 3.W0.7 mm Measuring rate 30 mm/min Chromatographic conditions: HPTLC plate, mobile phase chloroform/acetone 9O:lO v/v, twice, in each case 70 mm high; N-chamber; chamber saturation. 22 1

Table 2 summarizes the aflatoxin data

Substance Amount in pg

rel. approximate standard deviation

n‘

r’

a‘

22

0,9987

1,74 3.27

n

r

a

+0,04

6

0,997

1,78

-0,27

+0,65

6

0,998

3,39

+0,13 +0,38

b’

b

A f l a t o x i n B1

1000 500 200 pg + 2 , 5 % +3,0% & 1 2 , 5 %

A f l a t o x i n B2

+1,5% t3,2%

+3,5%

A f l a t o x i n G1

+3,6% +4,1%

+9,1%

24

0,9998

1,286+0,27

6

0,998

1,28

Aflatoxin G2

+2,4% +4,5%

+7,0%

23

0,9998

1,795

6

0,992

1,756+0,13

24

0,9995

The above data underline the accuracy with which nanogram analysis may be camed out on HFTLC plates, since the example reported has nothing to do with “dyestuff mixtures in the ranges”.

-0,29

Regression Line “Aflatoxins” and Example of Structure.

0 0

mbs

II

II

30

r/ Aflatoxin

GI

20

10

200

500

1000

pg

223

The standard deviations of the individual values for lo00 pg, corresponding to 100 nanoliters volume of sample, were between 1.5 and f 3.6% of the value, and for 200 pg, corresponding to 20 nanoliters volume of sample, between f3.5 and f 12.5%of the value.

*

It was to be expected that with 24 measurements or 8 values per concentration the correlation coefficient would come out better than with measurement of 6 neighboring bands or the equivalent 2 values per concentration. Gradient and coordinate section - i.e., passage through the origin - remained practically unchanged, however, so that for determination of the correction curve two determinations in each case from two or three concentrations affords adequate accuracy. The HPTLC plate could then accommodate 18 to 20 bands for the analytical determination itself.

Our interest in the future will continue to be directed towards simplification of analytical procedures, e.g., by “cleanup” directly on the HPTLC plate, and also towards improvement of the sensitivity of detection, possibly by chemical reactions on the plate. It is not impossible that in the near future the limits of detection of some classes of substances will be pushed into the femtogram range, as currently done with radio-labelled substances.

224

Appendix Interconversion of Rf, CRf and k

CRf

Rf

0,1

0,Ol

0,14

0,02

0,17 0,20

0,03 0,04

0,22

0,05

0,25

0,06 0,07 0,08 0,09

0,26 0,28 0,30 0,32 0,35 0,37 0,40 0,42

0,45 0,47 0,49 0,51 0,53 0,55

0,57 0,58

0 9 1

0,12 0,14 0,16 0,18 0,20 0,22

0,24 0,26 0,28 0,30 0,32 0,34

k 99 49 32,3 24 19 15,7 13,3 11,s 10,l 9 733 691 5,3 4,6 4 3,6 3,2 2,9 2,6 2 %3 231 199

CRf

Rf

0,60

0,36 0,38 0,40 0,42 0,44 0,46 0,48 0,50

0,62

0,63 0,65 0,66 0,68 0,69 0,71

0,75

0,52 0,54 0,56

0,76

0,58

0,77

0,60 0,65 0,70 0,75 0,80 0,85 0,90 0,95 1

0,72 0,73

0.81 0,84 0.87

0,89 0,92 0,95 0,97

1

k 1 ,a f,6 195 1,38 1,27 1,17 1,08 1 0,92 0,85

0,79 0,72 0,67 0,54 0,43 0,33 0,25 0,18 0.11 0,05

0

225

HP-65computer program for linear regression machine language 21 31 33 61 01 33 09 41 71 33 61 02 34 01 61 33 24 23 12 33 61 04 33 34 09 71 33 61 06 34 41 71 33 61 05 34

h 2 4 23 13 44 34 34 34 71 34 81 51 03 33 34 34 32 03 09 34 81 51 34 81 07 35 04 33 34 34 81 71 42 07 34 34 81 61 33 03 34

programing steps k 3 2 09 34 34 32 06 09 01 34 04 81 51 03 81 34 07 34 02 32 09 01 34 81 51 03 71 24 23 07 14 34 07 07 71 01 34 61 03 24 35 -35 04 03

load program DSP 0.3 D 0.000 05

04 03

Y1 inB D 1.000 X2 in A D 2.000 Y2 in B D 2.000 Xn in A D n.OOO

02 01

Yn in B D n.OOO D r2 press C

03

display of y: read in any value of x and press D. linearized y is displayed D y.yyy press RCL 7: the display shows D a press RCL 8: the display shows D b

08

y = ax+b

01

ol-

a = slope b = axis intercept

If more than two data pairs are read in ( 0 2 ) , D can be pressed any time.

08 07

Program Test x1=12 y1=4 x2 = 18 y 2 = 6 x3 = 2 4 y3 =7.6 consequently r2 = 0.996 (DSP.6 r2 = 0.995902) Press RCL 7 a = 0.300 Press RCL 8 b = 0.467 thus y = 0.3 x + 0.467 at r2 = 0.9959..

226

read X1 in B D 1.000

For critical evalutions in linear regression knowledge of statistical data is needed: standard deviation of a and b, regression angle ar. With the printing HP-97 lineare regression calculations and print out of r2, a, b, sa, sb, ar need only 18 seconds.

HP-97

HPTLC computer program

2 Calculates l i n e a r regression and p r i n t s r , a, bo, ?sb, y= ax t bo. any si from y, any Tj from x i n Calculates and p r i n t s

b l , bO, nreal

?sa, ar

and Separation Number

( O ) *

SN.

(according t o R . E . K a i s e r , CHROMATOGRAPHIA 2 (1976) 463-467;we d e t e c t e d t o o l a t e , t h a t formula (4), page 4 1 , J . B l o m e , is wrong. Most of t h i s book w a s p r i n t e d a t t h i s time. This program t h e r e f o r e corrects t h e error)

The program converts R f o r R R f t o k ( f o r m u l a ( 4 ) page 17, (9) page 27)

Procedure : HP-97

ON, RUN, NORM

Load program,

Press f e, Enter

zf(migration

d i s t a n c e s t a r t - f r o n t i n mn) STO 9. E n t e r y, press A

For c a l i b r a t i o n l i n e s :

E n t e r x, press B

c o n t i n u e w i t h more than two f u r t h e r p a i r s o f data p r i n t o u t see below Press C Enter z f

For HPTLC c a l c u l a t i o n s :

, press

STO 9

Enter peak w i d t h from recorded chromatogram,press e n t e r peak m i g r a t i o n d i s t a n c e from s t a r t , p r e s s B c o n t i n u e with more than two f u r t h e r p a i n o f d a t a Press C, p r i n t o u t see below data input

(data from page 30)

Press C 2 c o e f f i c e n t of determination, r a bO

?S

tS;

a

r e g r e s s i o n angle between t h e two r e g r e s s i o n l i n e s y+x, x-+y e n t e r 0, press D bO enter zf,press D -bl Press E

-

SN nreal

a t zf migration path

227

A

HP-97 HPTLC

0Q! $LELz F2.S 01.: ;;c rug oe; CL!: P S 0G5 a6 DSE RTN 087 006 # L E h 805 RCL9 010 0i 1 E N 1 t c: i RTN 8: 3 rLEL.4 614 EIl 1? Of5 Rlfl 616 xLELE 81 7 +: 01; STGl R TN 819 \

02h

02: 322 023 024

025 026 027 026 525 030 i;3; 032 03;

634 035 036 037 03s 039 048

computer program

01i

042 043 044 045 846 047 046 049 050 051 652 853 054 655 056 057

RCLS

RCL4

063

RiLi

864

FZS

RCL? -

-

RCLS 1

878

P S S x

-

RCL r ,

881 082 083

ENTt

064 085 086 BE?

A'z X3' RJ

086

14'

069 090 091 092 893 094 095 096 097 096 099 100 101

102 103 104

105 106 167

667 06s

069

STUC ENTS EN??

SPC

065 066

ENT;

-

PRTI:

858 G 59

060 661 062

#iKC

f ;t

STOD

1

671 0i 2 5 73

874 075 076 0;: 07s 579

-

088

x

A

e6

EUTt x'

-

Rcir

1i.x t

r:I' RCLE A.

x

STC4 PRTA'

s

X2

RCLl

A

J;; ;,/); RCLE x

ST05

10s 116 11;

FRTA'

i 12 113 114

XZ:'

S S? ,YE

115 116

RCLC

117 :ld 119

1AY RCLD

120

t

-

121 122 123 124 !25

E m 1

RCLD

-

x TRN-'

i26 127 FRTX 128 SPC 129 RTN 138 *LELC 131 RCL 1 X 132 133 RCL0 t 134 135 PRTX 136 RTN 137 rLBLd 138 RCL0 139 148 RCL 1 141 i 42 FRTX 143 RTN 144 8LBLE 145 DSF2 146 CLX 147 RCL 1 148 RCLS x 149 150 RC'LC t 151 152 S TC2 153 R X 154 155 LOG 156 ENT? 157 RCLS 156 RCLZ t 159 168 m a

161 162 163 164 165 166 167 166 164 170

171 172 173 174 175 176 177

178 179 i 66

RCLS RCL2

-

RCLO t

-

LOG

-

#

-

A

SFC FRTX RCLS ENTt RCL2 RCLB

--

181

x2

182 183 164

x

185 166 167 188

8 *

i

ih' x

FRTX

RTN

169 U L b 190 1 /.'A' 141 192

-

1

195 F R K 194 RTt; 195 #LELc 196 XZ 197 14' 198 1 199 206 PRT'; 281

228

-

ENT.:

urn

Subject Index

A

activity see layer activity absorption measurement see measurement aflatoxine . . . . . . . . . . . . . . . . . . . . . . . 177.179.22 0.223 anti-diagonal technique . . . . . . . . . . . . . . . . . . . . . . . 178 application techniques . . . . . . . . . . . . . . . . . . . . . . . 150 applicator platform . . . . . . . . . . . . . . . . . . . . . . . . 55 streak application . . . . . . . . . . . . . . . . . . . . . . . . . 54 see also dosage systems atmosphere see chamber atmosphere and solvent gas phase

B

Barollier pipettes . . . . . . . . . . . . . . . . . . . . . . . . . . . base line determination . . . . . . . . . . . . . . . . . . . . . . . bleeding effect . . . . . . . . . . . . . . . . . . . . . . . . . . . .

55 213 65

caffeine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172-173 chamber atmosphere . . . . . . . . . . . . . . . . . . . .52.53.65. 68 chambers CAMAG U-chamber . . . . . . . . . . . . . 73.77.80.91.134. 210 CAMAG U-chamber IfC . . . . . . . . . . . . . . . . . . . . 210 F-chamber . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 N-chamber . . . . . . . . . . . . . . . . . . . . . . . 108-119. 117 Schamber . . . . . . . . . . . . . . . . . . . . . . . 108-1 10. 117 134 trough chamber . . . . . . . . . . . . . . . . . . . . . . . . . Vario-KS-chamber . . . . . . . . . . . . . . . . . . . . . 110. 133 Petridish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 capacity factor; k definition of . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 relation to Rf . . . . . . . . . . . . . . . . . . . . . . . . . 16. 17 relation to RRf . . . . . . . . . . . . . . . . . . . . . . . . . . 67 chromatographiccharacteristics hRf. K - on temperature . . . . . . . . . . . . . . . . . . 116. 118 hRf. K - on migration distance . . . . . . . . . . . . . . . . . 122 Rs - on temperature . . . . . . . . . . . . . . . . . . . . . 117. 119 R, - on migration distance . . . . . . . . . . . . . . . . . . . 120 circular TLC comparison with linear TLC . . . . . . . . . . . . . . . 61.11 9-122 development of the ring TLC . . . . . . . . . . . . . . . . . . . 57 prerequisites for . . . . . . . . . . . . . . . . . . . . . . . . . . 52 working techniques . . . . . . . . . . . . . . . . . . . . . . . . 53 229

column chromatography see liquid chromatography 186 comparison TLC-HPTLC . . . . . . . . . . . . . . . . . . . . . . computer print-out for HPTLC evaluation . . . . . . . . . . . . . . . . . 211 appendix program . . . . . . . . . . . . . . . . . . . . . . . . . . continuous flow see flow

D

230

dansyl amino acids . . . . . . . . . . . . . . . . . . . . . . . . . 205 167-170 data pair technique . . . . . . . . . . . . . . . . . . . . . . . detection identification HPLC. TLC . . . . . . . . . . . . . . . . . . . . 129 iodine vapor . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 limitof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 129 limit of HPLC. TLC . . . . . . . . . . . . . . . . . . . . . . . limit of absorption . . . . . . . . . . . . . . . . . . . . . . . . 186 186 limit of fluorescence . . . . . . . . . . . . . . . . . . . . . . . 189 pre-coated plate F254 . . . . . . . . . . . . . . . . . . . . . . spectrophotometric . . . . . . . . . . . . . . . . . . . . . . . 171 spraying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 development time in relation to 106 velocity coefficient, K . . . . . . . . . . . . . . . . . . . . . . migration distance . . . . . . . . . . . . . . . . . . . . . . . . 106 diffusion 44 distance dependent . . . . . . . . . . . . . . . . . . . . . . . . longitudinal. . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 molecular . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 rest diffusion . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 space diffusion . . . . . . . . . . . . . . . . . . . . . . . . . . 42 three dimensional . . . . . . . . . . . . . . . . . . . . . . . . . 44 time dependent . . . . . . . . . . . . . . . . . . . . . . . . . . 44 dosage quality QD eccentric . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 definitionof . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 85,87, 107 optimalizationof . . . . . . . . . . . . . . . . . . . . . dosage systems applicator platforms . . . . . . . . . . . . . . . . . . . . . . . . 55 Barollier pipettes . . . . . . . . . . . . . . . . . . . . . . . . . 55 CAMAG plate holder . . . . . . . . . . . . . . . . . . . 3 1, 81, 84 CAMAG U-chamber . . . . . . . . . . . . . . . . . . . . . . 210 capillary Pt-Ir . . . . . . . . . . . . . . . . . . .30,88,90,150, 153 comparison . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

eccentric . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 EVA-Chrom applicator . . . . . . . . . . . . . . . 28.84.150. 153 Hamilton syringe . . . . . . . . . . . . . . . . . . . . . . 87. 150 Hamilton syringe with micrometer screw . . . . . . . . . . 88. 192 in continuous HPTLC . . . . . . . . . . . . . . . . . . . . . . . 82 macro dosage . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 nano dosage . . . . . . . . . . . . . . . . . . . . . . . . . 88. 192 optimized procedure . . . . . . . . . . . . . . . . . . . . 86. 167 reprojector Shandon . . . . . . . . . . . . . . . . . . . . . . . . 92 rocker applicator Merck . . . . . . . . . . . . . . . . . 88.185. 191 wet plate dosage . . . . . . . . . . . . . . . . . . . 31.81.91.92. 94 55. 86 dosage volume . . . . . . . . . . . . . . . . . . . . . . . . . . correlation to layer thickness . . . . . . . . . . . . . . . . . . 102 dynamicarea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 eccentric application . . . . . . . . . . . . . . . . . . . . . . 122. 203

E

EVA-Chrom applicator see dosage systems evaluation procedure quantitative . . . . . . . . . . . . . . . . . . . . . . . . . . . . rotational scanning . . . . . . . . . . . . . . . . . . . . . . . . see also photometric evaluation

69 83

F

flow continuous .Orlita pump type AE/O . . . . . . . . . . . 76.81. 82 continuous .CAMAG dosage unit . . . . . . . . . . . . . . . . 81 experimental examination . . . . . . . . . . . . . . . . . . . 24. 25 66 flowing around effect . . . . . . . . . . . . . . . . . . . . . . . function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 resistance . . . . . . . . . . . . . . . . . . . . . . . . . . 107. 108 fluorescence diminuition see measurement fluorescence quenching see measurement focusing chemical . . . . . . . . . . . . . . . . . . . . . . . . . . . 93. 94 thermal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

G

geometric progression . . . . . . . . . . . . . . . . . . . . . . . . . 45 glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174. 175 23 1

gradient see also solvent application of . . . . . . . . . . . . . . . . . . . . . . . . . . . effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . magnetic stirrer . . . . . . . . . . . . . . . . . . . . . . . . . . polarity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

69 64 59

60 59

H

heated metal ring . . . . . . . . . . . . . . . . . . . . . . . . . 76. 82 historyofTLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 HFTLC advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 circular . . . . . . . . . . . . . . . . . 52.53.57.193.200.20 3-208 combination with other systems . . . . . . . . . . . . . . . . . . 83 combination with GC . . . . . . . . . . . . . . . . . . . . . . . 83 combination with HPLC . . . . . . . . . . . . . 10.17.64.129. 149 comparison with TLC . . . . . . . . . . . . . . 61.119-122.129. 151 continuous . . . . . . . . . . . . . . . . . . . . . . . . . . 76. 82 three dimensional . . . . . . . . . . . . . . . . . . . . . . 193-197 twodimensional . . . . . . . . . . . . . . . . . . . . . . . 178. 198 HPTLC plate see plate pre-coated humidity and chromatographiccharacteristics . . . . . . . . . . . . . 108. 109 andRf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 see also mobile phase 215 hydrocortisone . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

isocratic conditions . . . . . .

L

layer 95.134. 135 activity . . . . . . . . . . . . . . . . . . . . . . . . . in dependence of H-Zf curves . . . . . . . . . . . . . . . . 99-101 in relation to spreading and sample volume . . . . . . . . . 101. 102 photometric measurement of . . . . . . . . . . . . . . . . . . . 97 preconditioning . . . . . . . . . . . . . . . . . . . . . . . 136. 142 totalvolume . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 volumeVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 lipophilic dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . 204 linear regression . . . . . . . . . . . . . . . . . . . . 30.49.142. 218 liquid chromatography . . . . . . . . . . . . . . . . . . . . 59.66. 67

232

.................

57. 64

M

measurement of fluorescence . . . . . . . . . . . . . . . . . . . 161.163.170. 187 fluorescence quenching . . . . . . . . . . . . . . . . 158.160. 187 photometrical . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 reflectance . . . . . . . . . . . . . . . . . . 114.147.156.169. 187 reflectance and transmission . . . . . . . . . . . . . . .157.168. 187 transmission . . . . . . . . . . . . . . . . . . . . . . . . . 154. 187 wavelength optimalization of reflectance . . . . . . . . . . . 172. 212 migration distance of mobile phase Zf in linear TLC . . . . . . . . . . . . . . . 21. 23 in circular TLC . . . . . . . . . . . . . . . . 21 of substance . . . . . . . . . . . . . . . . . . . . . . . . . . 15. 34 velocity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 speed in linear TLCkircular TLC . . . . . . . . . . . . . . . . . 78 migration distance in relation to base width of substance peak . . . . . . . . . . . . . . . . . . 123 development time . . . . . . . . . . . . . . . . . . . . . . . . 106 hRf-values . . . . . . . . . . . . . . . . . . . . . . . . . 105. 113 peakarea . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 plate height . . . . . . . . . . . . . . . . . . . . . . . . . 99. 100 velocity coefficient, K . . . . . . . . . . . . . . . . . . . . 105. 106 separation number . . . . . . . . . . . . . . . . . . . . . . . . 36 mobile phase volume constantflow . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 determination of . . . . . . . . . . . . . . . . . . . . . . . . 132 flow from above (in CTLC) . . . . . . . . . . . . . . . . . . . . 57 flow from below (in CTLC) . . . . . . . . . . . . . . . . . . . . 53 volume Vm of . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 see also solvent multiphase HPTLC . . . . . . . . . . . . . . . . . . . . . . . . . 193

N

N-chamber see chamber nomenclature see list of symbols

P

Particle size in relation to flow resistance . . . . . . . . . . . . . . . . . . . . . . . . . 107 H-Zfcurves . . . . . . . . . . . . . . . . . . . . . . . . . 99-101 Petri dish development . . . . . . . . . . . . . . . . . . . . . . . . 54 233

peak width correlation to RRF . . . . . . . . . . . . . . . . . . . . . . . . 46 starting . . . . . . . . . . . . . . . . . . . . . . . 29.30.394. 47 Phase ratio 21 determination of . . . . . . . . . . . . . . . . . . . . . . . . . 173 phenacetin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 phenylalanine in serum . . . . . . . . . . . . . . . . . . . . . . . photometric evaluation instrumental requirements . . . . . . . . . . . . . . . . . . . 180 212 procedure for HPTLC . . . . . . . . . . . . . . . . . . . . . . reproducibility. see reproducibility techniques . . . . . . . . . 154 see also measurement 31 plate height . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . effective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 real . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33. 36 32 theoretical . . . . . . . . . . . . . . . . . . . . . . . . . . . . plate height in relation to migration distance . . . . . . . . . . . . . . . . . . 99.100.103. 105 104 resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . separation efficiency. . . . . . . . . . . . . . . . . . . . . . . 106 comparison in TLC/HPTLC . . . . . . . . . . . . . . . . . . 100 plate number 32. 98 effective . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33. 36 real . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . theoretical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 comparison for linear and circular TLC . . . . . . . . . . . . . . 61 plate material . . . . . . . . . . . . 68.70.95.99.102.133.134.179. 189 plate pre-coated adjustment and dosage . . . . . . . . . . . . . . . . . . . . . . 84 advantagesof . . . . . . . . . . . . . . . . . . . . . . . . 96. 190 chromatographic performance of . . . . . . . . . . . . . . 102-108 125 specifications . . . . . . . . . . . . . . . . . . . . . . . . . . surface measurement . . . . . . . . . . . . . . . . . . . . . . . 97 plate preconditioning 68 bathing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . dipping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 82 gas phase saturation . . . . . . . . . . . . . . . . . . . . . . . . modification of stationary phase . . . . . . . . . . . . . . . . . 234 preliminaryrun . . . . . . . . . . . . . . . . . . . . . . . . . . 56 68 prewashing . . . . . . . . . . . . . . . . . . . . . . . . . . . . solvent pre-adsorption . . . . . . . . . . . . . . . . . . 109.135. 139 Poiseuille's equation . . . . . . . . . . . . . . . . . . . . . . . . . 19 preconditioning see layer preconditioning 234

Q

quantitative determination calibration curves . . . . . . . . . . . . . . . . . 162.163.173.177 quinine sulfate . . . . . . . . . . . . . . . . . . . . . . . . . 175. 176 quotient Rq definition of . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

R

reduction at vapor pressure . . . . . . . . . . . . . . . . . . . . . . 65 reflectance measurement see measurement reproducibility of hRf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 photometric evaluation . . . . . . . . . . . . . . . . . . . 166-170 RRf values . . . . . . . . . . . . . . . . . . . . . . . . . .68, 129 Rfvalues . . . . . . . . . . . . . . . . . . . . . . . . . . .26, 129 TLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130, 209 Zeiss KM3 spectrophotometer . . . . . . . . . . . . . . . . . . 165 resolution calculation of . . . . . . . . . . . . . . . . . . . . . . . . . 37, 62 definition of . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 in relation to R’f . . . . . . . . . . . . . . . . . . . . . . . . . 132 retention relative . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 time gross bs ........................... 16 timenet$ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Rf-value corrected (true) R’f . . . . . . . . . . . . . . . . . . . . . . . 131 correlation of linear and circular Rf-values . . . . . . . . . 26,62, 63 correlation for eccentric dosage . . . . . . . . . . . . . . . . . . 63 definition of . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 differences . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 function of migration distance . . . . . . . . . . . . . . . . . . . 25 hRf . . . . . . . . . . . . . . . . . . . . . . . . . . . 98,103, 112 real . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 ring development . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 rhodamin B . . . . . . . . . . . . . . . . . . . . . . . . . . . 217-219

s

S-chamber see chamber saccharose . . . . . . . . . . . . . . . . . . . . . . . . . . . sample volume layer surface and layer thickness . . . . . . . . . . . . . . in relation to spreading . . . . . . . . . . . . . . . . . . . .

174. 175

. . . 102 . 101 235

sampling. sampling techniques see dosage selectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . definitionof . . . . . . . . . . . . . . . . . . . . . . . . . . . .

62 34

separation capability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . performance . . . . . . . . . . . . . . . . . . . . . . . . . . . power . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

28 66 61 28

separation number 31 andRf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . calculation of, in linear TLC . . . . . . . . . . . . . . . . . . 40. 41 calculation of. in circular TLC . . . . . . . . . . . . . . . . . 44.48 comparison between linear and circular TLC . . . . . . . . . . . 61 definitionof . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 determination of . . . . . . . . . . . . . . . . . . . . . . . . . 31 solvent choice . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112. 113 chromatographic and physical parameters . . . . . . . . . . 126-127 front, see migration distance gas phases . . . . . . . . . . . . . . 23.54,65.68.108.118.130. 135 see also mobile phase sorbent volume . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 spot deformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 diameter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 eluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 164 shape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29.45. 92 width . . . . . . . . . . . . . . . . . . . . . . . . . . . spectrophotometer PMQ I1 Zeiss . . . . . . . . . . . . . . . . . . . . . . . . 96. 184 KM-3 Zeiss . . . . . . . . . . . . . . . . . . . . . . .165.181. 185 spreading in relation to sample volume. layer thickness and layer surface . . . . . . . . . . . . . . . . . . . . . . 101. 102 stationary phase see layer steorids .

............................

streak application see application techniques surface see layer

236

206. 207

temperature influence of chromatographic characteristics . . . . . . . . . 116-1 19 thiabendazole . . . . . . . . . . . . . . . . . . . . . . . . . . 176. 177 70 time consumption. . . . . . . . . . . . . . . . . . . . . . . . . . . transfer TLC-HFTLC . . . . . . . . . . . . . . . . . . . . . . . . 141 transmission measurement see measurement trough chamber CAMAG twin trough chamber . . . . . . . . . . . . . . . . . 134

v

W

Vario-KS chamber chromatographic characteristics of . . . . . . . . . . . .108-1 11. 133 Velocity coefficient K and migration distance . . . . . . . . . . . . . . . . 106.110. 111

wave length optimization . . . . . . . . . . . . . . . . . . . . . .

212

Zeiss KM3 chromatogram spectrophotometer. . . . . . . . . . . . 181 185 indicator unit of . . . . . . . . . . . . . . . . . . . . . . . . . 182 scanning stage of . . . . . . . . . . . . . . . . . . . . . . . .

237

List of Symbols (86) (21)

bl

B C

CTLC d d0,l dspot D F h hRf H Hreal HPLC HITLC IF1

bh k K L m n N’

N Nred P

q QD r R

factor, width of spot at half peak height width of spot at half peak height extrapolated width of the starting spot at half height of the concentration curve extrapolated width of spot at half height with Rf= 1 broadening of a peak concentration circular thin layer chromatography effective width of the flow resistance, thickness of absorption path radius of the spherical diffusion space spot diameter thickness of the layer flow of liquid effective plate height percentage of substance in mobile phase theoretical plate height real plate height high performance liquid chromatography high performance thin layer chromatography fluorescence intensity light intensity of excitation wavelength capacity factor, partition factor velocity constant lenght of the flow resistance (39) (a), number of effective plates number of effective plates number of theoretical plates number of real plates pressure drop quotient of the progression dosage quality radius of the disk gas constant, resolution Rf value for linear (laminar) TLC corrected (true) value

tf0

tm tmS

ts

T V

Vdos Vm VS Vsol Vt VV Wrny W n zf, z f y Zmobilephase Zfo zy

zx- za, Zsubstance

zf

a

P Po n

h K

quotient selectivity separation number separation number for spherical model separation number for circular TLC time, development time of chromatogram migration time of solvent from starting line to front migration time of solvent from immersion level to starting line dead time gross retention time net retention time temperature (3934,631 dosage volume volume of mobile phase, apparent volume of mobile phase volume of solid (stationary) phase dosage volume total volume of disc part of apparent mobile phase that was pre-adsorbed base peak width migration distance of mobile phase migration distance of solvent from immersion level to starting line migration distance of substance nominal value of migration distance after t second relative retention (45) (21) viscosity of the mobile phase wavelength velocity constant

239