Thin-Layer Chromatography

A Laboratory Handbook

Edited by

Egon Stahl

Second Edition, Fully Revised and Expanded

Translated by M. R.. F.Ashworth

With 241 Figures and 3 Plates in Color

Springer International Student Edition

Springer-Verlag Berlin Beideiberg GmbH 1969

English translation by Professor M. R. F. Ashworth, Institut für organische Chemie

Universität des Saarlandes, Analytische Abteilung, D-6600 Saarbrücken 15

ISBN 978-3-642-88490-0 ISBN 978-3-642-88488-7 (eBook)

DOI 10.1007/978-3-642-88488-7

Softcoverreprint ofthe hardcover2nd edition 1969

Distribution or sale of this Edition is authorized only in ASIA: Afghanistan, Bangladesh, Burma, Cambodia, Hong Kong. lndia, Indonesia, Japan, Korea, Laos, Malaysia, Pakistan, Philippines, Singapore, Sri Lanka, South Vietnam and Thailand.

Thls work is subject to copyright. All rights are re3erved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcastlng, repro­duction by photocopylng machine or similar means, and storage in data banks. Under § 54 of the German Copyright. Law wehre copies are made for other than private use, a fee is payable to the publisher, the. amount of the feetobe determined by agreement with the publisher. © by Springer­Verlag Berlin Heidelberg 1965, 1969. Library of Congress Catalog Card Nurober 69-14538. The use of general descriptive names, trade names. trade mark, etc. in this publication, even if the former are not especially identified, ls not to be taken as a slgn that such names, as understood by the Trade Marks and Merchandise Marks Act, may accordingly be used freely by

anyone.

Foreword to the 2nd Edition

The first edition, which came out four years ago, is recognised as a standard work on thin-layer chromatography. The subject has developed rapidly in the meantime and several thousand articles have since been published. With the exemplary cooperation of an appreciably widened group of contributors, the second edition has assimilated this mass of new data. The layout and presentation of the book, acclaimed as satisfactory, have been retained but new chapters and sections in­corporated. Special attention has been devoted to the adsorbents, an integral part of the method. The new gradient-, transfer- and coupling procedures, preparative TLC, direct quantitative evaluation, reactions at the start point and new isotope techniques have likewise been included. More space has been allotted in the special section to application of TLC in clinical diagnosis, 'in investigations of foodstuffs and in the analysis of products of the organic chemical industry. The number of reagents has been increased to 264.

Almost all chapters have been rewritten and only a small number of the former figures and tables taken over. Despite strenuous contrac­tion, the number of pages has almost doubled; a fourfold content of information has been provided, however. In other words, it is a new book which will be a reliable and indispensable laboratory aid for both beginner and specialist.

My thanks are due to all who have helped towards the reshaping of the book.

Saarbriicken, 18/10/1966 EGON STAHL

Translator's Note

As far as possible I have adopted English spelling usage, as befits my nationality. It has, however, often been necessary to consult American sources such as Chemical Abstracts or the Handbook of Chemistry and Physics (and, of course, American journals containing original work); some compounds, especially those which are less familiar, may thus turn out to be spelt in the American way. Moreover, I have left ChapterX, written by two American contributors, almost wholly unchanged. I.hope that the resulting "mid-Atlantic" mixture will not succeed merely in distressing purists and devotees of consistency on both sides of that ocean.

To use or not to use capital letters: trade names force the writer into a Hamlet role. No decision has been necessary with many pharma­ceuticals, antibiotics etc. which appear only in tables. I have used chiefly small letters, departing from this very rough rule with adsorbents, dyes, insecticides and other miscellaneous products which are newer and less "classical"; thus: silica gel, methyl red, aldrin but Sephadex, Palatine Fast Green, Rogor. Here, too, I hope to have offended neither firms nor individuals.

I should like to thank contributors in particular for most valuable help with terminology in ·their special subjects.

Saarbriicken, April 1969 M. R. F. AsHWORTH

Table of Contents

General Section

A. The Historical Development of the Method. EGON STAHL

B. Adsorbents for TLC. EGON STAHL . . . .

1

6

I. Silica Gel. H. W. KOHLSOHUTTER and K. UNGER . 7 1. Formation of Silica Gel and Construction of its Framework 8 2. The Cavity System of Silica Gel . . . . . . 13

a) Capillary and Firmly Combined Water. . . . . 20 b) Re-formation of Firmly Combined Water. . . . 21

II. Aluminas and Other Inorganic Adsorbents. H. ROSSLER 23 1. Alumina . 23 2. Kieselguhr . . . . . 27 3. Silicates . . . . . . 29

a) Magnesium Silicate 29 b) Calcium Silicate 29

4. Phosphates. . . 30 5. Calcium Sulphate . 30 6. Glass Powder. . . 30 7. Salts of Heteropoly-, Tungstic, Molybdic and Tetraboric Acids 31 8. Ferric and Chromic Oxides . . . . . 31 9. Zinc Carbonate and Zinc Ferrocyanide . . . . . . . 31

10. Active Carbon . . . . . . . . . . . . . . . . . 31 11. Zirconium Phosphate and Hydrous Zirconium Oxide. 32 12. Lanthanum Oxide. . . . . 32 13. Bentonites . . . . . . . . . . . 32 14. Combinations of Adsorbents . . . 32

III. Organic Adsorbents. P. WOLLENWEBER 32 1. Cellulose and Derivatives . . . 32

a) Normal Cellulose Powder. . . . 33 b) Acetylated Cellulose Powder . . 37 c) Cellulose Ion-Exchanger Powder. 37

2. Starch . 39 3. Sucrose . . . 40 4. Mannitol . . 40 5. Dextran Gels 40

IV. Polyamides as Adsorbents. H. ENDRES 41

V. Ion Exchangers in TLC. K. DORFNER . 44

VI. Modification of Adsorbents by Impregnation. EGON STAHL 48 1. Impregnation before Coating 48 2. Impregnation of the Ready, Dry Layer . . . . . . . 48

VITI Table of Contents

C. Apparatus and General Techniqnes in TLC. EGON STAJIL 52

I. Preparation of Thin, Uniform Layers 52 1. Pouring Procedures. . 53 2. Immersion Procedures . . . . . 53 3. Spreading Procedures. . . . . . 54

a) Fixed Spreader (Kirchner-type) 54 b) Movable Spreader (Stahl-type) 55 c) Spreading Rods and Home-made Apparatus 56

4. Spraying Procedures . . . . . 57 5. Automatic Coating . . . . . . 58 6. Ready-coated Plates and Sheets 59

II. Preparation of TLC-Plates . . . . 60 1. Drying, Storage and Handling . 60 2. Control and Marking of the Layer 61 3. Application of the Substance Mixture for Separation 63

a) Spot Application . . . . . . . 63 b) Band or Streak Application. . . . . . . . . . 64

III. Separation Chambers and Development . . . . . . . 65 1. Chamber Saturation and "Equilibration" of the Layer 66 2. Setting up the Chambers . . . . . . 68 3. Chambers for Ascending Development 68

a) Rectangular Chambers 68 b) S-Chambers. . . . . . . . 69 c) Moisture Chambers 71

4. Equipment for Descending TLC 72 5. Devices for Horizontal Development 73

a) Circular Technique ...... 73 b) Centrifugal Technique . . . . . 75 c) Horizontal Development, BN-Chamber and Automatisation . 75

IV. Aids for Visualisation of Colourless Substances on Chromatograms. 77 1. UV-Lamps for Exciting Fluorescence. 77 2. Spray Apparatus and Fume Cupboards 79 3. Apparatus for Heating the TLC-Plates 81 4. Further Aids for Visualisation . . . . 81

a) Direct Microsublimation . . . . . 81 b) SeH-recording Measurement of UV-absorption 82 c) Biological Visualisation Procedures 82

V. Laboratory and Basic Equipment for TLC . . . . . 83 1. Small Laboratories . . . . . . . . . . . . . . 83 2. Medium-Sized Research and Control Laboratories 84 3. Central TLC- and PC-Laboratories in Industry. 84

VI. Standard Conditions in TLC . . . 85

D. Special Techniques in TLC. EGON STAJIL 86

I. Special Development Procedures 86 a) Continuous Development 86 b) Multiple Development 86 c) Stepwise Development. . 87

Table of Contents

1. Two-Dimensional Separation and SRS-Technique 2. Multidimensional Technique of VON ARx and NEHER 3. Wedged-tip Technique .

II. Gradient-Techniques in TLC . . . . . . . 1. Gradient-Elution. . . . . . . . . . . 2. TLC on Gradient Layers (Gradient-TLC)

III. Temperature-TLC ...... . 1. Apparatus for Temperature-TLC 2. Possibilities of Application

IV. Preparative TLC . . . . . . . . 1. Improvement of Separation in Column Chromatography 2. Adaptation of TLC to Larger Applied Amounts .

a) Increase of the Layer Thickness and Plate Size b) Band Application of Substance Solutions c) Types of Development . . . . . . d) Visualisation of the Separated Zones e) Collecting the Zones and Extraction

V. Transfer Techniques 1. Transfer: TLC -+ Solution. 2. Transfer: TLC -+ Reagent . 3. Transfer: TLC -+ Paper .. 4. Transfer: Solution -+ Paper (PC) -+ TLC 5. Transfer: Gas Chromatography -+ TLC .

E. Thin-Layer Electrophoresis. K. HANNIG and G. PASCHER

I. Principle of Thin-Layer Electrophoresis

II. Theoretical Principles . . . . . . . . 1. General ............ . 2. Factors Influencing the Mobility of Particles in Electrophuresis

with Supporting Medium (Thin-Layer Electrophoresis) a) Adsorption b) Diffusion . . . . . . . . . . . c) Zone Flaws . . . . . . . . . . d) Zeta-potential and Electroosmosis e) "Suction Effects" . . . . .

3. Units of Reference . . . . . .

III. Details of Apparatus and Methods 1. Apparatus ......... . 2. Methods ......... .

a) Supporting Layers and Preparation of the Plates b) Buffer Solutions . . . . . . . . . . . . . c) Current ............... . d) One-dimensional Thin-Layer Electrophoresis . e) Two-dimensional Thin-Layer Electrophoresis f) Finger Print Technique ........ .

F. Coupling of Gas- and Thin-Layer Chromatography. R. KAISER.

I. Principle of the GC-TLC-Coupling Analysis

II. Equipment

IX

88 88 89

89 90 91

94 95 96

97 97 98 98 98

100 100 101

102 102 104 104 104 105

105

105

106 106

106 107 107 107 107 108 108 109 109 111 111 111 111 113 113 114

114

115

118

x Table of Contents

III. Information from Coupling Analysis. 120

IV. Details of Apparatus and Methods 121 a) Deposition . . . . 121 b) Example . . . . . 122

V. Examples of Application. 124

G. Documentation of Thin-Layer Chromatograms. H. GXNSRIRT 125

I. Rf-values in TLC . . . . . . . . . . . . 127

II. Preservation of Thin-Layer Chromatograms 127

III. Graphical Copying . . . . . . . . . . . 128

IV. Documentation with Light-Sensitive Paper. 129 1. Copies with Diazo-type Paper . . 129 2. Copies with Blue-Print Paper . . 130 3. Positive Copies and Photographs with Procedures Based on

Diffusion of Silver Salts . 130 4. Photography . . . . . . . . . . . . . . . . . . . . .. 131

a) General Points . . . . . . . . . . . . . . . . . . " 132 b) Black and White Photographs with UV-Source 366 or 254 nm. 132 c) Colour Photographs with UV Light Sources. . . . . .. 133

5. Electrophotography . . . . . . . . . . . . . . . . .. 133

H. Quantitative Evaluation of Thin-Layer Chromatograms. H. GANSRIRT 133

I. Quantitative Evaluation on the TLC-Layers . . 135 1. Visual Comparison . . . . . . . . . . . . . . . . . .. 135 2. Determination by Measurement of Spot Areas . . ... . .. 135 3. Transmission Measurements on Spots which are Coloured, Charred,

or which absorb UV-Light . . . . . . . . . . 138 4. Quantitative Determination of Fluorescing Spots 141

a) Direct Evaluation . . . . . . . . . . . . 141 b) Photographic Evaluation. . . . . . . . . . 142

5. Evaluation of Reflectance (Remission) Spectra. . 142

II. Quantitative Determination after Extraction from the Adsorbent Layer ............... . 1. Detection of the Separated Substances . . .

a) Use of Reference Chromatograms .... b) Use of Fluorescent Layers and Indicators. c) Non-Destructive Detection by Spraying with Water d) Localisation of Separated Spots with Iodine Vapour or

Spraying with Iodine Solution. . . . . . . . . . . e) Use of other Colour Reagents .......... .

2. Removal of Spots from the Plate and Elution Technique 3. Eluents (Solvents for Extraction) ..... 4. Methods of Determination Used after Elution

I. Isotope Technique. HELMUT K. MANGOLD

I. Layers, Solvents and Chemical Methods of Detection II. Procedures for Detection and for Measurement of Radioactivity

1. Autoradiography of Thin-Layer Chromatograms 2. Counting Tubes and Scintillation Counters ....... .

145 145 145 146 147

by 147 148 148 149 151

155

156 157 157 160

Ta.ble of Contents XI

III. Preparation of Radioactively Labelled Substances 167

IV. Isolation of Radioactive Compounds by TLC . 168

V. Analysis by Means of Radioisotopes . 170 1. Indicator Analysis 171 2. Isotope Dilution Method . 171 3. Activation Analysis 172 4. Isotopic Derivative Method 172

a) Fractionation before Radioactive Labelling 173 b) Separation of Radioactive Derivatives . . 173 c) Fractionation after Addition of a Radioactive Derivative to

the Mixture of Non·labelled Derivatives .......... 174 d) Separation after Addition of an Inactive Derivative to the

Mixture Containing the Radioactively Labelled Derivative of the Compound to be Determined 174

e) Use of Two Radioactive Isotopes . . . . . . . 174

VI. Directions for Radioactive Labelling .. . . . . . . 175 1. Esterification of Acids with Diazomethane (14CH.N.) 175 2. Acetylation of Alcohols with Acetic Anhydride (14CHaCO).O or

(caHsCO).O . . . . . . . . . . . . . . . . . . . . . .. 175

VII. Applications of TLC in Chemical and Biochemical Investigations with Radioisotopes . . . . . . . . . . . 176

Theoretical Fundamentals of TLC . . . . . . . . 179

Biblio~aphy of the General Section, Chapters A-I 180

Special Section

Introduction. EGON STAHL. . ............ 201

J. Terpene Derivatives, Essential Oils, Balsams and Resins. EGON STAHL and H.JORK . . . . . . . . . . . . . . . . . . . . . . . . . . .. 206

I. Separation of Lipophilic, Steam-volatile Mixtures . . . . . . .. 207 II. Chromatographic Separation of Lipophilic, Steam-volatile Mixtures 208

1. Mono- and Sesquiterpene Hydrocarbons . 210 2. Oxides, Epoxides and Peroxides . . 212 3. Steam-volatile Esters and Lactones 214 4. Aldehydes and Ketones. . . . . . 217

a) Separation of Free Carbonyl Compounds 217 b) Separation of Derivatives of Aldehydes and Ketones 219

5. Terpene and Sesquiterpene Alcohols 225 a) TLC on Silica Gel Layers. . . . . . . . . . . 225 b) Paraffin-impregnated Silica Gel Layers . . . . . 226 c) Silver Nitrate-impregnated Silica Gel Layers. . . 227 d) Separation of the Dinitrobenzoate Esters (DNBs) 228

6. Phenylpropane and Phenol Derivatives 229 a) Silica Gel Layers • . . . . . . . . . . . . . 229 b) Structure and hE/-Value . . . . . . . . . . . 231 c) TLC of Phenol Esters, Coupling Derivatives and Other Con­

densation Products . . . . . . . . . . . . . . . . . . . 231 d) Separation of the Methoxyallylbenzenes from their Cis-Trans

Propenyl Isomers . . . . . . . . . . . . . . . . . . . . 233

XII Table of Contents

III. Essential Oils 235 235 235 235 240

1. Mixtures of Terpene and Sesquiterpene Derivatives 2. Sulphur-Containing Oils. . . . . . . . . . 3. Essential Oils with Polyacetylene Compounds 4. Supplement . . . . . . . . . • .

IV. TLC of Involatile Terpene Derivatives. . . . 1. Diterpenes . . . . . . . . . . . . . . 2. Triterpene Derivatives and their Glycosides

a) Neutral Triterpenes . b) Triterpene Acids c) Triterpene Glycosides

3. Polyterpenes

240 240 241 241 243 245 247

V. Balsams and Resins. . . . 247

Bibliography for Chapter J. Terpene Derivatives . 250

K. Vitamins, Includiug Carotenoids, Chloropbylls and Biologically Active Quinones. H. R. BOLLIGER and A. KONIG 259

I. Method of Work and General Experience ............ 259

II. TLC of Fat-Soluble Vitamins, Carotenoids, Chlorophylls and Quinones 263 1. Mixtures of Fat-Soluble Vitamins 263

a) Separation . . . . . . . . 263 b) Detection and Determination 265

2. Carotenoids and Chlorophylls . 266 a) Separation . . . . . . . . 266 b) Detection and Determination 272

3. Vitamin A Group ..... . a) Separation . . . . . . . . b) Detection and Determination

4. Vitamin D Group . . . . . . a) Separation . . . . . . . . b) Detection and Determination c) Tested Assay of Vitamin D

5. Vitamin E Group ..... . a) Separation . . . . . . . . b) Detection and Determination

6. Vitamin K Group and Related Quinones a) Separation . . . . . . . . b) Detection and Determination . .

III. TLC of Water-Soluble Vitamins 1. Mixtures of Water-Soluble Vitamins

a) Separation . . b) Detection. . .

2. Vitamin Bl Group a) Separation . . b) Detection and Determination

3. Vitamin B. Group . . . • • . a) Separation . . . . . . . . b) Detection and Determination

273 273 275 275 276 278 280 283 284 286 288 288 291

292 292 292 294 295 295 296 296 296 297

Table of Contents

4. Pantothenic Acid Group a) Separation . . . . . b) Detection and Determination

5. Nicotinic Acid and Nicotinamide . a) Separation . . . . . . . . b) Detection and Determination

6. Vitamin Be Group . . . . . . a) Separation . . . . . . . . b) Detection and Determination

7. Vitamin Bll Group a) Separation . . b) Detection. . .

8. Folic Acid Group . a) Separation . . b) Detection and Determination

9. Vitamin C. . . . . . . . . . a) Separation . . . . . . . . b) Detection and Determination

10. Biotin .......... . , a) Separation . . . . . . . .

b) Detection and Determination 11. Other Vitamins . . . . .

Bibliography for Chapter K. Vitamins . .

L. TLC of Steroids and Related Compounds. R. NEHER

I. Nomenclature ............ .

XIII

297 298 298 299 299 299 300 300 301 301 302 303 303 303 304 304 304 306 306 306 307 307 308

311 312

II. Range of Application of TLC of Steroids in Comparison with other Chromatographic Methods 313

III. General Conditions 316 1. Adsorbents 316 2. Solvents . . . . 317 3. Procedures. . . 320 4. Reactions for Detection 322 5. Formation of Derivatives (Microreactions) 325

IV. Structure and Chromatographic Behaviour 326 1. Adsorption TLC . . . . . . . . . . . 326 2. Partition TLC . . . . . . . . . . . . 328 3. Improvement of the Separation of Similar, Polyfunctional Steroids 328

V. Sterols. . . . . . . . . . . . 329

VII. Cardiac Glycosides and Aglycones VIII. Saponins and Sapogenins ....

IX. Aminosteroids, Steroid Alkaloids and Glycosides . X. Phenolic Steroids (Oestrogens) ....... .

XI. Bile Acids and Conjugates, Steroid Carboxylic Acids and Steroid Conjugates ...... .

1. Bile Alcohols . . . . . . 2. Steroid Carboxylic Acids . 3. Steroid Conjugates . .

Bibliography for Chapter L. Steroids .

334 341 346 348 349

351 355 355 355 357

XIV Table of Contents

M. Aliphatic Lipids. HELMUT K. MANGOLD 363

I. Introduction . . . . . . . . . . 363 1. Neutral Lipids and their Hydrolysis Products 363 2. Phospholipids, Sulpholipids and Glycolipids 363 3. Older Methods of Lipid Analysis . . . . . 366 4. Newer Procedures for Separation of Lipids. 367 5. Processing of the Material for Analysis . 368

a) Homogenisation and Extraction. . . . 368 b) Separation of Lipids from Non-Lipids . 370 c) Degradation of Lipids and Preparation of Derivatives 370 d) Other Reactions. . . . . . . . . . . 374

6. Manufacturers and Suppliers of Pure Lipids . . . 374

II. Thin-Layer Chromatography of Lipids. . . . . . . 3U, 1. Separation of Lipids According to Compound Class 374

a) Neutral Lipids and Their Hydrolysis Products 375 b) Phospholipids, Sulpholipids and Glycolipids . 388

2. Fractionation of Pure Compound Classes . . . . 394 a) Argentation Chromatography . . . . . . . . 396 b) Chromatography of Mercuric Acetate Adducts . 402 c) Chromatography of Ozonides . . . . . . . . 406 d) Reversed Phase Partition Chromatography . . 409

3. Quantitative Evaluation of Thin-Layer Chromatograms. 414 a) Neutral Lipids and Their Hydrolysis Products 414 b) Phospholipids, Sulpholipids and Glycolipids . 415

Bibliography for Chapter M. Aliphatic Lipids 415

N. Alkaloids. F. SANTAvY. . . . . . . . . 421

I. Adsorbent Layers and Solvents for TLC 421 II. Visualisation of the Alkaloids 423

III. Separation Scheme for Alkaloids . . . 424 IV. Quantitative Determination of Alkaloids with TLC 425 V. Special Section . . . . . . . . . . . . . . . . 425

1. Colchicine Alkaloids . . . . . . . . . . . . 425 2. Pyrrolidine, Pyridine and Piperidine Alkaloids 430 3. Tropane Alkaloids. . . 432 4. Pyrrolizidine Alkaloids. . . . 435 5. Quinolizidine Alkaloids 435 6. Alkaloids of the Papaveraceae 436 7. Bis(benzylisoquinoline) Alkaloids 444 8. Ipecacuanha Alkaloids. . 444 9. Amaryllidaceae Alkaloids 445

10. Indole Alkaloids 446 a) Indolyl Alkylamines 446 b) Mavacurine, Fluorocurine, Ellipticine, Eburnamine, Aspido-

spermine and Strychnine Alkaloids . . . 447 c) Rauwolfia Alkaloids ......... 448 d) Vinca Alkaloids (Catharanthus Alkaloids) 449 e) Ergot Alkaloids 450 f) Oxindole Alkaloids . . . . . . . . . . 454

Table of Contents XV

ll. Cinchona Alkaloids . . . 454 12. Furanoquinoline Alkaloids 456 13. Purine Alkaloids 457 14. Sterol Alkaloids. . . . . 457

a) Alkaloids of the Genera Solanum and Lycopersicum 458 b) Alkaloids of the Genera Holarrhena and Funtumia 459 c) Alkaloids of the Benzofluorene Type. 461

General Bibliography on the Alkaloids . . . 461

Special Bibliography for Chapter N. Alkaloids 462

O. "Simple" Indole Derivatives and Plant Growth Regulators. Urine Meta-bolites, Auxins, Gibberellins and Cytokinins. HARALD KALDEWEY 471

I. Introduction . . . . . . . . . . . . 471

II. Preparation of the Material for Analysis 473 1. General Information 473 2. Free Auxins . . . . . . . 474 3. Diffusible Auxins. . . . . 474 4. Indole Derivatives in Urine 474 5. Gibberellins . . . . 475 6. Cytokinins. . . . . 475

III. Adsorbent and Solvents 475 1. General Information 475 2. Auxins and Urine Metabolites 476 3. Gibberellins . . . . . . . . 479

IV. Multiple Development, Stepwise Development and Two-Dimensional Separation . . . . . . . . . . . . . . . . . 481

V. Visualisation and Identification . . . . . . . . 483 1. Chemical and Physical Methods of Detection 483 2. Biological Methods of Detection . . . . . . 487

Bibliography for Chapter O. "Simple" Indole Derivatives 489

P. Amines and Tar Bases. EGON STAHL and P. J. SCHORN. 494

I. Aroines . .. . .. 494 1. Aliphatic Aroines. . . . . . . . . . . . . . 494 2. Nitrosamines . . . . . . . . . . . . . . . 497 3. AminoalcohoIs and Quaternary Ammonium Salts . 498 4. Catechol Amines (Phenylalkylamines) . 499 5. Aromatic and Heterocyclic Aroines . . 500 6. Thin-Layer Electrophoresis of Aroines 502

II. Tar Bases • . . . . . . . . . . . . . 503

Bibliography for Chapter P. Amines and Tar Bases. 505

Q. Synthetic Pharmaceutical Products. HERBERT G.!NSHIRT 506

1. Antihistamines, Anti-Allergics and Structurally Related Compounds with Psychic Activity . . . . . 507 a) Phenothiazines and Diazepines 508 b) Antihistamines ...... 518

XVI Table of Contents

2. Analeptics, Psychotherapeutic Agents with Antidepressive Activity, AppetiteDepressants andSomeCarbamateEsters of Varied Activity 518

3. Sympathomimetics of the Adrenaline Type . . . • 520 4. Analgesics, Antipyretics and Antirheumatic Agents 524

a) p-Aminophenol Derivatives, Pyrazolones etc. . 524 b) Analgesics with Narcotic Activ'ity ..... 527

5. Anticoagulants of the 4-Hydroxycoumarin Group 532 6. Hypnotics . . 533

a) Barbiturates . 533 b) Hydantoins . 537 c) Bromoureides 538 d) Other Hypnotics 538

7. Bactericidal and Bacteriostatic Substances 540 a) Phenols of Pharmaceutical Interest. . 540 b) Sulphonamides. . . . . . . . . . . 541 c) Chemotherapeutic Agents of the Nitrofuran Series, etc. 547

8. Diuretics. . . . . . . . . . . . . . 547 9. Purine Derivatives of Various Activities 548

10. Oral Antidiabetic Agents . 550 11. Laxatives. . . . . . . . . . . . . . 551 12. Local Anaesthetics . . . . . . . . . 551 13. Miscellaneous Other Active Substances . 554

I. Analysis of Various Drug Forms and of Commercial Preparations 554

II. Stability Tests on Pharmaceutical Materials . . . . . 558

Bibliography for Chapter Q. Synthetic Pharmaceutical Products 562

R. Antibiotics. K. H. W ALLHAUSSER • • • • • • 566

1. Execution of the Microbiological Test . 568 a) Direct Method .. . . . . . . . 570 b) Contact Method (Reprint or Filter Paper Print Process) 570

2. Execution of the Chemical Test . . . . . . . . . . 570 3. General Information about Layers and Solvents Used. 570

I. Polyenes and Polyacetylenes 571 II. Macrolides . . . . . . . . . . . . . . 572

III. Tetracyclines. . . . . . . . . . . . . 573 IV. Substances with Similar Structural Units 574 V. Basic, Water-Soluble, Non-extractable Antibiotics 574

VI. Nucleic Acid Derivatives 575 VII. Acyclic Compounds. . . 575

VIII. Heterocyclic Compounds. 575 IX. Macrocyclic Peptides . . 576

X. Other Peptides . . . . . 576 XI. Miscellaneous Antibiotics 576

Bibliography for Chapter R. Antibiotics 577

s. TLC in Clinical Diagnosis. NEPOMUK ZOLLNER and GUNTHER WOLFRAM 578

Introduction 578

Table of Contents

1. Investigation of Endogenous Substances . 1. Sugars, Their Derivatives and Metabolites .

a) Glycoproteins . . . . . . . . . . . . b) Ketone Bowes . . . . . . . . . . .

2. Amino Acids, Their Derivatives and Metabolites a) Amino Acids in Urine ..... . b) Amino Acids in Blood and Organs . c) Amino Acids in Other Body Fluids d) Iodoamino Acids ... e) e-Aminocaproic Acid .. f) Creatinine . . . . . . g) Amines and Metabolites h) Serum Proteins . . . .

3. Lipids and Related Compounds. a) Neutral Fat in Plasma ... b) Cholesteryl Esters in Plasma c) Phospholipids in Plasma .. d) Methods for Separating Other Medically Important Lipids e) Lipids in Secreted and Excreted Material. f) Lipids in Tissue

4. Steroids. . . . a) Cu-Steroids . b) Cn-Steroids . c) ClS"Steroids . d) Bile Acids . e) Sterols in Faeces

5. Porphyrins and Metabolites

II. Investigations of Exogenous Substances 1. Function Tests. . . 2. Poisoning . . . . . . . . . . . . 3. Control of Therapy . . . . . . . .

Bibliography for Chapter S. TLC in Clinical Diagnosis

TF. Synthetic Colouring Materials. H. SCHWEPPE •

I. Solvent Dyes. . . . . . Special Applications . . . . . . . . a) Dyes in Motor Fuels . . . . . . . b) Colouring Matter in Naturally Occurring Fats and Oils . c) Dyes in Polystyrene

II. Disperse Dyes • • III. Organic Pigments . IV. Basic Dyes .

V. Acid Dyes .. VI. Direct Dyes .

VII. Reactive Dyes VIII. Metal Complex Dyes

IX. Synthetic Food Colora.nts

XVII

579 579 580 581 581 582 585 586 586 587 588 589 591 592 593 593 596 597 597 598 599 600 602 602 603 603 604

604 605 605 606

607

612

612

. " 615 615 616 616

616 617 618 620 621 622 623 624

XVIII Table of Contents

X. Dye Intermediates 626

Bibliography for Chapter TF. Synthetic Colouring Materials. 628

TN. FoodstuJls and Their Additives. J. W. COPIUS-PEEREBOOlll • 630

I. General Applications 630 II. Antioxidants. 631

III. Preservatives. . . . 636 IV. Pesticides . . . . . 638

1. Phosphate Esters . 639 2. Chlorinated Hydrocarbons . 642 3. Pyrethrins and Synergists . 645 4. Herbicides ....... 647

V. Artificial Sweeteners 648 VI. Emulsifiers and Swelling Agents 649

VII. Alcohols and Glycols . . . . . 650 VIII. Organic Acids . . . . . . . . 650

Bibliography for Chapter TN. Foodstuffs and Their Additives 654

TS. Synthetic Organic Products. H.-J. PETROWITZ • 657

I. Plastics and Plasticisers . . . . . . . . . 657 1. Polymers and Polymerisable Compounds 657 la. Urethanes 658 2. Plasticisers . . . 659 3. Alcohols 660

a) Simple Alcohols 660 b) Polyalcohols . 662

4. Phenols. . . . . 663 5. Other Auxiliary Materials in the Plastics Industry 664

II. Organo-Metallic Compounds 664 1. Organo-Tin Compounds. . . . . 664 2. Ferrocenes ........ . . 665 3. Other Organo-Metallic Compounds 666

III. Polyphenyls and Fused Polynuclear Aromatic Hydrocarbons . 666 ~. Polyphenyls . . . . . . . . . . . 666 2. Polynuclear Aromatic Hydrocarbons 667

IV. Explosives. . . . . . . . . 669

V. Industrial Additives. . . . . 673 1. Inhibitors and Antioxidants 673 2. Detergents . . . . . . . 674 3. Optical Brighteners. . . . 675 4. Wood Protective Agents . 675 5. Chemicals for Photography 676 6. Special Mineral Oil Analyses. 676

VI. Intermediates in Organic Synthesis 677

Bibliography for Chapter TS. Synthetic Organic Products . 684

Table of Contents XIX

U. Hydrophllie Plant Constituents and Their Derivatives . 686

I. Plant Phenol Derivatives. KURT EGGER . . 687 1. Compound Classes and their Distribution 687 2. Concentration from Plant Material . . . 690

a) Acetone-extraction of Fresh Material . 690 b) Methanol-extraction of Dried Drugs 691 c) Extraction in Stages . . . . . . . . 691 d) Extraction with Water. . . . . . . 691

3. Chromatographic Separation (without TLC) 691 4. Experimental Conditions for TLC 692

a) Cellulose . . . . . . . . . . 692 b) Silica Gel. . . . . . . . . . 693 c) Polyamide and Other Polymers 699 d) 1;'olyacrylonitrile ...... 704 e) Ion Exchangers . . . . . . . 704

5. Visualisation of Phenol Derivatives . 705

II. TLC in the Characterisation of Animal and Plant Drugs. EGON STAHL and P. J. SCHORN. . . . 706 1. Anthraquinone Drugs. . . . . . . . 706 2. Lignan Drugs . . . . . . . . . . . 709 3. Drugs with Phloroglucinol Derivatives 712

a) Filix-Phloroglucinol Butanones 712 b) Hop Bitter Principles . . . . . . 714

4. Drugs Containing Bitter Principles . . 715 5. Constituents of Hashish. . . . . . . 715 6. Other Drugs and Mixtures of Natural Products. 716

III. TLC as a Legally Binding Method for Characteris!l.tion of Drugs. EGON STAHL and P. J. SCHORN. . . . . . . . 720 1. General Information for Practical Directions . 720 2. Two Examples of Special Procedures . . 722

a) Rhizoma Filicis (Male Fern Rhizome) 722 b) Radix Liquiritiae (Liquorice Root). . 723

Bibliography for Chapter U. Hydrophilic Plant Constituents. 724

V. Amino Aeids and Derivatives.l\f. BRENNER, A. NIEDERWIESER and G. PATAK! . . . . 730

I. Introduction . . . . . . . 730

II. General Technique . . . . 732 1. Preparation of the Layer 732 2. Chromatographic Techniques 734

III. Amino Acids . . . . . . . . . 734 1. Preparation of the Solution for Investigation 734 2. Hydrolysis of Proteins and Peptides . . . . 735 3. Free Amino Acids in Biological Material 736 4. Solvents and Separation Efficiency . . . . . 739 5. Detection of Amino Acids on the Chromatogram 746

IV. Peptides. . . . . . . . . . . . . . . . . . . 751

xx Table of Contents

V. N-(2,4-Dinitrophenyl)-amino Acids and 3-Phenyl-2-Thiohydantoins 756 A. Dinitrophenylamino Acids 756

1. Dinitrophenylation 757 a) Amino Acids. . . . 757 b) Peptides. . . . . . 758 c) Polypeptides and Proteins 759

2. Solvents and Separation Efficiency 760 a) Solvents for Chromatography of Acid- and Water-soluble

DNP-Amino Acids, not Extractable with Ether ..... 762 b) Solvents for Chromatography of Acid-insoluble DNP-Amino

Acids, Extractable with Ether 763 3. Documentation . . . . . . . . . . . . . . . . . . . . . 771

B. Phenylthiohydantoins . . . . . . . . . . . . . . .. . . . 772 1. Preparation of the Phenylthiocarbamyl-derivatives and their

Conversion into PTH-Amino Acids 773 2. Solvents and Separation Efficiency . . 774 3 .. Detection of the Phenylthiohydantoins . 777

C. Other Amino Acid Derivatives ..... 777 1. Dinitropyridyl-Amino Acids . . . . . 777 2. I-Dimethylamino-5-Naphthalenesulphonyl-Amino Acids (DANS-

Amino Acids) . . . . . . . . . . . . 778 3. Carbobenzoxy Compounds . . . . . . . . 778

D. Iodoamino Acids and Similar Comp~unds. . . 779

Bibliography for Chapter V. Amino Acids and Derivatives 781

w. Nucleic Acids and Nucleotides. HELMUT K. MANGOLD 786

I. Introduction . . . . . . . . . . . . . . . . 786 1. Nucleic Acids and their Hydrolysis Products 786 2. Nucleotide-Coenzymes . . . . . . . . . . 788 3. Older Methods of Nucleic Acid Analysis. . . 788 4. Newer Methods for Isolation of Nucleic Acids and Separation of

Their Constituents . . . . . . • . . . . . . . . . . . . . . 788 5. Colour Reactions for Distinguishing Ribo- and Deoxyribonucleic

Acids. . . . . . . . . . 789 6. Hydrolysis of Nucleic Acids 789

a) Alkaline Hydrolysis . 790 b) Acid Hydrolysis. . . . 790 c) Enzymatic Hydrolysis . 791

7. UV-Spectra of Nucleic Acid Constituents 791 8. Manufacturers and Suppliers of Pure Preparations 792

II. Thin-Layer Chromatography of Nucleic Acids and their ConstituentR 792 1. Purines, Pyrimidines and Nucleosides . 792 2. Nucleotides and Nucleotide-Coenzymes ............ 794 3. Oligonucleotides and Nucleic Acids . . . . . . . . . . . . . . 801

III. Thin-Layer Electrophoresis of Hydrolysis Products of Nucleic Acids 802

Bibliography for Chapter W. Nucleic Acids and Nucleotides 804

X. Sugars and Derivatives. B. A. LEWIS and F. SMITH 807

T. Introduction. . . . . . . . . . . . . . . 807

Ta.ble of Contents XXI

II. Preparation of Plates . . . . . . . . . . . . . . 807 1. Silica Gel G and Kieselguhr G Layers. . . . . . 808 2. Kieselguhr G, Impregnated with Sodium Acetate. 808 3. Kieselguhr G, Impregnated with pH 5 Phosphate Buffer 808 4. Impregnated Silica Gel G Layers. . . . . . . . 808 5. Cellulose Layers (a.) Cellulose MN 300. . . . . . 808 5b. "Avirin" ("Avicel") . . . . . . . . . . . . . 809 6. ECTEOLA·Cellulose Layers for Sugar Phosphates 809 7. Celite Layers (a) Filter·CeI and Hyflo Super-Cel 809 7 b. Celite 535-Starch . . . . . . . . . . 809

III. Visualization. . . . . . . . . . . . . . 810

IV. Chromatography of Sugars and Derivatives. 811 1. Sugars . . . . . . . . . . . . . . . 812

a) Separation of Sugars on Buffered Kieselguhr G Layers 812 b) Separation of Sugars on Buffered Silica Gel G . . . . 814 c) Separation of Sugars on Cellulose. . . . . . . . . . 814 d) Chromatography of Sugars on Miscellaneous Adsorbents . 815

2. Oligosaccharides .. . . . . . . . . . . . . 816 3. Amino Sugars ............... 817 4. Acids (Aldonic, Aldaric, Uronic and Saccharinic) 819 5. Sugar Alcohols . . . . 820 6. Methyl Glycosides. . . . 820 7. Sugar Phosphates. . . . 821 8. Acetates and Benzoates . 822 9. Hydrazones and Osazones 825

10. Methyl Ethers . . . . . 828 11. Miscellaneous Derivatives 830

V. Monitoring Reactions by TLC 831

Bibliography for Chapter X. Sugars and Derivatives 834

Y. Inorganic Ions. H. SEILER . . . . . . . . . . 837

I. Preparation of the Solutions for Analysis 838

II. TLC of the Cations Separated in this Preliminary Stage 839 1. Separation of the Cu-group (Solution I) . . . . . . 839 2. Separation of the (NH4).S-group (Solution II) . . . 840 3. Separation of the Ammonium Carbonate-group (Solution III) 841 4. Separation of the Alkali-group (Solution IV) . 842

III. Separation of Special Cation Mixtures . . . 843 1. UO~+ in a Mixture of Cations . . . . . . . 844 2. Ga3+ in Presence of Large Excess of Als+ . . 844 3. Sn, Cu, Hg, Pb,. Bi, Cd and Zn as Dithizonates . 844 4. Ag, Pd, Au and Pt as Dithizonates . . . . . . 844 5. Separation of Cations on Layers of Ion Exchangers . 845 6. Circular TLC of Cations. . . . . . . . . . . . . 845 7. Separation and Detection of Toxic Metals . . . . . 846

a) Qualitative Separation of Tl, Ni, Cu, Bi and Hg and of Ce, Ni, Cu, Be, Bi and Hg . 846

b) Determination of Hg. . . . . . . . . . . . . . . . . . . 846

XXII Table of Contents

8. Separation of cis-tra1!8 Co-Complex Isomers 846 9. Separation of Radionuclides 847

IV. Separation of Anions . . . 847 1. Separation of the Halides . 847 2. Separation of Phosphates . 848 3. Separation of Condensed Phosphates 848 4. Separation of Sulphates and Polythionates 850

V. Quantitative Determination . . . 851

Bibliography for Chapter Y. Inorganic Ions . . . . . 853

Z. Spray Reagents. K. G. KREBS, D. HEUSSER and H. WIMMER 854

I. Preparation and Application of the Spray Reagents . . 855

II. Compounds or Compound Classes and Reagents for their Detection . 905

III. Names and Abbreviations of Reagents. 909

Conversion table for HI into Hm and vice versa 910

Terms Frequently used in Thin-Layer Chromatography. HELMUT K. MANGOLD and M. BRENNER • • • • • . • 912

List of Manufacturers and Suppliers . 918

Author Index . 924

Subject Index 1023

Contributing Authors*

BOLLIGER, H. R., Dr., Hoffmann-La Roche u. Co., AG., CH - 4000 Basel

BRENNER, M., Prof. Dr., Institut ffir Organische Chemie der Universitat Basel, St. Johanns-Ring 19, CH - 4000 Basel

COPIUS-PEEREBOOM, J. W., Dr., Gouvernment Dairy Station, Vreewijkstraat 12 B Leiden/Niederlande

DORFNER, K., Dr., BadiBche Anilin- und Sodafabrik AG., 6700 Ludwigshafen

EGGER, K., Priv.-Doz. Dr., Botanisches Institut der Universitat, 6900 Heidelberg, HofmeiBterweg 4

ENDRES, H., Priv.-Doz. Dr., BadiBche Anilin- und Sodafabrik AG, Abt. A WETAfLe­der, 6700 Ludwigshafen

GlNSHlRT, HERBERT, Dr., Farbenfabriken Bayer AG, 5090 Leverkusen

lIANmG, K.,-Priv.-Doz. Dr., Max-Planck-Institut fUr EiweiB- und Lederforschung 8000 Miinchen 15, SchillerstraBe 42-46

KArSER, R., Dr., Badische Anilin- und Sodafabrik AG, Analytisches Labor M 310, 6700 Ludwigshafen

KALnEWEY, H., Prof. Dr., Botanisches Institut der Universitat des Saarlandes, 6600 Saarbriicken 15

KOHLSClIUTTER, H. W., Prof. Dr., Eduard-Zintl-Institut ffir AnorganiBche und Phy­sikalische Chemie der TechniBchen Hochschule Darmstadt, Lehrstuhl ffir An­organische und Analytische Chemie, 6100 Darmstadt, Hochschulstr. 4

KREBS, K. G., Prof. Dr., Direktor des Kontroll-Laboratoriums der E. Merck AG, 6100 Darmstadt

LEWIS, B. A., Dr., New York State College of Home Economics, A Statutory Col­lege of the State University, Cornell University Ithaca, NY 14850/USA

MANGOLD, H. K., Prof. Dr., The Hormel Institute, University of Minnesota, 801 16th Avenue N. E., P. o. Box 367, Austin, Minn. 55912/USA

NEHER, R., Dr., Ciba Aktiengesellschaft, CH - 4000 Basel

PETROWITZ, H. J., Dr.-Ing., Bundesanstalt fUr Materialpriifung, Fachgruppe 2,4 "Biologische Materialprlifung, Holzschutz und Holztechnologie" 1000 Berlin­Dahlem, Unter den Eichen 87

ROSSLER, H., Dr., E. Merck AG, Forschungsabteilungen, Hauptlaboratorium, 6100 Darmstadt

SANTAvY, F., Prof. Dr., Chemisches Institut der Medizinischen Fakultat, Palacky Universitat, Olomouc/Tschechoslowakei, Hnevotinska 3

* The names of the co-workers who collaborated with the various authors are given in the individual chapter headings.

XXIV Contributing Authors

SEILER, H., Priv.·Doz. Dr., Institut fiir Anorganische Chemie an der Universitii.t Basel, CH - 4000 Basel, SpitalstraBe 51

SCHWEPPE, H., Dr., Badische Anilin- und Sodafabrik AG, Abt. AWETA 1,6700 Ludwigshafen

SMITH, F., Prof. Dr. t, University of Minnesota, Department of Biochemistry, Synder Hall, St. Paul, MinnesotafUSA

STAHL, EGON, Prof. Dr., Direktor des Instituts fiir Pharmakognosie und Analytische Phytochemie der Universitat des Saarlandes, 6600 Saarbriicken 15

WALLHXUSSER, K. H., Dr., Farbwerke Hoechst AG, Mikrobiologisches Unter· Buchungslabor, 6230 Frankfurt·Hoechst

WOLLENWEBER, P., Dr., Machery, Nagel u. Co., 5160 Diiren

ZOLLNER, N., Prof. Dr., Medizinische Poliklinik der Universitii.t Miinchen, 8000 Miinchen 15, PettenkoferstraBe 6a