kowalska - preparative layer chromatography (crc, 2003)

Thin-Layer Chromatography: Techniques and Applications, Second Edition, Revised and Expanded, Bernard Fried and Joseph Sherma 36.. Chromatography in the Nonlinear Range: Selected Drawb

Preparative Layer

Chromatography

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14 Introduction to Analytical Gas Chromatography: History, Principles, and Practice, John A Perry

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17 Thin-Layer Chromatography: Techniques and Applications,

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24 High-Performance Liquid Chromatography in Forensic Chemistry, edited by Ira S Lurie and John D Wittwer, Jr.

25 Steric Exclusion Liquid Chromatography of Polymers, edited by Josef Janca

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27 Affinity Chromatography: Template Chromatography of Nucleic Acids and Proteins, Herbert Schott

28 HPLC in Nucleic Acid Research: Methods and Applications,

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29 Pyrolysis and GC in Polymer Analysis, edited by S A Liebman and E J Levy

30 Modern Chromatographic Analysis of the Vitamins, edited by

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31 Ion-Pair Chromatography, edited by Milton T W Hearn

32 Therapeutic Drug Monitoring and Toxicology by Liquid

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33 Affinity Chromatography: Practical and Theoretical Aspects,

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34 Reaction Detection in Liquid Chromatography, edited by Ira S Krull

35 Thin-Layer Chromatography: Techniques and Applications,

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37 Ion Chromatography, edited by James G Tarter

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39 Field-Flow Fractionation: Analysis of Macromolecules and Particles, Josef Janca

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41 Quantitative Analysis by Gas Chromatography, Second Edition, Revised and Expanded, Josef Novák

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44 Countercurrent Chromatography: Theory and Practice,

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45 Microbore Column Chromatography: A Unified Approach

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51 HPLC of Biological Macromolecules: Methods and Applications, edited by Karen M Gooding and Fred E Regnier

52 Modern Thin-Layer Chromatography, edited by Nelu Grinberg

53 Chromatographic Analysis of Alkaloids, Milan Popl, Jan Fähnrich, and Vlastimil Tatar

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55 Handbook of Thin-Layer Chromatography, edited by Joseph Sherma and Bernard Fried

56 Gas–Liquid–Solid Chromatography, V G Berezkin

57 Complexation Chromatography, edited by D Cagniant

58 Liquid Chromatography–Mass Spectrometry, W M A Niessen and Jan van der Greef

59 Trace Analysis with Microcolumn Liquid Chromatography,

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60 Modern Chromatographic Analysis of Vitamins: Second Edition, edited by André P De Leenheer, Willy E Lambert, and Hans J Nelis

61 Preparative and Production Scale Chromatography, edited by

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62 Diode Array Detection in HPLC, edited by Ludwig Huber

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63 Handbook of Affinity Chromatography, edited by Toni Kline

64 Capillary Electrophoresis Technology, edited by Norberto A Guzman

65 Lipid Chromatographic Analysis, edited by Takayuki Shibamoto

66 Thin-Layer Chromatography: Techniques and Applications:

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67 Liquid Chromatography for the Analyst, Raymond P W Scott

68 Centrifugal Partition Chromatography, edited by Alain P Foucault

69 Handbook of Size Exclusion Chromatography, edited by Chi-San Wu

70 Techniques and Practice of Chromatography, Raymond P W Scott

71 Handbook of Thin-Layer Chromatography: Second Edition,

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72 Liquid Chromatography of Oligomers, Constantin V Uglea

73 Chromatographic Detectors: Design, Function, and Operation,

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76 Introduction to Analytical Gas Chromatography: Second Edition, Revised and Expanded, Raymond P W Scott

77 Chromatographic Analysis of Environmental and Food Toxicants, edited by Takayuki Shibamoto

78 Handbook of HPLC, edited by Elena Katz, Roy Eksteen,

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79 Liquid Chromatography–Mass Spectrometry: Second Edition,

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80 Capillary Electrophoresis of Proteins, Tim Wehr,

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81 Thin-Layer Chromatography: Fourth Edition, Revised and Expanded, Bernard Fried and Joseph Sherma

82 Countercurrent Chromatography, edited by Jean-Michel Menet and Didier Thiébaut

83 Micellar Liquid Chromatography, Alain Berthod

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84 Modern Chromatographic Analysis of Vitamins: Third Edition,

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85 Quantitative Chromatographic Analysis, Thomas E Beesley,

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86 Current Practice of Gas Chromatography–Mass Spectrometry,

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87 HPLC of Biological Macromolecules: Second Edition,

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88 Scale-Up and Optimization in Preparative Chromatography:

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89 Handbook of Thin-Layer Chromatography: Third Edition,

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90 Chiral Separations by Liquid Chromatography and Related

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91 Handbook of Size Exclusion Chromatography and Related Techniques: Second Edition, edited by Chi-San Wu

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95 Preparative Layer Chromatography, edited by Teresa Kowalska and Joseph Sherma

edited by

Teresa Kowalska

University of Silesia Katowice, Poland

Joseph Sherma

Lafayette College Easton, Pennsylvania

Published in 2006 by

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Library of Congress Cataloging-in-Publication Data

Preparative layer chromatography / edited by Teresa Kowalska and Joseph Sherma.

p cm (Chromatographic science series ; 95) Includes bibliographical references and index.

ISBN 0-8493-4039-X (alk paper)

1 Preparative layer chromatography I Kowalska, Teresa II Sherma, Joseph III Chromatographic science ; v 95.

QD79.C52P74 2006

Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

Taylor & Francis Group

is the Academic Division of Informa plc.

This book has been designed as a practical, comprehensive source of information

on the field of classical preparative layer chromatography (PLC) It is organized intwo parts, the first of which covers the theory and up-to-date procedures of PLC(Chapter 1 to Chapter 8), while the second (Chapter 9 to Chapter 16) includesapplications to a selection of the most important compound classes and samplestypes Overall, the topics covered in the 16 chapters are evidence for the versatilityand wide use of PLC at the current time We have designed this first book everpublished on PLC to be valuable for scientists with a high degree of experience inthe separation sciences, but because most chapters include considerable introductoryand background information, it is also appropriate for the relatively inexperiencedchromatographer

The contributors to the book are experts on the topics about which they writeand include many of the best known and most knowledgeable workers in the field

of thin-layer chromatography and PLC throughout the world Rather than attempting

to adopt a uniform style, we have allowed chapter authors the freedom to presenttheir topics in a way that they considered most effective They have used figuresand tables as needed to augment the text, and selective reference lists include themost important new literature, as well significant older references, to set the basis

of their chapters

We had great cooperation from the authors in submitting their chapters in atimely fashion, so that the book has been completed about six months sooner thananticipated None of the chapters was unduly delayed, so all are equally up to date

in their coverage The authors represent laboratories in Germany, Poland, Romania,Norway, Canada, Japan, India, and the U.S and, therefore, have provided a globalperspective for the book

We hope that this book will be valuable for practitioners and teachers in diversescientific fields that make use of chromatographic methods and that it will promotebetter understanding of the field and lead to its even wider utilization

Teresa Kowalska Joseph Sherma

About the Editors

Teresa Kowalska is currently a professor in the Department of the PhysicochemicalBasis of Chromatography at the University of Silesia (Katowice, Poland) Herscientific interests include the physicochemical foundations of liquid chromatogra-phy and gas chromatography, with special attention focused on modeling of planarchromatography both in its analytical and preparative mode Over the past 37 yr Dr.Kowalska has directed the programs of over 70 M.Sc degree students who havecarried out their research on the theory and practice of different chromatographicand hyphenated techniques She has also supervised the research in the separationscience of 8 Ph.D students Dr Kowalska is the author of more than 200 scientificpapers, more than 300 scientific conference papers, and a vast number of the bookchapters and encyclopedia entries in the field of chromatography It is perhapsnoteworthy that she has authored (and then updated) the chapter on “Theory andMechanism of Thin-Layer Chromatography” for all three editions of the Handbook

published by Marcel Dekker

Dr Kowalska has acted as editor of Acta Chromatographica, the annual ical published by the University of Silesia (Katowice, Poland) and devoted to allchromatographic and hyphenated techniques, right from its establishment in 1992

format Its contributors originate from an international academic community, and it

is meant to promote the development in separation sciences It apparently serves itspurpose well, as can be judged from a wide readership, abundant citations throughoutthe professional literature, and also from the ISI ranking quota

Last but not least, in the course of the past almost 30 yr Dr Kowalska hasbeen active as organizer (and in recent years as a cochairperson, also) of the annualall-Polish chromatographic symposia with international participation, uninterrupt-edly held each year (since 1977) in the small mountain resort of Szczyrk in SouthPoland Integration of an international community of chromatographers throughthese meetings has been regarded by Dr Kowalska as a specific yet importantcontribution to chromatography

Joseph Sherma is John D and Frances H Larkin Professor Emeritus of Chemistry

at Lafayette College, Easton, Pennsylvania He is author or coauthor of over 500scientific papers and editor or coeditor of over 50 books and manuals in the areas

of analytical chemistry and chromatography Dr Sherma is coauthor, with BernardFried, Kreider Professor Emeritus of Biology at Lafayette College, of Thin Layer

Inc He served for 23 yr as the editor for residues and trace elements of the Journal

of AOAC International and serves currently on the editorial advisory boards of the

Uni-versity, New Brunswick, New Jersey

Faculty of Earth Sciences

The University of Silesia

Ocean Nutrition Canada Ltd

Halifax, Nova Scotia, Canada

Grzegorz Józwiak ´

Faculty of PharmacyMedical University of LublinLublin, Poland

Prof Krzysztof Kaczmarski

Faculty of ChemistryTechnical University of RzeszówRzeszów, Poland

Dr Angelika Koch

Frohme ApothekeHamburg, Germany

Prof Teresa Kowalska

Institute of ChemistryThe University of SilesiaKatowice, Poland

Dr Emi Miyamoto

Department of Health ScienceKochi Women’s UniversityKochi, Japan

Dr Ali Mohammad

Department of Applied Chemistry,Faculty of Engineering and TechnologyAligarh Muslim University

Aligarh, India

Dr Gertrud E Morlock

University of HohenheimInstitute of Food ChemistryStuttgart, Germany

Prof Bernd Spangenberg

Environmental Techniques SectionUniversity of Applied Sciences Offenburg, Germany

Anna Szymanczyk ´

Institute of ChemistryMilitary University of TechnologyWarsaw, Poland

Prof Monika Waksmundzka-Hajnos

Faculty of PharmacyMedical University of LublinLublin, Poland

Prof Fumio Watanabe

Department of Health ScienceKochi Women’s UniversityKochi, Japan

Prof Teresa Wawrzynowicz

Faculty of PharmacyMedical University of LublinLublin, Poland

SECTION I

Chapter 1 Introduction 3

Teresa Kowalska and Joseph Sherma

Chapter 2 Adsorption Planar Chromatography in the Nonlinear Range:

Selected Drawbacks and Selected Guidelines 11

Chapter 3 Sorbents and Precoated Layers in PLC 41

Heinz E Hauck and Michael Schulz

Chapter 4 Selection and Optimization of the Mobile Phase for PLC 61

Tadeusz H Dzido and Beata Polak

Chapter 7 Location of Separated Zones by Use of Visualization Reagents,

UV Absorbance on Layers Containing a Fluorescent Indicator, and Densitometry 163

Bernd Spangenberg

Chapter 8 Additional Detection Methods and Removal of Zones from

the Layer 177

Joseph Sherma

SECTION II

Chapter 9 Medical Applications of PLC 193

Chapter 10 PLC of Hydrophilic Vitamins 237

Fumio Watanabe and Emi Miyamoto

Chapter 11 Preparative Layer Chromatography of Natural Mixtures 251

Chapter 12 Application of Preparative Layer Chromatography to Lipids 299

Chapter 15 PLC in a Cleanup and Group Fractionation of Geochemical

Samples: A Review of Commonly Applied Techniques 369

Chapter 16 The Use of PLC for Isolation and Identification of Unknown

Compounds from the Frankincense Resin (Olibanum):

Strategies for Finding Marker Substances 391

Angelika Koch, Rita Richter, and Simla Basar

Index 413

Section I

Teresa Kowalska and Joseph Sherma

CONTENTS

1.1 Chromatography Background 3

1.2 Basics of PLC 4

1.3 Principles and Characteristics of PLC 5

1.4 Organization of the Book 8

1.5 Epilogue 10

References 10

1.1 CHROMATOGRAPHY BACKGROUND

The invention of chromatography can be traced to the milestone paper published in

1906 by the Russian botanist and plant physiologist Mikhail Semyonovitch Tswett (1872–1919) [1,2] In the experiments reported in this paper, Tswett separated chloroplast pigments from leaves in a column of precipitated chalk washed with carbon disulfide mobile phase During the 20th century and in the new millennium, chromatography has become an indispensable separation tool that is very widely used in natural and life science laboratories throughout the world

Tswett’s initial column liquid chromatography method was developed, tested, and applied in two parallel modes, liquid–solid adsorption and liquid–liquid parti-tion Adsorption chromatography, based on a purely physical principle of adsorption, considerably outperformed its partition counterpart with mechanically coated sta-tionary phases to become the most important liquid chromatographic method This remains true today in thin-layer chromatography (TLC), for which silica gel is by far the major stationary phase In column chromatography, however, reversed-phase liquid chromatography using chemically bonded stationary phases is the most pop-ular method

Preparative layer chromatography (PLC) was apparently first reported by F.J Ritter and G.M Meyer in 1962 [3] They used layers of 1-mm thickness Earlier preparative work, e.g., that reported by J.M Miller and R.G Kirchner (the inventors

of TLC as it is performed today by development of the layer in a closed tank, analogous to ascending paper chromatography) in 1951 and 1952 [4,5], was termed TLC but was carried out on adsorbent bars used as columns or on analytical layers after column chromatography In his classic TLC laboratory handbook, originally published in German in 1962 and translated to English in 1965 [6], Egon Stahl made only a few statements about the method he called “micropreparative TLC.” Layers

4 Preparative Layer Chromatography

of 0.5- and 0.7-mm thickness prepared with a spreading device were recommendedfor this method by Stahl, as well as streak (band) application of larger quantities ofmixture using a “specially designed instrument” (described in the Ritter and Meyerpaper [3]) or a “microspray gun” (used in Stahl’s laboratory)

1.2 BASICS OF PLC

PLC is used to separate and isolate amounts of material (e.g., 10 to 1000 mg) largerthan those used in analytical TLC The purpose of PLC is to obtain pure compoundsfor further chromatographic or spectrometric analysis or for determination of bio-logical activity “Classical” PLC (CPLC), involving mobile phase migration bycapillary action, requires relatively simple and inexpensive equipment, but thoroughcomprehension of the relevant chromatographic principles and techniques is critical.The required information for performing successful PLC is provided in this book

The sample dissolved in a weak (nonpolar for silica gel), volatile solvent is applied

as a narrow band across the plate Manual application can be achieved with a pipet

or syringe guided by a ruler, or round spots can be placed close together, side by side,

in a line Sample application instruments are available commercially, e.g., a ical streaker from Analtech and an automated spray-on apparatus from CAMAG

mechan-Plates with 0.5- to 2-mm layer thickness are normally used for increased loadingcapacity Layers can be self-made in the laboratory, or commercially precoatedpreparative plates are available with silica gel, alumina, cellulose, C-2 or C-18bonded silica gel, and other sorbents Resolution is lower than on thinner analyticallayers having a smaller average particle size and particle size range Precoated plateswith a preadsorbent or concentrating zone facilitate application of sample bands

The mobile phase is usually selected by trial-and-error guided by prior experience

or by performing preliminary analytical separations of the sample in a saturatedchamber PLC separations will be inferior to analytical TLC separations using thesame mobile phase because of the thicker layer, larger particle size, and overloadedsample conditions used for PLC A good general rule is that analytical TLC shouldachieve separations with least 0.1 Rf value difference if the PLC separations are to beadequate with the transferred mobile phase Isocratic development is usually used, butgradient development has been applied in certain situations for increased resolution

Rectangular glass tanks (N chambers) with inner dimensions of 21 ¥ 21 ¥ 9 cmare used most frequently for the ascending, capillary-flow development of PLC plates,which usually measure 20 ¥ 20 cm The tank is lined with thick chromatographypaper (e.g., Whatman 3 MM) soaked in the mobile phase and allowed to equilibratewith the mobile phase vapor for up to 2 h prior to development over a maximumdistance of 18 cm.A saturated chamber provides faster capillary flow of the mobilephase, more uniform bulk and alpha solvent fronts, and higher separation efficiency

A plate angle of 75° from horizontal is recommended for the fastest developmentwith minimum zone distortion Special taper plates (Analtech) with layer thicknessesranging from 300 mm (bottom) to 1700 mm (top) provide increased mobile phasevelocity compared to plates with uniform layer thickness Circular, multiple, andtwo-dimensional development, as well as development at temperatures other thanthe ambient, are also used for PLC in special applications

Introduction 5

Zones containing separated compounds can be detected nondestructively after

plate development and evaporation of the mobile phase by their natural color in

white light, natural fluorescence under 254-nm or 366-nm ultraviolet (UV) light, or

absorption of UV light on layers containing a fluorescent indicator (phosphor) This

method, termed fluorescence quenching, gives dark zones on a fluorescent

back-ground Postchromatographic chromogenic or fluorogenic reagents can be used to

detect compounds that are not naturally visible or fluorescent and do not quench

fluorescence One of the most widely used reagents is iodine vapor, which reversibly

detects many types of compounds as brown zones Destructive chromogenic or

fluorogenic reagents must be applied only to the side edges of the layer (the rest of

the layer is covered with a glass plate) to locate the areas from which to recover the

separated compounds

The zones containing the desired compounds are scraped from the plate backing,

the compounds are eluted with a strong solvent, any remaining sorbent particles are

separated, and the solution is concentrated

All of these steps are described in greater detail in the chapters in Section I of

this book

1.3 PRINCIPLES AND CHARACTERISTICS OF PLC

Successful separations in adsorption chromatography (adsorption TLC included)

are due to the difference in the energies of adsorption between the two separated

species The phenomenon of adsorption on a solid–liquid interface can be

charac-terized best by the empirical adsorption isotherm, which is unique for a given

compound with a particular stationary phase–mobile phase combination There is

one feature, however, that all mixture components share in common, namely, the

general nature of their adsorption isotherms Each empirical isotherm consists of

a linear and nonlinear part The linear part corresponds to the stepwise saturation

of active sites on the adsorbent surface, prior to its complete saturation For a

compound deposited on the adsorbent surface within the linear range of the

iso-therm, a circular TLC zone shape is expected and the densitometrically measured

mass distribution (i.e., the concentration profile of the zone) should be regular

(Gaussian) In the case of mass overload, the system operates within the nonlinear

sector of the isotherm, with an oval zone shape and tailing of the skewed

(non-Gaussian) concentration profile

There is no rigid demarcation line between adsorption TLC operating within the

linear or nonlinear range for the following reasons: (1) each individual solute is

characterized by its own adsorption isotherm, (2) for most analytes in most

chro-matographic systems, the respective adsorption isotherms remain unknown, and (3)

there is no need for steady control of the isotherm sector that is utilized in an

experiment When separating a compound mixture, it often happens that some of

the constituents are in the linear range of their adsorption isotherms, whereas others

are in the nonlinear (i.e., mass-overload) range

However, mostly because of the intuitive, trial-and-error approach of thin-layer

chromatographers, long ago the technique split into two subtechniques, one

bene-fiting from the linear range of the adsorption isotherm and the other utilizing the

6 Preparative Layer Chromatography

advantages of the nonlinear range Much less attention has been given to nonlinear

TLC, and no monograph covering this subject has ever appeared on the market until

now The remainder of this section will be focused on this very important, yet

considerably underestimated, method

Linear TLC is well suited to perform traditional analytical tasks With its use,

one can successfully separate mixtures consisting of a limited number of analytes

and, using more or less sophisticated supplementary techniques, identify the

con-stituents of such mixtures on the layer With more complex samples, linear TLC can

at least help to fractionate samples into the separate classes of compounds Owing

to the proportionality between the amount of solute contained in zones and their in

situ densitometric scan areas, a standard calibration plot that is useful for

quantifi-cation of an analyte in a sample can be established In summary, linear TLC conforms

very well with many separation-, identification-, and quantification-oriented

analyt-ical strategies that are focused on the individual chemanalyt-ical species Owing to its

flexibility, economy, and relative simplicity, linear TLC sometimes outperforms

certain other more expensive and less user-friendly analytical techniques

Optimi-zation of an analytical result of linear TLC can be attained easily with a number of

simple semiempirical, or even purely empirical, retention models (e.g., the

Martin-Synge or Snyder-Soczewinski approaches); in more difficult cases, a variety of more

advanced chemometric approaches are also available Knowledge of the operating

principles, techniques, and applications of linear TLC can be learned from books

published in many languages throughout the world, and also from a selection of

international scientific journals devoted exclusively, or partially, to the

chromato-graphic sciences

Contrary to the linear mode, nonlinear adsorption TLC was conceived, and to

a large extent remained, an “unofficial” and almost “underground” separation

tech-nique Among chromatography practitioners, it is known as PLC (or PTLC

[prepar-ative thin-layer chromatography] or PPC [prepar[prepar-ative planar chromatography]), and

it is generally accepted that this otherwise very useful separation tool does not serve

an analytical purpose, at least in the sense discussed in the previous paragraph As

described in the last section, in PLC much higher amounts of the mixtures are applied

to be separated than in linear TLC Therefore, the layer has to be considerably

thicker, and the optimization rules borrowed from the linear mode are either obeyed

to a much lesser extent or do not hold at all Thus, a simple statement that PLC

lacks a theoretical basis of its own is essentially true There is no book published

in English or any other language from which this technique can be learned In most

cases, it is individually “discovered” by those who need a simple, rapid, inexpensive,

and low-scale (i.e., microgram or milligram) separation tool and are fortunate enough

to be familiar with the more common analytical TLC The basic disadvantage of all

of these “amateur discoveries,” no matter how inquisitive and inventive their authors

might be, is that they are usually made in an unorganized trial-and-error manner

(sometimes aided by advice from a slightly more experienced colleague), and they

eventually prove far less beneficial than they could be if properly introduced in book

form by an expert, or group of experts

All researchers involved in natural and life sciences who wish to isolate or purify

microgram or milligram quantities of a given compound but, for whatever reason,

Introduction 7

cannot make use of a column technique would benefit from the correct use of PLC

For many scientists worldwide, the fully automated column approach can prove to

be too expensive, too complex, or both Nonpressurized open preparative column

chromatography can be chosen, but this is not very effective with low levels of

compounds, particularly not with difficult separations (i.e., in the case of closely

migrating or partially overlapping peak profiles)

PLC is well suited for micropreparative separations For example, consider the

simple scenario of someone involved in a multistage organic synthesis and needing

rapid spectrometric (e.g., mass, infrared, nuclear magnetic resonance, or x-ray)

confirmation of each step of the synthetic procedure (available today for trace

amounts of the compounds) In such cases, optimization of compound isolation with

an instrumental technique will almost certainly prove to be much more time

con-suming and expensive than the planar mode, e.g., using short, narrow strips of

aluminum- or plastic-backed adsorbent layers or microscope-slide-sized

glass-backed plates in a pilot procedure

Manufacturers of TLC materials and accessories are well prepared to satisfy the

needs for professionally performed PLC High-quality precoated preparative plates

are available from a number of commercial sources Alternatively, less expensive or

specialty preparative plates can be “homemade” in the laboratory, and loose sorbents

and coating devices can be purchased for this purpose More-or-less-automated

devices can also be purchased for band application of higher quantities of sample

solutions to preparative layers At least for some users, sophisticated densitometric

and other instrumental techniques are available as nondestructive tools for

prelimi-nary detection and identification of separated compounds in order to enhance the

efficiency of their isolation The only aid still missing, and maybe the most important

of all, is a comprehensive monograph on PLC that might encourage and instruct

many potential users on how to fully benefit from this very versatile, efficient,

relatively inexpensive, and rather easy to use isolation and purification technique

This book was planned to fill that void

The oldest, simplest, and most frequently employed method for feeding the

mobile phase to the layer in PLC is with the aid of capillary forces in the ascending

direction Forced-flow development can also be used for analytical TLC and PLC

The most popular forced-flow modes are overpressured layer chromatography

(OPLC; sometimes termed optimum performance laminar chromatography), in

which the mobile phase flow is due to mechanical force (pressure), and rotation

planar chromatography (RPC), for which the transport of the mobile phase occurs

due to a centrifugal force In this book, coverage is restricted to CPLC, which

operates with capillary flow and is accessible in all laboratories without the purchase

of quite expensive and rather complex instrumentation needed for preparative OPLC

and RPC CPLC is technically compatible with capillary flow analytical TLC, the

mode that is most widely used This means that for the two methods (analytical

and preparative), virtually the same laboratory equipment and procedures can be

used (e.g., sorbent type, coating device [if commercial precoated plates are not

used], chamber, sample application device, mobile phase, development mode

[ascending], reagent sprayer, etc.), and personnel can easily switch from one method

to the other or perform both in a parallel manner at the same time This technical

8 Preparative Layer Chromatography

compatibility of the two modes and the ease of switching between them represent

significant cost-saving advantages that for most laboratories throughout the world

are difficult to overestimate

Another reason for choosing CPLC as the sole option discussed in this book is

that the chromatographic behavior of compounds in this method resembles most

closely that observed in capillary flow analytical TLC This is particularly important

in view of the fact that, so far, the theoretical background of PLC has been practically

nonexistent, while the semiempirical rules valid for capillary flow analytical TLC

can be approximated relatively well for use in CPLC This means that an experienced

chromatographer well acquainted with the separation potential of capillary flow

analytical TLC has a good chance to (intuitively, at least) select working parameters

for CPLC that will produce a close to optimum result The same goal cannot,

however, be so easily reached with preparative OPLC and RPC For example, the

forced flow that operates in OPLC elevates quite drastically the chromatographic

activity of the adsorbent layer [7] This results from the mobile phase being pushed

through the narrow pores of the layer, which are impenetrable when capillary forces

alone are at play The elevation of adsorbent activity is equivalent to a corresponding

lowering of the mobile phase strength, leading to a considerable change in retention

of solutes in a given layer-mobile phase system and making an intuitive,

experience-based system optimization virtually impossible

1.4 ORGANIZATION OF THE BOOK

Section I of this book includes chapters on the principles and practice of PLC After

this introductory Chapter 1, Chapter 2 provides information on efforts undertaken

to date in order to establish the theoretical foundations of PLC With growing

availability and popularity of modern computer-aided densitometers, separation

results can be obtained in digital form as a series of concentration profiles that can

be relatively easily assessed and processed From these, relevant conclusions can be

drawn in exactly the same manner as in automated column chromatographic

tech-niques Efforts undertaken to build a theoretical foundation of PLC largely consist

of adaptation of known strategies (with their validity confirmed in preparative

col-umn liquid chromatography) to the working conditions of PLC systems

Chapter 3 through Chapter 8 deal with the basic aspects of the practical uses of

PLC Chapter 3 describes sorbent materials and precoated layers for normal or

straight phase (adsorption) chromatography (silica gel and aluminum oxide 60) and

partition chromatography (silica gel, aluminum oxide 150, and cellulose), and

pre-coated layers for reversed-phase chromatography (RP-18 or C-18) Properties of the

bulk sorbents and precoated layers, a survey of commercial products, and examples

of substance classes that can be separated are given

Chapter 4 discusses the selection and optimization of mobile phases for

suc-cessful separations in PLC Chapter 5 details procedures for sample application

and development of layers, and Chapter 6 complements Chapter 5 by dealing

specifically with the use of horizontal chambers for the development of preparative

layers, including linear, continuous, two-dimensional, gradient, circular, and

anti-circular modes

Introduction 9

In Chapter 7, approaches for visualization of zones in chromatograms are

dis-cussed, including use of nondestructive and destructive dyeing reagents, fluorescence

quenching on layers with a fluorescent indicator, and densitometry In Chapter 8,

additional detection methods, such as those used for biologically active and

radio-active zones, as well as the recovery of separated, detected zones by scraping and

elution techniques are covered

Section II of the book, encompassing Chapter 9 through Chapter 16, presents

practical applications of PLC in the chemical, biochemical, and life science fields

The great variety of these applications illustrates well the versatility and excellent

performance of PLC in solving a wide spectrum of micropreparative problems

Chapter 9 shows the importance of PLC in the critical field of medical research,

with representative examples of the applications to amino acids, carbohydrates,

lipids, and pharmacokinetic studies

Chapter 10 is devoted to the preparation and purification of hydrophilic vitamins

(C, B1, B2, B6, B12, nicotinic acid and nicotinamide, pantothenic acid, biotin, and

folic acid) in pharmaceutical preparations, food products, and biological samples

PLC of plant extracts is presented in Chapter 11, with sections on the choice of

systems, sampling, choice of the sample solvent, detection, and development modes

These applications in the field of pharmacognosy play a key role in the investigation

and understanding of the healing potential of the constituents of medicinal plants

PLC of lipids is discussed in Chapter 12 Lipids play a vital role in virtually all

aspects of human and animal life Many studies of food quality, human health,

metabolic and ageing processes, pheromone activity in animals, etc., benefit greatly

from the use of PLC for the separation and isolation of lipids

Chapter 13 is devoted to the PLC of natural pigments, which encompass

fla-vonoids, anthocyanins, carotenoids, chlorophylls and chlorophyll derivatives,

por-phyrins, quinones, and betalains Chromatography of pigments is especially difficult

because many are photo- and air-sensitive and can degrade rapidly unless precautions

are taken

In Chapter 14, one of the least-used applications of TLC and PLC is described,

namely inorganics and organometallics These separations in the analytical mode

often require quite unusual stationary phases (e.g., inorganic ion exchangers and

impregnated and mixed layers) combined with a variety of diverse mobile phases

This means that the use of the analogous systems in the preparative mode represents

an unusually difficult challenge

Chapter 15 allows a fairly broad insight into the areas of experimental

istry that can benefit from PLC Owing to the considerable complexity of

geochem-ical samples having an organic origin, PLC plays the role of a pilot separation

technique, enabling primary group fractionation of the respective natural mixtures,

followed by secondary fractionation of these groups with the aid of automated

column techniques, and, finally, followed by identification of the individual separated

species Examples related to oils, bitumens, coal liquids, and pyrolysates are given

The final chapter (Chapter 16) shows how PLC can be used to isolate and identify

unknown terpenoic compounds from the frankincense resin (olibanum) and to find

marker diterpenes The novel development at low temperatures is included in the

PLC methods described

10 Preparative Layer Chromatography

1.5 EPILOGUE

The editors wish to express their deepest conviction that this first book ever

pub-lished on PLC will prove to be a catalyst that inspires much wider use of the method

and is instructive for understanding the theory and correct operation of the various

steps We believe it will fill a serious void in the available information on planar

chromatography and that it will become an appreciated reference book or tutorial

for all those in need of the simple and cost-saving, yet very efficient, PLC

microsep-aration technique

REFERENCES

1 Tswett, M., Ber D Botan Ges., 24, 384–393, 1906 (Translated from German into

English by Strain, H.H and Sherma, J., J Chem Educ., 44, 238–242, 1967.)

2 Strain, H.H and Sherma, J., J Chem Educ., 44, 236–237, 1967.

3 Ritter, F.J and Meyer, G.M., Nature, 193, 941–942, 1962.

4 Miller, J.M and Kirchner, J.G., Anal Chem., 23, 428–430, 1951.

5 Miller, J.M and Kirchner, J.G., Anal Chem., 24, 1480–1482, 1952.

6 Stahl, E., Ed., Thin Layer Chromatography: A Laboratory Handbook,

Springer-Verlag, Berlin, 1965, pp 6, 13, and 39.

7 Pieniak, A., Sajewicz, M., Kowalska, T., Kaczmarski, K., and Tyrpien, K., J Liq.

Chromatogr Relat Technol., 28, 2479–2488, 2005.

Chromatography in the Nonlinear Range:

Selected Drawbacks and Selected Guidelines

Krzysztof Kaczmarski, Wojciech Prus,

CONTENTS

2.1 Adsorption on a Solid–Liquid Interface and the Experimental

Isotherms of Adsorption 122.2 TLC in the Linear and the Nonlinear Region of the Adsorption

Isotherm 162.3 Preparative Layer Chromatography as a Practical Usage of the

Nonlinear Region 212.4 Lateral Interactions and Their Impact on Separation in PLC 222.4.1 Self-Associative Lateral Interactions 242.4.1.1 Higher Fatty Acids 242.4.1.2 a,w-Dicarboxylic Acids 242.4.1.3 Phenyl-Substituted Monocarboxylic Acids 262.4.1.4 Phenyl-Substituted Alcohols 282.4.2 Mixed-Associative Lateral Interactions 282.4.3 Lateral Interactions in the Racemic Mixtures 312.5 The Meaning of the Retardation Factor (R F) in PLC 322.6 Modeling of Lateral Interactions 342.6.1 The Mass-Transfer Equations 342.6.2 Modeling of the Self-Associative Lateral Interactions 352.6.3 Modeling of Mixed-Associative Lateral Interactions 372.7 Final Remarks 39References 39

in high-performance liquid chromatography (HPLC), and the relationship betweencolumn chromatographic peak profiles and the isotherm models has been discussed

in depth by Guiochon et al [1]

The simplest isotherm model is furnished by Henry’s law

(2.1)

where q is the concentration of the adsorbed species, H is Henry’s constant, and C

holds for the concentration of this species in the mobile phase This isotherm is also

called the linear isotherm and, in this case, the concentration profiles resemble that

One of the simplest nonlinear isotherm models is the Langmuir model Its basicassumption is that adsorbate deposits on the adsorbent surface in the form of themonomolecular layer, owing to the delocalized interactions with the adsorbent sur-face The Langmuir isotherm can be given by the following relationship:

(2.2)

FIGURE 2.1 The linear isotherm of adsorption and the corresponding Gaussian distribution

of the analyte’s concentration in the chromatographic band.

direction of development

distance from the start line

q

C C

where q s is the saturation capacity and K the equilibrium constant To make use

of this particular model, ideality of the liquid mixture and of the adsorbed phasemust be assumed Concentration profiles of the chromatographic bands obtainedwhen adsorption can be best modeled with aid of the Langmuir isotherm aresimilar to that presented in Figure 2.2 The larger the equilibrium constant, themore stretched is the concentration tail (and, automatically, the chromatographicband, also)

With the adsorbate concentration low enough, the Langmuir isotherm transformsinto the linear equation and becomes the simplest isotherm of adsorption, asdescribed by Henry’s law

The case mathematically reciprocal to the Langmuir isotherm is represented bythe anti-Langmuir isotherm of adsorption, which, however, is not derived from anyphysical or physicochemical assumptions regarding the process of adsorption, aswas the case with the Langmuir isotherm The anti-Langmuir isotherm can be given

by the following relationship (Figure 2.3):

(2.3)

FIGURE 2.2 The Langmuir isotherm of adsorption and the corresponding nonsymmetrical

distribution of the analyte’s concentration in the chromatographic band.

FIGURE 2.3 The anti-Langmuir isotherm of adsorption and the corresponding

nonsymmet-rical distribution of the analyte’s concentration in the chromatographic band

direction of development

distance from the start line

q

C C

KC

s

=1

direction of development

distance from the start line

q

C C

Also, in this case, with the adsorbate concentration low enough, the muir isotherm transforms into the linear equation and becomes the simplest isotherm

anti-Lang-of adsorption, as described by Henry’s law

There are several isotherm models for which the isotherm shapes and peak

profiles are very similar to that for the anti-Langmuir case One of these models was

devised by Fowler and Guggenheim [2], and it assumes ideal adsorption on a set oflocalized active sites with weak interactions among the molecules adsorbed on theneighboring active sites It also assumes that the energy of interactions between thetwo adsorbed molecules is so small that the principle of random distribution of theadsorbed molecules on the adsorbent surface is not significantly affected For theliquid–solid equilibria, the Fowler-Guggenheim isotherm has been empiricallyextended, and it is written as:

(2.4)

where c denotes the empirically found interaction energy between the two molecules

adsorbed on the nearest-neighbor sites and q = q/qs is the degree of the surfacecoverage For c = 0, the Fowler-Guggenheim isotherm simply becomes the Langmuirisotherm

The Fowler-Guggenheim-Jovanovic model [3] assumes (as it was the earliercase also) the occurrence of intermolecular interactions among the moleculesadsorbed as a monolayer but is based on the Jovanovic isotherm The single-com-ponent isotherm is represented by the equation:

(2.5)

where a is a model constant.

Contrary to the last two isotherms, which take into the account interactionsbetween the neighboring molecules only, the Kiselev model assumes the single-component localized adsorption, with the specific lateral interactions among all theadsorbed molecules in the monolayer [4–6] The equation of the Kiselev isotherm

Assuming that the equilibria between the adjacent layers are depicted by the

same equilibrium constant K p and that K is the equilibrium constant between the

first layer and the active sites, the two-layer isotherm model can be expressed as:

In the simplest case to which the Langmuir isotherm is applicable, the component competitive model is given below [1]:

two-(2.8)

The two-component Fowler-Guggenheim model can be given as follows [3]:

(2.9)

where K i is the isotherm parameter, Qi is the fractional coverage of the ith component,

and Qi = q i /q s Terms c1 and c2 relate to the energy of lateral interactions between

s

p p

1 21

the molecules of the corresponding components Terms c12 and c21 take into theaccount cross-interaction between the separated components

Finally, for the two-component and the two-layer isotherm of adsorption, it iseasy to obtain the following isotherm model (Equation 2.10) by the method describedelsewhere [7]:

(2.10)

maximum capacity of the adsorbent, C i is the concentration of the component in

mobile phase, q i is the concentration of the adsorbed component, K i is the equilibrium

constant of adsorption of the ith component on the adsorbent surface, K ii is the

equilibrium constant of adsorption for the ith component on the same previously adsorbed ith component, and K ij is the equilibrium constant of adsorption for the ith component on the jth component It was also assumed that K ij = K ji

2.2 TLC IN THE LINEAR AND THE NONLINEAR

REGION OF THE ADSORPTION ISOTHERM

Chromatographic separations are often used for analytical purposes (in order toestablish the qualitative and quantitative composition of a given mixture of com-pounds) and, occasionally, one refers to this technique as the analytical one, which

is not correct, because chromatography has an indisputable importance as a versatileseparation tool, also, enabling isolation of preparative amounts of substances Suchpreparative chromatographic separations are most frequently carried out by means

of liquid chromatography (LC) on the nonpressurized columns filled with an bent and also with the aid of HPLC, TLC, and gas chromatography (GC)

adsor-The adsorption mechanism of solute retention is one of the two universal anisms in the chromatographic process (the other being the partition mechanism),and it operates on a purely physical principle In fact, virtually all solutes can adsorb

mech-on a microporous solid surface (or be partitimech-oned between the two immiscibleliquids) If the analytes are applied to a chromatographic system in the low-enoughaliquots, then the respective retention processes occur within the linear range of theempirical adsorption isotherms of the species involved and concentration profiles ofthe resulting chromatographic bands are Gaussian, as shown in Figure 2.1.The adsorption TLC operating in the linear range of the adsorption isotherm(sometimes dubbed as the linear adsorption TLC or simply as the linear TLC) isutilized for purely analytical purposes (which include establishing of a qualitativecomposition of a given mixture of analytes, often followed by their quantification

in the examined sample with aid of the calibration plot approach) In order tointroduce certain amount of rationale to the linear adsorption TLC (and enable

2 21 1 1

optimization of the separation result), several semiempirical models have beenelaborated, and the most popular of them is going to be briefly summarized in theforthcoming paragraphs of this section.

It is the main aim of semiempirical chromatographic models to couple theempirical parameters of retention with the established thermodynamic quantitiesgenerally used in physical chemistry The validity of a model for chromatographicpractice can hardly be overestimated, because it often and successfully helps toovercome the old trial-and-error approach to running the analyses, especially whenincorporated in the separation selectivity oriented optimization strategy

Partition (liquid–liquid) TLC was first among the chromatographic techniques

to gain thermodynamic foundations, owing to the pioneering work of Martin andSynge [9], the 1952 Nobel Prize winners in chemistry The Martin and Synge model

describes the idealized parameter RF (i.e., the parameter ) in the following way:

(2.11)

where t m and t s denote the time spent by a solute molecule in the mobile and stationary

phases, respectively, n m and n s are the numbers of the solute molecules equilibrially

contained in the mobile and stationary phases, and m m and m s are the respectivemole numbers

The most popular (and also most important) semiempirical model of the linearadsorption TLC, analogous to that of Martin and Synge, was established in the late1960s by Snyder [10] and Soczewinski [11] independently, and it is often referred

to as the displacement model of solute retention The crucial assumption of this

model is that the retention mechanism consists of a competition among the soluteand the solvent molecules to the adsorbent active sites and, hence, in the virtuallyendless acts of the solvent molecules displacing those of the solute on the solid

surface (and vice versa) Further, the authors assumed that certain part of the mobile

phase rests adsorbed and stagnant on the adsorbent surface This adsorbed mobilephase formally resembles the liquid stationary phase in partition chromatography.Thus, utilizing with imagination the main concept of the Martin and Synge model

of partition chromatography, Snyder and Soczewinski managed to define the R F

parameter valid for the adsorption TLC as given below:

(2.12)

where t m and t a denote the time spent by a solute molecule in the mobile phase and

on the adsorbent surface, respectively, n m and n a are the numbers of the solutemolecules equilibrially contained in the mobile phase and on the adsorbent surface,

m and m are the mole numbers of the solute molecules contained in the nonadsorbed

and the adsorbed moieties of mobile phase, c m and c a are the molar concentrations

of the solute in the nonadsorbed and the adsorbed moities of mobile phase, V m is

the total volume of the mobile phase, V a is the volume of the adsorbed mobile phase

per mass unit of an adsorbent, and W a is the mass of the adsorbent considered.Transformation of Equation 2.12 results in the following relationship:

The following Soczewinski equation is a simple linear relationship with respect

to logX S , linking the retention parameter (i.e., R M) of a given solute with quantitativecomposition of the binary eluent applied:

(2.14)

where C is in the first instance the equation constant (with a clear physicochemical explanation though), X s is the molar fraction of the stronger solvent in the nonaque-

ous mobile phase, and n is the number of active sites on the surface of an adsorbent.

Apart from enabling rapid prediction of solute retention, the Soczewinski equationallows a molecular-level scrutiny of the solute — stationary phase interactions The

numerical value of the parameter n from Equation 2.14, which is at least mately equal to unity (n ª 1), gives evidence of the one-point attachment of the solute molecule to the stationary phase surface The numerical values of n higher than unity

approxi-prove that in a given chromatographic system, solute molecules interact with thestationary phase in more than one point (the so-called multipoint attachment).The following Snyder equation is another simple linear relationship with respect

to j, which links the retention parameter (i.e., ln k) of a given solute with the volume

fraction of the organic modifier in the aqueous binary mobile phase (j):

where k is the solute’s retention coefficient (k = (1 – RF)/RF), kw is the solute’s

retention coefficient extrapolated for pure water as mobile phase, and S is the constant

characteristic of a given stationary phase

=+

Consequences of the Snyder and Soczewinski model are manifold, and theirpractical importance is very significant The most spectacular conclusions of thismodel are (1) a possibility to quantify adsorbents’ chromatographic activity and (2)

a possibility to define and quantify “chromatographic polarity” of solvents (known

as the solvents’ elution strength) These two conclusions could only be drawn onthe assumption as to the displacement mechanism of solute retention An obviousnecessity was to quantify the effect of displacement, which resulted in the following

relationship for the thermodynamic equilibrium constant of adsorption, K th, in thecase of an active chromatographic adsorbent and of the monocomponent eluent:

(2.16)

where a is the function of the adsorbent surface energy independent of the properties

of the solute (known as the activity coefficient of the adsorbent; practical nation of its numerical values can be regarded as quantification of the adsorbent’s

determi-chromatographic activity), S0 is the adsorption energy of the solute chromatographed

on an active adsorbent with aid of n-pentane as monocomponent mobile phase, A S

is the surface area of the adsorbent occupied by an adsorbed solute molecule,and is the parameter usually referred to as the solvent elution strength, or simplythe solvent strength (the adsorption energy of solvent per unit of the adsorbentsurface area)

Assuming that the adsorbent surface is occupied by an adsorbed solute molecule

(A S ) and a molecule of a stronger solvent (n B) which are equal to one another, theelution strength of a binary eluent, , shows the following dependence on itsquantitative composition:

(2.17)

where is the elution strength of the weaker component (A) of a given binary

eluent, is the elution strength of the stronger component (B) of the same eluent,

and is the molar volume of the component B.

Coupling Equation 2.16 and Equation 2.17, we can obtain the following

rela-tionship, which describes the dependence of the solute’s retention parameter, R M,

on the quantitative composition of a given binary eluent:

(2.18)Apart from the most widely utilized Snyder and Soczewinski semiempiricalmodel of linear TLC, several other physicochemically grounded approaches to thesame question exist as well [12] Also, a choice of the empirical rules in mathematical

1

and nonmathematical form [13] exist, which prove very helpful in selecting properexperimental conditions for running the developments in the analytical TLC mode.All these approaches inevitably share one common trait, namely, an absolute negli-gence of intermolecular interactions of the analyte-analyte type (i.e., of the so-calledlateral interactions).

The preparative (or nonlinear) adsorption TLC has never attracted enough tion from the side of theoreticians of the planar technique to result in a codifiedsystem of rules, helpful in an efficient carrying out of micropreparative isolation ofindividual compounds or compound groups Normally, it is taken for granted that tothe preparative (i.e., nonlinear) adsorption TLC, the same rules can be applied as tothe analytical (i.e., linear) variant, although it is also known in advance that perfor-mance of these rules in the former case is considerably worse than in the latter one.The primary reason that no valid rules for preparative TLC have so far beenelaborated is due to a rather limited access to densitometric detection, which is stillnot commonplace in many thin-layer chromatographic laboratories throughout theworld And the demand for densitometric detection in preparative TLC has one verysimple reason Working in the nonlinear range of the experimental isotherms ofadsorption of the analytes and because of the resulting mass overload of the respec-tive sorbents, the chromatographic bands cannot be Gaussian in terms of theirconcentration profiles (or circular, if the spot application and the traditional visual-ization methods are considered) In the preparative TLC mode, the skewed concen-tration profiles are dealt with exclusively, which can either demonstrate the backtailing or the front tailing, thus negatively affecting resolution and separation, basi-cally due to an increased chance of the bands overlapping

atten-Use of densitometric detection provides an insight into the concentration profiles

of chromatographic bands, thus furnishing an indispensable prerequisite, needed forproper assessment of the retention mechanisms in the preparative adsorption TLC.Figure 2.4 shows three types of the band concentration profiles The Gaussian peak(a) in this figure represents the linear isotherm of adsorption of a given species, peak

FIGURE 2.4 Three types of concentration profiles encountered among the thin-layer

chro-matographic bands: (a) symmetrical (Gaussian) without tailing, (b) skewed with front tailing, and (c) skewed with back tailing [14].

(b) is valid for the anti-Langmuir-type of the adsorption isotherm, and peak (c)results from the Langmuir-type of the adsorption isotherm.

It is generally assumed that the symmetrical concentration profiles withouttailing (which can be approximated by the Gaussian function; see Figure 2.4a) giveevidence of the fact that the adsorbent is not yet overloaded by the analyte (i.e., thatthe chromatographic band still appears within the linear range of the adsorptionisotherm) and from this particular shape, the chemical structure of the analyte itselfcannot be judged (i.e., the crucial question cannot be answered regarding whether

or not the analyte is equipped with such functionalities that allow its participation

in the lateral interactions)

From the asymmetrical concentration profile with front tailing (see Figure 2.4b),

it can correctly be deduced that (1) the adsorbent layer is already overloaded by theanalyte (i.e., the analysis is being run in the nonlinear range of the adsorptionisotherm) and (2) the lateral interactions (i.e., those of the self-associative type)among the analyte molecules take place The easiest way to approximate this type

of concentration profile is by using the anti-Langmuir isotherm (which has nophysicochemical explanation yet models the cases with lateral interactions in a fairlyaccurate manner)

From the asymmetrical concentration profile with back tailing (see Figure 2.4c),

it can correctly be deduced that (1) the adsorbent layer is already overloaded by theanalyte (i.e., again, the analysis is being run in the nonlinear range of the adsorptionisotherm), but (2) the lateral interactions (i.e., those of the self-associative type) arenegligible among the analyte molecules The simplest way to approximate this type

of the concentration profile is by using the Langmuir isotherm (i.e., the one whichhas a well-grounded physicochemical explanation)

2.3 PREPARATIVE LAYER CHROMATOGRAPHY AS A

PRACTICAL USAGE OF THE NONLINEAR REGION

Due to low aliquots of separated analytes (usually between 10 and 1000 mg) [15],preparative layer chromatography (PLC) can be regarded as one of the most versatilemicropreparative isolation techniques available in the arsenal of separation methods.Among those who most frequently utilize PLC, we can find, for example, the organicchemists who work at the microscale level and thus cannot isolate their intermediateand final products with aid of the classical nonpressurized chromatographic columns.Another group are researchers in the field of pharmacognosy, whose task is to isolatepharmacodynamically active constituents from the natural healing materials PLCcan also prove useful for those involved in geochemical and environmental studies,who attempt to isolate trace amounts of analytes often contained in the abundantlyavailable natural matrices One cannot forget about the applicability of PLC in thelife sciences, either, and the list of potential users of this technique is practicallyendless An additional and particularly valued feature of PLC (shared in commonwith the analytical TLC mode) is that, contrary to the advanced column techniques,

in adsorption TLC, various different samples of natural origin (e.g., body fluids,liquid environmental samples, etc.), which contain the suspended materials, do not

need any extra pretreatment (e.g., of the solid phase extraction [SPE] type), aimed

at their removal In fact, the adsorbent contained in the starting spot (or the startingline) of a typical thin-layer chromatogram purifies the applied sample from insolublesolid particles and also from other contaminants — in this sense acting as a specificinbuilt SPE device, also

In Chapter 1, we have clearly stated that the contents of this book are going to

be devoted exclusively to the capillary flow of the eluents employed in PLC, and wealso explained our reasons for making this particular decision Now let us repeatonce again that until now, the specificity of the multiple theoretical (and also practical)aspects of PLC have not been systematically introduced to the planar chromato-graphic community in form of a separate handbook, even if its procedures werebriefly described in several book chapters, e.g., in [16–18] In order to at least partiallyfill this undeserved void, we are now going to discuss selected theoretical aspectscharacteristic of developing chromatograms in the nonlinear adsorption TLC mode

2.4 LATERAL INTERACTIONS AND THEIR IMPACT ON

SEPARATION IN PLC

Lateral interactions — in other words, intermolecular interactions between the ecules of the same analyte or of the two different analytes — depend on the chemicalstructure of the species involved and more specifically, on the type of functionalitiespresent in their molecules Simplifying the problem, the analyte molecules can, upontheir structure and the resulting ability to form the hydrogen bonds, be divided intothe four categories first introduced by Pimentel and McClellan in their famous (andnow historical) monography of hydrogen bonding [19] This very useful classifica-tion is given in the aforementioned four categories of compounds

mol-N: The molecules from this category lack both the “acidic” and the “basic”

functionality and, hence, they remain unable to interact through the gen bonds Aliphatic hydrocarbons are the best examples of the analytesfrom this particular group of compounds

hydro-A: These molecules are equipped with acidic functionality only and, hence,

they can participate in the hydrogen bonds as proton donors The mostrepresentative examples from this group are chloroform (CHCl3) or dichlo-romethane (CH2Cl2)

B: The molecules from this group incorporate electronegative (i.e., basic)

heteroatoms (e.g., O, N, S, etc.) and p-electrons, and, hence, they are able

to participate in the hydrogen bonds as proton acceptors Ketones and ethersare among the most representative classes of analytes belonging to thisparticular group

AB: The molecules belonging to the last category are equipped with both

the acidic and the basic functionalities and, hence, they can interact throughthe hydrogen bonds both as proton donors and proton acceptors Acoholsand carboxylic acids are among the most representative examples fromthis group

Analytes from class N neither self-associate nor participate in the mixedhydrogen bonds Consequently, they cannot participate in lateral interactions ofany kind, either.

Analytes from classes A and B cannot self-associate through the hydrogen bondsand because of this, they cannot participate in the self-associative lateral interactions.However, they can participate in the mixed hydrogen bonds and take part in themixed lateral interactions For example, analyte A can laterally interact either withanalyte B or analyte AB In a similar way, analyte B can either interact with analyte

A or analyte AB

Analytes from class AB can self-associate through the hydrogen bonds and,

hence, under mild chromatographic conditions, they can participate in the

self-associative lateral interactions of the AB … AB type Moreover, they can participate

in the mixed lateral interactions of three different types (AB … A, AB … B, and

AB1 … AB2)

The enthalpy of the H-bonds among the majority of the organic compounds isrelatively low (usually within the range of about 20 kJ per one mol of hydrogenbonds) and therefore they can easily be disrupted In order to demonstrate the

presence of lateral interactions in chromatographic system, low-activity adsorbents

are most advisable (i.e., those having relatively low specific surface area, low density

of active sites on its surface, and low energy of intermolecular analyte–adsorbentinteractions, which obviously compete with lateral interactions) For the same reason,the most convenient experimental demonstration of lateral interactions can beachieved in presence of the low-polar solvents (basically those from the class N;

e.g., n-hexane, decalin, 1,4-dioxane, etc.) as mobile phases.

The adsorbent most suitable for demonstration of the existence of lateral actions among the analyte molecules is cellulose For planar chromatographic pur-poses, it is available either in the form of ready-made TLC plates precoated withmicrocrystalline cellulose or as chromatographic paper The examples that are going

inter-to be presented in the subsequent sections of this chapter refer inter-to the chromainter-to-graphic systems composed of cellulose as a stationary phase and either a single

chromato-hydrocarbon or ether (e.g., n-hexane, n-octane, decalin, 1,4-dioxan) as a

monocom-ponent mobile phase First, the examples from articles [8,20–24] will be presented.These examples focus on lateral interactions among the molecules of a single analyte(i.e., on the self-association through the hydrogen bonds) and on their impact onthe concentration profiles of the resulting chromatographic bands As convenientmodel analytes, selected alcohols and mono- and dicarboxylic acids were chosen,all of them representing the AB class of analytes, according to Pimentel and McClel-lan Shapes of the respective concentration profiles obtained in a nonlinear region

of the adsorption isotherm could be approximated with aid of the type adsorption isotherm

anti-Langmuir-Then, the examples from Reference 23, that focus on retention of the selectedbinary mixtures of the test analytes (one comprising carboxylic acid and ketone andthe other made of alcohol and ketone), chromatographed under the deliberately mildworking conditions (microcrystalline cellulose was used as adsorbent and either

decalin or n-octane as the monocomponent mobile phase) will be discussed One of

the test solutes in each binary mixture (either acid or alcohol) can be viewed as