Dyeing Reagents for Thin Layer and Paper Chromatography

Reagents MERCK

This most recent edition of a booklet of long standing lists 335 detectants for the visualisation of chromatograms. The dyeing reagents are listed in alphabetical order. Those for use in paper chromatography are marked additionally with "PC". An alphabetical index of compounds for which a detection reagent is being sought is given towards the end of this booklet. The directions given for each product in the main body of the text are supple- mented with appropriate literature references. Below the references we list the reagents needed to prepare the dyeing solution and include the Catalogue Number as in our General Catalogue of Reagents and Chemicals. There is an index at the end of the booklet summarising all the products referred to, together with details on available package sizes. For adsorbents, solvents, ready-to-use TLC products and miscellaneous items for chromatography, please refer to our various special brochures. Dyeing of thin layer chromatograms*

Spraying: Dry the chromatogram to remove the solvent,. then cool. Place vertically into a spraying box or into a fume cupboard and protect the surroundings by covering with filter paper or the like. Apply the spray solution from about 30 cm until the layer is evenly wened but not for so long that the chromatogram begins to run with liquid. In most cases the chromatogram is specially treated at this stage. For derails please refer to the directions quoted for the particular reagent concerned. Unless otherwise stated, subsequent treatment should be taken as meaning drying ar room temperature.

Ready-to-use spray reagents: Merck sells a number of spray reagents in handy aerosol form:

Aniline phthalate Spray reagent for chromatography Cat. No. 1266

Bromocresol green Spray reagent 0.05% for chromatography Cat. No. 1998

For detection of paper chromatograms see: I- M. Hais, K. Macek, Paper Chromatography, Publishing House Czechoslovak Academy of Sciences, Prague, and Academic Press, New York arid. London, 1963 F. Cramer, Paper-Chromatographic, Verlag Chemie, Weinheim, 5e edition, 1962 See also the relevant chapters in handbooks on the subject, e. g.: E. Stahl, Thin-Layer Chromatography, Springer Verlag and Academic Press, New York and London, 2nd edition, 1969 K. Randerath, Thin-Layer Chromatography, Verlag Chemie and Academic Press, New York and London, 2nd edition, 1966

V 2' ,7' -D ichlorofluoresce in Propellant gas sprayers: Spray reagent 0.1% for chromatography The spray gun type of equipment also offers a convenient means of applying Cat, No. 9677 reagent. The propellant gas container and the spray solution can be changed as desired. 4-Dimethylarninobenzaldehyde Spray reagent 0.5% for chromatography Cat. No. 3056

Molybdatophosphoric acid Spray reagent 3.5% for chromatography Cat. No. 531

N inhydrin Spray reagent 0.1% for chromatography Cat. No. 6758

Rhodamine B Spray reagent 0.1% for chromatography Cat. No. 7596

VI VII Glass spraying equipment: A further possibility is to spray the reagent from a glass vessel connected via a reducing valve to a compressed nitrogen bottle or to an air line. A small diaphragm pump can be used to provide oil-free compressed air. The rubber bulb sprayers formerly used for paper chromatography can no longer be recommended, since they rarely permit a uniform and finely atomised spray to be produced.

Fume cupboard: The fact that many of the reagents used for spraying in thin layer chromatography are aggressive as well as in many cases toxic means that a well functioning extrac- tion system (e.g. a fume cupboard) is essential. If a fume cupboard is not available in the laboratory, a small spraying cabinet made of acid-resistant material with an outlet connected to an extracting system can be used instead.

Heating: Frequently, optimum colour development after spraying with the detection reagent is only obtained by heating. An adjustable drying oven is generally used I for this purpose. For uniform heating the plate should be placed vertically into the 1 1 1 oven.

11 Immersion: For quantitative in-situ evaluation in particular it is essential for the reagent to be applied uniformly. For optical measurements (in reflectance or transmission mode), immersion of the chromatogram in the appropriate reagent usually pro- vides the best results. Pre-coated glass plates, aluminium sheets and plastic sheets are mostly better for this technique than user-coated plates on account of their superior layer adhesion.

Equipment for use in conjunction with the techniques mentioned is supplied by: CAMAG Chemie-Erzeugnisse and Adsorptionstechnik AG Muttenz, Homburger Str. 24, Switzerland; 1000 Berlin 45, Baseler Str. 65, Germany C. Desaga GmbH, 6900 Heidelberg 1, Postfach 407, Germany Shandon Scientific Comp. Ltd., 65 Pound Lane, Willesden, London NW 10, Great Britain

I I ' VIII IX 1. Acetic anhydride — sulfuric acid for 6,5-3-sterols, (cholesterol and esters), steroids and triterpene glycosides (Liebermann-Burchard reagent). Spray solution: Mix carefully and with cooling freshly before use .5 ml acetic anhydride with 5 ml 97"/o sulfuric acid and add the mixture with cooling to 50 ml ethanol. Treatment: Heat 10 min at 110°C. Characteristic .fluorescence in long-wave UV light. • Literature: C. Michalec, Biochim. et hiophys. Acta 19, 187 (19.56). R. Tschesch:, J. Chromatog. 5, 217 (1961). K. Tiaecla, S. Hara, A. Wada, N. Matsumoto, J. Chromatog. 11, 562 (1963), Chemicals: Acetic anhydride G R, Cot. No. 42 Sulfuric acid 95-97% (1.84) G R, Cat, No. 731 Ethanol af)s. G R, Cat. No. 972

2. Alizarin for cations. Spray sohitiou: Saturated ethanolic alizarin solution. Treatment: Place the moist chromatogram into a chamber with ammonia vapour. Literature: G. de Vries, G. P. Schiitze, E. van Dalen, J. Chromatog. 13, 119 (1964). Chemicals: Alizarin G R and indicator, Cat. No. 1016 Ethanol abs. G R, Cat. No. 972 Ammonia solution min. 25% (0.91) G R, Cat, No. 5432

3. Aluminium chloride for flavonoids. Spray solution: 1% ethanolic solution of aluminium chloride. Yellow fluorescence in long-wave LW light. Literature: T. G. Gage, C. D. Douglas, S. H, Wender, Anal. Chem. 23, 1582 (1951). Chemicals: Aluminium chloride extra pure cast., Cat. No. 1084 Ethanol :tills. G R, Cat. No. 972

4. 4-Aminoantipyrine — potassium hexacyanoferrate(I[I) for phenols (Emerson reaction). Spray solution 1: 2% ethanolic solution of 4-aminoantipyrine. Spray solution 11: 8% aqueous potassium hexacyanoferrate (III) solution. Procedure: Spray with 1, then with 11, and subsequently place the chromatogram into a chamber saturated with ammonia vapour. Red-orange to salmon pink spots_ Literature: G. Gabel, K. H. Muller, J. Schoknechr, Dtsch. Apotheker-Ztg., 102, 293, (1962).

1 9. Ammonium cerium(IV) nitrate - Chemicals: No. 7293 N,N-dimethy1-1,4-phenylenediammonium dichloride 4-Amino-2,3-dirnethy1-1-phenyl-3-pyrazolin-5-one, Cat. for polyalcohols. Potassium hexacyanoferrate(111) G R, Cat. No. 4973 Ammonia solution min. 25%, (0.91) GR, Cat. No. 5432 Solution a: 1% solution of ammonium cerium(1V) nitrate in 0.2 N nitric acid. Solution b: Dissolve 1.5 g N,N-dimethy1-1,4-phenylenediammonium dichloride in 5. o-Aminodiphenyl - phosphoric acid for sugars 128 nil methanol, 25 ml water and 1.5 ml glacial acetic acid. (modif. reagent acc. to Lewis-Smith). Spray solution: Mix 1 part a with 10 parrs b freshly before use. Spray solution: Dissolve 0.3 g o-aminodiphenyl and 5 ml 8S% phosphoric acid in Treatment: Heat 10 min ar 105'C. 95 ml ethanol. Yellowish green spots on red background. Treatment: Heat 15-20 min at 110'C. Literature: E. Knappe, D. Feted, J. Rohdewald, Z. anal. Chem. 199, 270 (1964). Sugars show brown spots. Chemicals: Literature: T. E. Timell, C. P. J. Glandemanns, Anal. Chem. 28, 1916 (1956). Ammonium cerium(11') nitrate G R, Cat. No. 2276 Chemicals: N,N-dimethy1-1,4-pbeirvIenediammonium dichloride G R, Cat. No. .3067 0-Aminodiphenyl Methanol G R, Cat. No. 6009 ortho- Phosphoric acid min. 85% (1.71) G R, Cat. No. 573 Acetic acid glacial min. 96% GR, Cat. No. 62 Ethanol abs. G R, Cat. No. 972 Nitric acid min. 65% G R, Cat. No. 454

6. 4-Aminohippuric acid for reducing sugars. 10. Ammonium cerium(1V) nitrate - nitric acid for Spray solution: 0.3'/0 ethanolic 4-arninohippuric acid solution, a-hydroxy acids, u-keto acids and mercaptans. PC. Treatment: Heat 8 min at 140'C. Dip solution: Dissolve 20 g ammonium cerium(IV) nitrate in 50 nil 0.5 N nitric acid. Characteristic spots in long-wave UV light. Dilute freshly before use 1 part of this solution with 3 parts water. Literature: L. Sat-tler, F. W. Zerban, Anal. Chem. 24, 1862 (1952). Procedure: After drying dip the chromatogram into the dip solution and place it on Chemicals: a dean filter paper. 4-Aminohippuric acid, Cat. No. 84 White spots on yellow background, Ethanol abs. G R, Cat. No. 972 Literature: M. Trop, M. Sprecher, A. Pinsky, J. Chromatog. 32, 426 (1968). Chemicals: 7. o-Aminophenol - phosphoric acid for sugars. Ammonium cerium(IV) nitrate GR, Cat. No. 2276 Spray solution: Dissolve 0.15 g o-aminophenol in 20 ml ethanol shortly prior to use. Nitric acid nri71, 65% (about 1.40) G R, Cat. No. 454 Add 10 50% phosphoric acid to the solution. Literature: L. Vigyaz-Var.,os, Magyar Kem. Folyorat 59, 253 (1953). 11. Ammonium cerium(IV) sulfate for Vinca alkaloids. 26, 183 (1953). S. Hirase, C. Araki, S, Nakanishi, Bull. Chem. Soc. (Japan) Spray solution: 1% solution of ammonium cerium(IV) sulfate in 85% phosphoric Chemicals: acid. No. 4 / 9 2-Aminophenol, Cat. Literature: 1. M. Jakovljevic, L. D. Seay, R. W. Shaffer, J. Pharm. Sci. 53, 5.53 (1964). Ethanol abs. G R, Cat. No. 972 Chemicals: ortho-Phosphoric acid min. 85% (1.71) GR, Cat. No. 573 Ammonium cerium(111) sulfate G R, Cat. No. 2273 ortho-Phosphoric acid. nun. 85% (1.4) G R, Cat. No. 573 8. Ammonia for tetracyclines. Place the chromatogram into a chamber saturated with ammonia vapour. Procedure: 12. Tetracyclines show yellow spots in long-wave UV light. Ammonium iron(III) sulfate for flavonoids. Spray solution: 0.2% aqueous solution of ammonium iron(I11) sulfate. Literature: M. Urx, J. Vondraaco% a, L. Kovaiik, O. Horsky, M. Herold, J. Chroma- tog. 11, 62 (1963). Literature: E. A. H. Roberts, D. J. Wood, Biochern. J. 49, 414 (1951). Chemicals: Chemicals: Ammonia solution mm. 25% (0.91) GR, Cat. No. 5432 Ammonium iron(111) sulfate G R, Cat. No. 3776

3 13. Ammonium iron(III) sulfate vor Vinca alkaloids. S. Burrows, F. S. M. Grylls, J. S. Harrison, Nature 170, 800 (1952.). C. W. Stanley, J. Chromatog. Spray solution: Dissolve 1 g ammonium iron(ill) sulfate in 100 ml phosphoric acid 16, 467 (1964). Chemicals; (75 or 85%). Spray the reagent on to heated chromatogram (100°C). Ammonium heptainolybdate G R, Cat. No. 1182 Hydrochloric acid min. 25% (1.125) G R, Cat. Literature: 1. M. Jakovljevic, L. D. Seay, R. W. Shaffer, J. Pharm. Sci. 53,553 (1964). No. 316 Percbloric acid about 70% (1.67) G R, Cat. No. 519 Chemicals: Acetone G R, Cat. No. 14 Anrmonium iron(111) sulfate G R, Cat. No. 3776 nth!. 85% (1.71) G R, Cat_ No.. 573 ortho-Phosphoric acid. 16. Ammonium molybdate — tin(II) chloride for phosphoric acids. 14. Ammonium molybdate — crystal violet for phosphoric acid. PC. Spray solution I. 1% aqueous ammonium molybdate solution. Spray solution 11: 1% Spray solution: Mixture of 5 ml l 'Xi aqueous ammonium molybdate solution, 5 ml solution of tin(11) chloride in 10% hydrochloric acid. 25% hydrochloric acid and 90 ml acetone. Procedure: Spray with I, dry the chromatogram and spray with II. Heat, if necessary, at 105°C for 3-5 minutes. Solution a: Dissolve 2 g crystal violet (or brilliant green or iodine green) in 350 nil water. Literature: H. Seiler, Helv. Chins. Acta 44, 1753 (1961). Solution b: Dissolve with heating 4 g ammonium molybdate in water, add 50 ml Chemicals: 10 N hydrochloric acid and fill up to 100 ml with water, Annnorrirmr heptamolybdate G R, Cat. No. 1182 Tin(11) chloride G R, Cat. No. 7815 Dip solution: Mix a and b, wait at least 3 hours and filter the solution. Hydrochloric acid min. 25% (1.125) G R, Cat, No, 31C, Procedure: Spray the chromatogram with the spray solution, heat 3-6 min at 85°C, dip into the dip solution and place immediately on a prepared clean filter paper. 17. Ammonium thiocyanate — iron(H) sulfate for peroxides. Note: 0.02 tg of phosphorus are detectable. Spray solution 1: Dissolve 0.4 g ammonium thiocyanate.in 30 ml acetone, Crystal violet = blue spots on yellow background Spray solution 11: Brilliant green = green spots on orange background Dissolve 1.2 g iron(II) sulfate in 30 ml water. Iodine green = turquois spots on colourless background Procedure: Spray with I, dry the chromatogram and spray with 11. Literature: Literature: F. ]ungnickel, J. Chromatog. 31, 617 (1967). M. H. Abraham, A. G. Davies, D. R. Llewellyn, E. M, Thain, Anal. Chiin. Acta 17, 499 (1957). Chemicals: Chemicals: Ammonium beptamolybdate G R, Cat. No. 1182 Crystal violet indicator, Cat. No. 1408 Ammonium tbiocyanate G R, Cat.. No. 1213 1ron(11) sulfate Brilliant green, Cat. No. 1310 G R, Cat. No. 3965 Acetone G R, Cat. No. 14 Iodine green Hydrochloric acid fuming min. 37% (1.19) G R, Cat. No_ 317 18. Acetone G R, Cat. No. 14 Aniline — diphenylamine — phosphoric acid for reducing sugars. Spray solution: Dissolve 4 g diphenylarnine, 4 ml aniline and 20 ml 85% phosphoric 15. Ammonium molybdate — perchloric acid (Hanes reagent) acid in 200 ml acetone. for phosphate esters (sugar phosphates). Treatment: Heat 10 .min at 85 °C. Spray solution: Dissolve 0.5 g ammonium molybdate in 5 ml water, add 1.5 ml 25% Characteristic colours: I,4-aldohexose oligosaccharides turn blue. perchloric acid. After cooling to room temperature hydrochloric acid and 2.5 nil 701/4 Literature: R. W. Bailey, E. J. Bourne, J. Chromatog. 4, 206 (1960). fill up to 50 ml with acetone. Allow the solution to stand for at least one day prior to J. L. Buchan, R. J. Savage, Analyst 77, 401 (1952). use. The solution is stable for about three weeks. S. Schwimmer, A. Bevenne, Science 123, 543 (1956), Treatment: Irradiate the chromatogram for 2 min with an IR lamp from a distance of Chemicals: 30 cm and subsequently with long-wave UV light for 7 min or hear 5-10 min at Aniline G R, Cat. No. 1261 110 °C. Dipbenylamine G R, Cat. No. 3086 Literature; C. S. Hanes, F. A. Isherwood, Nature 164, 1107 (1949). Acetone G R, Cat. No. 14 T. H. Bevan, G. 1. Gregory, T. Malkin, A. G. Poole, J. Chem. Soc.. 1951, 841. ortbo-Phosphoric acid min. 85% (1.71) G R, Cat. No. 573

4 5 19. Aniline - phosphoric acid for sugars. Chemicals: Spray solution: Mix 1 part 2 N aniline solution in 1-butanol saturated with water Anisaldehyde Cat. No. 1450 with 2 parts 2 N phosphoric acid in 1-butanol. Acetic acid glacial min. 96% (1.06) G R, Cat. No. 62 Treatment: Heat the chromatogram 10 min at 105°C. Sulfuric acid 95-97% (1.84) G R, Cat. No. 731. Ethanol abs. G R, Cat. No. 972 Literature: I. L. Bryson, T. 1. Mitchell, Nature 167, 864 (1951). Chemicals: 23. p-Anisidine for reducing sugars. Aniline G R, Cat. No. 1261 Spray solution: ortho-Phosphoric acid min. 8S% (1.71) G R, Cat. No. 573 Dissolve 1 g p-anisidine hydrochloride in 10 ml methanol, fill up the solution ro 100 ml with 1-butanol and shake well after addition of 1 g sodium dirhio- 1-Butanol G R, Cat. No. 1990 nite. Treatment: Heat IO min ar 30°C, Literature: R. C. Bean, G. G. Portwe, Anal. Chem. 31, 1929 (1959). 20. Aniline phthalate. L. Hough, J. K. Spray reagent for chromatography. N. Jones, W. H. Wadman, J. Chem. Soc. 1950, 1702. Visualisation reagent ready for use in aerosol cans. Chemicals: p-A Ilisidine hydrochloride Treatment: Heat the chromatogram 10 min at 105°C. Sodium ditbionite G R, Cat. No. 6507 Cat. No. 1266 Methanol G R, Cat. No. 6009 1-Butanol G R, Cat. No. 1990

21. Aniline phthalate for reducing sugars and anions 24. p-Anisidine phthalate for reducing sugars. of halogen oxy-acids. Spray solution: 0.1 M solution of p-anisidine and phthalic acid in 96% ethanol, Spray solution: Dissolve 0.93 g aniline and 1.66 g phthalic acid in 100 nil 1-butanol Treatment: Heat 10 min at 100°C. saturated with water. Chemicals: Treatment: Heat 10 min at 105°C. p-Anisidine, Cat. No. 458 Literature: S. M. Partridge, Nature 164, 443 (1965). Phtlialic acid G R, Cat. No. 96/1 W. Peschke, J. Chromatog. 26, 572 (1965). Ethanol abs. G R, Cat. No, 972 Chemicals: Aniline G R, Cat. No. 1261 25. Anthrone for ketoses. Phthalic acid G R, Cat. No. 9611 Spray solittion: Dissolve 0.3 g anthrone in 10 ml acetic acid and add to the solution 1-Butanol G R, Cat. No. 1990 20 nil 96%. ethanol, 3 nil 85% phosphoric acid and 1 ml water. The solution is stable for several weeks in the refrigerator. Treatment; Heat .5-6 min at 110°C. 22. Anisaldehyde - sulfuric acid for sugars, steroids, terpenes. Ketoses and oligosaccharides containing ketoses show yellow spots. Spray solution: Prepare freshly before use a solution of 0.5 ml anisaldehyde in SO ml Literature: R. Johanson, Nature 172, 956 (1953). glacial acetic acid and 1 ml 97% sulfuric acid. Chemicals: Treatment: Heat to lO0-105°C until maximal visualisation of the spots. The background may be brightened by water vapour. Lichen constituents, phenols, terpenes, Anthrone for synthesis, Cat. No. 801461 sugars and steroids turn violet, blue, red, grey or green. Acetic acid glacial min. 96% (1.06) G R, Cat. No. 62 Ethanol abs. G R, Cat_ No. 972 Modified spray solution: For visualisation of sugars mix freshly before use 0.5 ml ortho-Phosphoric acid min. 85% (1.71) G R, Cat. No. 573 anisaldehyde, 9 ml ethanol, 0.5 ml 97% sulfuric acid and 0.1 ml acetic acid. Treatment: Heat the sprayed chromatogram 5-10 min at 90-100°C. 26. Antimony(III) chloride for flavonoids. Literature: E. Stahl, U. Kaltenbach, J. Chromatog. 5, 351 (1961). Sprat solution: 10% solution of antimony(I11) chloride in chloroform. B. P. Lisboa, J. Chromatog. 16, 136 (1964). Fluorescing spots in long-wave UV light.

6 7 235 (1964). Literature: L. Horhammer, H. Wagner, K. Hein, J. Chromatog. 12, Chemicals: R. Neu, P. Hagedorn, Naturwissenschaften 40, 411 (1953). Antimony(111) chloride G R, Cat. No. 7838 Chemicals: 1-Butanol G R, Cat. No. 1990 Antimony(l11) chloride G R, Cat. No. 7838 Acetic acid glacial min. 96% (1,06) G R, Cat. No. 62 Chloroform G R, Cat. No. 2445 Sulfuric acid 9.5-97% (1.84) G R, Cat. No. 731

30. Antimony(V) chloride for vitamin A, D and E, terpenes, 27. Antimony(111) chloride for vitamin A and D, carotenoids, steroids, oils, resins, steroid sapogenins. sapogenins, steroid glycosides, terpenes (Carr-Price reagent). Spray solution: Mix freshly before use 1 part antimony(V) chloride with 4 parrs Spray solution: Dissolve 2.5 g antimony(M) chloride in 75 ml chloroform; generally a carbon tetrachloride or chloroform. saturated solution of antimony (111) chloride.in chloroform or carbon tetrachloride is Treatment: Heat the chromatogram until the spots appear. used. Inspect in long-wave UV light. Treatment: Heat 10 min at I00°C. Literature: J. M. MacMahon, R. B. Davis, G. KaInitzky, J. Am_ Chem. Soc. 74,4483 Inspect the chromatogram in long-wave UV light. (1952). Literature: E. Stahl, Chemiker-Ztg. 82, 323 (1958). E. Stahl, Chemiker-Ztg. 82, 323 (1958). K. Takeda, S. Hata, A. Wada, N. Matsumoto, J. Chromatog. 11, 562 (1963). R. Ikan, J. Kashman, E. D. Bergmann, J. Chromatog. 14, 275 (1964). 1-1 Chemicals: G. Henkel, W. Ebing, J. Chromatog. 14, 285 (1964). Antimony(111) chloride G R, Cat. No. 7838 Chemicals: Chloroform G R, Cat. No. 2445 Antimony(V) chloride G R, Cat. No. 7837 Carbon tetrachloride G R, Cat. No. 2222 Carbon tetrachloride GR, Cat. No. 2222 Chloroform G R, Cat. No. 2445

Antimony(111) chloride — acetic acid for steroids and diterpenes. 31. Aurin tricarboxylic acid (Aluminon) for aluminium, 28. chromium, and lithium ions. Spray solution: Dissolve 20 g antimony trichloride in a mixture of 20 ml glacial acetic Spray solution: acid and 60 ml chloroform. 0.1% solution of aurin tricarboxylic acid -ammonium salt in 1% aqueous ammonium acetate solution. Treatment: Heat 5 min at 100°C. Treatment: Diterpenes show red-yellow to blue-violet spots. Place the chromatogram into a chamber saturated with ammonia vapour. Inspect in Long-wave UV light. Literature; G. P. Heisig, F. H. Pollard, Anal. Chico. Acta 16, 234 (1957), Literature: H. P. Kaufmann, A. K. sen Gupta, Chem. Ber. 97, 2652 (1964). Chemicals: Chemicals: Aurin tricarboxylic acid ammonium salt G R, Cat. No. 128 Antimony(111) chloride G R, Cat. No. 7838 Ammonium acetate G R, Cat. No. 1116 Acetic acid glacial min. 96% (1.06) G R, Cat. No. 62 Ammonia solution min. 25% (0.91) G R, Cat, No. 5432 'Chloroform G R, Cat. No. 2445 32. Benzidine for persulfates. Spray solution: Dissolve 0.05 g benzidine in 100 ml 1 N acetic acid. 29. Antimony(111) chloride — sulfuric acid for bile acids. Persulfates show blue spots immediately after spraying. Dissolve 20 g antimony(III) chloride in 50 ml anhydrous 1-butanol, Spray solution: Literature: Y, Servigne, C. Duval, Compt. Rend. 245, 1803 (1957). and mix this solution with 10 ml 97% sulfuric acid and 20 ml glacial acetic acid. The Chemicals: solution should be prepared freshly before use. Benzidine After drying for 15 min in the air heat the chromatogram: conjugated bile Treatment: Acetic acid glacial min. 96% (1.06) GR, Cat. No. 62 acids for 25-30 min, free bile acids for 45-50 min at 110°C. Colours from yellow to green. 33. Benzidine for terpene aldehydes, flavonoids, carbohydrates. L. Anthony, W. T. Behr, J. Chromatog. 13, 567 (1964). Literature: W. Spray solution: Dissolve 0.5 g benzidine in 20 ml glacial acetic acid and 80 ml ethanol. 8 9 Chemicals: Treatment: Heat 15 min at 100°C. Benzidine. Spraying with dilute hydrochloric acid after hearing intensifies the colour of the spots Trichloroacetic acid G R, Cat. No, 807 of some substances, Acetic acid glacial min. 96% (1.06) G R, Cat. No, 62 Literature: J. K. N. Jones, J. B. Pridham, Biochem. J. 58, 288 (1954). Ethanol abs. G R, Cat. No. 972 Chemicals: Benzidine 37. 2,2%Bipyridine — iron(III) chloride for phenols, vitamin E Acetic acid glacial min. 96% (1.06) G R, Cat. No. 62 and other reducing compounds. Ethanol abs. G R, Cat, No. 972 Solution a: 0.5% ethanolic iron(III) chloride solution. Keep in the dark. Hydrochloric acid min. .25% (1.125) G R, Cat. No. 316 Solution b: 0.5% ethanolic solution of 2,2'-bipyridine. Spray solution: Mix equal parts of a and h before use. 34. Benzidine dia.zotised for phenols. Literature: G. M. Barron, J. Chromatog. 20, 189 (1965). Benzidine solution: Dissolve 5 ghenzidine in 14 ml 37% hydrochloric acid and fill up R. Strohecker, H. M. Henning, Vitaminbestininitingen, Verlag Chentie Wcinhcim to 100 ml with water. 1963, p. 311. Nitrite solution: Freshly prepared 10% aqueous sodium nitrite solution. Chemicals: 2,2'-Bipyridine G R, Spray solution: Mix 20 ml of the henzidine solution with 20 ml of the nitrite solution Cat. No. 3098 at 0°C with constant stirring. Iron(111) chloride G R, Cat. No. 39-13 Ethanol abs. G R, Cat. No. 972 Note: The reagent is stable for 2-3 hours. The colours appear very rapidly or after some hours depending on the phenol present. 38. Bismuth chloride for sterols. Literature: J. Sherma, L. V. S. Hood, J. Chrornatog. 17, 307 (1965). Spray solution: Chemicals: 337i, ethaimIic bisinuth(III) chloride solinion, Benzidine Treatment: Heat at 110°C until maximal fluorescence of the spurs in long-wave Sodium nitrite cryst. G R, Cat. No. 6549 UV light. Hydrochloric acid min. 37% (1.19) G R, Cat. No. 317 Literature: J. W. Copius-Peereboom, Thin Layer Chromatography, Ed. G. B. Marini-Bettolo, Elsevier Publ. Comp. Amsterdam, 1964, p. 199. Chemicals: 35. Benzidine — peroxide for chromium and manganese ions. Bismu t h (111) chloride Spray solution I: 5% aqueous sodium peroxide solution. Ethanol abs. G R, Cat. No. 972 Spray solution II: 1% benzidine solution in glacial acetic acid. Procedure: Spray consecutively with I and IL. 39. Boric acid — citric acid for quinolines. M. Ladenbauer, L. K. Bradacs, F. Hecht, Mikrochim. Acta 1954, HS. Literature: I. Spray solution: Dissolve 0.5 g boric acid and 0,5 g citric acid in 20 ml methanol. Chemicals: Treatment: Heat at 100°C. Sodium peroxide G R, Cat. No. 6551 Inspect in UV light. Benzidine Literature: R. Neher, A. Wettstein, Hely. Chins. Acta 35, 276 (1952). Acetic acid glacial min. 96% (1.06) G R, Cat. No. 62 Chemicals: Boric acid cryst. G R, Cat. No. 165 36. Benzidine trichloroacetic acid for sugars. Citric acid G R, Cat. No_ 244 Spray solution: Dissolve 0.5 g benzidine in 10 ml glacial acetic acid, add 10 ml 40% Methanol G R, Cat. No, 6009 aqueous trichloroacetic acid solution and fill up to 100 ml with ethanol. Treatment: Irradiate the chromatogram 15 min with UV light. 40. Bromine — fluorescein — silver nitrate for insecticides. Sugars show greyish-brown to deep reed-brown spots. Spray solution: Fil] up 1 ml of a 0.25% solution of fluorescein in N,N-dimethyl- Literature: J. S. D. Bacon, J. Edelmann, Biochem. J. 48, 114 (1951). forinamide to 50 nil with ethanol. G. Harris, I. C. Macwilliam, Chem. & Ind. (London) 1954, 254. 11 10 44. Spray solution II: Dissolve 1.7 g silver nitrate in 5 ml water, add 10 ml ethylene glycol Bromocresol purple for dicarboxylic acids on polyethyleneglycol monophenyl ether and fill up the solution to 200 ml with acetone. impregnated layers. Spray solution: Procedure: Place the chromatogram 30 sec into a chamber with a 5% solution of Dissolve 0.04 g bromocresol purple in 100 ml 50% ethanol and bromine in carbon tetrachloride. Spray the chromatogram with 1, then with II and adjust the solution to pH 10.0 with sodium hydroxide solution 0.1 mol/I (glass irradiate 7 min with long-wave UV light. electrode). Procedure: Literature: K. C. Walker, M. Beroza, J. Assoc. Off. Agr. Chemists 46, 250 (1963). Develop the chromatogram with the eluent di-iso-propyl ether — formic acid—water (90+7+3) and heat subsequently 10 min at 100 °C. Spray after cooling Chemicals: to room temperature. Bromine G R, Cat. No. 1948 Yellow spots on blue background. Fluorescein Cat. No. 3990 N,N-Dimethylforinamide, Cat. No. 3034 Literature: E. Knappe, D. Peteri, Z. anal. Chem. 188, 184 (1962). Ethylene glycol monophenyl ether G R for synthesis, Cat. No. 807291 Chemicals: Ethanol abs. G R, Cat. No. 972 Bromocresol purple indicator, Cat. No. 3025 Acetone G R, Cat. No. 14 Ethanol abs. G R, Cat. No, 972 Sodium hydroxide solution 0.1 mo1/1 Titrisol, Cat. No. 9959 Bromocresol green. 41. 45. Spray reagent 0.05% for chromatography. Bromocresol purple for halogen ions. Visualisation reagent for use in aerosol cans. Cat. No. 1998 Indicator reagent for use of acetone — 1-butanol — ammonia (25%) — water (65+20+10+5) as eluent. Spray solution; Bromocresol green — indicator reagent. 0.1% ethanolic bromocresol purple solution. Adjust the solution 42. with some drops of 10",4) ammonia solution until the colour change just appears. Dissolve 0.04 g bromocresol green in 100 ml ethanol. Add sodium Spray solution: Literature: H. Seiler, T. Kaffenberger, hydroxide solution 0.1 mol/l until blue colour appears. Hely. Chim. Acta 44, 1282 (1961). Chemicals: T. Overell, Biochim. et biophys. Acta 10, 471 (1953). Literature: F. Bryant, B. Bromocresol purple indicator, Cat. No. 3025 Chemicals: Ethanol abs. G R, Cat. No. 972 Bromocresol green indicator, Cat. No. 8121 Ammonia solution abt. 10% (0.96) G R, Cat. No. 5423 Ethanol abs. G R, Cat. No. 972 s, Cat. No. 9959 Sodium hydroxide solution 0.1 moll! Titrisol 46. Bromocyan — 4-aminobenzoic acid (reagent acc. to Koenig) for tertiary pyridine compounds with at least one free a-position. 43. Bromocresol green — bromophenol blue — potassium Primary treatment: Before spraying place the chromatogram for 1 hour into a cham- permanganate for organic acids. ber with a solution of bromocyan (Caution, very poisonous!). For preparation of the Solution a: Dissolve 0.075 g bromocresol green and 0.025 g bromophenol blue in bromocyan solution add 10% aqueous solution of sodium cyanide to saturated bro- 100 ml ethanol. mine water, cooled in ice, until the colour of bromine has disappeared. Spray solution: Solution b: Dissolve 0.25 g potassium perrnanganate and 0.5 g sodium carbonate in Dissolve 2 g 4-aminobenzoic acid in 75 ml 0.75 N hydrochloric acid 100 ml water. and fill up the solution to 100 ml with ethanol. Literature; E. Spray solution: Mix 9 parts a and 1 part b prior to use and spray immediately. The Kodicek, K. K. Reddi, Nature 168, 475 (1951). mixture is stable for 5-10 minutes only. Chemicals: Literature: J. Paskova, V. J. Munk, J. Chromatog. 4, 241 (1960). Bromine G R, Cat. No. 1948. Sodium cyanide powder, Cat. No. Chemicals: 6439 Bromocresol green indicator, Cat. No. 8121 4-Aminobenzoic acid pore, Cat. No. 102 Bromophenol blue indicator, Cat. No. 8122 Hydrochloric acid 1 mo1/1 Titrisol®, Cat. No. 9970 Potassium permanganate G R, Cat. No_ 5082 Ethanol abs. G R, Cat. No. 972 Sodium carbonate 10-hydrate G R, Cat. No. 6391 Variation: Mixture of 2% ethanolic 4-aminohenzoic acid solution and phosphate Ethanol abs. G R, Cat. No. 972 buffer 0.1 mok/I pH 7.0 (1+1).

12 13 Chemicals: Procedure: After spraying dry the thromatogram 15 min at room temperature and place subsequently into a chamber with some crystals of brornocyan. N-Bromosteccinimide for synthesis, Cat. No. 801949 Fluorescein, Cat. No. 3990 Literature: E. Hodgson, E. Smith, F. E. Guthrie, J. Chromatog. 20, 176 (1965). Sodium hydroxide solution 0.1 moll! Titrisoric, Cat. No. 9959 Chemicals: Ethanol airs. G R, Cat, No. 972 Bromocyate 1,1,1-Trichlornetbane 4-Aminobenzoic acid pure, Cat. No. 102 Ethanol abs. G R, Cat. No. 972 50. Bromothymol blue for lipoids. Buffer Titrisol pH 7.0 (phosphate), Cat. No. 9.887 Spray solution: Dissolve 0.04 g bromothymol blue in 100 nil sodium hydroxide solution 0_01 Bromophenol blue — methyl red — Pauly reagent for phenols. 47. Literature: H. Jatzkewitz, E. Mehl, Hoppe-Sevlers Z. physiol. Chem. 320, 251 Spray solution l: Mix 100 nil 0.12% aqueous Bromophenol blue solution, 100 ml {1960). 0.06% ethanolic methyl red solution and 100 nil phosphate buffer acc. to Sorensen Cbetnicals: (pH 7.2). Bromothymol blue indicator, Cat. No. .3026 11: See sulfanilic acid diazorised (p. 90), Spray solution Sodium hydroxide solution 0.01 moll! Titrisol*, Cat. No. 9961 Procedure: Spray the chromatogram consecutively with 1 and IL Literature: J. W. Copus-Peereboom, H. W. Beekes, J. Chromatog. 14, 417 (1964). 51. Cacotheline for vitamin C. Chemicals: Spray solution: 2% aqueous cacotheline solution. Bromophenol blue indicator, Cat. No. 8122 Treatiprent: Hear at 110°C. Methyl red indicator, Cat. No. 6076 Violet spots. Potassium &hydrogen phosphate solution llis Cat. No. 4875 Literature: 6587 B. Tegethoff, Z. Narurforsch. 8b, 374 (195.37. di-Sodium hydrogen phosphate solution 'Its n7o111, Cat, No. Chemicals: Ethanol abs. G R, Cat. No, 972 Cacotheline G R, Cat. No. -1795

48. Bronaosuccinimide — fluorescein for lipids. 52. Carbazole — sulfuric acid for sugars. Dissolve 0_01 g N-bromosuccinimide in 100 ml glacial acetic acid Spray solution I: Spra)' solutions: Dissolve 0.5 g carbazole in 95 nil ethanol and add 5 ml 97% sulfuric Spray solution II: Dissolve 0.01 g fluorescein in 100 ml ethanol. acid. Prepare freshly before use. Procedure: Spray consecutively' with I and 11. Treatment: Heat 10 min at 120°C. Inspect in day light and in long-wave UV light. Violet spots on blue background. Literature: A. Popov, V. Gadeva, J. Chromatog. 16, 256 (1964). Chemicals: J. Micev, A. Popov, L. Nedelceva, J. Chromatog. 24, 432 (1966). Carbazole Chemicals: Ethanol abs. G R, Cat, No. 972 'N-Bromosuccinimide, Cat. No. 1949 Sulfuric acid 95-97% (1.84) G R, Cat. No. 73I Fluorescein, Cat, No. 3990 Acetic acid glacial min. 96% (1.06) G R, Cat. No. 62 Ethanol abs. G R, Cat. No. 972 53. Carmine for polysaccharides. PC. Stock solution: Heat 1 g carmine, 0.5 g anhydrous aluminium chloride and 2 nil 49. Bromosuccinirnide — fluorescein for sulfur containing compounds. water 2-3 min, add the solution to 100 m150". ethanol and filter after 24 hours. The Spray solution I: 0.035 "/;, solution of N-bromosuccinimide in 1,1 ,1-trichloroethane. filtrate must- he stored at 5 °C. Spray solution II: Fill up 3 nil 0.33% solution of fluorescein in sodium hydroxide Spray solution: Dilute 5 ml of stock solution with 17 ml ethanol and .3 nil water. solution 0.1 moll to 100 ml with ethanol. Procedure: Before drying it is advantageous to fix the polysaccharides. Dip the Procedure: Spray with I, dry at room temperature and spray with 11. chromatogram 15 min into a mixture of 20 ml formaldehyde and'80 ml ethanol and Literature: J. W. Cook, J. Assoc. Off. Agr. Chemists 37, 983 (1954). dry at room temperature.

14 15

J. F. Heremans, J. P Vaerman, Clin. Chim. Acta 3, 430 (1958). Chemicals: Literature: Cerium(117) sulfate GR, Cat. No. 2274 D. Hamerman, Science 122, 924 (1955). Nitric acid min. 65% (1.4) G R, Cat. No. 454 Chemicals: Carmine, Cat, No. 2233 56. Cat. No. 801082 Cerium(IV) sulfate — sulfuric acid for alkaloids and iodo-organic Aluminium chloride anhydrous, compounds (modified reagent acc. to Sormen.schein). Ethanol abs. G R, Cat. No. 972 Spray solution: ForMaldehyde solution 35% G R, Cat. No, 4003 Slurry 0.1 g cerium(1V) sulfate in 4 ml water. After addition of 1 g trichloroacetic acid boil and add dropwise 97% sulfuric acid until the solution becomes clear. 54. Cerium(IV) sulfate — arsenite for organic and inorganic Treatment: Heat some minutes at 110°C until the spots appear. iodine containing compounds. PC. Note: The reagent dyes the alkaloids apornorphine, brucine, colchicine, papaverine Solution a: Add 10 g cerium(IV) sulfate to 100 ml 1 N sulfuric acid, which has been and physostigrnine. Organic iodine compounds also can he detected. cooled to 0-5 °C. The mixture is cooled for another hour and then filtered or centri- Literature: O.-E. Schultz, D. Strauss, Arzneimittel-Forseh. S, 342 (1955). fuged. Store the clear solution until use in the refrigerator. Chemicals: Solution b: Dissolve .5 g sodium arsenite in 30 ml sodium hydroxide solution 1 mol/l. Cerirr►n(V) sulfate G R, Cat. No. 2274 Add the solution dropwise with stirring to 65 ml 2 N sulfuric acid cooled to 0-5 °C Tricbloroacetic acid G R, Cat. No. 807 and fill up to 100 ml with water. Sulfuric acid 95-97% (1.84) G R, Cat. No. 731 Spray solution: Mix equal pars of a and b prior to use. 57. Cerium(IV) sulfate — sulfuric acid for solanum steroid alkaloids Procedure: Spray the chromatogram with the spray solution by placing it on a glass plate. This permits uniform spraying. Place a second glass plate of equal size over the and steroid sapogenins. moistened chromatogram and press. Within 30 minutes white spots on yellow back- Spray solution: Saturated solution of cerium(IV) sulfate in 65% sulfuric acid. ground will appear at the sites of iodine compounds. Potassium iodide turns Treatment: Heat 15 min at 120°C. chocolate-brown. Note: Not applicable with aluminium oxide layers. Treatment: For greater contrast the chromatogram may be sprayed before drying Literature: K. Schreiber, O, Aurich, G. Osske, J. Chromatog. 12, 63 (1963). with 1% solution of o-phenylenediamine in acetone. Thus the entire chromatogram Chemicals: turns brown and the white spots are more pronounced. Dry the chromatogram in Cerizon(IV) sulfate G R, Cat. No. 2274 iodine-free air. Sulfuric acid 95-97`3;, (1,84) G R, Cat. No. 731 Literature: C. H. Bowden, N. F. MacLagan, J. H. Wilkinson, Biochem. J. 53, 93 (1955). 58. Chloramine T for caffeine. Chemicals: Spray solution I: 10% aqueous chloramine T solution. Cerium(1V) sulfate G R, Cat. No. 22 74 Spray solution 11: 1 N hydrochloric acid. Sodium metaarsenite G R, Cat. No, 6287 Procedure: Spray with 1 and after short drying with II. Heat at 96-98 °C until the o-Phenylenediamine, Cat. No. 809721 smell of chlorine has disappeared. Place the chromatogram into a chamber sarurated Sulfuric acid 0.5 na!ll Titrisol'1,. Cat. No. 9984 ► with ammonia vapour and heat subsequently for a short time until the maximal visua- Sulfuric acid 95-97% (1.84) G R, Cat. No. 731 lisation of the spots. Sodium hydroxide solution 1 niola Titrisol®, Cat. No. 9956 Literature: Acetone G R, Cat. No. 14 H. Gnshirt, A. Malzacher, Arch. Pharm. 293, 925 (1960). Chemicals: Hydrochloric acid 1 moll! Titrisoll, Cat. No. 9970 55. Cerium(IV) sulfate — nitric acid for polyphenyls. Chloranrine T G R, Cat. No. 2426 Spray solution: Dissolve 0.3 g cerium(TV) sulfate in 100 ml 65% nitric acid. Ammonia solution min. 25% (0.91) GR, Cat. No. 5432 Treatment: Heat 15-20 min at 120 °C. 59. Inspect in long-wave UV light. Chloramine T — trichloroacetic acid for digitalis glycosides. Spray solution: Mix Literature: F. Geiss, H. Schlitt, Euratom-Bericht EUR-l-19 d (Nov. 1961). 10 ml of a freshly prepared 3% aqueous chloramine solution

16 17 with 40 ml 25% solution of trichloroaccric acid in ethanol. Trichloroacetic acid 62. Chlorine — tolidine (modif. ace. to Greig and Leaback). solution is stable for several days_ Spray solution I: 2% aqueous solution of potassium hypochlorite. Procedure: Heat 7 min at 110°C_ Bluish and yellow fluorescence in long-wave UV light. Spray solution II: Mix before use equal volumes of saturated solution of o-tolidine in 2% acetic acid and 0.85% aqueous potassium iodide solution. Literature: D. Waldi, Arch. Phartn. 292, 206 (1959). Procedure: Spray lightly with I, dry at room temperature for 1-2 hours, and spray Chemicals: with U. Chloramitte T G R, Cat. No. 2426 Literature: Trichloroacetic acid G R, Cat. No. 8(17 C. C. Greig, D. H. Leaback, Nature 188, 310 (1960). Ethanol abs. G R, Cat. No. 972 Chemicals.. Acetic acid glacial Min. 96% (1.06) G R, Cat. No. 62 60. Chlorine — pyrazolone — cyanide for indoles, amides, Potassium iodide G R, Cat. No. 50-13 sulfonamides. ?Grasse-toll hyp och lord c Chlorination: Place the chromatogram for abour 2-3 min into a chlorine atmosphere R, Cat. No. S311 (prepared from potassium and 2.5% hydrochloric acid). To remove excess chlorine heat the plate at 100'C. 63 Chlorocyan — 4-aminobenzoic acid for tertiary pyridine Spray solution: Equal volumes of 0.2 :N.1 solution of 3-methyl- l -phenyl-3-pyrazo- compounds with at least one free at-position. lon-5-one in pyridine and potassium cyanide 1 nwlll solution. Spray solution: 5"/,, merhanolic solution of 4-aminobenzoic acid, Procedure: After removal of the excess chlorine spray the chromatogram until be- Procedure: Place the sprayed chromatogram into a chainber with a ginning transparency. Caution, poisonous! freshly prepared mixture of 20 inl 28% aqueous slurry of chloramine, 20 nil 1 N hydrochloric acid and The respective compounds show bright red spots which turn blue after 2 min. 10 ml 10% aqueous potassium cyanide solution. Literature: Private communication G. Bohnstedt, Inst. f. Organ. Cheinie, Universitat Caution, poisonous! des Saarlandes. The spots will appear after a short time. Chemicals: Literature: E. Nurnberg, Dtsch. Apotheker-Ztg. 101, 142 (1961). 5082 Potassium permanganate G R, Cat. Nu. Chemicals: 316 Hydrochloric acid min. 2Š% (1.125; G R, Cat. No. 4-Aminobenzoic acid G R, Cat. No, 102 Pyridine G R, Cat. No. 9728 Chloramine T G R, Cat. No. 2426 G R, Cat. No. 4967 Potassium cyanide Potassium cyanide G R, Cat. No. 4967 61_ Chlorine — tolidine for compounds convertible into chloramines. Hydrochloric acid 1 mob! TitrisoP., Cat. No. 9970 Methanol G R, Cat, No. 6009 Chlorination: Place the chromatogram inro chlorine atmosphere; 5-10 min with chlorine from a bomb, 15-20 min with chlorine prepared from a 1.5% solution of potassium permanganate and l0% hydrochloric acid (1+ I), 64. 1-Chloro-2,4-dinitrobenzene -- indicator reagent. air. For removing excess chlorine allow the plate to stand for 5 min in the Spray solution: 0.5% ethanolic solution of 1-chioro-2,4-dinitrobenzene. Spray solution: Dissolve 0.16 g o-rolidine in 30 nil glacial acetic acid, fill up the Chetnica/s: solution to 500 nil with water and add l g potassium iodide, 1-Chloro-2,4-dinitrohenzene G R, Cat. No. 2427 Note: Spray a corner of the chromatogram ro establish that chlorine has been Ethanol abs. G R, Cat. No. 972 removed completely. If no blue colour appears spray the whole plate. 65. Literature: F. Reindl, W. Hoppe, Chem. Fier. 87, 1103 (1954). 1-Chloro-2,4-dinitrobenzene for nicotinic acid, nicotin.arnide, pyridoxol. Chemicals: Potassium permanganate G R, Cat. No. 5082 Spray solution 1: 1%, merhanolic solution of 1-chloro-2,4-dinitrobenzene. Hydrochloric acid min. 35';, (1.12.5) G R, Cat. No. 376 Spray solution Sodium hydroxide solution 3 mail. Acetic acid glacial milt. 96% (1.06) G R, Cat. No. 62 Procdure: Spray subsequently with I and o-Tolidine G R, Cat. No. 8311 Literature: L. Maiwald, H. Maske, Hoppe-Seylers Z. physiol. Chem_ 306, 143 Potassium iodide G R, Cat. No. 50-13 (1956).

18 19 Treatment: Heat 30 min at 105 °C. Chemicals: Literature: M. Beroza, Agricult. and Food Chemistry 11, 51 (1963), 1-Chloro-2,4-dinitrobenzene G R, Cat. No. 2427 Chemicals: Sodium hydroxide .solution min. 27% (1.3) G R, Cat. No. 5591 Chrornotropic acid sodium salt G R, Cat. No. 2498 Methanol G R, Cat, No. 6009 Sulfuric acid 95-97% (1.84) G R, Cat. No. 731 66. Chlorophenol red — indicator reagent. 70. Cinnamaldehyde — acetic anhydride — sulfuric acid for steroid Spray solution: 0.04% ethanolic solution of chlorophenol red. Adjust the solution sapogenins. with sodium hydroxide solution 0.1 rnol/l to pH 7.0. Spray solution 1: 1% ethanolic cinnamaldehyde solution. Literature: A. R. Jones, F.. J. Dowling, W. J. Skroba, Anal. Chem. 25, 394 (1953). Spray solution 11: Prepare freshly before use a mixture of 12 parts acetic anhydride Chemicals: and 1 part 97% sulfuric acid, Chloropheirol red indicator, Cat. No. 3024 Procedure: Sodium hydroxide solution 0.1 moll! Titriso14, Cat. No. 9959 Spray with I, dry 5 min at 90°C and spray with II. After 1-2 min at room temperature, the chromatogram is heated at 90°C until the spots appear. 67. Chlorosulfonic acid — glacial acetic acid for triterpenes, Chemicals: sterols, steroids. Cinnamaldehyde, Cat- No. 2505 Sulfuric acid 95-97% (1,84) G R, Cat, No. 731 Spray solution: Dissolve 5 nil chlorosulfonic acid in 10 nil glacial acetic acid with Acetic anhydride G R, Cat. No. 42 cooling. Ethanol abs. G R, Cat. No. 972 Treatment: Hear 5-10 min at 130°C. Inspect in long-wave UV light. 71. Cinnamaldehyde — hydrochloric acid for indole derivatives. Literature: R. Tscheche, C. Wulf, Chem. Ber. 94, 2019 (1961). Spray solution: Dissolve S ml cinnamaldehyde in 100 ml ethanol and add 5 ml 37% R. Tschesche, J. Chromatog. 5, 217 (1961). hydrochloric acid freshly before use. K. Takeda, S. Hara, A. Wada, N. Matsumoto, J. Chromatog. 11, 562 (1963). Treatment: Place the plate into hydrogen chloride atmosphere. Red spots. Chemicals: Literature: Chlorosulfonic acid, Cat. No. 220 D. Jerschel, R. Muller, Naturwissenschaften 38, 561 (1951), Acetic acid glacial min. 96% (1.06) G R, Car. No. 62 Chemicals: Cinnamaldehyde, Cat. No. 2505 68. Chromosulfuric acid as universal detectant for organic Ethanol abs. G R, Cat. No. 972 compounds. Hydrochloric acid fuming min. 37% (1.19) C R, Cat. No. 317 Dissolve 5 g potassium dichromate in 100 ml 40% sulfuric acid. Spray solution: 72. The reagent is suitable for charring organic compounds, in particular, lipids, by Cobalt(u) chloride for organic phosphate esters. Note: Spray solution: heating the chromatogram at 150'C. 1% anhydrous cobalt(11) chloride solution in acetone. Treatment: Hear at 40-50°C. Literature: J. Bertetti, Ann. Chim, (Rome) 44, 495 (1954). Blue spots. The reaction is not sensitive. Chemicals: Literature: Potassium dichromate G R, Cat. No. 4864 R. Donner, K. Lohs, J. Chromatog. 17, 349 (1965). Chemicals: Sulfuric acid 95-97% (1.84) G R, Cat. No. 7.31 Cobalt(II) chloride G R, Cat. No. 2539 69. Chroraotropic acid for methylenedioxyphenyl-type compounds Acetone G R, Cat. No. 14 (e.g. narcotine, hydrastine, sesamine and other compounds splitting off formaldehyde). 73. Cobalt(II) — lead nitrite for ammonium and 100% aqueous solution of chromotropic acid sodium salt. potassium ions. PC. Solution a: Spray solution l: Add 5 parrs 97% sulfuric acid to 3 parrs water and cool to room tempera- Dissolve 5 g cobalt(II) nitrate and 5 g lead nitrate in 100 ml water Solution b: and add 1-2 drops nitric acid. ture. Spray solution IL Saturated sodium nitrite solution in Spray solution: Prepare freshly before use a mixture of 1 part a and 5 parts h. acetic acid 2 mol/1

20 21 Spray with 1, and after drying with 11. Then rinse with water and dry 77. Copper acetate — potassium hexacyanoferrate(II) for the Procedure: identification of higher fatty acids acc. to Kaufmann. PC. again. Drp solution 1: Literature: E. Beerstecher, Anal. Chem. 22, 1200 (195.0). Mix 10 ml saturated aqueous copper acetate solution with 240 ml R. U. Magee, J. B. Headridge, Analyst 82, 95 (1957). water. Drp solution 11: Freshly prepared 1.5% aqueous potassium hexacyanoferrate(II) Chemicals: solution. Lead(11) nitrate G R, Cat. No. 7398 Cobalt(11) nitrate G R, Cat. No. 2536 Procedure: After separation of the fatty acids on petroleum — or undecane — im- Sodium nitrite G R, Cat. No, 6549 pregnared paper heat the chromatogram 2 hours at 120°C to remove the impreg- Acetic acid glacial min. 96r% (1.06) G R, Cat. No. 62 nation. Then place the chromatogram 45 min into dip solution I. Subsequently Nitric acid min. 65% (1.4) G R, Cat. No. 454 remove the excess copper acetate with running water by rinsing for 15 min. Then place the chromatogram into dip solution II where the acids show red-brown spots. Cobalt(II) nitrate — ammonia for barbiturates (Zwikker reagent). Literature: H. P. Kaufmann, W. H. Nietsch, Fate u. Seifen, Anstrichrnittel 56, 1.54 74. (1954). 1% ethanolic cobalt(11) nitrate solution. Spray solution: Chemicals: Treatment: Dry and place into a chamber sarurated with ammonia. Copper(11) acetate G R, Cat. No. 2711 Literature: E. J. Shellard, J. V. Osisiogu, Lab. Practice 13, 516 (1964). Potassium hexacyanoferrate(11) G R, Cat. No. 4984 Chemicals: Cobalt(I1) nitrate G R, Cat. No. 2536 78. Copper acetate — rubeanic acid for the identification Ethanol abs. G R, Cat. No. 972 of higher fatty acids ace. to Kaufmann. PC. Ammonia solution 2S% (0.91) G R, Cat. No. 5432 Dip solution 1: Dilute 10 ml saturated copper(11) acetate solution to 1 l with water. Dip solution 11: 0.1% ethanolic rubeanic acid solution with' 0.5% ammonia. 75. Cobalt(II) nitrate — lithium hydroxide for barbiturates. Procedure: Place the chromatogram 45 min into dip solution I and remove excess Spray solution 1: 2% cobalt(11) nitrate solution in absolute methanol. copper salt by rinsing with water for 1.5 hours. Dip the moist chromatogram 30 min into II, then rinse again 30 min with running water and dry. Spray solution 11: 0.5% methanolic lithium hydroxide solution. Literature: P. E. Ballance, W. M. Crombie, Biochem. J. 69, 632 (1958). Spray with 1 and after drying at room temperature with 11, Procedure: Chemicals: Weidmann, Dissertation, Berlin 1961. Literature: H. Copper(11) acetate G R, Cat. No. 2711 Chemicals: Rubeanic acid (dithiooxamide) G R, Cat. No. 629 Cobalt(II) nitrate G R, Cat. No. 2536 Ethanol abs. G R, Cat. No. 972 5691 Lithium hydroxide, Cat. No. Ammonia solution min. 25% (0.91) G R, Cat. No. 5432 Methanol G R, Cat. No. 6009 Methanol dried G R, Cat. No. 6012 79. Copper chloride for oximes. Spray solution: 0.5% aqueous copper(II) chloride solution. 76. Cobalt(II) thiocyanate for alkaloids and amines. Note: (3-Oxime complex compounds show green spots immediately after spraying, Spray solution: Dissolve 3 g ammonium thiocyanate and 1 g cobalt(11) chloride in u-oxime complex compounds show weak green spots after hearing 10 min at 110'C. 20 ml water. Literature: M. HranisavlieviC-JacovljeviC, I. PexjkoviC-TadiC., A. StojiljkoviC, Note: Alkaloids and amines show blue spots on white to pink background. J. Chromatog. 12, 70 (1963). The colours grow pale after 2 hours and can be restored by spraying with water or Chemicals: placing the chromatogram into water vapour. Copper(11) chloride G R, Cat. No. 2733 Literature: E. S. Lane, J. Chrounatos. 18, 426 (1965). 90, Copper sulfate — benzidine for pyridine Chemicals: monocarboxylic acids. Cobalt(11) chloride G R, Cat. No. 2539 Ammonium thiocyanate G R, Cat. No. 1213 Spray sohit/on 1: Dissolve 0.3 g copper(II) sulfate in 100 ml 45% ethanol,

22 23 0.1°/0 solution of benzidine in 50% ethanol. Spray solution 117 Literature: D. C. Malins, H. K. Mangold, J. Am. Oil Chemists Soc. 37, Spray with I, dry the chromatogram at 60 °C and spray with 11. Blue spots. 576 (1960). Procedure: H. K. Mangold, J. L. Gellermann, H. Schlenk, Federation Proc. 17, 269 (1958). Chemicals: H. K. Mangold, B. G. Lamp, H. Schlenk, J. Am. Chem. Soc. 77, 6070 (19.55). Copper(11) sulfate G R, Cat. No. 2790 Chemicals: Benzidine G R, Cat. No. 1762 Iodine resublimed, G R, Cat. No. 4761 Ethanol abs. G R, Cat. No. 972 Ethanol abs. G R, Cat. No. 972 a-Cyclodextrin 81. Copper sulfate — quinine — pyridine for barbiturates and thiobarbiturates. 84. Cysteine — sulfuric acid for desoxyribonucleosides Spray solution I: Dissolve 0.2 g eopper(ll) sulfate and 0.02 g quinine hydrochloride in 50 ml water, add 2 ml pyridine and fill up to 100 ml with water. (modif. reagent acc. to Dische). Spray solution: 0.5% aqueous potassium permanganate solution. Mix freshly before use 1 part of a 0.5% cysteine hydrochloride solu- Spray solution 11: tion in 3 N sulfuric acid with 9 parts acetone. a: Spray with I and dry at room temperature. White, yellow or violet spots Procedure Procedure: in daylight, dark spots on fluorescent background in long-wave UV light. Spray the chromatogram with the solution or dip into it, then heat 5-10 min at 85°C. Spray subsequently with 11. Procedure b: Desoxyrihonucleosides and their phosphates turn green or grey, purincs are dyed Yellow or white spots- more rapidly than pyrirnidines. M. Frahm, A. Gottesleben, K. Soehring, Pharm. Acta Hely. 38, 785 Literature: Literature: G. Buchanan, Nature 168, 1091 (1951). (1963). Chemicals: Chemicals: L(+)Cysteinium hydrochloride, Cat. No. 2839 2790 Copper(II) sulfate G R, Cat. No_ Sulfuric acid 95-97% (1.84) G R, Cat. No. 731 Potassium permanganate G R, Cat. No. 5082 Acetone G R, Cat. No. 14 Quinine hydrochloride Pyridine G R, Cat. No. 9728 85. 3,5-Diaminobenzoic acid — phosphoric acid for 2-deoxy-sugars. 82. Copper(II) sulfate — sodium citrate for flavonoids and Spray solution: Dissolve 1 g 3,S-diaminobenzoic acid dihydrochloride in 25 ml 80% cournarins with o-dihydroxy groups (Benedict's reagent). phosphoric acid and dilute with 60 ml water, Treatment: Spray solution: Dissolve 1.3 g copper(II) sulfate, 17.3 g sodium citrate and 10 g an- Heat 15 min at 100'C. hydrous sodium carbonate in water and fill up to 100 ml. The spots fluoresce green-yellow in long-wave UV. light. Amounts more than 2 lig are visible as brown spots in daylight. Note: The fluorescence in long-wave UV light of cournarins with o-dihydroxy groups is quenched by Benedict's reagent. Compounds without o-dihydroxy groups keep or Literature: M. Pesez, Bull. soc. chim. biol. 32, 701 (1950). show stronger fluorescence, often connected with change of colour. Chemicals: Literature: H. Reznik, K. Egger, Z. anal. Chem. 183, 196 (1961). ortbo-Phosphoric acid min. 85% (1,71) G R, Cat. No. 573 3,5-Diaminobenzoic acid dihydrochloride Chemicals: Copper(11) sulfate G R, Cat. No. 2790 Sodium carbonate anhydrous G R, Cat. No. 6392 86. o-Dianisidine for aldehydes and ketones. 6448 tri-Sodium citrate G R, Cat. No. Spray solution: Saturated solution of o-dianisidine in glacial acetic acid. Note: In some cases 2,7-d iaminofl uorene may be used instead of o-dianisidine. Good 83. a-Cyclodextrin for straight-chain lipids. differentiation of colours_ 30% ethanolic solution of ct-cyclodextrin. Spray solution: Literature: R. Wasicky, O. Frehden, Mikrochim. Acta 1, K. Freudenberg et al., Liebigs Ann. Chem. 558, 1 (1947)- 55 (1937). Preparation: Chemicals: D. French et al., J. Am. Chem. Soc. 71, 353 (1949). Dry the chromatogram at room temperature and place it into a chamber o-Dianisidine (3,3'-dimetboxybenzidine) G R, Cat, No. 2953 Treatment: Acetic acid glacial min. 96% (1.06) G R, Cat. No. 62 containing iodine vapour. 2,7-Diaminofluorene

24 25 Note: 87. Diazotisation and coupling with 1-naphthol for aromatic After drying wirh warm air it is sometimes advisable to place the chromatogram in a current of steam, or to spray it with water. primary amines and sulfonamides. Inspect in long-wave UV light. I: Freshly prepared 1% sodium nitrite solution in hydrochloric acid Spray solution Literature: D. C. Malins,1-1. K. Mangold, J. Am. Oil Chemists Soc. 37, 576 (1960). 1 mol/l. P. J. Dunphy, K. J. Whittle, J. F. Pennock, Chem. & Ind. (London) 1965, 1217. IL Freshly prepared 0.2% 1-naphthol solution in potassium hydrox- Spray solution Chemicals: ide 1 mol/l. 2',7'-nrchlorofiztorescein, Cat. No, 9676 111 Spray with 1 and after I min with 11. Dry the chromatogram at 60°C. ' Procedure: III I Ethanol abs. G R, Cat. No. 972 Instead of 1-naphthol a 0.4% methanolic solution of N-(1-naphrhyl)ethylene- Note: diammonium dichloride may be used as coupling agent. 128, 537 (1939). 91. 2',7'-Dichlorofluorescein — aluminium chloride — iron(III) chloride Literature: A. C. Brarton, E. K. Marshall, J. Biol. Chem. A. Wanktniiller, Naturwissenschafren 39, 302 (1952). for free fatty acids (specific detection). Spray solution G. Wagner, Arch. Pharm. 285, 409 (1952). /: 0.05% ethanolic solution of 2',7"-dichlorofluorescein. T. Bit'an-Fister, V. K.ajganovie, J. Chromatog. 11, 492 (1963). Spray solution IL ethanolic solution of aluminium chloride. Chemicals: Spray solution 111: 1% aqueous solution of iron(III) chloride. 1-Naphthol G R, Cat. No. 6223 Procedure: Spray with 1, dry some minutes at 100°C, spray with 11, dry again some Sodium nitrite G R, Cat. No. 6549 minutes at 100°C and spray with 111. No. 6237 N-(1-Naphthyl)ethyleliediainntonium dichloride G R, Cat. Pink-violet spots on fallow background. TitrisoM Cat. No_ 9970 Hydrochloric acid 1 Literature: A. E. Dudzinsky, J. Ch romatog. 31, 560 (1967). Potassium hydroxide solution 1 molll Titrisora', Cat. No. 9915 Cheinicals: Methanol G R, Cat. No. 6009 2',7"-Dichlorofitrorescein, Cat. No. 9676 Aluminium chloride anhydrous, Cat. No. 1052 88. 2,6-Dibromoquinone chlorimide for phenols (Gibbs reagent). lron(111) chloride G R, Cat. No. 3943 Freshly prepared 0.4% methanolic solution of 2,6-dibromoquinone •Ethanol abs. G R, Cat. No. 972 Spray solution: I ,I II chlorimide. Spray the chromatogram with 10% aqueous sodium carbonate solution Treatment: 92. 2,6-Dichlorophenolindophenol — silver nitrate for alkali chlorides. I '11 1 or place it in a chamber saturated with ammonia. Spray solution: 0.2% ethanolic solution of 2,6-dichlorophenolindophenol sodium Literature: E. NUrnberg, Dtsch. Apotheker-Ztg. 101, 268 (1961). I 11 salt. Filter after addition of 3 g silver nitrate and shaking_ Prepare freshly before use! I Chemicals: Literature: 1,1 HI Cat. No. 3035 T. Barnahas, M. G. Badve, J. Barnabas, Naturwissenschaften 2,6-Dihroinoquinove chlorimide G R, (1954). 41, 478 I II R, Cat. No. 6391 Sodium carbonate 10-hydrate G Chemicals: Ammonia solution min, 25% (0.91) G R, Cat. No. 5432 II 2,6-Dichlorophenolindophenol sodium G R, Cat. No. 3025 I I Methanol G R, Cat. No. 6009 1 1 Silver nitrate G R, Cat. No. 1512 Ethanol abs. G R, Cat. No. 972 89. 2',V-Dichlorofluorescein. Spray reagent 0.1% for chromatography. 93. 2,6- Visualisation reagent ready for use in aerosol cans. Cat. No. 9677 Dichlorophenolondophenol sodium salt for organic acids and keto acids. Spray solution: 2',7'-Dichlorofluorescein fluorescence indicator for saturated 0.1 % ethanolic solution of 2,6-dichlorophenolindophenol sodium 90. salt. and unsaturated lipids. Treatment: 0.2 ethanolic solution of 2',7'-dichlorofluorescein. After brief warming the acids appear as red spots on light blue back- A. Spray solution: ground. 0,01% ethanolic solution of 2',7'-dichlorofluores- B. Spray solution (for vitamin E): Literature: C. Passera, A, Pedrorti, G. Ferrari, J. Chromarog. cein. 14, 289 (1964).

26 27

94. Diethylamine copper(II) sulfate for thiobarbiturates. II Chemicals: Spray solution: Dissolve 0.5 g copper(11) sulfate in 100 ml methanol. 2,6-Dichlorophenolindopherrol sodium G R, Cat. No. 3028 Add 3 ml 11 diethylamine to the solution. Ethanol abs. G R, Cat. No. 972 Note: Shake prior to use; stable for only a few days. 2,6-Dichlorophenolindophenol sodium salt for vitamin C Thiobarbituric acids show green spots. 94. Literature: (Tillman reagent). W. Dietz, K. Soehring, Arch. Pharm. 290, 80 (1957), 0.05% solution of 2,6-dichlorophenolindoPhenol sodium salt in Chemicals: Spray solution: Copper(11) sulfate G R, Cat. No. 2790 50% ethanol. Methanol G R, Cat. No. 6009 Note: Colourless spots on blue background.. Biochern. J. 55, 821 (1953). Diethylarnine for synthesis, Cat. No. 803010 Literature: Y.-T. Chen, F. A. Isherwood, L. W. Mapson, Chemicals: 95. Diethyl malonate for 3,5-dinitrobenzoic acid esters. 2,6-Dichloropbenolindopbenol sodium G R, Cat. No. 3028 Spray solution 1: 10% ethanolic solution of diethyl malonate. Ethanol abs. G R. Cat. No. 972 Spray solution II: 10% aqueous sodium hydroxide. 2,6-Dichloroquinone chlorimide for antioxidants, adrenaline Procedure: Spray with 1 and then with 11. 95. and derivatives, cyanamide and derivatives. Heat 5 min at 95°C. Red-violet spots. Prepare freshly before use a 0.1-1% solution of 2,6-dichloroquinone Spray sol ution: Literature: J. Cerny, Chem. listy 49, 1899 (19.55). chlorimide in 100 nil absolute ethanol. The spots appear after about 15 minutes. Not to be.used for urea. Some antioxidants Chemicals: show characteristic change of colours after being sprayed with a 2% solution of so- Diethyl malonate, Cat. No. 898 dium tetraborate in 40% ethanol.. Sodium hydroxide solution 10% (7.109) G R, Cat. NO. 5588 Ethanol abs. G R, Cat. No. 972 Literature: A. Seher, Fette u. Seifen, Anstrichmittel 61, 345 (1959). v. d. Heide, O. Wouters, Z. Lebensm.-Linters. u. Forsch. 115, 129 (1962), R. F. 96. Dimedone — phosphoric acid for keto sugars. K. Segura-Cardona, K. Soehring, Med. Exp. 10, 251 (1964). Spray solution: Dissolve 0.3 g .5 Chemicals: ,5-dimerhylcydohexane-1,3-dione (dimedone) in 2,6-Dichloroquinone cblorimide G R, Cat. No. 3037 90 ml ethanol and add 10 ml 85% phosphoric acid. Treatment: Ethanol abs. G R, Cat. No. 972 Heat 15-20 min at 110°C. di-Sodium tetraborate G R, Cat_ No. 6308 In daylight yellow spots on a white background, in long-wave UV light blue fluorescent spots. Literature: 96. Dicobalt octacarbonyl for acetylene compounds. S. Adachi, Anal. Biochem. 9, 224 (1964). Dissolve 0.5 g dicobalt octacarbonyl in 100 ml petroleum benzine. Chemicals: Spray solution I: Dimedone G R, Cat. No. 6013 Spray solution II: Hydrochloric acid 1 mol/l. Spray with I, wait 10 min, spray with 11 and remove the layer with ortho-Phosphoric acid min. 85% (1.71) G R, Cat. No. 573 Procedure: Ethanol abs. G R, Cat. No. 972 Neatan after drying. Wash out excess reagent with water and place the chromatogram into a bromine atmosphere. 97. 4-Dimethylaminobenzaidehyde. The spots show yellow colours. Spray reagent 0.5% for chromatography. K. E. Schulte, F. Ahrens, E. Sprenger, Pharrn. Ztg. 108, 1165 (1963). Literature: Visualisation reagent ready for use in aerosol cans. Chemicals: Cat. No, 3056 Hydrochloric acid 1 moll! Titrisola, Cat. No. 9970 Bromine G R, Cat. No. 1948 98. 4-Dimethylaminobenzaldehyde — acetic acid — phosphoric acid Neatan® for thin layer chromatography, Cat. No. 6746 for proazulenes and azulenes (EP reagent). Dicobalt octacarbonyl Spray solution: Dissolve 0.25 g 4- Petroleum benzine boiling range 100-140°C, Cat. No. 1770 dimethylaminobenzaldehyde in a mixture of 50 g

29 28 is complete, add glacial acetic acid and 5 g 85% phosphoric acid. After dissolution Chemicals: 20 ml water. Stable for months in a brown bottle. 4-Dimethylaminobenzaldehyde G R, Cat. No. 3058 show blue spots Note: Azulenes turn deep blue at room temperature. Proazulenes Hydrochloric acid min. 2.5`)/0 (1.12S) G R, Cat. No. 316 become green to only after heating for 10 min ar 80°C. The colours grow pale and Hydrochloric acid fuming min. 37% (1.19) G R, Cat. No. 317 yellow. By exposure ro steam over a water bath the spots show again their intense hluc Methanol G R, Cat. No. 6009 colour. Ethanol abs, G R, Cat, No, 972 Literature: E. Stahl, Dtsch. Apotheker-Ztg. 93, 197 (1953). H. Kaiser, G. Hasenmayer, Arch. Pharm. 287, 503 (.1954). 104. 4-Dimethylaminobenzaldehyde — hydrochloric acid according to Chemicals: Stahl for indole derivatives (van Urk reagent). 4-Dionethylamirtobenzaldehyde G R, Cat. No. 3058 Spray solution: Dissolve I g 4-Dimethylantinobenzaidehyde in .50 rn1 37% hydro- Acetic acid glacial min. 96% (1.06) G R, Cat. No. 62 chloric acid and add 50 ml ethanol. ortho-Phosphoric acid min. SS% (1.71) G R, Cat. No. 573 Note: In case of eluents with volatile alkaline reacting components it is necessary to hear the plate to ahour 50°C, until these compounds have disappeared. 102. 4-Dimethylaminobenzaldehyde — acetylacetone Procedure: Spray intensively until transparency.. Subsequently blow vapours of aqua for amino sugars (Morgan-Elson reagent). regia over the layer. Spray solution Add 5 ml of a mixture of 5 nil 50% aqueous potassium hydroxide Literature: E. Stahl, H. Kaldewey, Hoppe-Seylers Z. physiol. Chem. 323, 182 (1961). 0.5 ml acetyl- and 20 ml ethanol immediately prior to use to 10 ml of a solution of Chemicals: acetone and 50 ml 1-butanol. 4-Dirnethylaminobenzaldehyde G R, Cat. No. 3058 Dissolve 1 g 4-dimerhylarninobenzaldehyde in 30 nil ethanol. Add Spray solution If: Hydrochloric acid finning Min. 37% (1.19) G R, Cat. No. 317 30 ml 37% hydrochloric acid. If required dilute with 180 nil l-huranol. Ethanol abs. G R, Cat. No. 972 Procedure: After spraying with 1 heat 5 min at 105 °C, spray with 11 and dry S min at Nitric acid min. 6.5% (1.4) G R. Cat. No. 4.5.1 90°C. 105. Red spots. 4-Dimethylaminobenzaldehyde — sulfuric acid for ergot alkaloids. Literature: L. A. Elson, W. T. J. Morgan, Biochem. J. 27, 182.4 (1933). Spray solution: Dissolve 0.125 g 4-dimethylaminobenzaldehyde in a cooled mixture R. Belcher, A. J. Mutten, C. M. Sabrook, Analyst 79, 201 (1954). of 65 ml 97% sulfuric acid and 35 ml water and add 0.05 mI 5% aqueous iron(III) chloride solution. Stable for about a week. Chemicals: 4-Dimethylaminobenzaldehyde G R, Cat. No. 3058 Literature; M, Zinser, C. Baumgartel, Arch. Pharm. 297, 158 (1964). Acetylacetone G R, Cat. No. 9600 Chemicals: Potassium hydroxide G R, Cat. No. 5033 4-Dimethylarninobenzaldebyde G R, Cat. No. 3058 Ethanol abs. .G R, Cat. No. 972 Sulfuric acid 95-97% (1.84) G R, Cat. No. 731 1-Butanol G R, Cat. No. 1990 Iron(III) chloride G R, Cat. No. 3943 Hydrochloric acid fuming min. 37% {1.19) G R, Cat. No. 317 106. Dimethylaminobenzylidenerhodanine for silver, copper and mercury ions. 103. 4-Dimethylaminobenzaldehyde — hydrochloric acid for amines (Ehrlichs reagent). Spray solution: 1% ethanolic solution of 5-(4'-dimethylaininobenzylidene)- rhodanine. Spray solution A: Dissolve 1 g 4-di methylaminobenzaidehyde in a mixture of 25 ml 37% hydrochloric acid and 75 mi methanol. Treatment: Spray with 25% ammonia solution or place into a cham•her saturated Treatment: In some cases it is necessary to warm the plate. with ammonia. Pink to violet spots. Spray solution B: 1% ethanolic solution of 4-dimethylarninobenzaldehyde. Literature: F. W. H. M. Merkus, Pharm. Weekblad 98, 955 (1963): Treatment: Place the sprayed chromatogram 3-5 min in a chamber saturated with hydrochloric acid vapours or spray with 25% hydrochloric acid. Sometimes it is Chemicals: necessary to warm the plate. .5-(4.-Dimethy/aminobenzyfideite)rhodaiiiize G R, Cat, No. 30.59 Ethanol abs. G R, Cat. No, 972 Literature: R. A. Heacock, M. E. Mahon, J. Chromatog. 17, 33.8 (1965). Ammonia solution min. 25% (0.91) G R, Cat. No. 5432 30 31 Treatment: Heat 1-2 min at 120°C. 107. Dimethyla-minocinna.maldehyde for in.doles. .100 ml Note: Stock solution: Dissolve g 4-dimethylaminocinnamaldehyde in a mixture of The colour spots may be eluated for colorimetric determination. hydrochloric acid 6 moll' and 100 ml ethanol, Store the solution in the refrigerator. Literature: W. C. Schaefer, J. W. van Cleve, Anal. Chem. 28, 1290 (1956). Spray solution: 1 part stock solution and 4 parts ethanol. L. Boggs, L. S. Cuendet, I. Ehrenrhal, R. Koch, F. Smith, Nature 166, 520 (1950). Chemicals: Treatment: Heat 5 min at 105°C. The colours of the spots are intensified by blowing vapours of aqua regia over the N,N-Dimethy1-1,4-phenylenediantinonizon dichloride G R, Cat. No. 3067 layer. Trichloroacetic acid G R, Cat. No. 807 Note: Unsuitable with ammonia-containing eluents because the background becomes coloured. By brief hearing (10 min) at 105 °C this can be evaporated before 111. 1,3-Dinitrobenzene for 17-ketosteroids. spraying. Solution a: 2% ethanolic solution of 1,3-dinitrobenzene. Literature: J. Harley-Mason, A_ A. P. G. Archer, Biocheni. J. 69, 60 (1958). Solution b: Merhanolic potassium hydroxide solution 2.5 cool/l. Chemicals: Spray solution: 4-Dimetbylaminocinnanialdehyde Mix equal parts of a and h. Hydrochloric acid min. 25% (1.125) G R, Cat. No, 316 Treatment: Pleat 1-2 min at 80°C. Violet spots. Nitric acid Min. 65% (1.4) G R, Cat. No. 45.4 Literature: T. Feher, Mikrochirn. Acta 1965, 105. 108. N,N-Dimethy1-1,4-phenylenediammonium dichloride for B. P. Lisboa, J. Chromatog. 16, 136 (1964), bromine-containing hypnotics and chlorinated insecticides. R. Neher, Steroid Chromatography, Elsevier 1964, Amsterdam, London, Spray solution: Dissolve 0.5 g N,N-dimethy1-1,4-pheny lenedi am in onium dichloride New York. in 100 ml sodium ethoxide )l g sodium in 100 nil ethanol). Chemicals: Procedure: After spraying moisten the chrumatogrant with a water spray and 1,3-Dinitrobenzetre, Cat. No. 3114 irradiate 1 min with unfiltered UV light. Sodium hydroxide G R, Cat. No. 5033 This liberates free halogen which oxidises the reagent to Wurster's red. Methanol G R, Cat. No. 6009 Literature.' J. Baumler, S. Rippstein, Hely. Chico. Acta 44, 1162 (1961). Ethanol abs. G R, Cat. No. 972 Variation for PC: Chemicals: N,N-Dimetlry1-1,4-phenylenediammonium dichloride G R, Cat. No. 3067 Dip solution I: Mix 1 part 30% aqueous potassium hydroxide with 1 part ethanol. Sodium G R, Cat. No. (i261 Dip solution II: 2% erhanolic 1,3-dinitrobenzene solution. Ethanol abs. G R, Cat. No. 972 Procedure: After dipping into l press off excess between filter paper. Then dip into II, press off and heat slowly at 65 'C. N,N-Dimethy1-1,4-nhenylenediammonium dichloride for 109. 17-Ketosteroids turn violet, 2-ketosteroids blue-violet and 20-ketosteroids brown. peroxides. Literature: J. Bart- Dissolve 1.5 g N,N-dimethyl-1,4-phenylenediammonium dichloride oiler, J. Heilmann, Z. physiol. Chem. 309, 221 (1957). Spray solution: O. Schindler, T. Reichsrein, Hely. Chim. in a mixture of 128 ml methanol, 25 nil water and 1 nil glacial acetic acid. ACIa 34, 108 (1951). Chemicals: Peroxides show purple spots. 1,3-Dinitrobenzeire, Cat. No. 3114 E. Knappe, D. Peteri, Z. anal. Chem. 190, 386 (1962). Literature: Potassium hydroxide G R, Cat. No, 503 3 Chemicals: Ethanol abs. G R, Cat. No. 972 N,N-Dimethy1-1,4-pheilylenediammonium dichloride G R, Cat. No. 3067 Acetic acid glacial min. 96% (1.06) G R. Cat. No. 62 Methanol G R, Cat. No. 6009 112. 3,5-Dinitrobenzoic acid for cardiac glycosides. A. Spray solution: Dissolve 1 g 3,5-dinitrobenzoic acid in a mixture of SO nil metha- 110. N,N-Dimethy1-1,4-phenylenediammonium dichloride — trichloroacetic acid for methyl-sugars. nol and 50 ml potassium hydroxide solution 2 mo1/1. B. Spray solution I: 2% methanolic solution of 3,5-dinitrobenzoic acid. Spray solution: Dissolve 0.4 g N,N-dimethy1-1,4-phenylenediammonium dichloride in 100 ml 2% aqueous trichloroacetic -acid solution. Spray solution II: 5.7% methanolic potassium hydroxide solution.

32 33 Procedure: Spray lightly with I and then with excess 11. The spots show blue violet colours. drochloric acid 2 mol/l. Saturated keto-DNPH show blue colour immediately, satu- Seehofer, Chem. Ber. 86, 1235 (1953). Literature: R. Tschesche, G. Grimmer, F. rated aldehyde-DNPH show olive-green colour more slowly. Unsaturated carbonyl M. L. Lewbart, W. Wehrli, T. Reichstein, Hely. Chico. Acta 46, 505 (1963). derivatives change only slowly or not at all. Chemicals: Literature: A. Mehlitz, K. Gierschner, T. Minas, Cheiniker-Ztg. 87, 573 (1963). 3,5-Dinitrobenzoic acid, Cat. No. 135 Chemicals: Potassium hydroxide G R, Cat. No. 5033 2,4-Dinitrophenylhydrazine G R, Cot. No. 3073 Methanol G R. Cat. No. 6009 Hydrochloric acid fuming min. 37% (1.19) G R, Cat. No. 317 Ethanol abs. G R, Cat. No. 972 113. 3,5-Dinitrobenzoic acid for reducing sugars. Potassium bexacyanoferrate(111) G R, Cat. No. 4973 .Spray solution: 1% solution of 3,5-dinitrobenzoic acid in sodium carbonate solution 2 cool/l. 116. 3,5-Dinitrosalicylic acid for reducing sugars. Treatment: Dry 5-10 min at I00°C. Spray solution: 0,5% solution of 3,5-dinitrosalicylic acid in 4% sodium hydroxide Literature: F. Weygand, H. Hofmann, Chem. Ber. 83, 405 (1950). solution. Treatment: Chemicals: After brief pre-drying at room temperature heat 4-5 min at 100°C. 3,5-Dinitrobenzoic acid, Cat. No. 138 Literature: A. Jeanes, C. S. Wise, R. J. Dirnier, Anal. Chem. 23, 415 (1951). Sodium carbonate G FL, Cat. No. 6392 Chemicals: acids. acid, Cat. No. 800141 114. 2,4-Dinitrofluorobenzene for amino Sodium hydroxide G R, Cat. No. 6495 Spray solution 1: Dissolve 8.4 g sodium hydrogen carbonate in 80 nal water, add 2.5 ml N sodium hydroxide solution and make up to 100 nil with water. 117. Diphenylamine for glycolipids. Spray solution 11: 10% methanolic solution of 2,4-dinitrofluorobenzene. Spray solution: Mixture of 20 nil 10% ethanolic diphenylamine solution, 100 nil Treatment: Spray with l and subsequently with II. 37"A. hydrochloric acid and 80 ml glacial acetic acid. ' Procedure: Scrape off 5 mm from both sides of the plate. Place two polyethylene strips Treatnient: Heat 5-10 min at 100.°C. of same breadth on the margins so that a second glass plate can be laid on the layer. Blue-grey spots. Heat 1 hour at 40°C in the dark, cool the plate and place 10 min in an ether bath. Literature: H. Jatzkewitz, Hoppe-Seylers Z. physiol. Chem. 320, 251 (1960). After drying the spots are outlined. Chemicals: Literature: G. Pataki, J. Chromatog. 16, 541 (1964). Diphenylainine G R, Cat, No. .3086 Chemicals: Ethanol abs. G R, Cat. No. 972 1-Fluoro-2,4-dinitrobenzene G R, Cat. No. 2966 Hydrochloric acid fuming Min. 37% (1.19) G R, Cat. No. 317 Sodium hydrogen carbonate G R, Cat, No. 6329 Acetic acid glacial min. 96% (L06) G R, Cat. No. 62 Sodium hydroxide solution 1 moll' Titrisolg, Cat. No. 9956 118. Methanol G R, Cat. No. 6009 Diphenylamine — palladium(II) chloride for nitrosamines. Diethyl ether G R, Cat. No. 921 Spray solution: Mix .5 parts 1.5% ethanolic diplienylamine solution and 1 part 0.2% sodium chloride solution containing 0.1 g palladium(II) chloride. 2,4-Duthrophenylhydrazine for free aldehyde and. keto groups 115. Treatment: After exposure to short-wave UV light the substances show violet spots. and ketoses. Literature: R. Preussmann, D. Daiber, H. Hengy, Nature 201, 502 (1964). 0,4% solution of 2,4-dinitrophenylhydrazine in hydrochloric acid A. Spray solution: R. Preussmann, G, Neurath, G. Wulf-Lorentzen, D. Daiber, H. Hengy, Z. anal. 2 cool/l. Chem. 202, 187 (1964), 13. Spray solution: Add 10 ml 37% hydrochloric acid to 1 g 2,4-dinitrophenylhydra- Chernic.als: zinc in 1000 ml ethanol. Diphenylamine G R, Cat. No. 3086 Treatment: For distinction of the formed 2,4-dinittophenylhydrazones (DNPH) Palladium(11) chloride, Cat. No. 7110 spray consecutively with 0.2% solution of potassium hexacyanoferrate(11I) in hy- Ethanol abs. G R, Cat. No. 972 Sodium chloride G R, Cat. No. 6404 34 35 Chemicals: 119. Diphenylamine — zinc chloride for chlorinated insecticides. Diphenylcarbazone, Cat. No. 3087 Ethanol abs. G R, Cat. No. 972 solution: Dissolve 0.5 g diphenylamine and 0.5 g zinc chloride in 100 ml Spray Nitric acid min. 65% (1.4) G R, Cat. No. 454 acetone. Treatment: Heat 5 min at 200°C. 123. Colour reaction. Diphenylcarbazone for cations. Spray solution: Literature: D. Kath, J. Chromatog. 15, 269 (1964). Saturated solution of diphenylcarbazone in methanol. Literature: Chemicals: G. B. Heisig, F. H. Pollard, Anal. Chim. Acta 16, 234 (1957). Diphenylatnitte G R, Cat. No, 3086 Chemicals: Zinc chloride G R, Cat. No. 8816 Diphenylcarbazone, Cat. No. 3087 Acetone G R, Cat. No. 14 Methanol G R, Cat. No. 6009

120.I3-Aminoethyl diphenylborate for u.- and ,1-pyrones 124. Diphenylpicrylhydrazyl for essential oils. (Neu reagent). Spray solution: solution: 1% methanolic ii-aminoethyl diphenylborate solution. Dissolve 0.06 g diphenylpicrylhydrazyl in 100 nil chloroform, Spray Treatment: Spray about 10 ml of the solution and inspect the fluorescence in long- Heat 5-10 niin ar 110°C. Procedure: Yellow spots on violet background. wave UV light. Literature: C. Bergstrom, C. Lagercrantz, Acta Chem. Scand. Literature: R. Neu, Naturwissenscliaften 44, 181 (1957). 18, 560 (1964). E. Stahl, P. J. Schorn, Hoppe-Seylers Z. physiol. Chem. 325, 263 (1961). Chemicals: 2 ,2'-Dipheny1-1-picrythydrazyl Chemicals; Chloroform G R, Cat. No. 2445 p-Aminoethyl diphenylborate Methanol G R, Cat. No. 6009 125. 2,5-Dipheny1-3-(4-styrylphenyl)tetrazolium chloride (TPTZ) 121. Diphenylcarbazide for silver, lead, mercury, copper, tin, zinc, for reducing steroids (corticosteroids). and calcium ions. Solution a: Freshly prepared 1% methanolic solution of TPTZ. ethanolic diphenylcarbazide solution. Spray solution I: 1-2% Solution b: 3% aqueous sodium hydroxide solution. Spray solution II: 25% ammonia solution or a chamber saturated with ammonia. Spray solution: Mix equal parts of a and b freshly before use. For mercury acetate adducts heat some minutes at 80°C, causing the spots to Note: Literature: P. J. Stevens, J. Chromatog. 14, 269 (1964}. turn blue-violet. Chemicals: Literature: F. W. M. H. Merkus, Pharm.Vreekblad 98, 947 (1963). 2,5-Dipbeny1-3-(4-styrylphenyl)tetrazoliton Chloride Chemicals: Methanol G R, Cat. No. 6009_ Diphenylcarbazide G R, Cat. No, 3091 Sodium hydroxide solution 10% (1.109) G R, Cat. No. 5588 Ethanol abs. G R, Cat. No. 972 Ammonia solution min. 25% (0.91) G R, Cat. Nu. 5432 126. Dipicrylainine for choline (non-specific). 122. Diphenylcarbazone for addition compounds of unsaturated Spray solution: Dissolve 0.2 g dipicrylarnine in a mixture of 50 nil acetone and 50 ml fatty acids. water. Note: Spray solution; 0.2% ethanolic solution of diphenylcarbazone. Choline and its derivatives appear as red spots on yellow background. Addition compounds of unsaturated acids (e, g. with Hg) are dyed purple. Literature: K. B. Augustinsson, M. Grahn, Acta Chem. Scand. 7, 906 (1953). Note; in Colour intensification may be obtained by respraying with nitric acid 0.05 molll Chemicals: ethanol. Dipicrylarnine G R, Cat. No. 3089 Literature.' Y. Inouye, M. Noda, O. Hirayama, J. Am. Oil Chemists Soc. 32, 132 Acetone G R, Cat. No. 14 (1955). 37 36 -t present is filtered off and the solution made up to 100 ml with water. The.solution is 127. Dipicrylamine for vitamin B. stable for several weeks in the refrigerator when stored in an amber flask. Stock solution: Add 1 g dipicrylamine to 0.12 g magnesium carbonate and 15 ml Spray solution: Mix in this order: 20 ml water, 5 ml hydrochloric acid 6 moll], 2 nil water, heat the mixture 15 min on a boiling water bath and filter. stock solution and 6 ml sodium hydroxide solution 6 mo1/1. In case bismuth hydrox- Spray solution: Add to 0,2 ml of the dipicrylamine solution 50 inl methanol, 49 ml ide is not completely dissolved by shaking, add several drops of hydrochloric acid water and 1 ml 2.5% ammonia solution. 6 mo1/1. Note: Literature.- K. B. Augustinsson, M. Grahn, Acta Chem. Scand. 7, 906 (1953). The spray solution is stable for about 10 days in the refrigerator. Literature Chemicals: : H. M. Bregoff, E. Roberts, C. C. Delwiche, J. Biol. Chem. 205, 565 Dipicrylamine G R, Cat. No. 3089 (1953). Magnesium carbonate Chemicals: Methanol G R. Cat. No. 6009 Bismuth(Ill) nitrate basic G R, Cat. No. 1878 Ammonia solution min, 25'% (0.91) G R, Cat. No. 5432 Nitric acid min. 6.C'f,. (1.4) G R, Cat, No. 414 Hydrochloric acid min. 25•%. (1,125) G R, Cat. No. 316 128. Dithizone for ions of heavy metals. Potassium iodide G R, Cat. No. 5043 Spray solution 0.05% solution of dithizone in carbon tetrachloride. Sodium hydroxide solution 10% (1.109) G R, Cai. No. 1588 Spray with 25 % ammonia solution or place the chromatogram in a Spray solution 11: 131. Dragendorff reagent acc. to Munier for alkaloids and other chamber saturated with ammonia. nitrogen-containing compounds. T. Barnahas, J. Barnahas, Naturwissenschaften 44, 61 (1957).. Literature: Solution a: F. W. H. M. Merkus, Pharin. Weekblad 98, 955 (1963). Dissolve 1.7 g bismuth(lII) nitrate and 20 g tartaric acid in 80 ml water. Solution b: Chemicals: Dissolve 16 g potassium iodide in 40 ml water. Dithizone (diphenylthiocarbazone) G R, Cat. No. 3092 Stock solution: Mix equal parrs of a and b. Stock solution is stable for several months, Carbon tetrachloride G R., Cat. No, 22 22 if refrigerated. Ammonia solution min, 25% (0.91) G R, Cat. No. 5432 Spray solution: Dissolve 10 g tartaric acid in 50 nil water and add 10 nil of the stock solution. 129. Dragendorff reagent for polyethylene glycols, polyethylene Note: For detecting vitamin Bi spray with the stock solution. glycol ethers and polyethylene glycol esters. Literatur: R. Munier, Bull. soc. chim. biol. 35, 1225 (1953), Dissolve 1.7 g hismuth(lll) nitrate in a mixture of 20 oil glacial acetic acid Solution a: Chemicals: and 80 nil water, add a solution of 40 g potassium iodide in 100 ml water and 200 nit Bismuth(111) nitrate basic G R, Cat. No. 1878 glacial acetic acid and make up to 1000 ml with water. Potassium iodide G R, Cat: No. 5043 Solution b: 20% aqueous barium chloride solution. L(+)Tartaric acid G R, Cat. No. 804 Spray solution: Mix 2 parts a with 1 part h before use. R. Thoma, R. Rombach, E. Ullmann, Sci. Pharm. 32, 216 (1964). Literature: 132. Dragendorff reagent acc. to Munier and Macheboeuf for Chemicals: alkaloids and other nitrogen-containing compounds. Bisnnith(111) nitrate basic G R, Cat. No. 1878 Solution a: Potassium iodide G R. Car. No. 5043 Dissolve 0.85 g bismuth(III) nitrate in 10 m] glacial acetic acid and 40 ml water. Barium chloride G R, Cat. No. 1719 Acetic acid glacial min. 96% (1.06) G R, Cat. No. 62 Solution b: Dissolve 8 g potassium iodide in 20 ml water. Stock solution: Mix equal parrs of a and b. The mixture can be stored in a dark bottle 130. Dragendorff reagent ace. to Bregoff-Delwische for a long time. for quaternary bases. Spray solution: Mix 1 mi stock solution with 2 ml glacial acetic acid and 10 ml water Stock solution: Dissolve 8.0 g bismuth(III) nitrate in 20-25 ml 25% nitric acid. Add before use. this solution slowly with stirring to a slurry of 20 g potassium iodide and 1 nil 6 N Literature: R. Munier, Al. Macheboeuf, Bull. soc. chim. biol. 33, 846 (1951). hydrochloric acid and 5 ml water. Add water to the dark precipitate until an orange- jatzkewitz, Hoppe-Seylers Z. physiol, Chem. 292, 99 (1953). red colour develops. The volume of the solution should be 95 ml. Any solid residue

38 39 Chemicals: Chemicals: BistnuthalI) nitrate G R, Cat. No. 1878 Ethylenediamine, Cat. No_ 947 Potassium hexacyanoferrate(111) G R, Cat. No. 4973 Potassium iodide G R, Cat. No. 5043 G R, Cat. No. 62 Ethanol abs. G R, Acetic acid glacial min. 96% (1.06) Cat. No. 972 133. Dragendorff reagent acc. to Thies and Reuther, 136. Fast blue salt B for phenols and coupling amines raodif. by Vagujfalvi, (diazonium reagent). for alkaloids and other nitrogen-containing compounds. Spray solution 1: Boil 2.6 g bismuth carbonate and 7 g sodium iodide with 25 ml glacial A freshly prepared 0.5% aqueous fast blue salt B solution. Stock solution: r off the precipitated sodium acetate. Spray solution 11: Sodium hydroxide solution 0.1 mo1/1 acetic acid for a few minutes. After 12 hours filte Then mix 20 ml of the red-brown filtrate with 80 nil ethyl acetate and add 0.5 ml Treatment: Spray with l and then with 11. Literature: water. Store in a dark bottle. H. Jatzkewitz, U. Lenz, Hoppe-Seylers Z. physiol. Chem. 305, 53 (1956), Mix 10 ml stock solution with 100 ml glacial acetic acid and 240 ml Chemicals: solution: Spray Fast blue salt 13, Cat. No. 3191 ethyl acetate. After spraying of 5-10 ml alkaloids and some other compounds containing no Sodium hydroxide solution 0.1 mol./1 Titrisole', Cat. No. 9959 nitrogen show orange spots. A more sensitive detection.is availably by subsequent spraying with sul- 137. Treatment: Fluorescein for lipids. furic acid 0.025-0.05 moI/1. The spots are bright orange to red on a grey background. 230 (1954). Spray solution: 0.01% ethanohc solution of fluorescein. Thies, F. W. Reuther, Naturwissenschaften 41, Literature: H. Treatment: Dry with warm air and handle subsequently with water vapour or spray Vaguifalvi, Planta Med. 8, 34 (1960). lightly with water. D. 14, 125 (1964). E. Tyihak, J. Chromatog. Chemicals: Chemicals: Fluorescein, Cat. No. 3990 pure, Cat. No. 1854 Bisimith(111) carbonate basic extra Ethanol abs. G R, Cat. No. 972 Sodium iodide extra pure, Cat. No. 6520 Cat. No. Acetic acid glacial min. 96% (1.06) G R, Cat. No. 9623 138. Fluorescein — ammonia for purines, pyrimidines Ethyl acetate G R, 9984 Sulfuric acid 0.025 mol/lTitrisoM Cat. No. and barbiturates. Spray solution: 0.005% solution of fluorescein in 0.5 N ammonia solution, 134. Ethylenediamine for catecholamines. Inspect the chromatogram in long- and short-wave UV light. Mixture of equal parts .of ethylenediamine with water or diluted Spray solution: Literature: T. Wieland, L. Bauer, Angew. Chem. 63, 511 (1951). sodium hydroxide solution. C. inspection in short- or Chemicals: Hear the chromatogram 20 min at 50-60 ° Procedure: Fluorescein, Cat. No, 3990 long-wave UV light. Ammonia solution min. 25% (0.91) G R, Cat. No. 5432 R. Segura-Cardona, 1-:. Soehring, Med. Exp. 10, 251 (1964). Literature: Chemicals: 139. Fluorescein — bromine for unsaturated compounds. No. 947 Ethylenediamine, Cat. 9136 Spray solution: Sodium hydroxide solution 2 molli, Cat. No. 0.1% ethanolic fluorescein solution. - Bromine solution: 5% bromine in carbon tetrachloride, 135. Ethylenediamine — potassium hexacyanoferrateall) for catechol Procedure: ne, noradrenaline and acetyl derivatives). After spraying with the fluorescein solution place the chromatogram into amines (adrenali - a chamber containing the bromine solution. Fluorescein is converted to eosin which Solution of 0.1 g potassium hexacyanoferrate(M) in 5 ml ethylene Spray solution: shows no fluorescence in long-wave UV light. Compounds adding on bromine prevent the formation of eosin and the fluorescence remains. diamine, 45 ml ethanol and 50 ml water. inspection under UV light. Larger amounts of substance show yellow spots on reddish background. Treatment: Heat the chromatogram 10 min yet05 J. S. Stern, M. J. Franklin, J. Ma, J. Chromatog. 30, 637 (1967). Literature: F. Runge, A. Jumar, F. KOhler, J. prakr. Chem, Literature: 21, 39 (1963).

40 41 Chemicals: 142. Fluorescence indicators and luminescent substances Fluorescein, Cat. No. 3990 as general visualisation reagents. Bromine G R, Cat. No. 1948 A. Spray reagents: Ethanol abs. G R, Cat. No. 972 1. 2',7'-Dichlorofluorescein, spray reagent no. 8.9. Carbon tetrachloride G R, Cat. No. 2222 2. Fluorescein, spray reagent no. 137. Variation: With self-coated plates take 0.04% aqueous fluorescein sodium solution 3. Methylumbelliferone, spray reagent no. 189. instead of water. 4. Morin, spray reagent no. 195. Treatment: After development of the chromatogram blow bromine vapours over the 5. Rhodamine B, spray reagent no. 260, 261. dried plate. B. Additives to adsorbents: Literature: E. Stahl, Chcmiker-Ztg. 82, 323 (1958). 6. Fluorescein sodium 0.04% in water added to the adsorbent suspension, Cat. No. 3922. Chemicals: 7. Fluorescein sodium, Cat. No. 3992 Fluorescence indicator F254 for thin layer chromatography (1-2% added to the adsorbent) for detection in short-wave UV light (A max 254 rim), Cat. No. 9182. Bromine G R, Cat. No. 1948 The following adsorbents contain an additional fluorescence indicator for detection in long- and short-wave UV light (A max 254 nm and A niax 366 nm). . Silica gel 60 HF23-4,15,Cat. No. 7741 Silica gel 60 140. Fluorescein — hydrogen peroxide for hypnotics containing PF2s4+36,5, Cat. No. 7748 Aluminium oxide 60 PF2.4 bromine. 3nn (Type E), Cat. No. 1104 Aluminium oxide 50 PE,54.4.3„ (Type T), Cat. No. 1065 Spray solution 1: 0.1%fluorescein solution in 50% ethanol. Spray solution 11: Mix equal parts of 30% hydrogen peroxide and glacial acetic acid. 143. Folin Ciocalteau reagent for phenols. Spray with 1 and then with 11, heat finally 20 min at 90°C. Procedure: Stock solution: Dissolve 10 g sodium tungstate and 2.5 g sodium molybdate in 70 ml Note: Bromine formed by oxidation reacts with fluorescein under formation of eosin. water, add 5 ml 85% phosphoric acid and 10 ml 37% hydrochloric acid and reflux Literature: H. Weichsel, Mikrochirn. Acta 1965, 325, the mixture for 10 hours. Add subsequently 15 g lithium sulfate, 5 ml water and Chemicals: 1 drop bromine, heat again IS min and make up to 100 ml with water after cooling. Fluorescein, Cat. No. 3990 The solution shall not show green colouring. Acetic acid glacial min. 96% (/.106) G R, Cat, No. 62 Spray solution 1: 20% aqueous sodium carbonate solution. PerhydroP' (30% by weight H202) G R, Cat. No. 7209 Spray solution 11: Dilute freshly before use 1 part of the stock solution with 3 parts Ethanol abs. G R, Cat. No. 972 water. Procedure: Spray with I, dry for a short while and spray with 11. Literature: R. W. Keith, D. le Turneau, D. Mahium, J. Chromatog, 1, 534 (1958). 141. Fluorescein — rhodamine B — sodium carbonate for chlorinated hydrocarbons and heterocyclic compounds. Sodnon molybdate G R, Cat. No. 6521 Sodium tungstate G R, Cat. No, 6673 0.5% ethanolic rhodamine B solution. Spray solution l: Lithium sulfate, Cat. No. 5694 10% aqueous sodium carbonate solution. Spray solution 11: Sodium carbonate G R, Cat. No. 6392 Procedure: Using plates impregnated with fluorescein sodium spray the chro- Bromine G R, Cat. No. 1948 matograms after development first with 1, dry and spray liberally with IL ortho-Phosphoric acid min. 85% (1,711G R, Cat. No. 573 Inspect in daylight and in long-wave UV light. Hydrochloric acid fuming min. 37% (1.19) G R, Cat. No. 317 Chemicals: Fluorescein sodium, Cat. No. 3992 144. Formaldehyde — hydrochloric acid for indoles and indole Sodium carbonate G R, Cat. No. 6392 derivatives (Prochazka reagent). Rhodamine B G R, Cat.. No. 7599 Spray solution: Freshly prepared mixture of 10 nil formaldehyde solution (35%), Ethano! abs. G R, Cat. No. 972 10 ml hydrochloric acid (1.125) and 20 ml ethanol,

42 43

Treatment: Heat 5 min at 100°C. The yellow-orange-greenish fluorescence colours become stronger by blowing vapours of aqua regia over the layer. 148. Glucose — aniline for acids (Schweppe reagent). Prochazka, Chem. Listy 47, 1643 (1953). Literature: Z. Spray solution: Dissolve 2 g glucose in 20 ml warer and also 2 ml aniline in 20 ml E. Stahl, H. Kaldewey, Hoppe-Seylers Z. physiol. Chem. 323, 182 (1961). ethanol. Mix both solutions and make up to 100 ml with 1-butanol. Chemicals: Procedure: After spraying heat the chromatogram 5 —10 min at 125 °C. Formaldehyde solution (35%) G R, Cat. No. 4003 Dark brown spots on white background. Hydrochloric acid Min. 2S% (1.125) G R, Cat. No. 316 Literature: H. Schweppe, Dissert. Munster 1954. Ethanol abs. G R, Cat. No. 972 Chemicals: Nitric acid min. 65% (1.4) G R, Cat. No. 454 D-Glucose, Cat. No. 8342 Aniline G R, Cat. No. 1261 145. Formaldehyde — phosphoric acid for steroid alkaloids, steroid sapogen.ins and phenothiazine derivatives. Ethanol abs. G R, Cat. No. 972 1-Butanol G R, Cat. No. 1990 Spray so/art/on: Dissolve 0.03 g paraformaldehyde in 100 nil 85% phosphoric acid

with stirring at room temperature. 149. Glucose — phosphoric acid for aromatic amines. The reagent is stable for several weeks. Spray solution: K. Schreiber, O. Aurich, G. Osske, J. Chromarog. 12, 63 (.1963). Dissolve 2 g glucose in 10 ml 85% phosphoric acid and 40 ml water. Literature: Add 30 nil ethanol and 30 nil 1-butanol. E. G. C. Clarke, Nature 181, 1152 (1958). Treatment: Heat for about 10 min at 45°C. Chemicals: Literature: Paraformaldebyde, Cat. No. 4005 F. Micheel, H. Schweppe, Microchim. Acta 1954, 53. ortbo-Phosphoric acid min. 85% (1.71) G R, Cat. No. 573 Chemicals: D-Glucose, Cat. No. 8342 146. Formaldehyde — sulfuric acid for aromatic compounds. ortho-Phosphoric acid min. 85% (1.7) G R, Cat. No. S73 Spray solution: Mixture of 0.2 ml 37% formaldehyde solution and 10 ml 97% Ethanol abs. G R, Cat. No, 972 1-Butanol G R, Cat. No. 1990 sutfuric acid. Spray the chromatogram directly afrer taking our of the developing Procedure: 150. Glyoxalbis-(2-hydroxyanil) for cations chamber. Spray solution: Dissolve 1 g glyoxalbis-(2-hydrox Varicoloured spors. -yanil) and 3 g potassium hydroxide in 100 nil methanol. Literature: N. Kucharczyk, J. Fohl, J. Vymeral, J. Chromatog. 11, 55 (1963). Procedure: Spray the dried chromatogram and dry again with a srream of air at 50 Chemicals: °C. Literature: H. G. Moller, N. Zeller, J. Chromarog. Formaldehyde SOlUt1/272 Min. 37%, Gat. No, 4003 14, 560 (1964). Sulfuric acid 95-97% (1.84) G R, Cat. No. 731 Chemicals: Glyoxalbis(2-hydroxyanil) G R, Cat. No. 4191 147. Furfural — sulfuric acid for carbamate esters. Potassium hydroxide pellets G R, Cat. No. 5033 Methanol G R,. Cat. No. 6009 Spray solution I: 1% solution of furfural in acetone. Spray solution II: 10% solution of sulfuric acid in acetone. 151. Hydrazine sulfate for piperonal, vanillin and ethyl vanillin. Spray with 1 and subsequently with 11. Procedure: Spray sohition: Mix 90 ml of a saturated aqueous solution of hydrazine sulfate with Literature: A. Heyndrickx, M. Schauvliege, A. 13lommel, J. Pharm. Belg. 20, 117 10 ml hydrochloric acid 4 mo1/1. (1965). Note: Inspect the moist chromatogram in long-wave UV light before and after 1. Sunshine, A. J. Clin. Pathol. 40, 576 (1963). exposure to ammonia vapour. Chemicals: Literature: K. G. Bergner, H. Sperlich, Dtsch. Lebensm.-Rundschau 47, 134 (1951). Furfural G R, Cat. No. 4013 Chemicals: Sulfuric acid 95-97% (1.84) G R, Cat. No_ 731 Hydrazinium sulfate G R, Cat. No. 4603 Acetone G R, Cat. No. 14 Hydrochloric acid min. 2S% (1.125) G R, Cat. No. 316 Ammonia solution nun. 25% (0.91) G R, Cat. No. 5432 44 45 152. Hydrochloric acid for glycals. Chemicals: Spray solution: Mix 1 part 36% hydrochloric acid with 4 parts ethanol. Hydroxylammonium chloride GR, Cat. No. 4616 Procedure: Glycals appear as pink spots on heating to 90°C. Potassium hydroxide G R, Cat, No. 5033 tron(111) chloride GR, Cat. No. 3943 Note: To be used also as general spray reagent for TLC. Hydrochloric acid fuming min. 37% (1.19) G R, Cat. No, 317 Literature: J. T. Edward, D. M. Waldron, J. Chem. Soc. 1952, 3631 Ethanol abs. G R, Cat. No. 972 Chemicals: Diethyl etlier G R, Cat. No. 921 Hydrochloric acid fuming men_ 37% (1.19) G R, Cat. No. 317 Ethanol abs. G R, Cat. No. 972 156. 8-Hydroxyquinoline for barium, strontium 153. Hydrogen peroxide for aromatic acids. and calcium ions. Spray solution: Dissolve 0-5 g 8-hydroxyquinoline in 100 ml 60% ethanol, Spray solution: 0.3% aqueous hydrogen peroxide solution. Treatment: Respray with 25% Irradiate the chromatogram with long-wave UV light until maximal blue ammonia solution or place the chromatogram into a Treatment: chamber with ammonia. fluorescence. Inspect in long-wave UV light. D. W. Grant, J. Chromatog. 10, 511 (1963). Literature: Literature; W. A. Reeves, T. B. Crumler, Anal. Chem. 23, 1576 (1952). Chemicals: T. V. Arden et al., Nature 162, 691 (1948), PerhydroP2' (30% by weight H,02) G R, Cat. No. 7209 Chemicals: 8-Hydroxyquinohne G R. Cat. No. 7098 154. 4-Hydroxybenzaldehyde — sulfuric acid for sapogenins Ethanol abs. G R, Cat. No. 972 and corticosteroids (Komarowsky reagent). Ammonia solution Mill. 25% (0.91) GR, Cat. No. 5432 Solution a: 50% sulfuric acid. 2% methanolic solution of 4-hydroxybenzaldehyde. Solution b: 157. solution: Mix freshly before use 5 ml a with 50 ml h. 8-Hydroxyquinoline — hypobromite for arginine and other Spray guanidine derivates (Sakaguchi reagent). Treatment: Heat 3-4 min at 105°C or 10 min at 60°C. Yellow to pink spots. Spray solution I: 0.1 % solution of 8-hydroxyquinnline in acetone. Literature: P. J. Stevens, J. Chromatog. 14, 269 (1964). Spray solution 11: Mixture of 0.2 ml bromine and 100 nil sodium hydroxide solution Chemicals: 0.5 rno1/1. 4-Hydroxybeizzaldehyde Procedure: Sulfuric acid 95 —97% (1.84) G R, Cat. No. 731 Spray with I and after drying with II. The spots show orange to red. colour. Literature: J. Methanol G R, Cat. No. 6009 B. Jepson, J. Smith, Nature 172, 1100 (1953). J. Kalouiek, M. KutiZek, J. Bilek, Ceskoslov. farm. 4, 188 (1955). 155. Hydroxyla.mine — iron(111) chloride for lactones, esters, Chemicals: amides and anhydrides of carboxylic acids. 8-Hydroxyquinoline G R, Cat. No. 7098 Solution a: Dissolve 20 g hydroxylammonium chloride in .50 m] water, make up to Bromine G R, Cat. No. 1948 200 ml with ethanol. Store the solution in the refrigerator. Sodium hydroxide solution 0.5 mo1/1 Titrisol, Cat. No. 9957 Acetone G R, Cat, No. 14 Solution b: Dissolve 50 g potassium hydroxide in as little water as possible and make up to 500 ml with ethanol. Spray solution 1: Mix equal parts of a and b and filter off the precipitated potassium 158. 8-Hydroxyquinoline — kojic acid for aluminium, magnesium, chloride. Place the solution in the refrigerator (stable for about 2 weeks). calcium, strontium and barium ions. Dissolve 10 g finely powdered iron(IIl) chloride in 20 ml 36% Spray solution II: Spray solution I: Solution of 2.5 g 8-hydroxyquinoline and 0.5 g kojic acid in 500 ml hydrochloric acid. Shake with 200 ml diethyl ether until a homogenous mixture is 90% ethanol. obtained. The solution 11 is stable for some time only well sealed. Spray solution II: 25% ammonia solution. Procedure: Spray with I, dry at room temperature and spray with 11. The spots fluoresce in long-wave UV light. Literature: V. P. Whittaker, S. Wijesundera, Biochem. J. 51, 348 (1952). Literature: F. H. Pollard, J. F. W. McOmie, I. I. M. Elbeih, J. Chem. Soc. 1951, 466.

46 47 Procedure: Spray with 1 and subsequently- with 11. Chemicals: Literature: 8-Hydroxyquinoline G R, Cat. No. 7098 E. Chargaff, C. Levine, C. Green, J. Biol. Chem. 175, 67 (1948), Kojic acid, Cat. No. 5193 W. Awe, 1. Reinecke, J. Thum, Naturwissenschaften 41, 528 (1954). Ethanol abs. G R, Cat Na 972 Chemicals: Ammonia solution min 25% (0.91) G R, Cat. No. 5432 Sodium azide, Cat. No. 6688 Iodine solution 0.05 mo1.12/17-itriso14, Cat. No. 9910 159. Indanedione for carotenoid aldehydes. Starch soluble GR, Cat. No. 1252 Spray solution: Dissolve 0.5 g 2-diphenylacery1-1,3-indanedione-1-hydrazone in 20 nil water, filter after shoit warming and add 0.3 ml 36% hydrochloric acid. 162. Iodine — potassium iodide acidic for alkaloids. Treatment: Dry with cold air. Spray solution: H. Thommen, O. Wiss, Z. Erngirungswiss. 1963, Suppl. 3, 18. Dissolve 1 g iodine and 10 g potassium iodide in 50 ml water and add Literature: 2 ml glacial. acetic acid, Make up this solution to 100 ml with water. Chemicals: Literature: F. Sanravy, not published. 2-Dipbenylacetyl-1,3-indanedione-1-hydrazone Chemicals: Hydrochloric acid fuming min. 37% (1.19) G R, Cat. No. 317 Iodine G R, Cat. No. 4761 Potassiunz iodide G R, Cat. No. 5043 as general detection reagent. 160. Iodine Acetic acid glacial min. 96% (1.06) G R, Cat. No. 62 Place the chromatogram into a chamber in which some crystals of iodine have been placed. Iodine vapour is more quickly generated by gently warming the chamber. Many organic compounds show brown spots. 163. Iodine — potassium iodide for organic compounds. Modification: Place the chromatogram 5 min into a strong iodine atmosphere or Spray solution: Dissolve 0.2 g iodine and 0.4 g potassium iodide in 100 nil water. solution of iodine in chloroform. Excess iodine evaporates on spray with a 5% Literature: A. Zaffaroni, R. B. Burton, H. Kentmann, Science 111, 6 (1950). standing in the air. After spraying with 1% aqueous starch solution the spots turn A. Bettschart, H. Fliick, Pharm. Acta Helv. 31, 260 (1956). blue. The background also turns blue if there is too much iodine still on the layer (test J. BUchi, H. Schumacher, Pharm. Acta Helv. 32, 194 (1957). on a corner or part of the covered layer). • Chemicals: Literature: G. C. Barret, Nature 194, 1171 (1962). Iodine G R, Cat. No. 4761 A. Bettschart, H. Fliick, Pharm. Acta Hely. 31, 260 (1956). Potassium iodide GR, Cat. No. 5043 G. Brame, Nature 163, 651 (1949). R. Munier, M. Macheboeuf, Bull. Soc. chim. biol. 31, 1144 (1949). R. Munier, Bull. soc. chim. France 19, 852 (1952). 164. Iodine — sulfanilic acid — N-(1-naphthyl)ethylenediamine Chemicals: for hydroxylamines (Csaky reagent). Iodine G R, Cat. No. 4761 Solution a: 1.3% solution of iodine in acetic acid. Chloroform G R, Cat. No. 2445 Solution b: 1% Starch soluble G R, Cat. No. 1252 su]fanilic acid solution in 30% acetic acid. Spray solution I: Prepare freshly before use a mixture of equal parts of a and b. 161. Iodine azide for sulfur-containing amino acids, sulfides Spray solution II: 0.1% aqueous solution of N-(1-naphthyl)ethylenediammonium and penicillins. dichloride. Iodine azide solution: Freshly prepared solution of 3 g sodium azide in 100 ml iodine Procedure: Spray with I and subsequently with II. solution 0,05 molI2/1. Literature: J. M. Bremmer, Analyst 79, 138 (1954). Dry iodine azide is explosive! Chemicals: Iodine azide-starch reagent: Iodine G R, Cat. No. 4761 Spray solution I: Freshly prepared solution of 1 g sodium azide in 100 ml 0.005 N N-(1-NaphthAethylenediammonium dichloride G R, Cat. No, 6237 iodine solution. Sulfanilic acid G R, Cat. No. 686 Acetic acid glacial min. 96% (1.06) G R, Cat. No. 62 Spray solution II: 1% aqueous starch solution. 49 Literature: A. Norrfalise, M. H. Grosjean, J. Chromatog. 16, 236 (1964). 165. Iodine — sulfuric acid for organic compounds containing nitrogen, polyethylene glycols and polyethylene glycol derivatives. Chemicals: 1ron(111) chloride G R, Cat. No. 3943 Mix equal parts of iodine solution 0.5 mol 12 /land 10% sulfuric acid. Spray solution: Perchloric acid about 20% (1.12) G R, Cat. No. 516 Literature: H. Feltkamp, F. Koch, J. Chromarog. 15, 314 (1964). Nitric acid min. 65% (1.4) G R, Cat. No. 454 Chemicals: Iodine solution 0.05 mol 12" Titrisol, Cat. No. 9910 170. Iron(III) chloride — potassium hexacyanoferrate(III) arsenite Sulfuric acid 95— 97% (1.84) G R, Cat. No. 731 for thyroid hormones and other iodine containing compounds. Solution a: Dissolve 2.7 g iron(1111) chloride in 100 ml hydrochloric acid 0.2 mol/l, 166. Iron(III) chloride for phenols and hydroxamic acids. Solution h: 3.5% aqueous potassium hexacyanoferrate(111) solution. Spray solution: 1-5% solution of iron(111) chloride in hydrochloric acid 0.5 mol/1.. Solution c: Dissolve 5 g sodium memarsenite in 30 mI sodium hydroxide solution Hydroxamic acids turn red, phenols blue or greenish. Note: 1 mold at 0°C and niix with 65 ml hydrochloric acid 2 mol/1 with stirring. Fink, Proc. Soc. Expl. Bic. Med. 70, 654 (1949). Literature: K. Fink, R. M. Spray solution: Mix 5 parts a, 5 parts 13 and 1 part c. Chemicals: Dry the chromatogram with precaution at 50 °C and spray, cover with a second glass 1ron(111) chloride G R, Cat. No. 3943 plate and store in darkness for 15 min. Iodine containing compounds show light blue Hydrochloric acid 0,5 molll Titrisoff , Cat. Nn. 9971 spots on yellowish background. Literature: E. Zappi, J. Chrornatog. 31, 241 (1967). 167. Iron(III) chloride — iodine for xanthine derivatives. Spray solution: Dissolve 5 giron(11I) chloride and 2 g iodine in a mixture of 50 nil lron(111) chloride G R, Cat. No. 3943 acetone and 50 nil 20% aqueous tartaric acid solution_ Potassium hexacyanoferrate(11) G R, Cat, No. 4973 Literature: J. Zaroak, S. Pfeiffer, ['hormone 19, 216 (1964), Sodium inetaarsenite G R, Cat. No. 6287 Chemicals: Sodium hydroxide solution 1 molt!, Cat. No, 9956 _ Iron(III) chloride G R, Cat. No_ 3943 Hydrochloric acid min. 25% (1.125) G R, Cat. No. 316 L(+)Tartaric acid G R, Cat. No. 804 Iodine G R, Cat. Nc. 4761 171. Iron(111) chloride — sulfosalicylic acid for thiophosphate esters. Acetone G R, Cat. No. 14 Spray solution 0.1 % solution of iron(1II) chloride in 80% ethanol. Spray solution II: 1 1 ., solution of sulfosalicylic acid in 80% ethanol. 168. Iron(III) chloride — perchloric acid for indoles Procedure: (Salkowsky reaction). Place the chromatogram 10 min into a bromine atmosphere and spray subsequently with L Dry 15 niin at room temperature and spray with IL Spray solution: Mix 1 ml aqueous iron(III) chloride solution 0.5 mol/1 with 50 ml White spots on violet background. 35% perchloric acid. Literature: M. Salome, J. Chromatog. 16, 476 (1964), Treatment: Hear 5 min at 60°C. Chemicals: Blow vapours of aqua regia over the layer for intensivarion of the colours. Iron(III) chloride G R, Cat. No, 3943 S. A. Gordon, R. P. Weber, Plant. Physiol. 26, 192 (1951). Literature: Sulfosalicylic acid G R, Cat. No. 691 Chemicals: Bromine G R, Cat. No. 1948 Iron(111) chloride G R, Cat. No. 3943 Ethanol abs. G R, Cat. No. 972 Perchloric acid about 60% (1.35) G R, Cat. No. 518 Hydrochloric acid min. 25% (1.125) G R, Cat. No. 316 172. Iron(III) chloride — sulfuric acid for bile acids. Nitric acid min.. 65% (1.4) G R, Cat. No. 'i54 Spray solution: Dissolve 2 g iron (III) chloride in 83 nil anhydrous 1-butanol and mix with 15 ml 97% sulfuric acid. 169. Iron(III) chloride — perchloric acid for phenothiazines. Treatment: After drying for 15 min at room temperature heat 25-30 min with con- Mix 5 ml 5% aqueous iron(111) chloride solution with 45 ml 20% Spray solution: jugated bile acids, 45-50 min with free bile acids. perchloric acid and 50 ml nitric acid. Literature: W. L. Anthony, W. T. Beher, J. Chromatog. 13, 567 (1964). Colour reaction,

50 51 Treatment: Heat 30 min at 80-85°C or better inspect the chromatogram after Chemicals: standing 20 hours at room temperature. Iron(111) chloride G R, Cat. No. 3943 Literature: J. Barrolier, J. Heilman, E. Watzke, Hoppe-Seylers Z. physiol. Chem. 1-Butanol G R, Cat. No. 1990 304, 21 (1956). Sulfuric acid 95-97% (1.84) G R, Cat. No. 731 Chemicals: Isatin G R, Cat. No. 4734 173. Iron(III) chloride — sulfuric acid for indole derivatives (Salkowsky reaction). Zinc acetate G R, Cat. No. 8802 Acetic acid glacial mite. 96% (1.06) Spray solution: Mix 3 ml aqueous iron(Ill) chloride solution 1.5 mo1/1 with 100 nil G R, Cat. No. 62 2-Propanol G R, Cat. No. water and add 60 ml 97% sulfuric acid. 9634 Heat 5 min at 60°C. Blow vapours-of aqua rcgia over the layer for inten- Treatment: 177. Isonicotinic acid hydrazide for A4-3-Ketosteroids. sivation of the colours. Spraysolution: Dissolve 1 g isoniconnic acid hydrazide (INH) and 1 nil glacial acetic Literature: P. E. Piles, Rev. gen. Got. 64, l (1957). acid in 100 ml ethanol. Chemicals: Procedure: Dry after spraying at room teinperature. 3943 Iron(111) chloride G R, Cat. No. Spots show yellow fluorescence in long-wave UV light. —97% (L84) G R, Cat. No. 731 Sulfuric acid 95 Literature: Hydrochloric acid min. 25% (1.125) G R. Cat. No. 316 B. P. Lisboa, Acta Endocrinol. 43, 47 (1963), B. P. Lisboa, J. Chromatog. 16, 136 (1964). Nitric acid min. 65% (1.4) G R, Cat. No. 454 Chemicals: 174. Iron(III) thiocyanate for peroxides. lsonicotinic acid hydrazide Acetic acid glacial min. Solution a: 4% aqueous iron(III) sulfate solution. 96% (1.06) G R, Cat. No. 62 Ethanol abs. G R, Cat. No. 972 Solution b: 1.3% solution of ammonium thiocyanate in acetone. Mix freshly before use 10 nil a and 15 ml b. Spray solution: 178. Kojic acid for metal ions. Fast appearence of brown-red spots (iron(111) thiocyanate) shows the presence Note: Spray solution: Dissolve 0.1 kojic.acid in 100 ml 64% ethanol. of peroxide compounds. Note: Inspect fluorescence under UV light. E. Stahl, Cherniker-Ztg. 82, 323 (1957). Literature: Literature: E. knappe, D. Peteri, Z. anal. Chem. 190, 386 (1962). F. H. Pollard, J. F. W. McOmie, I. I. M. Elbeih, Nature 163, 292 (1949). Chemicals: Chemicals: Koji( acid 15- lron(II) sulfate G R, Cat. No. 3965 hydroxy-2-Iiydroxymethylpyrone-(4)1 Cat. No. 5193 Ethanol abs. G R, Cat. No. 972 Anuninuni thiocyanate G R, Cat. No. 1213 Acetone G R., Cat. No. 14 179. Lead acetate basic for flavonoids. 175. Isatin — sulfuric acid for thiophene derivatives. Spray solution: 25% aqueous solution of basic lead acetate. Spray solution: Dissolve 0.4 g isatin in 100 nil 97% sulfuric acid. Fluoresceing spots in long-wave UV light. Literature: Treatment: Heating to 120°C is occasionally needed. L. Horhammer, H. Wagner, K. Hein, J. Chromatog. 13, 235 (1964). Variously coloured spots. R. Neu, P. Hagedorn, Narurwissenschaften 40, 411 (1953). Chemicals: Literature: R. F. Curtis, G. T. Phillips, J. Chromatog. 9, 366 (1962). Lead(II) hydroxide acetate, Cat. No. 7414 Chetnicals: G R, Cat. No_ 4734 [satin 180. Lead(IV) acetate for 1,2-diol groups. Sulfuric acid 95-97% (L84) G R, Cat. No. 731 Spray solution: 1% solution of lead(IV) acetate in benzene. (Prepare freshly!) 176. 'satin — zinc acetate for amino acids. Treatment: Heat 5 min at 110°C. Spray solution: Dissolve 1 g isatin and 1.5 g zinc acetate in 100 ml 95% isopropanol White spots on brown background. by warming to 80°C and add 1 nil glacial acetic acid after cooling. The reagent is Literature: J. Wright, Chem. & Ind. (London) 1963, 1125. stable stored in refrigerator. 53 52 Chemicals: Solution b: 0.2% ehtanolic diphenylcarbazone solution. Lead(IV) acetate, Cat. No. 807418 Spray solution: Mix freshly before use equal parts of a and b. Benzene G R, Cat. No. 1783 Pink spots on violet background. Literature: F. K. J. Christensen, T. Vos, T. Huizanga, Pharm. Weekblad 100, .517 181. Lead(IV) acetate - rosaniline for 1,2-dial groups. (1965). Spray solution]: Dissolve 3 g lead (11, IV) oxide in 100 nil acetic acid with occasional B. Spray solution 0.1% ethanolic diphenylcarbazone solution. stirring until completely dissolved. Spray solution II: 0.33% mercury(II) nitrate solution m nitric acid 0.05 mo1/1. Dissolve 0.05 g rosaniline base in a mixture of 10 parts glacial Spray solution II: Procedure: Spray with l until the plate is faintly pink, then spray with H. acetic acid and 90 parts acetone. Pink spots on violet background, the latter is bleached by sunlight or UV light and the 0.1% merhanolic fuchsine solution may be used equally. spots turn violet. Procedure: Spray with I. and after 4-5 min with IL Literature: J. Lehmann, V.Karamustafauglu, Scand. J. Clin. & Lab. Invest. 14, 554 Literature; K. Sampson, F. Schild, R. J. Wicker, Cehtn. & Ind. (London) 1961, 82. (1962). Bergner, H. Sperlich, Z. Lebensin.-Untersuch. u. Forsch. 97, 253 (1953). K. G. C. Spray solution 1 (Mercury/ II 1. sulfate solution): Suspend 5 g mercury{11) oxide in Chemicals: 100 ml water and add 20 ml 97% sulfuric acid with stirring. After cooling fill up to Lead(11,IV) oxide, Cat. No. 6080 250 ml with water. Fuchsin (NB), Cat. No. 4041 Spray solution II: 0.01% diphenylcarbazone solution in chloroform. Acetic acid glacial min. 96`;'; (1,06) G R, Cat. No. 62 Procedure: Spray with I, dry and spray with 11. Acetorge G R, Cat_ Nel. 14 Methanol G R, Cat. No. 6009 Literature: 1. Sunshine, E. Rose, J. Le Beau, Clin, Cheni. 9, 312 (1963). Chemicals: 182. Leukomethylene blue for ubi-, plasto- and tocopherylquinones. Mercury(11) chloride G R, Cat. No. 4419 Dipbenylcarbazone G R, Cat. No. 3087 Spray solution: Add a suspension of 0.25 g zinc powder in 1 ml glacial acetic acid to Ethanol abs. G R, Cat. No. 972 0.02% solution of methylene blue in acetone. 5 ml Mercury(II) nitrate G R, Cat. No. 4435 Literature: T. W. Goodwin, Lah. Practice 1964, 295. Nitric acid min. 65"/0 (1.4) G R, Cat. No. 454 Chemicals: Mercury(II) oxide yellow G R. Cat. No. 4461 Zinc powder G R, Cat. No. 8789 Sulfuric acid 95-97% (1.84) G R, Cat. No. 731 Methylene blue B, Cat, No. 1283 Chloroform G R, Cat. No. 2445 Acetic acid glacial mnr, 96% (1.06) G R, Cat. No.. 62 Acetone G II, Cat. No. 14 185. Mercury(II) chloride - potassium iodide for steroid alkaloids 183. Magnesium acetate for anthraquinone glycosides and (Meyer reagent). PC. their aglucones. Spray solution 1: Dissolve 13.55 g mercury(II) chloride and 49.8 g potassium iodide methanolic magnesium acetate solution. Spray solution: 0.5% separately each in 20 nil water. Mix both solutions and fill up with water to 11. Before Procedure: After spraying dry 5 min at 90°C. spraying add 1 parr 17% hydrochloric acid to 10 parts of this solution. Orange to violet colour. Spray solution ff: Dissolve .5 g zinc chloride in 80 ml water and add 15 ml 36% Literature: S. Shibita, M. Takido, O, Tanaka, J. Am, Chem. Soc. 72, 2789 (1950). hydrochloric acid. Chemicals; Spray solution III: 15% ammonia solution.. Magnesium acetate G R, Cat. No. 5819 Procedure; After spraying with 1, the steroid alkaloids appear as faint yellow spots. Methanol C R, Cat. No. 6009 Rinse the chromatogram 10 min with water and, after removal of the water, spray with II and subsequently with 111. 184. Mercury(II) chloride - diphenylcarbazone for barbiturates. Note; The resulting dark brown spots are not stable for a prolonged period. A. Solution a: 2% ethanolic mercury(II) chloride solution. Literature: R. Tschesche, R. Petersen, Chem. Ber. 87, 269 (1953).

54 cc Chemicals: 189. Methylumbelliferone for heterocyclic compounds containing Mercury(II) chloride G R, Cat. No. 4419 nitrogen (fluorescence indicator). Potassium iodide G R, Cat. No. 5043 Spray so/zit/on: Dissolve 0.02 g 4-methylumbelliferone in 35 nil ethanol and fill up to Hydrochloric acid fuming min. 37% (1.19) G R, Cat. No_ 317 100 ml with water. Zinc chloride G R, Cat. No. 8816 Treatment: Ammonia solution min. 25% (0.91) G R, Cat. No_ 5432 Place the chromatogram into a chamber sarurared with ammonia and inspect in long-wave UV light. Literature: I. M. Hais, K. Macek, Handbuch der Papierchroinatographie 1, p. 759, 186. Mercury(I) nitrate for barbiturates. G. Fischer, Jena 1958. Spray solution: 1% aqueous mercury(1) nitrate solution. Chemicals: Literature: J. Baumler, Mi.rr. Gehiete Lebensm. u. Hygiene 48, 135 (1957). 4-Methylumbelliferone R. Deininger, Arzneimittel-Forsch. 5, 472 (1955). Ethanol ahs. G R, Cat. No. 9'2 Chemicals: Ammonia solution min. 25% (0.91) G R, Cat. No. 5432 Mercury(1) nitrate G R, Cat. No. 4417 190. Methyl yellow for chlorinated insecticides. 4-Methoxy-2-nitroaniline diazotised for the identification 187. Spray solution: Dissolve 0.1 methyl yellow in 100 ml 75% ethanol. of vitamin C. Procedure: After spraying dry the chromatogram ar room temperature and irradiate Solution a: Dissolve 0.5 g 4-methoxy-2-nitroaniline in 125 ml glacial acetic acid. with UV light without filter for 5 min. Dilute the solution to 250 ml with 10% sulfuric acid. Red spots on yellow background. 0.2% aqueous sodium nitrite solution. Solution b: Literature: L. F. Krzeminsky, W. A. Landmann, J. Chromatog. 10, 525 (1963). Spray solution 1: Mixe equal parts of a and b. Chemicals: Spray solution 11: Sodium hydroxide 2 mo1/1, 4-Dimethylaminoazobenzene (methyl yellow), Cat. No. 3055 Procedure: Spray with 1, and then with Ethanol abs. G R, Cat. No. 972 Blue spots on orange background. Literature: N. Schmall, C. W. Pifer, E. G. Wollish, Anal. Chem. 25, 1486 (1953). 19i. Millon's reagent for phenols, phenol ethers and phenol ether Cheinic.als: glycosides. 4-Methoxy-2-nitroaniline, Cat. No. 6225 Spray solution: Dissolve 5 g mercury in 10 g fuming nitric acid and add 10 ml water. 96",,, (1.106) G R, Cat. No. 62 Acetic acid glacial min. Treatment: Heating at 100-110°C often produces colour changes. Sodium hydroxide solution 2 moil!, Cat. No. 9136 Literature: Sulfuric acid 95-97% (1.84) G R, Cat. No. 731 E. Stahl, P. J. Schorn, Hoppe-Seylers Z. physiol. Chem. 325, 263 (1961). Chemicals: Sodium nitrite G R, Cat. No. 6549 Mercury G R, Cat. No. 4403 Nitric acid Ironing 100% (1_52) G R, Cat. No. 455 188. Methylene blue for sulfate esters of steroids. Spray solution: Dissolve 0.025 g methylene blue in 100 nil sulfuric acid 0.025 mo1/1. 192. Before use dilute 1 part of the spray solution with 1 parr acetone. Molybdatophosphoric acid Spray reagent 3.5% for chromatography. Note: The sulfate esters show differently coloured spots on blue background. On development with chloroform the formed colour complexes migrate and leave white Visualisation reagent ready for use in aerosol cans. Cat. No. 331 spots on blue background. 193. Literature: O. Crepy-, O. Judas, B. Lachese, J. Chromatog. 16, 340 (1964). Molybdatophosphoric acid for reducing compounds, lipids, sterols and steroids. Chemicals: 4-Methylene blue B, Cat. No. 1283 A. Spray solution: 5-10% ethanolic rriolybdatophosphoric acid. Acetone G R, Cat. No. 14 Treatment: Heat at 120`C until maximal visualisation of the spots. Chloroform G R, Cat. No. 2445 Note: Treatment with ammonia vapour produces a colourless background.

56 57 B. Spray solution: 20% solution of molybdatophosphoric acid in ethanol or ethylene 197. 1,3-Naphthalenediol — sulfuric acid for sugars. glycol monomethylether (2-rnethoxyethanol). Solution a: 0.2 ethanolic solution of 1,3-naphthalenediol. Antioxidants show blue spots after 1-2 min. Solution b: 20% sulfuric acid. Literature: D. Kritschevsky, M. C. Kirk, Arch. Biochem_ Biophys. 35, 346 (1952). Spray solution: Prepare freshly before use a mixture of equal parts a and h. A. Scher, Fette u. Seifen, Anstrichmittel 61, 345 (1959). Treatment: Heat 5-10 min at 100-105°C. Chemicals: Literature: M. Molybdatophosphoric acid G R, Cat. No. 532 Lato, E. Brunelli, G. Ciuffini, J. Chromarog. 34, 26 (1968). Ethanol abs. G R, Cat. No. 972 Chemicals: Ethylene glycol monornethyl ether for synthesis, Cat. No. 800858 1,3-Naphthalenediol G R, Cat. No. 6252 Ammonia solution min. 25% (0.91) G R, Cat. No. 5432 Sulfuric acid 95-97% (1.84) G R, Cat. No. 731 Ethanol abs. G R, Cat. No. 972 194. Molybdatophosphoric acid alkaline for estrogens. Spray solution 1: 8% methanolic solution of molybdatophosphoric acid. 198. 1,3-Naphthalenediol — trichloroacetic acid for sugars Spray solution 11: 2.5% aqueous potassium hydroxide or 3% aqueous sodium and uronic acids. hydroxide solution, Solution a: 0.2% ethanolic 1,3-naphthalenediol solution. Procedure: Spray with 1 and subsequently with II. Solution b: 20% aqueous trichloroacetic acid solution. Note: Instead of spraying with 11 place the chromatogram into a chamber saturated Spray solution: Mix freshly before use equal parts of a and b. with ammonia. Treatment: For ketoses heat 5-10 min at 100-105 °C, for uronic acids 10-15 min in B. Hoffmann, J. Chromatog. 34, 269 (196S). Literature: moist atmoshphere (water bath) at 70-80 °C. Chemicals: Note: The presence of collidine and pyridine interferes with the colour reaction. Molybdatophosphoric acid G R, Cat. No. 532 Instead of 1,3-naphthalenediol resorcinol, orcinol, phloroglu,cinol or 1-naphthol may Potassium hydroxyde G R, Cat. No. 5033 be used. One part trichloroacetic may be replaced by Vail part phosphoric acid. Sodium hydroxide G R, Cat. No. 6498 Literature: S. M. Partridge, Biochem.']. 42, 238 (1948). Ammonia solution min. 25% (0.91) G R, Cat. No, 5432 Chemicals: Methanol G R, Cat. No. 6009 1,3-Naphthalenediol G R, Cat. No. 6252 Trichloroacetic acid G R, Cat. No. 807 195. Morin for aluminium ions. Ethanol abs. G R, Cat. No. 972 solution of morin in glacial acetic acid. Spray solution: l% 1-Naphthol G R, Cat. No. 6223 Pronounced light green fluorescence in long-wave UV light. Orcinol, Cat. No. 7093 1954 (1953). Literature: T. V. Toribara, R. E. Sherman, Anal. Chem. 25, Phlorogliwinol G R, Cat. No. 7266 Chemicals: Resorcinol G R, Cat. No. 7593 Morin G R, Cat. No. 6098 ortho-Phosphoric acid. min, 85% (1.71) G R, Cat. No. 573 Acetic acid glacial min. 96% (1.06) G R, Cat. No. 62

196. 1,3-Naphthalenediol — phosphoric acid for sugars. 199. 1-Naphthol — hypobromite for arginine and other guanidine derivatives (Sakaguchi reagent). Spray solution: Mixture of 100 ml 0.2% ethanolic 1,3-naphthalenediol solution with 10 ml 85% phosphoric acid. Spray solution 1: Solution of 0.1% 1-naphthol in sodium hydroxide solution 1 mo1/1, Spray Treatment: Heat 5-10 min at 100-105 °C. solution IL: Mixture of 100 ml 5% aqueous sodium hydroxide and 2 ml. Literature: V. Prey, H. Berbaek, M. Kausz, Mikrochim, Acta 1961, 968. Procedure: Spray with I and then with 11. Note: Chemicals: For the detection of streptomycine it is recommended to spray with a mixture 1,3-Naphthalenedio1 G R, Cat. No_ 6252 of 50 ml aqueous sodium hypochlorite solution (13% activated chlorine) and 50 nil ortho-Phosphoric acid. min. 85% (1.71) G R, Cat. No. 573 ehtanol instead of spraying with H. Ethanol abs. G R, Cat. No. 972 Literature: R. Acher, C. Crocker, Biochem. biophys. Acta 9, 704 (1952).

58 59 Procedure: Chemicals: Heat at 70-80°C and inspect the development of the spots. First pink, after prolonged heating blue spots. 1-Naphthol G R, Cat. No. 6223 Sodium hydroxide G R, Cat. Nu. 6498 Literature: E. Richter, J. Chromatog. 18, 164 (1965). Bromine G R, Cat. No. 1948 C. W. M. Adams, Nature 192, 331 (1961). • Sodium hypochlorite solution Chemicals: Ethanol abs. G R, Cat. No. 972 1,2-Naphthoquinone-4-sulfonic acid sodium salt G R, Cat. No. 6531 Perchloric acid alit. 601/4 (1.53) G R, Cat. No. 518 1-Naphthol — sulfuric acid for sugars. Formaldehyde solution min. 37% G R, Cat. No. 4003 200. ethanolic solution of 1-naphthol, 6_5 nil 97% Spray solution: Mix 10.5 ml 151/4 Ethanol abs. G R, Cat. No, 972 sulfuric acid, 40.5 ml ethanol and 4 ml water. 204. Treatment: Heat 3-6 min at I00°C. 1-Naphthylamine for 3,5-dinitrobenzoic acid esters and dinitrobenzamides. Literature: H. jacin, A. R. Mishkin, J. Chromatog. 18, 170 (1965). Spray solution 1: 0.5 % ethanolic 1-naphthylamine solution, Cherincals: Spray solution 11: 10% 1-Naphthol G R, Cat. No. 6223 methanolic potassium hydroxide solution. Sulfuric acid. min. 95-97'li (1.84) G R, Cat. No. 731 Procedure: Spray with 1 and then with 11. Ethanol abs. G R, Cat. No. 972 Spots show red-brown colour. Literature: R. G. Rice, G. J. Keller, J. G. Kirchner, Anal. Cheni. 23, 194 (1951). 201. 1-Naphthoquinone-sulfonic acid sodium salt for amino acids Chemiea/s: (Folio reagent). sulfonic 1-Ni2phthylamine G R. Cat, No. 6245 Prepare freshly a solution of 0,2 g 1,2-naphthoquinone-4- Spray solution: Ethanol abs. G R, Cat. No. 972 sodium salt in 100 ml 5% aqueous sodium carbonate solution. acid Methanol G R, Cat. No. 6009 Spray and dry the chromatogram at room temperature. No further treat- Procdure: Potassium hydroxide G R, Cat. No. 503 3 ment. Amino acids show various colours. 205. Nessler's reagent for alkaloids. Literature: D. Muting, Naturwissenschaften 39,303 (1952), Spray so/le/ion: Nessler's reagent (s. spray reagent no. 284). K, V. Giri et al., Naturwissenschaften 39, 548 (1952), Note: Apomorphine, hydrastinine and physostigmine show colour reaction. Literature: Chemicals: 6531 O. E. Schultz, D. StrauS, Arzneimittel-Forsch. 5, 342 (1955). 1,2-Naphthoquinone-4-sulfonic acid sodium salt G R, Cat. No. Chemicals: 6391 Sodium carbonate 10-hydrate G R, Cat. No. Nessler's reagent

202, Naphthoquinone-sulfonic acid sodium salt for aromatic amines. 206. Ninhydrin Dissolve 0.5 g 1,2-naphthoquinone-4-sultonic acid sodium salt in Spray solution: Spray reagent 0.1% for chromatography. 95 ml water and add 5 ml glacial acetic acid. Filter off from insoluble parts. Visualisation reagent ready for use in aerosol cans. Note: inspect the colour of the spots after 30 min. Cat. No. 6758. Literature: R. B. Smyth, G. G. McKeown, J. Chromatog. 16, 454 (1964). 207. Ninhydrin for amino acids, amines and amino-sugars. Chemicals: 1,2-Naphthoquinone-4-sulfonic acid sodium salt G R, Cat. No. 6531 A. Spray solution: Dissolve 0.3 g ninhydrin in 100 ml I-butanol and add 3 ml glacial 62 acetic acid. Acetic acid glacial min. 96% (1.06) G R, Cat. No. B. Spray solution: 0.2% ethanolic ninhydrin solution. 203. Naphthoquinone-sulfonic acid — perchloric acid sterols. Treatment: Heat at 110°C until maximal visualization of the spots. Dissolve 0.1 g 1,2-naphthaquinone-4-sulfonic acid in mixture of For pantothenic acid heat at 160°C. Spray solution: 50 ml ethanol, 25 ml 60% perchloric acid, 25 ml 37% formaldehyde solution and Literature: R. A. Famv, A. Niederwieser, G. Pataki, M. Brenner, Hely. Chim. Acta 44, 2022 (1961). 22.5 ml water.

60 61 209. Ninhydrin — copper(I1) nitrate for amino acids A. R. Patton, P. Chism, Anal. Chem. 23, 1683 (1951). (polychromatic detection). Chemicals: Solution a: 6762 Dissolve 0.1 g ninhydrin in 50 ml ethanol and add 10 rill glacial acetic acid Ninhydrin G R, Cat. No. and 2 ml 2,4,6-trimethylpyridine. Acetic acid glacial min. 96% (1.06) G R, Cat. No. 62 Solution b: 1% ethanolic copper(II) nitrate solution. 1-Butanol G R, Cat. No. 1990 Spray solution: Ethanol abs. G R, Cat. No. 972 Before use mix solution a and b in the proportion 50:3_ Treatment: Heat the chromatogram until the colour development is just- beginning. In transmitted light the gradual intensification of colours on the warm plate can be Stabilisation of ninhydrin spots. observed. Some amino acids show first small points of colours only, they should be Mix 1 nil saturated aqueous copper(11) nitrate solution with 0.2 nil Spray solution: marked with a sharp pencil. In this way one can often detect individual spots which 10% nitric acid and 100 nil ethanol. later merge into each other. Some amino acids show characteristic colours. They Procedure: Spray the ninhydrin spots with the spray solution and place the chroma- differ amongst themselves also in the speed with which coloured products a re formed. togram into a chamber with ammonia. The red copper complex is stable as long as no Literature: M. Brenner, A. Niederwieser, Experientia 16, 378 (1960). free hydrogen ions or strong complex forming compounds are present. Chemicals: 77 (1951). Literature: E. Kawerau, T. Wieland, Nature 168, Ninhydrin G R, Cat. No. 6762 Chemicals: Copper(II) nitrate G R, Cat. No. 2752 Copper(II) nitrate G R, Cat. No. 2752 Acetic acid glacial min. 96% (1.06) G R, Cat. No. 62 Nitric acid min. 65% (1.4) G R, Cat. No. 454 2,4,6-Trintetbylpyridine G R, Cat. No. 2635 Ethanol abs. G R, Cat. No. 972 Ethanol abs. G R, Cat. No. 972 Ammonia solution min. 25% (0.91) G R, Cat. No. 5432 210. Ninhydrin — tin(II) chloride for amines. 208. Ninhydrin — cadmium acetate for amino acids and amines. Stock solution: Dissolve by heating 2 g ninhydrin in 40 ml water. Add a solution of Lill up to 500 ml with ethanol a solution of 1 g ninhydrin, 2.5 cad- Spray solution: 0.08 g tin(II) chloride in 50 ml warer and allow to stand. After filtration fo the precipi- mium acetate and 10 ml glacial acetic acid. tate store in the refrigerator. Treatment: Heat 20 min at 120°C. Spray solution: Add 50 ml water and 450 ml 2-propanol to 25 nil of the stock This method is more suitable for detecting heterocyclic amines than the procedure solution. using reagent no. 207. Literature: R. J. Block, Anal. Chem. 22, 1327 (1950). Chemicals: Alternative: Ninhydrin G R, Cat. No. 6762 Dip solution: Dissolv.. 0.1 g cadmium acetate in 10 ml water, add 5 ml glacial acetic acid and 100 ml acetone and dissolve 1 g ninhydrin. This order of the reagents for Tin(II) chloride G R, Cat. No. 7815 the preparation of the dip solution must he observed. The solution is stable in the 2-Propanol G R„ Cat. No. 9634 refrigerator. 211. Procedure: After dipping place the chromatogram for colour development 30 min Nitric acid for alkaloids and amines. into a chamber containing concentrated sulfuric acid. Spray solution: Add 50 drops 65% nitric acid to 100 ml ethanol. Literature: J. Barrolier, J. Heilmann, E. Watzke, Hoppe-Seylers Z. physiol. Chem. Note: Inspect in UV light. 309, 219 (1957). The spray solution may be used in this or higher concentration also in TLC for the Chemicals: identification of other organic compounds. Frequently fluorescent spots appear only Cadmium acetate G R, Cat. No. 2003 after prolonged heating at 120 °C. Ninhydrin G R, Cat. No. 6762 Literature: H. Schmid, J. Kebrle, P. Karrer, Helv. Chim. Acta 35, 1864 (1952). Cat. No. 62 Acetic acid glacial min. 96% (1.06) G R, Chemicals: Acetone G R, Cat. No. 14 Ethanol abs. G R, Cat. No. 972 Ethanol abs. G R, Cat. No. 972 Nitric acid min. 65% (1.4) G R, Cat. No. 545 Sulfuric acid 95-97% (1.84) G R, Cat. No. 731

63 62 hydrochloric acid and 30 ml water by warming. After cooling at 5 °C add a solution o 212. 4-Nitroaniline dia.,ctised (acidic) for plasticisers. f Potassium hydroxide solution 0.5 mol/l in ethanol. 8 g sodium nitrite in 20 ml water and then 60 ml 40% hydrofluoboric acid. Filter off Spray solution I: the yellow precipitate, wash with hydrofluoboric solve 0.8 g 4-nitroaniline in 250 ml water, add 20 ml 25% acid, ethanol and ether and dry in a Spreay solution IL Dis vacuum desiccator. hydrochloric acid and dropwise 5% aqueous sodium nitrite solution until the Spray solution 1: Prepare freshly a 1% 4-nitrophenyldiazonium fluoborate solution solution is colourless. M acetone. Spray with I, dry 15 min at 60'C and spray with 11. Procedure: Spray solution II: 0.1% methanolic potassium hydroxide .solution.. Yellow to orange spot• Procedure: Spray with I, then with II. pius-Peerehoom, J. Chromatog. 4, 323 (1960). Literature: J. W. Co Literature: 77 (1965). J. H. Freeman, Anal. Chem. 24, 955 (1952). D. Braun, Chimia 19, H. Seehoth, H. Gbrsch, Chem. Techn. 15, 294 (1963). Chemicals: Chemicals: • 4-Nitroaniline, Cat. No. 6760 4-Nitroaniline, Cat_ No. 6760 Sodium nitrite G R. Cat. No. 6.549 G R, Cat. No. 316 Sodium nitrite G R, Cat. No. 6549 Hydrochloric acid 25% (1.125) lirdrofluoboric acid, Cat. No. 171 Potassium hydroxide G R, Cat. No. 5033 cat. No_ 972 Potassium hydroxide 0.5 moll! in methanol, Cat, No. 9.3,51 Ethanol abs. G R, Acetone G R, Cat. No_ 14 213. 4-Nitroaniline dia.zotised for phenols, phenol carboxylic acids, Ethanol abs, G R, Cat. No. 972 coupling amines And heterocyclic compounds. Diethyl ether G R. Cat. No, 921 Hydrochloric acid fuming aim, 36% (1.18) G R, Cat. Spray solution: Mix 1 0 nil 0.1% aqueous 4-nitroaniline solution with 10 ml 0.2% No. 320 aqueous sodium nitrite solution and 20 ml 10% aqueous potassium carbonate solution. 216. 2-Nitroso-1-naphthol-4-sulfonic acid for iron ions. Coloured products are formed. Spray solution: O.05% solution of 2-nitroso-1-naphthol-4-sulfonic acid in 70% Literature: A. Sturm, H. W. Scheia, J. Chromatog. 16, 194 (1964). ethanol_ Chemicals: Treatment: Spray with 25% ammonia solution or place the chromatogram into a 4-Nitroaniline, Cat. No, 6760 chamber with ammonia.. Sodium nitrite G R, Cat. No. 654 9 Green spots. G R, Cat. No. 4928 Potassium carbonate Literature: G. B. Heisig, F. H. Pollard, Anal. Chitn. Acta 16, 234 (1957). Chemicals: 214. 4-Nitroaniline diazotised (buffered) for phenols. 2- Nitroso-l-naphthol-4-sulfonic acid Spray solution: Mix Linder cooling 5 ml 0.5% 4-nitroaniline solution in hydrochloric acid 2 moi/1 with 0.5 ml 5% aqueous sodium nitrite solution and add 15 ml 20% Ammonia solution Mill- 25% (0.91) G R, Cat. No. .5432 Ethanol abs. G R, Cat. No. 972 aqueous sodium acetate solution. Literature: H. G. BrM!, W. V. Thorpe, K, White, Biochem. J. 46, 271 (1950). 217. T. Swain, Biochem. J. 53, 200 (1953). Orcinol — iron(III) chloride — sulfuric acid for sugars. C. F. van Sumerc, G. Wolf, H. Teuchy, J. Kint, J. Chromatog. 20, 48 (1965). Solution a: Dissolve 1 g iron(III) chloride in 100 nil 10% sulfuric acid. Chemicals: Solution b: 6% ethanolic orcinol solution. 4-Nitroaniline, Cat. No. 6760 Spray solution: Mix freshly before use 10 ml a and 1 ml b, No. 6549 Sodium nitrite G 14., Cat. Treatment: Heat 10-15 min at 100°C. No. 6267 Sodium acetate G R, Cat. Chemicals: Hydrochloric acid 25% (1.125) G R, Cat. No. 316 Orcinol, Cat. No. 7093 Iron(111) chloride G R, Cat. No_ 215. 4-Nitrophenyldiazonium fluoborate for phenols 3943 and coupling alines. Sulfuric acid 95-97% (1.84) G R, Cat. No. 731 Ethanol abs. GR, Cat. No. 972 4-Nitrophenyldiazorium fluoborate: Dissolve 14 g 4-nitroaniline in 30 nil 36%

64 65 218 Palladium(II) chloride for thiophosphate esters Treatment: Heat 10-15 min at 110°C. Brown spots. and other sulfur compounds. Literature: Spray solution: Dissolve 0.5 g palladium(II) chloride in100 ml water containing a few S. Adachi, J. Chromatog. 17, 295 (1965). drops 25% hydrochloric acid. Chemicals: Phenol G R, Cat. No. 206 Literature: J. Baumler, S. Rippstein, Helv. Chim. Acta 44, 1162 (1961). Sulfuric acid abt. 95-97% (1.84) G R, Cat_ No. 731 Chemicals: Ethanol abs. G R, Cat. .No, 972 Palladium (11) chloride, Cat. No. 7110 Hydrochloric acid min. 25% (1.12.5) G R, Cat. No, 316 223. rn-Phenylenedia.mine for reducing sugars. Spray solution: Dissolve 3.6 g m-phenylenedi.amine dihydrochloridc in 100 ml 219. Paraformaldehyde phosphoric acid for --9"lanum,., steroid 70% ethanol. alkaloids and steroid sapogenins. Treatment: Heat briefly 105°C. g paraforrnaldehyde in l00 m1 85% phosphoric acid Spray solution: Dissolve 0.03 Note: Intensely fluorescent colours in UV light. under shaking. The reagent is stable for several weeks. Literature: S. S. Chernick, I. L. Chaikoff, S. Abraham, J. Biol. Chem. 193, 793 Literature: K. Schreiber, O. Aurich, G. Osske, J. Chromatog. 12, 63 (1963). (1951). Chemicals: Chemicals: Paraformaldebyde, Cat. No. 4005 »r-Phenylenedtainine dihydrochloride G R, Cat. Cat. No. 573 No. 7244 ortbo-Phosphoric acid min. 85% (1.71) G R, Ethanol abs. G R, Cat. No. 972

220. Perchloric acid for steroids and bile acids. 224. p-Phenylenediamine - phthalic acid for conjugated 3-ketosteroids. A. Spray solution (for steroids): 20% aqueous perchloric acid solution. Spray solution: B. Spray solution (for bile acids): 60% aqueous perchloric acid solution. Dissolve 0.9 g p-phenylenediamine and 1.6 g phrhalic acid in 100 ml 1-butanol, saturated with water. Treatment: Heat the chromatogram for about 10 min at 150°C until maximal visu- Treatment: alisation of the spots. Heat 100-110°C. Inspect also in long-wave UV light. Yellow to orange spots. .961). Literature: Literature: H. Metz, Naturwissenschaften 48, 569 1 B. P. Lisboa, Acta Endocrinot 43, 47 (1963).. S. Hara, M. Takeuchi, J. Chromatog. 11, 565 (1963). B. P. Lisboa, J. Chromatog. 16, 136 (1964). Chemicals: Chemicals: Perchloric acid abt. 20% (1.12) C R, Cat, No. 516 p-Phenylenediainine dihydrochloride G R, Cat. No. 7247 Perchloric acid abt. 60% (1.53) G R, Cat. No. 518 Phthalic acid G R, Cat. No. 9611 1-Butanol G R, Cat. No. 1990 for indole derivatives. 221. Perchloric acid - iron(III) chloride 225. 1,2-Phenylenediamine - sulfuric acid for dehydroascorbic acid.. Spray solution: Mix 100 nil 5'Yi aqueous perchloric acid solution with 2 ml 0.05 M Spray solution: Dissolve 0.1 g 1,2-phenylenediamine in a mixture of 50 ml sulfuric iron(III) chloride solution. acid 0.05 mol/1 and 50 ml ethanol. No reaction with isatin and other oxindole derivatives. Note: Literature: S. Ogawa, J. Pharm. Soc. Japan 73, 59 (1953). Literature: T. A. Bennet-Clark, M. S. Tamhiah, N. P. Kefford, Nature 169, 452 Chemicals: (1951). 1,2-Phenylenediarnine, Cat. No. 809721 Chemicals: Sulfuric acid 0.05 moll!, Cat. No. 9984 Perchloric acid abt. 20% (L12) G R, Cat. No. 516 Ethanol abs. G R, Cat. No. 972 Iron(111) chloride C R, Cat. No. 3943 226. 1,2-Phenylenediamine - trichloroacetic acid for u-keto acids. 222. Phenol - sulfuric acid for sugars. Spray solution: Dissolve 0.05 g 1,2-phenylenediamine in 100 ml 10% aqueous trichloroacetic acid solution. Spray solution; Dissolve 3 g phenol and .5 ml 97°/0 sulfuric acid in 95 mi ethanol.

66 67 Procedure: Heat the chromatogram at 100°C for not more than. 2 min. at 120 °C and spray lightly with iL Glycosides of the series A show now orange, of the Green fluorescent spots in long-wave UV light. series C grey-green to grey-blue fluorescence in UV light. Literature: T. 'Wieland, F. Fischer, Naturwissenschaften 36, 219 (1949), Literature: L. Fauconnet, M. Waldesbiihl, Pharm. Acra Hely. 38, 423 (1963). O. Wiss, Hoppe-Seylers Z. physiol. Chem. 293, 106 (1953). Chemicals: Chemicals: ortho-Phosphoric acid nzin. 85% (1.71) G R, Cat. No. 573 1,2-Phenylenediamine, Cat. No. 809721 Potassium bromide G R, Cat. No, 4912 Trichloroacetic acid G R, Cat. No. 807 Potassium bromate G R, Cat. No. 4905 Hydrochloric acid ?nil:. 2S% (1.125) G R, Cat. 227. Phenylfluorone for germanium. No. 316 0.05% solution of phenylfluorone in a mixture of 3 parrs ethanol and Spray solution: 231. Picric acid for epoxides. 1 part 37% hydrochloric acid. Spray solution: Literature: I. M. Ladenhauer, K. Bradacs, F. Hecht. Mikrochim. Acra 1954, 388. 0.05 M ethanolic picric acid solution. Treatment: Chemicals: Place the sprayed chromatogram 30 niin into a chamber with ether/ Phenylfluorone, Cat. No.. 7252 ethanol/glacial acetic acid (80-20-1) and subsequently 1-2 min into a chamber with ammonia. Ethanol abs. G R, Cat. No. 972 Orange spots on yellow background. Hydrochloric acid finning 7f/in. 37% ( / .19) G R, Cat. No..317 Literature: J. A. Fioriti, R. J. Sims, J. Chrornatog. 32, 761 (1968). 228. Phenylhydrazine for dehydroascorbic acid. Chemicals: Spray solution: Dissolve 0.3 g phenylhydrazine and 0.45 g sodium. acetate in 10 nil Picric acid moistened with about SO% water G R, Cat. No. 623 water. Ethanol abs. C R, Cat, No. 972 Diethyl ether G R, Cat. No. 921 Chemicals: Pheylhydrazine G R, Cat. No. 7251 Acetic acid glacial min. 96% (1.06) G R, Cat. No, 62 Sodium acetate G R, Cat. No. 626S Ammonia solution min. 25% (0.91) G R, Cat. No. 54.32' 229. Phosphoric acid for sterols and steroids. 232. Picric acid — alkali for creatinine, glycocyamidine A. Spray solution: Mix 85% phosphoric acid with water 1:1 (volume). (Jaffe reagent). B. .Spray solution: 15% niethanolic phosphoric acid solution. Spray solution 1: I% ethanolic picric acid solution. Spray Procedure: Spray the layer thoroughly until transparent and heat 15-30 min at so/titian 11: 5% ethanolic potassium hydroxide solution. 120 °C. The individual sterols or steroids require varying heating times for attainment Procedure: Spray with I, dry and spray with of maximal colour intensity or fluorescence, Orange colour, Note: All compounds of this class show fluorescence in long-wave UV light. Larger Literature: R. Williams, Iiiochem. Inst. Stud. IV, University of Texas, Publ,, amounts of substance yield spots which are visible in daylight. Austin/Texas No. 5 109, 2.05 (1951). Literature: R. Neher, A. Wettstein, Hely. Chico. Acta 34, 2278 (1951). Chemicals: Chemicals: Picric acid moistened with about 50% water G R, Cat. No. 623 ortho-Phosphoric acid min. 85% (1.71) G R, Cat. No. 573 Potassium hydroxide G R, Cat. No. 5033 Methanol GR, Cat. No. 6009 Ethanol abs. G R, Cat. No. 972

230. Phosphoric acid — bromine for digitalis glycosides. 233. Picric acid — perchloric acid for A5-30-hydroxysteroids. Spray solution 1: 10% aqueous phosphoric acid solution_ Spray solution: Dissolve 0.1 g picric acid in a mixture of 36 ml glacial acetic acid and Spray solution 11: Mix 2 ml saturated aqueous potassium bromide solution, 2 nil 6 nil 70% perchloric acid. saturated aqueous potassium bromate solution and 2 ml 25% hydrochloric acid. Procedure: Heat 3-5 min at 70-80°C. Procedure: Spray with 1 and heat the plate 12 min at 120 'C. Digitalis glycosides of the Yellow-red spots. series B, D and E show blue fluorescence in long-wave UV light. Heat the plate again Literature! W. R. Eherlein, J. C]in. Endocrinol. 25, 288 (1965).

68 69

II Chemicals: Chemicals: Picric acid moistened with about 50% water G R, Cat. No. 623 Potassium hexacyanoferrate(11) G R, Cat. No. 4984 Acetic acid glacial min, 96% (1.06) G R, Cat. No. 62 Ammonium. chloride G R, Cat. No. 1145 Perchloric acid abt. 70% (1.67) G R, Cat. No. 519 Potassium carbonate G R, Cat, No. 4928 Hydrochloric acid fuming min. 37% (1.19) G R, Cat. No. 317 034. Picryl chloride for hydroxylamines, hydrazines Perhydrole (30% by weight H2 02) G R, Cat. No. 7209 and pyridine derivatives. Spray solution: 0.5-1.5% ethanolic picryl chloride solution. 238. Potassium hexacyanoferrate(III) for adrenaline and derivatives. Treatment: Place the chromatogram into a chamber with ammonia. Spray solution: 55, 669 Dissolve 0.1 g potassium hexacyanoferrate(111) in 100 nil 0.S% Literature: W. F. J. Cuilibertson, D. M. Ireland, W. Wolff, Biochem. J. sodium hydroxide solution. (1953). Spots show red colour. J. M. Bremner, Analyst 79, 198 (1954). Literature: A. H. Beckett, M. A. Beaven, A. E. Robinson, J. ['harm. Pharmacol_ 12, Chemicals: 203 T (1960). Picryl chloride (2-chloro-1,3,5-trinitrobenzene) Chemicals: Ethanol abs. G R, Cat. No. 972 Potassium hexacyanoferrate(111) G R, Cat. R, Cat. No. 5432 No. 4973 Ammonia solution min. 25% (0.91) G Sodium hydroxide solution 10% (1.109) G R, Cat, No. 5588 235. Pinaciyptol yellow for alkyl- and arylsulfonic acids. Spray solution: 0.05-0.1% aqueous pinacryptol yellow solution. 239. Potassium hexacyanoferrate(III) for vitamin B1 Yellow to orange fluorescence in long-wave UV light. (thiochrome reaction). Literature: J. Borec4, J. Chromatog. 2, 612 (1959), Solution a: 1% aqueous potassium hexacyanoferrate(III) solution. Chemicals: Solution b: 15% aqueous sodium hydroxide solution. Pinacryptol yellow, Cat. No. 9723 Spray solution: Mix 1.5 ml a with 20 ml water and add 10 nil h. Inspect after drying in long-wave UV right. 236. Potassium hexacyanoferrate(II) for iron(III)ions. Literature: D. Siliprandi, N. Siliprandi, Biochim. et biophys. Acta 14, 52 (1954). Spray solution: Freshly prepared 2% aqueous solution of potassium Chemical's: hexacyanoferrate (II). Potassium hexacyanoferrate(///) G R, Cat. No. 4973 Davies, R. P. Linstead, R. A. Wells, J. Chem. Soc. Literature: F. H. Burstall, G. R. Sodium hydroxide solution min. 27% (1.30) G R, Cat. No. 5591 1950, 516. Chemicals: 240. Potassium hexacyanoferrate(II) G R, Cat. No. 4984 Potassium hexacyanoferrate(III) — iron(III) chloride for reducing compounds, phenols, amines, thiosulfates, isothiocyanates. 237. Potassium hexacyanoferrate(II) — hydrogen peroxide Solution a: 1% aqueous poassium hexacyanoferrate(III) solution. for barbiturates. Solution b: 2% aqueous iron(III) chloride solution. Spray solution!: Dissolve 0.1 g potassium hexacyanoferrateill) in 100 ml water con- Spray solution: Mix freshly before use equal parts of a and b, 5 g ammonium taining 0.5 ml 37% hydrochloric acid. Add to 10 ml of this solution Treatment: Spray with hydrochloric acid 2 mo1/1 for intensivation of colours. chloride and make up to 100 rid with water. Literature: G. M. Barton, R. S. Evans, J. A. F. Gardner, Nature 1970, 249 (1952). Spray solution If: 30"/. hydrogen peroxide solution. M. Gillio-Tos, S. A. Previtera, A. Vimercati, J. Chromatog. 13, 571 (1964), Spray solution 111: 10% aqueous potassium carbonate solution. H. Wagner, L. Hen-hammer, H. Nufer, Arzneimittel-Forsch. 15, 453 (1965). Treatment: Spray with 1 and dry at 100°C. After cooling spray with II and heat Chemicals: 30 min at 150°C. Spray with 111 for intensivation of the yellow and red spots. This Potassium hexacyanoferrate(111) G R, Cat. No. 4973 reaction may be applied afrer detection with mercury(I) nitrate. Iron(111) chloride G R, Cat. No. 3943 Hydrochloric acid Literature: H. Weichsel, Mikrochim. Acta 1965, 325, rlli72. 25% (1.125) G R, Cat. No, 316

70 71 Potassium hexacyan.oferrate(III) — phosphate buffer Sulfidoge0 (sulfur paraffin rods) for generation of hydrogen sulfide, Cat. No, 241. 7992 for adrenaline. Spray solution: 0.44% solution of potassium hexacyanoferrate(Ill) in phosphate 245. Potassium iodide — starch for peroxides. buffer solution, pH 7,8. Spray solution I.- Add to a mixture of 40 ml glacial acetic acid and 10 ml 4% aqueous Noradrenaline appears as brown red spots, adrenaline as light red and methyl- Note: potassium iodide solution a spatula-ripful of zinc powder. adrenaline as white spots on yellow-brown background. Spray solution 11: Freshly prepared 1% aqueous starch solution. Literature: S. Senoh, B. Witkop, J. Am. Chem. Soc. 81, 6222. (1959), Procedure: After filtering off zinc powder, spray with I, dry 5 min at room tempera- Chemicals: ture and spray with 11 until the layer is transparent. Peroxides show blue spots by Potassium hexacyanoferrate(111) C R, Cat. No. 4973 formation of free iodine. di-Sodium hydrogen phosphate solution 1115 mall, Cat. No. 6587 Literature: E. Stahl, Chemiker-Zug. 82, 323 (1958). 242. Potassium hexacyanoferrate(M) —potassium hexacyanoferrate(II) Chemicals: for morphine. Potassium iodide G R, Cat. No. 5043 Zinc powder G R, Cat. No. 8789 Spray solution: Dissolve 57 mg potassium hexacyanolerrate(111) and 7.8 mg potas- Starch soluble G R, Cat. No. 1251 sium hex-acyanoferrate(I1) in 100 nil water. Acetic acid glacial min_ 96% (1,06) G R, Cat. No, 62 Literature: H. J. Kupferberg, A. Burghalter, E. L. Way, J. Chromatog. 16, 558 (1964). 246. Potassium iodine platinate for alkaloids. Chemicals: Spray solution: Add to 5 ml 5% hexachloroplatinic(IV) acid solution 45 nil I0% Potassium hexacyanoferrate(111) G R, Cat. No. 4973 aqueous potassium iodide solution and 100 mi water. Prepare freshly before Potassium hexacyanoferrate(II) G R, Cat, No. 4984 use. Literature: J. Smith, Chromatographic and Electrophoretic Techniques, W. Heine- mann, London 1969, Vol. I, p. 243. Potassium hydroxide methanolic for coum.arins, 519, anthraquinone glycosides and their aglucones. Chemicals: Potassium iodide C R, Cat. No. 5043 Spray solution: 5% rnethanolic potassium hydroxide solution. Inspect the chromato- Hexachloroplatinic(I V) acid solution 10% G R, Cat. No. 7341 gram after drying in daylight and long-wave UV light. M. Hais, Ceskolov. farm. 9, 401 (1960). Literature: Z. Ledinova, 247. Potassium iodine platinate for alkaloids and other organic 13, 537 (1963). L. Horhammer, H. Wagner, G. Burner, Arzneimirtel-Forsch. compounds containing nitrogen. Chemicals: Spray solution: Add to .3 ml 10% hexachloroplatinic(IV) acid solution 97 ml water Potassium hydroxide G R, Cat. No. 5033 and 100 ml 6% aqueous potassium iodide solution. Prepare freshly before use. Methanol G R, Cat. No. 6009 Literature: R. Munier, Bull soc. Chilli. France 19, 852 (1952). 244. Potassium iodide — hydrogen sulfide for heavy metal ions. R. Hilz, F. F. Castano, G. A. Lightbourne, J. Lab. Clin. Med. 54, 632 (1959).. Chemicals: Spray solution: 2% aqueous potassium iodide solution. Hexachloroplatink(IV) acid solution 10% G R, Cat, No. 7341 Dry the plate after spraying and place it into a chamber saturated with Procedure: Potassium iodide G R, Cat. No. 5043 ammonia. After a few minutes place the plate into a second chamber with hydrogen sulfide. 248. Potassium iodine platinate for ketosteroids. PC. it Caution! Hydrogen sulfide is poisonous and explosive! Spray solution: Add to 5 nil 5% hexachloroplatinic(IV) acid solution in hydrochloric H. Seiler, M. Seiler, Helv. Chim. Acta 43, 1939 (1960). Literature: acid 1 mol/l 45 ml 10% aqueous potassium iodide solution and 100 ml water. Chemicals: The reagent is stable for some time when stored in the dark. Potassium iodide G R, Cat, No. 5043 Treatment: After spraying rinse out the excess reagent with water. Ammonia solution min. 25% (0.91) G R, Cat. No. 5432 Literature: R. Iron(11) sulfide fused, Cat. No. 3954 T. Burton, A. Zaffaroni, E. H. Keutniann, J. Clin. Endocrinol. 8, 618 (1958). Hydrochloric acid min. 25% (1.125) G R, Cat. No. 3/6

73 72

Literature: H. Seiler, M. Seiler, Hely. Chim. Acta 44, 939 (1961). Chemicals: F. W. H. M. Merkus, Pharm. Weekblad 98, 947 (1963). Potassium iodide G R, Cat. No. 5043 Chemicals: Hexachloroplatinic(1V) acid, Cat. No. 734() 1-(2- Hydrochloric acid 1 moll! Titrisol ®, Cat. No. 9970 Pyridylazo)-2-naphthol (PAN), Cat. No. 7531 Ethanol abs. G R, Cat. No. 972 249. Potassium permanganate alkaline for reducing compounds Ammonia sohition min. 25% (0.91) G R, Cat. No. 5432 and aromatic polycarboxylic acids. Add to 1"/, aqueous potassium permanganate solution an equal Spray solution: 254. 1(2-PyridyIazo)-2-naphthol (PAN) — cobalt(II) nitrate for volume of 5% aqueous sodium carbonate solution. glucuronides of steroids. B. Maximoy, L. S. Panthinkbina, J. Chromatog, 20, 150 (1965). Literature: O. Spray solution 1: Mix freshly before use 1 part 0.4%, erhanolic PAN solution and I. M. Hais, K. Macek, Papierchromarographie I, G. Fischer, Jena 1958, p, 735. 4 parts methylene chloride (by volume). Chcinicals: Spray solution 11: Mix 8 nil 0.8% aqueous cobalt(II) nitrate solution wirh 4 nil Potassium permanganate G R, Cat. No. .50S2 acetate buffer solution 0.2 mold (pH 4.6) and fill up to 100 ml with water. Sodium carbonate ankvitroirs G R, Cat. No. 6192 Procedure: Spray wirh I until the layer is evenly yellow, dry and spray with 11. Glucuronides show rapidly fading violet spots, the colours of which turn greenish on 250. Potassium permanganate alkaline for sugars and polyalcohols. drying, Spray solution: Dissolve 0.5 g potassium permanganate in 100 inl sodium hydroxide Literature: O. Crept', O. Judas, B. Lachese, J. Chromatog. 16, 340 (1964). solution 1 cool/1. Chemicals: Treatment: After spraying heat the plate at 100'C• 1-(2-Pyridylazo)-2-naphthol (PAN), Cat, No. 7531 , F. Smith, J. Chromatog. 11, 479 (1963). Literature: G. W. Hay, B. A. Lewis- Cobalt(11) nitrate G R, Cat. No. 2536 Chemicals: Standard acetate buffer solution pH 4.66, Cat. No. 78.27 Potassium permanganate G R, Cat. No. 50S2 Ethanol abs. G R, Cat. No. 972 Sodium hydroxide solution 1 roof/1 Titrisol %[ Cat. No. 9956 Dichlorometbane (methylene chloride) Cat. No. 6050

251. Potassium permanganate neutral for easily oxidisable 255. Quercetin for cations of the hydrogen sulfide group, compounds. aluminium, magnesium, uranyl and tungstate ions. 0.05% aqueous potassium permanganate solution. Spray solution: Spray solution: 0.2% ethanolic quercetin solution. Chemicals: Treatment: Spray wirh 25% ammonia solution or place into a chamber with ammo- Potassium permanganate G R, Cat. No. 5082 nia. In long-wave UV light fluorescing spots. 252. Potassium permanganate — sulfuric acid (universal reagent). Literature: A. Weif;, S. Fallab, Hely. Chim. Acta 37, 1253 (1954). acid. Spray solution: Dissolve 0.5 g potassium permanganate in 15 nil 97% sulfuric E. Pfeil, A. Friedrich, T. IX/Rebmann, Z. anal. Chem. 158, 429 (1957). Cautionl Manganese heptoxide is explosive: Chemicals: (1962).. Literature: H. Ertel, L. Horner, J. Chromatog. 7. 268 Quercetin, Cat. No. 7546 Chemicals: Ethanol abs. G R, Cat. No. 972 Potassium permanganate G R, Cat. No. 5082 Ammonia solution min. 25% (0.91) G R, Cat. No. 5432 Sulfuric acid 95 —97% (1,84) G R, Cat. No, 731 256. Quinalizarin for cations. 1-(2-Pyridylazo)-2-naphthol (PAN) for lead, cadmium, cobalt, 253. Spray solution: copper, manganese, nickel, zinc and uranyl ions. 0.05% solution of quinalizarin in 70% ethanol. Treatment: Place the chromatogram into a chamber saturated with ammonia vapour. Spray solution: 0.25% ethanolic solution of PAN. Literature: O. H. Johnson, H. H. Krause, Anal. Chim. Acta 11, 128 (1954). Treatment: Place the plate into a chamber with ammonia.

74 75 260. Rhodamine B Chemicals: 1,2,5,8-Tetrahydroxyantbraquinone (quinalizarin) G R, Cat. No, 8126 Spray reagent 0.1% for chromatography Ethanol abs. G R, Gat. No. 972 Visualisation reagent ready for use in aerosol cans, Car. No. 7596 Ammonia solution Mill. 25% (0.91) G R, Cat. No. 5432 261. Rhodamine B, general spray reagent. 257. p-Quinone for ethanolamine. Spray solution: 0.025-0.25% ethanolic solution of rhodamine B. Spray solution: Dissolve 0.5 g p-benzoquinone (p-quinone) in a mixture of 10 m1 Inspect in long-wave UV light. pyridine and 40 ml 1-butanol. Literature: H. P. Kaufmann, J. Budwig, Ferte u. Seifen, Anstrichmittel 53, 390 Note: After spraying red spots of ethanolamine will appear immediately. Cholinc (1951). shows no reaction. Chemicals: Chemicals: Rhodamine B G R, Cat. No. 7599 p-Benzoquinone, Cat. No. 802410 Ethanol abs, G R, Cat. No. 972 1-Butanol G R, Cat. No, 1990 Pyridine G R, Cat. No. 9728 262. Rhodamine 6 G for lipids. Resorcinol — zinc chloride — sulfuric acid for plasticisers Spray solution: Dissolve 0.001 g rhodamine 6 G in 100 ml acetone. 258. - Inspect in long-wave UV light. (especially phthalate esters). Add to a 20% ethanolic resorcinol solution some zinc powder. Literature: R. F. Witter,G. V. Marinetti, A. Morrison, Arch. Biochem. Biophys. 68, Spray solution 1: 15 (1957). Sulfuric acid 2 mo1/1. Spray solution 11: Chemicals: Spray solution 111: 40% aqueous potassium hydroxide solution. Rhodamine 6 G Procedure: Spray with 1, heat 10 min at 150°C, spray with 11, heat 10 min at 120°C Acetone G R, Cat. No. 14 and spray with 111. Orange spots on yellow background. 263. Rhodanthe for carotenoid aldehydes. Literature: J. W. Copius-Peereboom, J. Chromatog. 4, 323 (1960). Spray solution 1: 1-5% ethanolic solution of rhodamine. D. Braun, Chimia (Switz.) 19, 77 (1965). Spray solution 11: 25% ammonia solution or 27% aqueous sodium hydroxide Chemicals: solution. Resorcinol G R, Cat. No, 7593 Procedure: Zinc chloride G R, Cat. No. 8816 Spray with 1, then with II and dry the chromatogram. Ethanol abs. G R, Cat. No. 972 Literature: A. Winterstein, B. Hegediis, Chimia (Switz.) 14, 18 (1960). Sulfuric acid 95-97% (1.84) G R, Cat. No. 731 Chemicals: Potassium hydroxide G R, Cat. No. 5033 Rhodanine Ammonia solution min. 25% (0.91) G R, Cat. No. 5432 259. Resorcyl aldehyde — sulfuric acid for 16-dehydrosteroids. Sodium hydroxide solution min. 27% (1,3) GR, Cat. No. 5591 Solution a: 0.5% solution of resorcyl aldehyde in acetic acid. 264. Solution b: 5% sulfuric acid solution in glacial acetic acid. Rhodizonic acid sodium salt for barium and strontium ions. Spray solution: Mix freshly before use equal parts of a and b. Spray solution 1.: 1"/0 aqueous solution of sodium rhodizonate. Treatment: Heat at 100-110°C until maximal visualisation of the spots. Spray solution 11: 25 % ammonia solution. Literature: D. B. Gower, J. Chromatog. 14, 424 (1964). Literature: T. V. Arden, F. H. Burstall, G. R. Davies, J. A. Lewis, R. P. Linstead, Nature 162, 691 (1948). Chemicals: Resorcyl aldehyde Chemicals: Acetic acid glacial min. 96% (1.04) G R, Cat. No. 62 Rhodizonic acid sodium salt G R, Cat. No. 6595 Sulfuric acid 95-97% (1.84) GR, Cat. No. 7.31 Ammonia solution min. 25% (0.91) GR, Cat. No. 5432

76 77 7IP

Rubeanic acid for lead, cobalt, copper, manganese, nickel, 265. Solution b: Ammonia solution 5 mo1/1. mercury and bismuth ions. Spray solution: Mix a and b in the ratio 1:5 freshly before use. Spray solution 1: 0.5% ethanolic solution of rubeanic acid. Caution! Formation of explosive silver azide by prolonged .standing. Spray solution 11: 25% ammonia solution. Treatment: Heat 5-10 min at 105 °C until the dark spots have become most intense, Procedure: Spray with I, dry briefly, then spray with II or place the chromatogram Literature: A. C. Bath-Smith, R. G. Wesrall, Biochim. et hiophys. Acta 4, 427 (1950). into a chamber with ammonia. Chemicals: Literature: F. W. H. M. Merkus, Pharm. Weekblad 98, 955 (1963). Silver nitrate solution 0.1 moll!, Cat. No. 9081 J. A. Lewis, J. M. Griffiths, Analyst 76, 388 (1951). Ammonia solution Min. 25% (0.91) G R. Cat. No, 5432 Chemicals: Rubeauic acid G R, Cat. No. 629 269. Silver nitrate — ammonia — fluorescein for halogen ions. Ethanol abs. G R, Cat. No. 972 Spray solution 1: Dissolve 1 g silver nitrate in 100 nil ammonia solution 0.5 moo/1. Ammonia solution MM. 25% (0.91) G R, Cat. No. 5432 Spray solution 11: 0.1% ethanolic fluorescein solution. 266. Silver nitrate for phenols. Procedure: Spray with I, dry briefly and spray with II. Spray solution: Add with stirring 1 ml saturated aqueous silver nitrate solution to Literature: H. Seiler, T. Kaffenberger, Helv. Chin). Acta 44, 1282 (1961). 20 ml acetone, then add water dropwise until the precipitated silver nitrate has just Chemicals: dissolved. Silver nitrate G R, Cat. No. 1512 Light pink to deep green spots are yielded. Fluorescein, Cat. No. 3990 Literature: W.J. Burke, A.D. Potter, R.M. Parkhurst, Anal. Chem. 32, 727 (1960). Ammonia solution min. 25% (0.91) G R, Cat. No. 5432 Ethanol abs. G R, Cat. No. 972 Chemicals: Silver nitrate G R, Cat. No. 1512 270. Silver nitrate — ammonia — sodium chloride for thioacids. Acetone G R, Cat. No. 14 Spray solution 1: Mix freshly before use 50 ml silver nitrate solution 0.1 mul/1 with 50 ml 10% ammonia solution. 267. Silver nitrate — ammonia for sugars and sugar_alcohols Longer standing may lead to formation of explosive silver azide! (Dedonder reagent). PC. Spray solution 11: 10% aqueous sodium chloride solution. Spray solution: Add with stirring 1 nil saturated aqueous silver nitrate solution to Procedure: Spray with I, then dry and spray with II. Expose the chromatogram to 20 nil acetone, then add water dropwise until the silver nitrate just dissolves. daylight until the yellow-brown spots have attained maximum colour intensity. Procedure: Spray the chromatogram liberally from both sides. Chemicals: Treatment: Place the moist chromatogram 1 hour into a chamber saturated with Silver nitrate solution al moll, Cat. No. 9081 ammonia (protected against light). Then heat the chromatogram at 80°C until the Ammonia solution aht. 10% (0.96) GR, Cat. No. 5423 paper background has turned light brown, and remove the excess silver nitrate with Sodium chloride G R, Cat. No. 6404 10% sodium thiosulfare solution. After rinsing for 2 hours under running water dry the chromatogram. 271. Silver nitrate — ammonia — sodium methoxide for sugars. Literature: C. Petronici, C. Safina, Chem. Abstr. 47, 11297 (1953). Solution a: 0.3% methan.olic silver nitrate solution. Chemicals: Solution b: Ammonia gas saturated methanol. Silver nitrate G R, Cat. No. 1512 Solution c: Dissolve 7 g sodium in 100 ml methanol. Sodium thiosulfate G R, Cat. No. 6516 Spray solution: Mix freshly before use 20 ml a, 4 ml b and 8 ml c. Acetone G R, Cat. No. 14 Treatment: Heat 10 min at 110°C. Ammonia solution Min. 25% (0.91) G R, Cat. No. 5432 Chemicals: 268. Silver nitrate — ammonia for reducing substances Silver nitrate G R, Cat. No. 1512 (Tollens or Zaffaroni reagent). Sodium G R, Cat. No. 6261 Methanol G R, Cat. No. 6009 Solution a: Silver nitrate solution 0.1 molt]. Ammonia solution aht. 10% (0.96) G R, Cat. No. 5423 78 79 272. Silver nitrate — bromophenol blue for purines (Wood reagent). 275. Silver nitrate — hydrogen peroxide for chlorinated hydrocarbons. Spray solution: Dissolve 0.2 g bromophenol blue in 50 ml acetone and add 50 ml 2"/i, Spray solution: Dissolve 0.1 g silver nitrate in 1 ml water, add 10 m] ethylene glycol aqueous silver nitrate solution. The reagent is stable for about one week. monophenyl ether, fill up to 200 ml with acetone and add 1 drop 30% hydrogen Procedure: After development in acidic solvents dry the chromatogram and place into peroxide. a chamber with ammonia. Then remove the excess ammonia by hot air and spray. Treatment; Irradiate with unfiltered UV light. Literature: H. Michl, F. Harberler, Mh. Chem. 85, 779 (1954). If long-wave UV light- is used expose alumina layers about 50 min and silica gel layers Chemicals: up to 15 min. Bromopherrol blue indicator, Cat. No. 8122 Dark spots are formed. Silver nitrate G R, Cat. No. 1512 Literature: M. F. Kovacs, J. Assoc. Off. Agr. Chemists 46, 884 (1963). Acetone G R, Cat. No. 14 Chemicals: Silver nitrate G R, Cat. No. 1512 273. Silver nitrate — fluorescein for alkyl- and arylsulfonic acids. Acetone G R, Cat, No, 14 PerhydroN (30% by weight H Solution a: 10% aqueous silver nitrate solution, 2 02) G R, Cat. No. 7209 Ethylene glycol monophenyl ether G R, Cat. No. 807291 Solution b: 0.2% ethanolic fluorescein sodium solution. Spray solution: Mix freshly before use 10 ml a and 50 ml b. Yellow spots on salmon-pink background. 276. Literature: F. H. Pollard, G. Nicklas, K. W. C. Burton,J, Chromatog. 8,507 (1962). Silver nitrate — potassium dichromate for barbiturates. C. M. Coyne, G. A. Maw, J. Chromatog. 14, 552 (1964). Spray solution 1: Add 25 ml saturated aqueous silver nitrate solution to a mixture of 50 ml acetone and 2 nil water, Chemicals; Silver nitrate G R, Cat, No. 1512 Spray solution 11: 0.3% aqueous potassium dichromate solution. Fluorescein sodium, Cat. No. 3992 Spray solution 111: 2% methanotic sodium hydroxide solution. Ethanol abs. G R, Cai. No. 972 Procedure: Spray liberally with I and dry in the air. Then spray with 11, dry, re-spray with it and re-dry again in the air. Then spray with 111. 274. Silver nitrate — formaldehyde for chlorinated insecticides Literature: H. Weidmann, Dissertation Berlin 1961. (e.g. dieldrin, aldrin and Undone). Chemicals: Spray so/talon 1: Silver nitrate solution 0.05 moll]. Silver nitrate G R, Cat. No. 1512 Spray solution 11: 35% formaldehyde solution. Potassium dichromate G R, Cat. No. 4864 Sodium hydroxide G R, Cat. No. 6498 Spray solution III: Methanolie potassium hydroxide solution 2 moll. Acetone G R, Cat. No. IV: Fr.shly prepared mixture of equal volumes of 30% hydrogen 14 Spray solution Methanol G R, Cat, No. 6009 peroxide and 65% nitric acid. Procedure: Spray with 1, dry 30 min, spray with it and dry again 30 min. Spray with Ill and heat 30 min at 130'C. Spray with IV, allow the chromatogram to stand in the 277. Silver nitrate.— potassium permanganate for reducing • dark for 12 hours, and expose to daylight. compounds. Dark green spots on light grey background. Solution a: Literature: L. C. Mitchell, J. Assoc. Off. Agr. Chemists 35, 920 (1952.). Mix freshly before use 1 part silver nitrate solution 0.1 mol/1, 1 part ammonia solution 2 mo1/1 and 2 parts sodium hydroxide solution 2 mo1/1. Chemicals: Solution b: Silver nitrate solution 0.1 mo111, Cat. No. 9081 Dissolve 0.5 g potassium permanganate and 1 g sodium carbonate in 100 rnl water, Formaldehyde solution 35% G R, Cat, No. 4003 Potassium hydroxide G R, Cat, No. 5033 Spray solution: Mix freshly before use equal parts of a and b. Methanol G R, Cat. No. 6(i09 Note: Reducing compounds show light yellow spots on green-blue background Perbydrol a (30% H2 02) G R, Cat. No. 7209 immediately after spraying. Nitric acid min. 65% (1.4) G R, Cat. No. 454 Literature: J, Kellen, Chem. listy 51, 973 (1957).

80 81 Chemicals: Potassium permanganate G R, Cat. No. 50S2 A4-3-ketosteroids show yellow fluorescence in long-wave UV light. Sodium carbonate 10-hydrate G R, Cat_ No_ 6391 Literature: I. E. Bush, Biochem. J. 50, 370 (1951). Silver nitrate solution 0_1 moll!, Cat, No. 9081 Chemicals: No. 9136 Sodium hydroxide solution 2 moll!, Cat. Sodium hydroxide G R, Cat. No. 6498 Armnonia solution min, 25% (0.91) G R, Cat. No. 5432 Methanol G R, Cat. No, 6009

278. Silver nitrate — sodium dichromate for pur ines. PC. 2% aqueous silver nitrate solution. Dip solution 1: 262. Sodium meta-periodate benzidine for compounds with Dip solution 11: 0.5% aqueous sodium dichromate solution. 1,2-diol groups (sugars, polyalcohols). Dip solution 111: Nitric acid 0.5 niol/l. Spray solution 1: 0.5% aqueous sodium meta-periodate solution, 10 min and dip into ll. Dip the Procedure: Dip into I, dry the chromatogram in the air Spray solution 11: Add 50 ml water, 20 ml acetone and 10 nil 0.2 N hydrochloric acid chromatogram dyed red into Ill, thus discolouring the background, leaving the to a solution of 1.8 g benzidine in 50 ml ethanol. purines as red spots. Procedure: Spray with I and after 5 min with 11. Soc. 73, 5781 (1950). Literature: R. M. Reguera, I. Asimov, J. Am. Chem_ White spots 011 blue background. Chenricals: Literature: J. A. Cifonelli, F. Smith, Anal. Cheni. 26, 1132 (1954). Silver nitrate G R, Cat. No. 1512 Chemicals: No. 6.136 Sodium dichromate G R, Cat. Sodium meta-periodate, Cat. No, 6597 (1.4) G R, Cat. No. 454 Nitric acid min. 65% Benzidine G R, Cat.. No. 1762 Hydrochloric acid 1 inollITitrisoll, Cat. No. 2'79. Silver nitrate — sodium hydroxide for sugars and polyalcohols. 9970 Ethanol abs. G R, Cat. No. 972 Spray solution 1: Fill up 1 rill saturated aqueous silver nitrate solution to 200 ml with Acetone G R, Cat. No. 14 acetone and add 5-10 nil water to dissolve the resulting precepitate. Spray solution 11: Sodium hydroxide solution 0.5 mo1/1 in aqueous methanol (dissolve 20 g sodium hydroxide in a minimum of water and fill up to l lwith methanol). 283. Sodium meta-periodate benzidine — silver nitrate for Procedure: Spray with 1 and 11 and heat 1-2 inin at 100'C. substances with 1,2-diol groups (sugars, polyalcohols). Chemicals: Spray sohrtion 1: 0.1 u/” aqueous sodium meta-periodate solution. Silver nitrate G R, Cat. No, 1512 Spray solution 11: Add 70 ml water, 30 ml acetone and 1.5 ml hydrochloric acid Sodium hydroxide G R, Cat. No. 6495 1 mo1/1 to a solution of 2.8 g benzidine in 80 ml ethanol. No, -I Acetone G R, Cat. Spray solution 111: Mix 1 ml aqueous saturated silver nitrate solution with stirring No. 6009 Methanol G R, Cat. with 20 ml acetone and add water dropwise until the precipitated silver nitrate dissolves. 280. Sodium dithionite for antimony, arsenic, mercury, silver and Procedure: Spray with I and dry the chromatogram at room temperature. Spray with bismuth ions. 11 and plate it into a chamber saturated with ammonia. Additionally you can spray Spray solution: 0.1% aqueous sodium dithionite solution. with 111, the white spots turn dark, F. H. Pollard, J. F. H. N1cOmie,. Chromatographic Methods of Inorganic Literature: Literature: D. Waldi, J. Chromatog. 18, 417 (1965). Analysis, Butterworths Scientific Publications, London, 1953, p_ 47. Chemicals: Chemicals: Sodium meta-periodate, Cat. No. 6597 Sodium dithionite, Cat. No. 65()7 Benzidine G R, Cat. No. 1762 Silver nitrate G R, Cat. No. 1512 4-3-ketosteroids. 278. Sodium hydroxide for A Hydrochloric acid 1 mola Titrisol% Cat. No. 9970 Spray solution: 101/4 sodium hydroxide solution in 60% methanol. Ethanol abs. G R, Cat. No. 972 Treatment: Heat 10 min at 80°C. Acetone G R, Gat. No. 14 Ammonia solution min, 25% (0.91) G R, Cat. No. 5432 82 83 284. Sodium meta-periodate — Nessler's reagent for hydroxyamino Literature: d. v. Denffer, M. Behrens, A. Fischer, Naturwissenschaften 39, 258 acids (serine, threonine). (1952). Spray solution 1: I aqueous sodium meta-periodate solution. Chemicals: Sodium nitrite C R, Cat. No. 6549 Spray solution 1!: Nessler's reagent. Hydrochloric acid 1 mo1/1 Titriso1 Make a paste with 10 g mercury (11) iodide and. a little water and add .5 g potassium 8, Cat. No: 9970 iodide. Add a solution of 20 g sodium hydroxide in 80 nil water. After complete Hydrochloric acid fuming min. 37% (1.19) G R, Cat. No. 317 dissolution fill up to 100 nil with water. Allow to stand for some days and decant after deposition of the precipitate. 287. Sodium nitroprusside for compounds with SH-group (cysteine), Procdrire: Spray with 1, dry the chromatogram at room temperature and spray with S-S-group (cystine) and arginine. with IL Sprav solution I: Dissolve 1.5 g sodium nitroprusside in s nil hydrochloric acid 2 m1/1. Lireratirre: R. Consden, A. H. Gordon, A. J. P. Martin, Biochini. J. 40, 33 (1946)- Filter after addition of 95 nil methanol and 10 nil 25% ammonia solution. Chemicals: Note: 5H-Compounds show red spots, arginine turns orange and later grey-hlue. Sodium meta-periodate, Cat. No. 6597 Spray solution II: Dissolve 2 g sodium cyanide in 5 ml water arid fill up to 100 nil with Mercury(11) iodide, Cat. No. 4420 methanol. Potassium iodide G R, Cat. No. 50+3 Non!: Spraying with II visualises compounds with -S-S-linkage as red spots on yelloW Sodium hydroxrde G R, Cat. No. 6495 background. Caution when using this highly toxic reagent! 285. Sodium meta-periodate — 4-Nitroaniline for deoxy-sugars. Variation for -S-5-compounds: Mix 1 part saturated aqueous sodium meta-periodate solution with Spray solution 1; Spray solution /: Dissolve .5 g sodium cyanide and 5 g sodium carbonate in 100 nil 2 parts water. 25% ethanol. Spray 11: Mix 4 parts 1% ethanolic-4-nitroaniline solution with 1 part 37% sohrtion Spray solution 11: Dissolve 2 g sodium nitroprusside in 100. ml 75% ethanol. hydrochloric acid. Procedure: Spray with I, dry briefly in the air and spray with II. Procedure: Spray with I, wait 10 min, then spray with 11. . Caution when using this highly toxic reagent! Deoxy-sugars and glycals show yellow spots which fluorescence strongly in Note: Literature: G. Tonnies, J. J. Kolb, Anal. Chem. 23, 823 (1951). long-wave UV light. The colour changes to green by spraying with 5% methanolic Variation for thiolacrones: sodium hydroxide solution. Spray solution !: Sodium hydroxide solution 1 mo1/1. Literature: J. T. Edward, D. M. Waldron, J. Chem. Soc. 1952, 3631. Spray solution 1!: Dissolve 2 g sodium nitroprusside in 100 nil ethanol. Chemicals: 75% Sodium meta-periodate, Cat. No. 6597 Procedure: Spray with I, dry briefly in the air and spray with II. 4-Nitroaniline, Cat. No_ 6760 Literature: F. Korte, J. Vogel, J. Chromatog. 9, 381 (196-2 1. Etbaiio/ abs. G R. Cat. No. 972 Chemicals: Hydrochloric acid Porting min. 37% (1.19) G R,Cat. No. 317 Sodium nitroprusside G R, Cat. No. 6541 Sodium hydroxide. Cat. No. 6495 Sodium carbonate 10-hydrate G R, Cat. No. 6391 Methanol G R. Ca- No. 6009 Hydrochloric acid min. 25% (1.125) G R, Cat. No. 316 Ammonia solution min. 25% (0,91) G R, Cat. No. 5432 286. Sodium nitrite — hydrochloric acid for indoles and thiazoles. Methanol G R, Cat. No. 6009 SPra.Y solution:- Freshly prepared solution of 1 g sodium nitrite in 100 ml hydrochloric Ethanol abs. G R, Cat. No. 972 acid 1 mol/l. Sodium cyanide, Cat. No. 6437 Heat at 100°C. Sodium hydroxide solution 1 mol/! Titrisol5, Cat. No. 9956 Note: Indo]es turn red and thiazole derivatives light green. Alternative: 288. Sodium nitroprusside — acetaldehyde for secondary aliphatic Spray solution: 0.5". aqueous sodium nitrite solution. and alicyclic amines. Treatment: Place the chromatogram into a chamber with hydrogen chloride vapours. Spray solution: Dissolve .5 g sodium nitroprusside in 100 ml 10% aqueous acetal-

84 85

dehyde solution. Before use mix 1 part of this solution with 1 part 2% aqueous 292. Sodium nitroprusside — potassium hexacyanoferrate(III) for sodium carbonate solution. aliphatic nitrogen compounds, cyanamide, guanidine, urea, Literature: F. Feigl, Spot Test in Organic Analysis, Elsevier Pub. Co., 7,1, Ed., 1966, thiourea and derivatives, creatine and creatinine. p. 251. Spray solution: Mix in the ratio 1:1:1:3 10% aqueous sodium hydroxide solution, K. Macek, J. Hacaperkova", B. Kaka, Pharmazie 11, 533 (1956). 10% aqueous sodium nitroprusside solution, 10% aqueous potassium hexacyano- E. Stein, V. Kamicnski, Planra Med. 50, 291 (1957). ferrate(III) solution and water. The mixture is allowed to stand at least 20 min at Chemicals: room temperature before use. stable for several weeks when stored in the refrigerator. Sodium nitroprusside G R, Cat. No. 6541 Before use mix with an equal part of acetone. Sodium carbonate 10-hydrate G R, Cat. No. 6391 Literature: J. Roche et al., Biochim. et biophys. Acra 14, 71 (1954). Acetaldehyde, Cat. No. 800004 L. Fishbein, M. A. Cavanaugh, J. Chromatog. 20, 283 (1965).. L. Fishhein, Rec. tray. chin]. 84, 465 (1965). 289. Sodium nitroprusside — ammonia for hemlock alkaloids. Chemicals: aqueous sodium nitroprusside solution. Spray solution 1: 1% Sodium nitroprusside G R, Cat. No. 6541 Spray solution 11: 10% amnionia solution. Potassium hexacyanoferrate(111) G R, Cat. No. 4973 Procedure: Spray with l and then with 11. 10% Sodium hydroxide solution (1.109) G R, Cat. No. 5588 Note: y-Coniceine turns red. Acetone G R, Cat. No. 14 Literature: F. Moll, Arch. ['harm. 296, 205 (1963). Chemicals: Sodium nitroprusside G R, Cat. No. 6541 293. Sodium nitroprusside — potassium permanganate for Ammonia solution abt. 10% (0.96) G R, Cat. No. 5423 sulfonamides (Roux reagent). Spray solution: Sodium nitroprusside — hydrogen peroxide for guanidine, Dissolve 10 g sodium nitroprusside in 100 ml warer, add 2 ml 33% 290. aqueous sodium hydroxide and urea, thiourea and derivatives, creatine and creatinine. 5 ml potassium permanganate solution 0.02 rno1/1 and filter after mixing. Mix 2. ml 5% aqueous sodium nitroprusside, 1 nil 10% aqueous Spray reagent: Procedure: Spray and inspect in UV light. sodium hydroxide and 5 m13% aqueous hydrogen peroxide and dilute with 15 ml water. The solution can be stored several days in the refrigerator. Literature: E. Vitolo, Bull, Chim. Farm. 89, 351 (1950). G. Wagner, Pharmazie 9, 979 (1954). Literature: E. Hofmann, A. Wiinsch, Naturwissenschaften 45, 338 (1958). Chemicals: Chemicals: Sodium nitroprusside G R, Cat. No. 6541 Sodium nitroprusside G R, Cat. No. 6541 Sodium hydroxide G R, Cat. No. 6498 Sodium hydroxide 10% solution 0.109; G R, Cat. No. 5558 Potassium permanganate solution 0.02 mo111 Titrisol®, Cat. No. 9935 Perhydrol® (30% by weight H202), Cat, No. 7209 291. Sodium nitroprusside hydroxylamine for thiourea derivatives (Grote reagent). 294. Sodium nitroprusside — sodium hydroxide for methyl ketones Spray solution: Dissolve 0.5 g sodium nitroprusside in 10 ml water, add 0.5 g hyd- and activated methylene groups. roxylamine hydrochloride and 1 g sodium hydrogen carbonate. After gas generation is Spray solution: Dissolve 1 g sodium nitroprusside in 100 ml of a mixture of sodium complete, add 2 drops bromine and fill up to 25 ml with water. The reagent is stable hydroxide 2 mot/1 and ethanol (1+1). for about 2 weeks. Red ro violet spots. Literature: 1. W. Grore, J. Biol. Chem. 93, 25 (1931). Literature: F. Feigl, Spot Tests in Organic Analysis, Elsevier Publ. Co., 1966, 701 Ed., Cheinicalc: p. 208. Sodium nitroprusside G R, Cat. No. 6.541 Chemicals: Sodium hydrogen carbonate G R, Cat. No. 6329 Sodium nitroprusside G R, Cat. No. 6541 Bromine G R, Cat. No. 194 Ethanol abs. G R, Cat. No. 972 Hydroxylamine hydrochloride G k, Cat. No. 4616 Sodium hydroxide solution 2 mob'', Cat. No. 9136

86 87 295. Sodium nitroprusside — sodium meta-periodate for Spray solution 1!: 0.015% deoxy-sugars. methanolic solution of fisetin or quercetin. Procedure: Spray solution 1: 2.5% aqueous sodium meta-periodate solution. Spray with I, dry at room temperature, then spray with 11 and dry again at MOM temperature. Mixture of 1 part 7% aqueous sodium nitroprusside solution, Spray solution H: Orange to red spots which fluoresce in long-wave UV light. 3 parts water and 20 parts of a saturated solution of piperazine in ethanol. Literature.' R. Neu, J. Chrom.atug. 11, 364 (1963). Procedure: Spray with 1, dry 10 min at room temperature, then spray with II. Chemicals: • Literature: J. T. Edward, D. M. Waldron, J. Chein. Soc. 1952, 3631. kalignost''' (Sodium tetraphenylboron) G R, Cat. No. 6669 Chemicals: Quercetin, Cat. No. 7546 Sodium nitroprusside G R, Cat. No. 6541 Fisetin Sodium meta-periodate, Cat. No. 6597 Methanol G R, Cat. No. 6009 Prperazine Cat. No. 7327 Ethyl methyl ketone C R, Cat, No. 9708 Ethanol abs. G R, Cat_ No. 972 299. Sodium tetraphenylboron(Kalignost®) rhodamine B Sodium pentacyanoamineferrate(11) for urea, thiourea 296. for potassium ions. and guanidines (Fearon reagent). Spray solution: Sodium hydroxide solution 0.1 moll]. Sodium pentacyatioamineferrate(11): Dissolve 10 g sodium nitroprusside in 40 ml 25% ammonia solution. Allow the solution to stand at 0°C until all nitroso iron(111) Spray solution 11: 1% ethanolic Kalignost"' solution. cyanide is decomposed. This is the case if several drops of the mi xturc added to a solu- Spray solution 111: 0.5% ethanolic rhodamine B solution. tion of crearinine in sodium carbonate solution 0.5 mol/l produce no longer any red Procedure Spray with 1, dry, spray with II, and then with 111. colour. Then filter and add ethanol to the clear filtrate until no further precipitate is Intense dark blue fluorescence in long-wave UV light. formed. Filter off the resulting precipitate, wash with absolute ethanol and dry over Larger amounts of potassium appear in daylight as light red spots on dark red back- sulfuric acid in a vacuum desiccator. The salt is stable when stored protected from ground. light and moisture. Chemicals: Spray solution: Add to 5 ml 10% sodium hydroxide IS nil 1% aqueous sodium pen- kalignost5 (Sodium tetraphenylboron) G R, Cat. No. 6669 tacyanoaminoferrate(11) solution and 1 drop Perhydror. Stable for about 24 hours. Sodium hydroxide solution 0.1 moll! Titrisol4; Cat. No. 9959 Literature: P. H. List, Hoppe-Seylers Z. physiol. Chein. 305, 27 (1956): Rhodanzine BGR, Cat. No. 7599 Chemicals: Ethanol abs. G R, Cat. No. 972 Sodium nitroprusside G R, Cat. No. 6541 300. Sodium thiosulfate — copper(II) acetate for antimony ions. PC. Sodium carbonate G R, Cat, No. 6392 Ethanol abs, G R, Cat. No. 972 Spray solution 1: Saturated aqueous sodium thiosulfate solution. Ammonia solution min, 25% (0.91) G R, Cat. No. 5432 Spray solution II: Dissolve 0.4 g copper(11) acetate in a mixture of 2 nil glacial acetic Perhydrole (30% by weight H2 Oa) G R, Cat. No. 7209 acid and 48 ml water. Creatinine, Cat. No. 5208 Procedure: Spray with 1, hear briefly, rinse our excess sodium thiosulfate with water Sulfuric acid 95-97% (84) G R, Cat. No. 731 and spray with II. Literature: G. P. Heisig, F. H. Pollard, Anal, Chim. Acta 16, 234 (1957). 297. Sodium sulfide solution for ions of the hydrogen sulfide group. Chemicals: Spray solution: Freshly prepared 0.5% aqueous sodium sulfide solution. Copper(11) acetate G R, Cat. No. 2711 Literature: F. W. H. M. Merkiis, Pharm. Weekhlad 98, 957 (1963). Sodium thiosulfate G R, Cat. No. 6516 Chemicals: Acetic acid glacial min, 96% (1,06) G R, Cat. No. 62 Sodium sulfide G R, Cat. No. 6638 301. Starch for amylases. 298. Sodium tetraphenylboron (Kalignost) for alkaloids. Spray solution 1: 2% aqueous starch solution. Spray solution 1: 1% sodium tetraphenylhoron solution in ethyl methyl ketone, Spray solution II: Iodine solution 0.005 mol 12/1. saturated with water. Procedure: Spray with I, then place the chromatogram into a moist chamber at 88 89 Note: Aliphatic nitrosamines show red-violet spots, aromatic nitrosamines turn 40-50°C for 1 hour. After drying at room temperature spray with II. green to blue. Amylases will appear as white spots on violet or brown background. Note: Literature; R. Preussmann, D. Daiber, H. Hengy, Nature 201, 502 (1964). K. Wallenfels, E. v. Pechmann, Angew. Chem. 63, 44 (1951). Literature: R. Preussmann, G. Neurath, G. Wulf-Lorentzen, D. Daiber, H. Hengy, Z. anal. Chemicals: Chem. 202, 187 (1964). Starch soluble G R, Cat. No. 1252 Chemicals: 9910 Iodine solution 0.005 ;not 1,11, Cat. N. Sulfanilic acid, Cu. No. 686 1-Naplithylamine G R, Cat. No. 6245 Sulfanilamide diazotised for phenols, coupling amines and 302. Acetic acid glaci41 min. 96% (1.06) G R, Cat. No. 62 heterocycles (Pauly reagent acc. to Kutacek). Spray solution 1: Dissolve 3 g sulfanilamide in 200 nil water, 6 m136% hydrochloric 305. Sulfuric acid as general visualisation reagent (in particular acid and 14 ml 1-butanol. Add freshly before use to 20 nil 0.3 g sodium nitrite. for sterols, steroids, bile acids and gibberellins). Spray solution 11: 10% aqueous sodium carbonate solution. Spray solutions: A: Procedure: Spray with 1, and after 5-10 min with 11. Mix equal parts of 95% sulfuric acid and methanol with cooling. B: 5% ethanolic solution of 95% sulfuric acid. Literature: 1. M. Hais, K. Macek, Handhuch der Papierchromatographie I, G. Fischer, Jena, 1958, p. 743. .C: 15% solution of 95% sulfuric acid in 1-butanol. D: solution of 95% sulfuric acid in acetic anhydride. Chemicals: E: Mix equal parts of 95% sulfuric acid and glacial acetic acid. Sulfanilamide, Cat. No. sais Procedure: Sodium nitrite G R, Cat. No. 6549 Spray the chromatogram with one of these reagents, allow to dry for Sodium carbonate 10-hydrate G R, Cat. No. 6391 15 min in the air and heat to 110°C until maximal visualisation of the spors. Hydrochloric acid fuming min. 37% (1.19) G R, Cat. No. 317 Note: Cholesterol and vitamin A, their esters and many isoprenoid lipids show 1-Butanol G R, Cat. No. 1990 characteristic colours after spraying with spray solution A and subsequently heating: cholesterol and esters turn red, then red-violet and brown; vitamin A and esters turn 303. Sulfanilic acid diazotised for phenols, coupling amines and blue. Most compounds may be subsequently charred, yielding black spors. Heating heterocycles (Pauly reagent). with sulfuric acid on layers impregnated with silver nitrate may be followed by oxidation to CO2. Spray solution: Dissolve 4.5 g sulfanilic acid in 45 ml hydrochloric acid 12 mold with warming and fill up the solution to 500 ml with water. Cool 10 ml of the diluted solu- Literature: D. F. Jones, J. McMillan, M. Radley, Phytoehernistry 2,307 (1964) gib- tion with ice and add 10 ml of cold 4.5% aqueous sodium nitrite solution. Allow to berellins). stand for 15 min ar 0 °C (it is stable for 1-3 days or this temperature) and add freshly W. L. Anthony, W. T. Beher, J. Chromatog. 13, 570 (1964). before use equal parts of 10%, aqueous sodium carbonate solution. H. Jatzkewitz, E. Mehl, Hoppe-Seylers Z. physiol. Chem. 320, 251 (1960). H. Metz, Naturwissenschaften 48, 569 1961). Literature: H. Jatzkewitz, Hoppe-Seylers Z. physiol. Chem. 292, 99 (1953). N. R. Griminett, E. L. Richards, J. Chromatog. 20, 171 (1965). Chemicals: Sulfuric acid 95-97% (1.84) G R, Cat. Chemicals: No. 731 Ethanol abs_ G R, Cat. No. 972 Sulfanilic, Ccit. No. 686 1-Butanol Hydrochloric acid fuming min. 37% (1.19) G R, Cat, No. 317 G R, Cat. No. 1990 Acetic anhydride G R, Cat. No. 42 Sodium nitrite C R, Cat. No. 65-19 Acetic acid glacial min. Sodium carbonate 10-hydrate G R, Cat. No. 639/ (1.06) G R, Cat. No. 62 306. Sulfuric acid — hypochlorite for digitalis glycosides. 304. Sulfanilic acid — 1-naphthylamine for nitrosamines. sulfanilic acid solution in 30% aqueous acetic acid. Spray solution: Mix 10 ml sulfuric acid 1 mol/l and 3 ml sodium hypochlorite Solution a: 1% solution. Solution b: 0.1% 1-naphthylainine solution in 30% aqueous acetic acid. Treatment; Heat 10-15 min at 125°C. solution: Mix freshly before use equal parts of a and b. Spray Note; irradiate the chromatogram for about 3 min with short-wave UV light, Digitalis glycosides of series A—E show fluorescence of various colours in Procedure: long-wave UV light, then spray with the spray solution.

90 91 Chemicals: Literature: L. Fauconnet, R. Fazan, Bull. Soc. vaud, sci. nat. 66, 307 (1956). Kalignoste (Sodium tetraphenylboron) G R, Cat. No. 6669 L. Fauconnet, M. WaldesbiThl, Pharm. Acta Hely. 38, 423 (1963). Hydrochloric acid min. 25% (1.123) G R, Cat. No. 316 Chemicals: Petroleum benzine G R boiling range 40-60°C G R, Cat. No. 1775 Sulfuric acid 95-97% (1.84) G R, Cat. No. 731 Sodium hypochlorite solution 310. Tetrazolium blue for corticosterids and other reducing compounds. 307. Tetracyanoethylene for aromatic hydrocarbons, phenols and Spray solution: Mix freshly before use equal parts of 0.5"1, methanolic tetrazolium herocyclic compounds. blue solution and sodium hydroxide solution 6 mol/1 in water or water-methanol Spray sohitiou: 10% solurion of tetracyanoethylene in benzene. mixture. Procedure: Spray directly after development of the chromarogram. Violets spots ar room temperature or after short warming. Note: Aromatic hydrocarbons show various colours, some of then) for a short time Literature:. O. Adarnec, Steroids 1, 495 (1963). only. Janak recommends warming at 100°C. T. Feller, Mikrochint. Acta 1965, 105, U. Literature: P. V. Peurifoy, S. C. Slaymaker, M. Nager, Anal. Chem..31, 1740 (1959). Freimurh, B. Zawta, M. Bilchner, Acta Biol. et Med. Ger. 13, 624 (1964). J, janak, J. Chromatog, 15, 15 (1964). O. Nishikaze, R. Abraham, H. Staudinger, J. Biochem. (Tokyo) 54, 427 (1963)- N. Kucharczyk, F. Fohl, J. Vymetal, J. Chromatog. 11, 55 (1963). 1. E. Bush, M. Willoughby, Biochern. J. 67, 689 (1957). Chemicals: Chemicals: Tetrazolium hhre, Cat. No. 8103 Benzene G R, Cat. Nn. 1783 Sodium hydroxide G R, Cat. No. 6498 1etr.rcvairoethylen', Cat. Nn. 8240 Methanol G R, Cat. No. 6009 308. Tetranitrodiphenyl for cardiac glycosides. 311. Thiobarbituric acid for sorbic acid. Spray solution 1: Saturated solution of 2,2',4,4'-tetranitrodiplienyl in benzene. Spray solution: Saturated aqueous solution of thiobarbitutic acid. Spray solution II: 10% potassium hydroxide solution in 50% aqueous methanol. Sorbic acid show red spots. Procedure: Spray with I, dry at room temperature and spray with 11. Literature: J. W. Copius-Peereboom, H. W. Beekes, J. Chromatog. 14, 417 (1964). Blue spots. Chemicals: J. Binkert, F. Angliker, A. v. Wartburg, Hely. Chins. Acta 45, 2122 Literature: 2-Thiobarbituric acid, Cat. No. 8180 (1962). Chemicals: 312. Thymol — sulfuric acid for sugars. No. _5033 Potassium hydroxide G R, Cat. Spray solution: Dissolve 0.5 g thymol in 95 nil ethanol and add 5 inl 97% sulfuric acid Benzene G R, Cat. No. 1783 with caution. 2,2',4,4'-Tetranitrodiphenyl Treatment: Heat 15-20 min at 120°C. Methanol G R, Cat. No. 6009 Sugars show pink spots. 309. Tetraphenyldiboroxide for flavones. PC. Literature: S. Adachi, J. Chromatog. 17, 295 (1965). Prepare tetraphenyldiboroxide according to the directions by R. Neu from 3 g Chemicals: sodium tetraphenylboron (Kalignost*), 8.5 nil 2 N hydrochloric acid and 8.5 nil Thymol, Cat. No. 8167 water. For details see R. Ncu, Chem. Ber. 87, 802 (1954). Ethanol abs. G R, Cat. No. 972 Sulfuric acid 95-97% (1.84) G R, Cat. No 731 Dip solution 1: Saturated solution of tetraphenyldiboroxide in petroleum benzine. Dip solution 11: 1-2% aqueous solution of a quaternary ammonium base (e. g. 313. Thymol blue for diemthylamino acids. Laudace). Spray solution: Dissolve 0.04 g thymol blue in a mixture of 25 ml 1-butanol, 25 ml Dip into 1, dry briefly at room temperature and then dip into 11. Suh- Procedure: ethanol and 50 mI sulfuric acid 0.005 moll]. sequenrly dry at room temperature, Yellow spots on red background. R. Neu, Z. anal. Chem, 143, 30 (1954). Literature: Literature: V. M. Ingram, J. Biol. Chem. 202, 193 (1953). R. Neu, Z. anal. Chem. 151, 321 (1956).

93 92 Chemicals: 317. Titan yellow for cadmium ions. Thymol blue indicator, Cat. No. 8176 Spray solution: 0.1% aqueous titan yellow solution. Ethanol abs. G R, Cat. Na. 972 Treatment: Spray either with 25% ammonia solution or place the chromatogram 1-Butanol G R, Cat. No. 9990 sprayed with titan yellow solution into a chamber with ammonia. Sulfuric acid 0.005 moll! Titriso16-', Cat. No. 998? Literature: I. I. M. Elbeih, M. A. Abou-Elnaga, Anal. Chico. Acta 17, 397 (1957). 314. Tin(II) chloride — hydrochloric acid — 4-dimethylamino- Chemicals: benzaldehyde for aromatic compounds containing nitro groups. Titan yellow G R, Cat. No. 1307 Spray solution l: Prepare freshly before use a mixture of 3 rn1 15% aqueous tin(11) Ammonia solution 'Olin. 25% (0.91) G R, Cat. No. 5432 chloride and 15 ml 37`V., hydrochloric acid and dilute with 180 nil water. 318. p-Toluenesulfonic for steroids, flavonoids and catechins. Spray solution !1: Dissolve I g4-dimethylarninoberizaldehyde in a mixture of 3Q nil ethanol, 3 ml 37% hydrochloric acid and 180 nil 1-butanol. Spray solution: 20% solution of p-toluenesulfonic acid in chloroform. Treatment: Spray with 1, dry at room temperature and spray with II. Treatment: After spraying heat a few minutes at I 00°C. Yellow spots after re-drying at room temperature. Inspect the spots in long-wave UV light, Literature: M. Jurecek, J. Churacek, V, Ccrvinka, Mikrochim. Acta 1960, 102. Literature:. D. G. Roux, Nature 180, 973 (1957), H. J. Zeitler, J. Chromatog. 18, 180 (1963). Chenricals: H. Silbermann, R. H. Tin(II) chloride G R, Cat. No. 7815 Thorp, J. Pharm. Pharmacol. 6, 546 (1954). 4-Dimethylaminobenzaldebyde G R, Cat. No. 3058 Chemicals: Ethanol abs. G R, Cat, No. 972 4-Tohierresulfonic acid G R, Cat. No. 9613 Hydrochloric acid fuming min. 37% (1.19) G R, Cal. No. 317 Chloroform G R, Cat. No. 2445 1-Butarrol G R, Cat. No. 1990 319. Toluidine blue for acidic polysaccharides. PC. 315. Tin(II) chloride — potassium iodide for gold ions. Fixing solution: 20 ml 3.5% formaldehyde solution in 80 ml ethanol. Spray solution: Dissolve 5_6 g tin (11) chloride in 10 nil 37% hydrochloric acid. After Spray solution: Dissolve 0,04 g toluidine blue in 80 nil acetone and 20 ml water. dilution with water to 100 rnl, add 0.2 g potassium iodide to the solution. Dip solution: 5% acetic acid solution. Black spots. Procedure: Place the chromatogram 15 min into the fixing solution. After drying, Burstall, G. R. Davies, R. P. Linstead, R. A. Wells, J. Chem. Soc. Literature: F. H. spray with the spray solution and rinse the excess dye first with dip solution, then with 1950, 516. water. Chemicals: Literature: D. Hamerman, Science 122, 924 (1955), Tin(11) chloride G R, Cat. No. 7815 Chemicals: Potassium iodide G R, Cat. No. 5043 Formaldehyde solution 35% G R, Cat. N. 4003 Hydrochloric acid fuming min. 17% (1. /9) G R, Cat, No. 317 Toluidine blue 0, Cat. No. 1273 316. Tin(IV) chloride for triterpenes, sterols, steroids, phenols Acetone G R, Cat. No. 14 and polyphenols. Ethanol abs. G R, Cat. No. 972 Acetic acid glacial miry. 96% (1.06) G R, Cat. No. 62 Spray solution.. Add 10 ml tin(IV) chloride to 160 ml a mixture of equal volumes of chloroform and glacial acetic acid. 320. Treatment: Heat the layer 5-10 min at 100°C and inspect subsequently in daylight Trichloroacetic acid for steroids, digitalis glycosides, Veratrum alkaloids and vitamin and in long-wave UV light. D. A. Spray solution: Literature: J. J. Scheidegger, E. Cherbuliez., Hely. Chico. Acta 38, 547 (1955). 25% solution of trichloroacetic acid in chloroform. B. Spray solution (for vitamin D): Chemicals: 1% trichloroacetic acid solution in chloroform. Tin(1V) chloride fuming, Cat,. No. 7811 C. Spray solution (for digitalis glycosides): Dissolve 3.3 g trichloroacetic acid in Chloroform G R, Cat. No. 2445 10 ml chloroform and add 1-2 drops hydrogen peroxide. Acetic acid glacial min. 96% (1.06) G R, Cat. No. 62 Treatnrent: Heat 5-10 min at 120°C.

94 95 Chemicals: Inspect the spots in daylight and in long-wave UV light. 2,3,5-Triphenyltetrazolium chloride, Cat. No. 8380 Literature: B. J. Aldrich, M. L. Frith, S. E. Wright, J. Pharm. Pharmacol, 8, 1042 Methanol G R, Cat. No. 6009 (1956). Sodium hydroxide solution 1 molll Titrisor3, Cat. No. 9956 H. J. Zeitler, J. Chromatog. 18, 180 (1963). Chemicals: 324. Tungstophosphoric acid for reducing compounds, lipids, Trichloroacetic acid G R, Cat. No, 807 sterols and steroids. Chloroform G R, Cat, No. 2445 Spray solution; 20% ethanolic solution of tungstophosphoric acid. Perhvdroll' (30% by weight H) 0,) G R, Cat. No. 7209 Treatment: Heat at 120 °C until maximal visualisation of rhe spots. Literature: H. P. Martin, Biochim. et biophys. Acta 25, 408 (19.57). 321. Trifluoroacetic acid for steroids. Chemicals: Spray solution: 1% trifluoroacetic acid in chloroform. Tungstophosphoric acid G R, Cat. No. 583 Treatment: Heat 5 min at 120°C. Ethanol abs. G R, Cat. No, 972 Chemicals: Trifluoroacetic acid, Cat. No. 8082.60 325. Urea — hydrochloric acid for sugars. Chloroform G R, Cat. No, 244.5 Spray solution: Dissolve 5 g urea in 20 ml hydrochloric acid 2 mold and add 100 nil ethanol. 322. 2,4,6-Trinitrobenzoic acid for cardiac glycosides. Treatment: Heating at I00°C promotes reaction. Ketoses and oligosaccharides containing keroses turn blue. Spray solution I: 0.1% solution of 2,4,6-trinitrobenzoic acid in a mixture of water and dimethylformainide, Literature: R. Dedonder, Bull. soc. chim. biol. 34, 44 {1952). Spray solution 11: 5% aqueous sodium carbonate solution. Chemicals: Urea GR, Cat. No. 8487 Spray solution 5% aqueous sodium dihydrogen phosphate solution. II Hydrochloric acid inin. 25% (1.125) G R, Cat. No. 110 Procedure: Spray with I, then with II, heat 4-5 min at 90-110 °C, cotil and spray Ethanol abs. G R, Cat. No. 972 finally with III. Cardiac glycosides show orange-red spots. 326. Vanillin — hydrochloric acid for catechins. Literature: T. Momose, T. Matsukunia, Y. Ohkura, J. Pharin. Soc. Japan 84, 783 Spray solution: Dissolve 0.5 g vanillin in SO ml 37 hydrochloric acid. (1964). Treatment: Dry the chromatogram at room temperature_ Chemicals: Catechols show red spots. N,N-Dimethylfortnamide, Cat. No. 3034 Sodium carbonate 10-hydrate GR, Cat. No. 639/ Literature: E. A. H. Roberts, R. A. Cartwright, D. J. Wood, J. Sci, Food Agr. 7, 637 Sodium dihydrogen phosphate C R, Cat. No. 6346 (1957). 2,4,6-Trinitrobenzoic acid Chemicals: Vanillin, Cat. No. 8510 Hydrochloric acid fuming min. 37% (1.19) G R, Cat. No. 317, 323. 2,3,5-Triphenyltetra.zolium chloride (TTC) for reducing sugars, corticosteroids and other reducing compounds. 327. Vanillin — phosphoric acid for steroids. Spray solution: Mix freshly before use one parr 4% methanolic TTC solution with Spray solution: Dissolve 1 g vanillin in 100 ml SO% aqueous phosphoric acid. one part sodium hydroxide solution 1 mo1/1. Treatment: Hear 10-20 min at 120°C. Treatment: Heat 5-10 min at I00°C. Literature: H. Metz, Naturwissenschaften 4S, 569 (1961). Reducing compounds show red spars. Tetrazolium b]ue is more sensitive, Chemicals: Vanillin, Cat. No. 8510 Literature: F. G. Fischer, H. Doric), Hoppe-Seylers Z. physiol Chem. 297, 164 ortho-Phosphoric acid min. 85% (1.71) G R, Cat, No. 573 (1954).

96 97

Alt 331. Xanthydrol for tryptophan and other indole derivatives. 328. Vanillin — potassium hydroxide for amino acids Spray solution: Dissolve 0.1 g xanthydro] in 90 rril ethanol and add 10 ml 37% (ornithine, lysine, proline) and amines. hydrochloric acid freshly before use. 2% vanillin solution in 2-propanol. Spray solution I: Treatment: Hear at 110 CC until maximal visualisation of the spots. ethanolic potassium hydroxide solution. Spray solution 11: l% Literature: S. R. Dickmann, A. L. Crockett, J. Biol. Chem. 220, 957 (1956). Procedure: Spray with I and heat the chromatogram 10 min at 110 °C. Ornithine then Chemicals: fluoresces intensively green-yellow in long-wave UV light, lysine only weakly green- Xanthydrol abt. 10%, in methanol, Cat. No. 8696 yellow. Ethanol abs. G R. Cat. No. 972 After spraying with 11, heat again in the same manner. Ornithine first shows a salmon Hydrochloric acid fuming min. 3 7% (1.19) G R, Cat. No. 317 colour and then fades, while proline, hydroxyproline, pipecolic acid and sarcosine turn red after several hours. Glycine turns brown-green, the other amino acids faintly brown. 332. Zinc chloride for steroid sapogenins and steroids. Literature: G. Curzon, J. Giltrow, Nature 172, 356 (1953). Spray sohrtion: Dissolve 30 g zinc chloride in 100 ml methanol and filter off from the Chemicals: insoluble matter. Vanillin, Cat. No. 8.510 Treatment: Heat 1 hour at 105 CC and cover the layer immediately with a glass plate Potassium hydroxide G R, Cat. No. 5033 for protection against the influence of moisture. Ethanol abs. G R, Cat. No. 972 The spots fluoresce in long-wave UV light. 2-Propanol G R, Cat. No. 9634 Literature: P. J. Stevens, J. Chroinatog. 14, 269 (1964). Chemicals; 329. Vanillin — sulfuric acid for higher alcohols, phenols, steroids Zinc chloride G R, Cat. No. 8816 and essential oils. Methanol G R, Cat. No. 6009 A. Spray reagent: Dissolve 1 g vanillin in 100 ml 97% sulfuric acid. Treatment: Heat the chromatogram at 120 °C until the spots attain maximum colour 333. Zinc uranyl acetate for sodium ions. intensity. Spray solution: Dissolve 10 g uranyl acetate in 6 ml 30% acetic acid and fill up to Literature: E. Tyiliak, D_ Vagujfalvi P. L. Higony, J. Chromatog. 11, 45 (1963). 50 ml with water. Mix 30 g zinc acetate with 3 m130% acetic acid and fill up to 50 ml A. L. le Rosen, R. T. Moravek, J. K. Carlton, Anal. Chem. 24, 1335 (1952). with water. Mix equal volumes of both solutions, allow to stand for one day and filter B. Spray reagent: Dissolve 0.5 g vanillin in 100 ml of a mixture of 97% sulfuric acid off. and ethanol (40+10). Note: Inspect in UV light. Treatment: Heat the chromatogram at 120 °C until the spots attain maximum colour Literature: H. H. Barber, I. M. Kolthoff, J. Am. Chem. Soc. 50, 1625 (1928). intensity. Chemicals: Literature: J. S. Matthews, Biochim. et biophys. Acta 69, 163 (1963). Uranyl acetate G R, Cat. No_ 8473 Chemicals: Zinc acetate G R, Cat. No. 8802 Vanillin, Cat. No. 8510 Acetic acid glacial min. 96% (1.06) GR, Cat. No, 62. Sulfuric acid 95-97% (1.84) G R, Cat. No. 731 Ethanol abs. G R, Cat. No. 972 334. Zirconyl chloride — alizarin — hydrochloric acid for fluorine ions. Spray solution: 330. Violuric acid for alkali and alkaline earth metal ions. Dissolve 0.05 g zirconyl chloride and 0.05 g alizatinsulfonic acid sodium salt in 100 nil hydrochloric acid 2 mold. Spraysolution: 1.5% aqueous viol uric acid solution. Viuluric acid must not be heated Literature.. H. Seiler, T. Kaffenberger, Helv. Chico. Acta 44, 1282 (1961). above 60°C. Chemicals: Treatment: Heat 20 min at 120°C_ Zirconium(1V) oxide chloride G R, Cat. No. 8917 H. Erlenmeyer, H. v. Hahn, E. Sorkin, Hely. Chico. Acta 34, 1419 (1951) Literature: Aliz..-arinsulfonic acid sodium salt G R, Cat. No. 6279 Chemicals: Hydrochloric acid min. 25% (1.125) G R, Cat. No. 316 Violuric acid, Cat. No. 9616 99 98 335. Zirconyl chloride - citric acid for glycosides. PC. Compounds or Compound Classes and Spray so/ution 1: 2% methanolic zirconium(IV( oxide chloride solution. Reagents for their Detection Spray solution II: 5% aqueous citric acid solution. Procedure: Glycosides are first hydrolysed on the chromatogram which has been Acetylene compounds No. 96 placed into a covered beaker with boiling 25% hydrochloric acid. After drying, spray Acids, aromatic No. 153, 249 with 1, dry again and spray vigorously wirh 11. Acids, organic No. 42, 43, 64, 66, 77, 78, 91, 93, 122, Literature: L. Hrirhammer, K. H. Midler, Arch. Pharm. 287, 310 (1954). 148 Adrenaline and derivarives No. 95, 135, 238, 241 Chemicals; Alcohols, higher Zirconiuni(1V) oxide chloride, G R, Cat, No. 8917 No. 329 Aldehydes No. 244 No. 86, 115 Citric acid G R, Cat. Alkali chlorides Hydrochloric acid min. 25"'A, (1.125) G R, Cit. No. 316 No. 92 Alkali ions Methanol G R, Cat. No. 6009 No. 330 Alkaline earth metal ions No. 330 Alkaloids No. 56, 76, 131, 132, 133, 162, 205, 211, 246, 247, 298 Aluminium ions No. 31, 158, 195, 255 Amides No, 69, 155 Amines No. 76, 102, 207, 208, 211, 240, 288 Amines, aromatic No. 87, 136, 149, 202, 213, 215, 302, 303 Amines, quaternary No. 130 Amino acids No. 114, 176, 201, 207, 208, 209, 210, 328 Amino acids, sulfur containing No. 161, 287 Aminosugars No. 102, 207 Ammonium ions No. 73 Amylase No. 301 Anhydrides No. 155 Anthroquinone glycosides No. 183, 243 Antimony ions No. 280, 300 Antioxidants No. 95 Arginine No. 157, 199, 287 Arsenic ions No. 280 Azulenes No. 101

Barbiturates No. 74, 75, 81, 138, 184,186,237,276 Barium ions No. 156, 158, 264 Bile acids No. 29, 172, 220, 305 Bismuth ions No. 265, 280

Cadmium ions No 253, 317 Caffeine No. 58 Calcium ions No. 121, 156, 158

101 100 Carbamate esters No. 147 Glycocyanamidine No. 232 Cardiac glycosides No. 112, 308, 322 Glycolipids No. 117 Carotenoids No. 27 Glycosides No. 27, 59, 112, 230, 306, 308, 320, Carotenoid aldehydes No. 159, 263 322, 335 Catechins No. 318, 326 Gold ions No. 315 Catecholamines No. 134, 135 Guanidine and derivatives No. 157, 199, 290, 29Z 296 Cations, inorganic No. 2, 123, 150, 256 Chloramines No. 61, 62 Halogen ions No. 45, 269 Cholesterol, -esters No. 1 Halogen oxyacids No. 21 Choline No. 126 Heavy metal ions No. 128, 244 Chromium ions No. 31, 35 Hemlock alkaloids No. 289 Cobalt ions No. 253, 265 Heterocyclic compounds No, 141, 189, 213, 302, 303, 307 Copper ions No. 106, 121, 253, 265 Hydrastine No. 69 Corticosteroids No. 125, 154, 310, 323 Hydrazine No. 234 Coumarines No. 82, 243 Hydrocarbons, aromatic No. 146, 307 Creatine No. 232, 290, 292 Hydrocarbons, chlorinated No. 141, 275 Creatinine No. 290, 292 Hydrocarbons see also sugars Cyanamide No. 95, 292 Hydroxamic acids No. 166 Cysteine, Cystine No. 287 Hydroxyamino acids No. 284 Hydroxyl amine No. 164, 234 Dehydroascorbic acid No. 225, 228 a-Hydroxy acids No. 10 Deoxy sugars No. 85, 285, 295 Hypnotics, bromine-containing No. 108, 140 Deoxyribortucleosides No. 84 Dicarboxylic acids No. 44 Indole and derivatives No. 60, 71, 104, 107, 144, 168, 173, Digitalis glycosides, No. 59, 230, 306, 320 221, 286, 331 Dimethylamino acids No. 313 Insecticides No. 40, 108, 119, 190, 274 3,5-Dinitrobenzoic acid esters No. 98, 204 Iodine-containing compounds No. 54, 170 1,2-Di ols No. 180, 181, 282, 283 Iron ions No. 216, 236 Diterpenes No. 28 Isothiocyanates No. 240

Epoxides No_ 231 Keto acids No. 10, 93, 226 Ergot alkaloids No. 105 Ketones Nr. 86 Essential oils No. 124, 329 Ketoses No. 25, 99, 115 Esters No. 155 Ketosteroids No. 11, 177, 224, 248, 281 Estrogens No. 194 Ethanolamine No. 2,57 Lactones No. 155 Ethylvanillin No. 151 Lead ions No. 121, 253, 265 Lipids No. 48, 83, 90, 137, 193, 262, 324 Flavonoids No. 3, 26, 33, 82, 179, 309, 318 Lipoids No. 50 Fluorine ions No. 334 Lithium ions No. 31 Lysine No. 328 Germanium ions No. 227 Gibberellins No. 305 Magnesium ions No. 158, 255 Glycals No. 152 Manganese ions No. 35, 121, 253, 265

102 103 Mercaptans No. 10 Purines No. 138, 272, 278 Mercury ions No. 106, 121, 265, 280 Pyridine compounds No. 46, 63, 80, 234 Meta! ions No. 178 Pyrimidines No. 138 Methyl ketones No. 294 Pyrones No. 120 Methyl sugars No. 110 No. 242 Morphine Quinolines No. 39

Narcotine No. 69 Reducing compounds No. 233, 265 No. 37, 193, 240, 249, 268, 277, 310, Nickel ions 323, 324 No. 65 Nicotinic acid Resins No. 30 Nicotinamide No. 65 Nitro compounds, aromatic No. 314 Sapogenins No. 27, 154 Nitrogen compounds No. 131, 132, 133, 165, 189, 247 Serine No. 284 Nitrogen compounds, aliphatic No. 292 Sesamine No. 69 Nitrosamines No. 118, 304 Silver ions No. 106, 121, 280 Sodium ions No. 333 Oils No. 30 Sorbic acid No. 311 No. 68, 163 Organic compounds Steroids No. 1, 22, 27, 28, 67, 193, 220, 229, Ornithine No. 328 233, 259, 282, 283, 305, 316, 318, Oximes No. 79 320, 321, 324, 327, 329, 332 Steroid alkaloids No. 57, 145, 185, 219 Penicilli ns No. 161 Steroid glucuronides No. 254 Peroxides No. 17, 109, 174, 245 Steroid glycosides No. 27 Persulfates No. 32 Steroid sapogenins No. 30, 57, 70, 145, 219, 332 Phenols No. 4, 34, 37, 47, 88, 136, 143, 166, Steroid sulfates No. 188 191, 213, 214, 215, 240, 266, 302, Sterols No. 1, 38, 67, 193, 203, 229, 305, 316, 307, 316, 329 324 Phenol carboxylic esters No. 213 Strontium ions No. 156, 158, 264 Phenol ethers No. 191 Sugars No. 5, 7, 19, 22, 33, 36, 52, 196, 197, Phenothiazines No. 145, 169 198, 200, 217, 222, 250, 267, 271, Phosphate esters No. 72 312, 325 Phosphoric acids No. 14, 16 Sugar phosphates No. 15 Phthalate esters No. 258 Sugars, reducing No. 6, 18, 21, 23, 24, 113, 116, 223, Piperonal No. 151 323 Plasticisers No. 212, 258 Sulfides No. 161 Plastoquinones No. 182 Sulfonamides No. 60, 87, 293 Polyalcohols No. 9, 250, 279, 282, 283 Sulfonic acids No. 235, 273 Polyethylene glycols and derivatives No. 129, 165 Sulfur-containing compounds No. 49, 168, 218, 287 Polyphenols No. 316 Polyphenyls No. 55 Te rpenes No. 22, 27, 30 Poliysaccharides No. 53, 319 Terpene aldehydes No. 33 Potassium ions No. 73, 299 Tetracyclines No. 8 Proazulenes No. 101 Thiazoles No, 286 Proline No. 328 Thioacids No. 270

104 105 Thiobarbiturates No. 81, 97 Thiophene derivatives No. 175 Solvents and reagents Thiophosphate esters No. 171, 218 Thiosulfates No. 240 800004 Acetaldehyde for synthesis 500 ml, 1 L, 2 L Thiourea and derivatives No. 290, 291, 292, 296 7827 Acetate buffer solution pH 4,66 Threonine No. 284 (0.1 M acetic acid, 0.1 M sodium acetate) 1L Thyroid hormones No. 170 42 Acetic anhydri de GR 1 L, 21/2 L, 33 K Tin ions No. 121 14 AcetoneGR 1 L, 21/2 L, 20 K Tocopherylquinones No. 182 9600 Acetylacetone GR 100 m], 500 m] Triterpenes No. 67, 316 1016 Alizarin GR and indicator 25 g, 100 g, 1 K Triterpene glycosides No. 1 6279 Alizarinsulfonic acid sodium salt GR and Tryptophan No. 331 indicator 25 g, 100 g Tungstate ions No. 255 801082 Aluminium chloride anhydrous sublimed for synthesis 100 g, 500 g, 1 K, 50 K Ubiquinones No. 182 1084 Aluminium chloride tryst. exrra pure 1 K, 50 K Unsaturated compounds No. 139 4-Aminoantipyrine see 1-rheny1-2,3-dimethy1- Oran)/ ions No. 253, 255 4-amino-5-pyrazolone 102 4-Aminobenzoic acid pure Urea No. 290, 292, 296 250 g, 1 K, 25 K Uronic acids No. 198 84 4-Aminohippuric acid [N-(4-aminobenzoy1)- aminoaceric acid] 25 g 800419 2-Aminophenol for synthesis No- 151 250 g, I K, 50 K Vanillin 5432 Ammonia solution min. 25% (0.91) GR No. 320 1 L, 2'/2 L, 28 K Veratrum alkaloids 1116 Ammonium acetate GR No. 13 SOO g, 1 K, 5 K, .50 K Vinca alkaloids 2276 Ammonium cerium (IV) nitrate GR No. 27, 30 100 g,1 K Vitamin A 2273 Ammonium cerium (IV) sulfate qR No. 127, 239 100 g Vitamin Bi 1145 Ammonium chloride GR No. 65 500 g, I K, 5 K, 50 K Vitamin Bo 1182 Ammonium hepramolybdate GR No. 51, 94, 187 250 g, 1 K, 5 K, 25 K Vitamin C 3776 Ammonium iron(11I) sulfate GR No. 27, 30, 320 500 g, 1 K, 5 K, 50 K Vitamin D 1213 Ammonium thiocyanareGR 500 g, 50 K No. 30, 37 Vitamin E 1261 Aniline GR 250 ml, 1 L 1266 Aniline phthalate spray reagent for chromato- Xanthine derivatives No. 167 graphy 1 pack Anisaldehyde see 4-Methoxybenzoldehyde Zinc ions No. 121, 253 800458 p-Anisidine for synthesis 500 g 1468 Anthrone GR TO g 7838 Antimony(III) chloride GR 100 g, 250 g, 1 K 807837 Anrimony(V) chloride for synthesis 25 ml, 100 ml 128 Aurintricarboxylic acid ammonium salt GR 25 g 1719 Barium chrorideGR 500 g, 1 K, 5 K, 50 K 1783 Benzene GR 1 L, 21/2 L, 22 K 802410 p-Benzoquinone (p-quinone) for synthesis 100 g, I K, SO K 3098 2,2'-Bipyridine (a, a'-dipyridyl)GR ..... . 5 g, 25 g 1854 Bismuth(III) carbonate basic extra pure 250 g, 1 K, 25 K 1878 Bismuth (III) nitrate basic GR 100 g, 500 g 165 Boric acid tryst. GR SOO g, 1 K, 5 K, 50 K 1310 Brilliant green SO g

106 107

1948 Bromine GR 50 ml, 250 ml, 1 L 800898 Diethyl malonate for synthesis 500 ml, I L, 21/2 L, 8121 Bromocresol green indicator 1 g, 5 g, 25 g 30 K, 200 K 1998 Bromocresol green spray reagent 0.05% 1,3-Dihydroxynaphthalene for chromatography 1 pack see 1,3-Naphthaienediol 3025 Bromocresol purple indicator 5 g, 25 g 820933 3,5-Dihydroxytoluene (monohydrate) 8122 Bromophenol blue indicator 5 g, 25 g for synthesis 5 g, 25 g, 100 g 801949 N-I3rornosuccinimide for synthesis 250g, 1 K, 25 K 6013 Dimedone GR 25 g 3026 Bromothymol blue indicator 5 g, 25 g 4-Di meth ylaminoazobenzene see Methyl yellow 9887 Buffer Titrisol ® pH 7.00 (phosphate) 1 pack 3058 4-Dimethylaminobenzaldehyde GR 25 g, 100 g, 1 K 1990 I-Butanol (n-butyl alcohol)GR 1 L, 2'/2 L, 22 K 3056 4-Dimethylaminobenzaldehyde spray reagent 0.5% for chromatography 1 pack 4795 Cacothehne GR 5g 3059 5-(4'-Dimethylaminobenzylidene)rhodanine GR 5 g 2003 Cadmium acetate GR 100 g, 500 g 3034 N,N-Dimethylformamide 1 L, 2.'/2 L, 27 K 2222 Carbon tetrachloride GR 1 L, 2V2 L, 44 K 3067 N,N-Ditnethy1-1,4-phenylenediammonium 2233 Carmine for microscopy 5 g, 25 g dichloride GR 25 g 2274 Cerium (IV) sulfate GR 25 g, 100 g, 250 g 3114 1,3-Dinitrobenzene 50 g, 250 g 2426 ChloramineT (trihydrate)GR 250 g, 1 K 138 3,5-Dinitrobenzoic acid 100 g, I K 2427 1-Chroro-2,4-dinirrobenzene G R 100 g 3081 2,4-Dinitrophenylhydrazine GR 25 g, 100 g 2445 Chloroform GR 1 L, 23/2 L, 40 K 3,5-Dinitrosalicylic acid see 2-Hydroxy- 3024 Chlorophenol red indicator 5g 3,5-dinitrobenzoic acid 800220 Chlorosul ionic acid for synthesis 1L 3086 Diphenylamine GR and redox indicator 100 g, 250 g 2498 Chromotropic acid disodium sal tGR 25 g 3091 1,5-DiphenylcarbazideGR and redox indicator • 25 g, 100 g 802505 Cinnamaldehyde forsynthesis 250 ml, 1 L 3087 1 ,5-Dipbenylearbazone GR 5 g, 25 g 244 Citricacid 1-hydrate GR 500 g, 1 K, 5 K, 50 K 3089 Dipicrylamine with 50% water GR 10 g 2539 Cobalt(11) chloride GR 100 g, 250 g, 1 K 2,2'-Dipyridyl see 2,2'-Bipyridine 2536 Cobalt(11) nitrate GR 100 g, 250 g, 25 3092 Dithizone (diphenylrhiocarbazone) GR 5 g, 25 g 2,4,6-Collidine GR see 2,4,6-Trimethylpyridine 983 Ethanol abs.GR 1 L, 21/2 L, 22 K GR 9623 Erhyl acetate GR 1 L, 2'/2 L, 25 K 2711 Copper(11) acetate GR 250 g 800947 Ethylenediamine for synthesis 500 ml, 1 L, 21/2 L, 50 K 2733 Copper(1I) chloride GR 250 g, I K 859 Ethylene glycol mon ometh yl ether GR 1 L, 21/2 L, 25 K 250 g, 1 K 2753 Copper(II) nitrate GR 807291 Ethylene glycol mon °phenyl ether for synthesis I L, 21/2 L 2790 Copper(II) sulfate 5-hydtateGR 250 g, 1 K, 5 K, 50 K 9708 Ethyl methyl ketone GR 1 L, 21/2 L 5208 Creatinine 10 g, 100 g 1408 Crystal violet indicator 25 g, 100 g 3191 Fast blue salt B for microscopy 25 g 2839 L-Cysteinium chloride 25 g, 100 g, 1 K 3990 Fluorescein 100 g 3992 Fluorescein sodium powder 50 g, 250 g, 1 K 3035 2,6-Dibromoquinone chlorimide G R 5 g, 25 g 9182 Fluorescent indicator F254 for thin layer 9676 2',7'-Dich]orofluorescein GR 5 g chromatography 50 g 9677 2',7'-Dichlorofluorescein spray reagent 0.1% 2966 1-Fluoro-2,4-dinitrobenzene GR 20 m], 100 m] for chromatography I pack 4003 Formaldehyde solution min. 35% GR 6050 Dichloromethane GR 1 L, 2V2 L, 37 K (stabilised with about 10% methanol) . 1 L, 5 L, 32 K 3028 2,6-Dichlorophenolindophenol sodium salt 4002 Formaldehyde solution min. 37% 1 L, 5 L, 32 K (dihydrate) GR 5 g, 25 g 4013 Furfural GR 20 ml, 100 ml 3037 2,6-Dichloroquinone chlorimideGR 10 g, 100 g 8342 D(+)Glucose (monohydrare) 803010 Diethylamine for synthesis 500 ml, 1 L, 23/2 L, 35 K for biochemistry and microbiology 500 g, 1 K, 2'/2 K, 50 K 921 Diethyl etherGR 1 L, 5 L, 20 K 4191 Glyoxal bis-(2-h ydroxyanil) GR 25 g

108 109 7341 Hexachloroplatinic(IV) acid solution 10% 6252 1,3-Naphrhalenediol GR 1 g, 5 g (abt. 3.9% Pt) GR 5 011, 25 ml 6223 1-Naphthol CR 50 g, 250 g 4603 Hydrazinium sulfateGR 100 g, 500 g 6531 1,2-Naphrhoquinone-4-sulfonic acid sodium 316 Hydrochloric acid min. 25% (abt. 1.125) GR 1 L, 2'/2 L, 33 K salt GR 5 g, 25 g 317 Hydrochloric acid fuming min. 37% Naphthoresorcinol see 1,3-Naphthalenediol (abt.1.19)GR I L, 2112 L, 35 K 6245 1-Naphrhylamine G R 100 g, 500 g 9970 Hydrochloric acid 1 mold Titrisor ampoule 6237 N-(1-Naphthyl)ethylene diammonium 9971 Hydrochloric acid 0.5 mol/1Titrisols ampoule dichlorideGR 5 g, 25 g 9973 Hydrochloric acid 0.1 mol/ITirrisoPL ampoule 6746 Neata0 for thin layer chromatography 1 L Hydrofluoroboric acid see Tetrafluoroboric acid 6762 Ninhyd rin GR 10 g, 100 g, 1 K Hydrogen peroxide GR see Perhydrola 6758 Ninhydrin spray reagent 0.1% for 800141 2-H ydroxy-3,5-dinitrobenzoic acid chromarography 1 pack (rnonohydrare) for synthesis 25 g, 100 g 456 Nitric acid min. 65% (about 1.40) 4616 hydroxylammonium chloride GR 100 g, 250 g, 1 K tested by the dithizone method GR 1 L, 2'/2 L, 40 K 7098 8-Hydroxyquinoline GR 50 g, 250 g 455 Nitric acid fuming 100% (abr. 1.52)GR 1 L, 30 K 4761 iodine resublimed GR 100 g, 500 g 6760 4-Nitroaniline for derermination of phenol 50 g 9099 Iodine solution 0.05 mot 12/1 1 L Orcinol crust. see 3,5-Dihydroxyroluene 3943 Iron(111) chloride GR 250 g, 1 K, 50 K (monohydrate) for synthesis 3965 Iron(1I) sulfate GR 500 g, 1 K, 5 K, 50 K Orthophosphoric acid see ortho-Phosphoric acid 3954 Iron(II)sulfide fused small lumps 1 K, 5 K 807110 Palladium(1I) chloride (59% Pd) anhydrous 807418 Lead(IV) acetate forsynthesis 100 g, 2.5 K for synthesis 1 g, 5 g, 10 g 7414 Lead (11) hydroxide acetate anhydrous, 4005 Paraformaldehyde extra pure 1 K, 5 K, 50 K for Horne analysis ofglucoses 1 K, 5 K, 50 K 518 Perchloricacid abt. 60% (aht. 1.53) GR 1 L , 21/2 L, 35 K 7398 Lead(11) nitrareGR 1K 519 Perchloric acid abt. 70% (abt. 1.67) G R 1 L, 21/2 L, 35 K 6080 Lead(II, IV) oxide 1 K, 50 K 7209 Perhydrola (30% H202)GR 250 ml, 1 L 5691 Lithium hydroxide abt. 98% LiOH (LAB) 100 g, 250 g, 1 K 1770 Petroleum benzine boiling range 100-140°C 5694 Lithium sulfate GR 250 g extra pure 1 L, 5 L, 20 K, 4.5 K 5819 Magnesium acetate GR 250 g, 1 K 1775 Petroleum benzine GR boiling range 40-60 °C 1 L, 5 L, 18 K 4403 Mercury, GR 250 g, 1 K 206 Phenol GR 250 g, 1 K, 25 K 4419 Mercury(II) chloride GR 50 g, 250 g, K 7293 1-Pheny1-2,3-dimerhyl-4-amino- 4420 Mercury(11) iodide red extra pure DAB 6 100 g, 1 K, 20 K 5-pyrazoloneGR 10 g, 100 g 50 g, 250 g 4437 Mercury (I) nitrate Gt1 809721 1,2-Phenylenediamine forsynthesis 100 g, 250 g, 1 K 50 g, 250 g 4439 Mercury(1I) nitrate 1-hydrareGR 7244 1,3-Phen ylenediammonium dichloride GR 50 g 4461 , Mercury (II) oxide yellow GR 50 g, 250 g, 1K 822297 1,4-Phenylenediammonium dichloride 1 L, 21/2 L, 20 K 6009 Methanol GR for synthesis 250 g, 1 K 6012 Methanol dried (max. 0.01`!,,, H2O) GR 1 L, 21/2 L 7252 Phenylfluorone 1 g, 5 g 250 ml, 1L 822314 4-Methoxybenzaldehyde for synthesis 7251 Phenylh ydrazine G R 100 ml, 1 L 806225 4-Meth ox y-2-n itroaniline for synthesis 250 g 7266 Phloroglucinol (dihydrate)GR 25 g, 100 g 1283 Methylene blue for microscopy 25 g, 100 g Phosphomolybdic acid see Molybdato- Methylene chloride see Dichloromethane phosphoric acid 25 g, 100 g 6078 Methyl red sodium salt indicator 573 ortho-Phosphoric acid min. SS% (abt. 1.71) GR 1 L, 21/2 L, 45 K 3055 Methyl yellow indicator 10 g, SO g 9611 Phrhalic acid GR 500 g 532 Molybdatophosphoric acid GR 25 g, 100 g 623 Picricacid (with 0,5 ml H20/g) GR 100 g, SOO g 531 Molybdatophosphoric acid spray reagent 3.5% 9723 Pinacryptol yellow (LAB) 1 g for chromatography , 1 pack 807325 Piperazine anhydrous for synthesis 250 g, 1 K 6098 MorinGR 5 g 4912 Potassium bromate GR 250 g, 50 K

110

—di 4905 Potassium bromide GR . . . 500 g 9956 Sodium hydroxide solution 1 mol/1 Titrisol® ampoule 4928 Potassium carbonate GR 500 g, 1 K, 5 K, 50 K 9957 Sodium hydroxide solution 0,5 moUlTitrisol® . ampoule 4967 Potassium cyanide GR 100 g, 250 g, 1 K 9959 Sodium hydroxide solution 0.1 mol/ITitrisc0 ampoule 4864 Potassium dichromate GR 500 g, 1 K, 5 K 6523 Sodium iodide GR 100 g, 250 g, 1 K, 50 K 4875 1/15 M Potassium dihydrogen phosphate 6287 Sodium meta-arsenite GR 250 g solution 1 L 6597 Sodium meta-periodare 50 g, 250 g, 1 K 4984 Potassium hexacyanoferrare(I1) GR SOO g 6521 Sodium molybdate GR 100 g, 250 g, 1 K 4973 Potassium hexacyanoterrate(111)GR 250 g, 1 K, 50 K 6549 Sodium nitrite cryst.GR 500 g 5033 Potassium hydroxide pellets GR 500 g, l K, 5 K, SO K 6541 Sodium nitroprusside GR 25 g, 100 g, 500 g 9918 1 N Potassium hydroxide solution Titrisoe' • ampoule 6551 Sodium peroxide powder GR 500 g 9351 Potassium hydroxide solution 0.5 mo1/1 6638 Sodium sulfide GR 250 g, 1 K in methanol 1L 6308 di-Sodium tctraborate 10-hydrate tryst. GR 500 g, 1 K, .50 K 5043 Potassium iodide neutral G R 250 g, 1 K, 21/2 K, 50 K 6516 Sodium thiosulfate 5-hydrate GR 500 g, 1 K, 5 K, 50 K 6673 Sodium tungstate GR 5082 Potassium permanganate GR 250 g, 1 K 100 g, 250 g, 1 K, 50 K 9121 Porassiuin permanganate solution 0.02 ino1/1 1L Standard acetate buffer solution 9634 2-Propanol GR 1 L, 21/2 L, 20 K see Acetate buffer solution pH 4.66 9728 Pyridine GR 500 nil, 1 L, 21/2 L 1252 Starch soluble GR 100 g, 250 g, 1 K 7531 1-(2-Pyridylazo)-2-naphthol (PAN) metal (pM) 8035 Sulfanilamide extra pure 1 K, 50 K indicator 1 g, 5 g 686 Sulfanilic acid GR 100 g, 250 g 7546 Quercetin crysr. (LAB) 10 g 7996 Sulfidogen® (sulfur paraffin grated) Quinalizarin see 1,2,5,8-Tetrahydroxyanth ra- for generation of hydrogen sulfide 500 g qui none 691 5-Sulfosalicylic acid (dihydrare) GR 100 g, 250 g, 1 K, 50 K p-Quinone see p-Benzoquinone 731 Sulfuric acid 95-97% (abt. 1.84) GR 1 11, 21/2 L, 40 K 7593 Resorcinol GR 100 g, 250 g 9981 Sulfuric acid 0.5 mol/l Titrisol® ampoule 7599 Rhodamine B GR and for microscopy 25 g, 100 g 9984 Sulfuric acid 0.05 mol/I Titrisol® ampoule 7596 Rhoda mine B spray reagent (0.1%) I pack 9982 Sulfuric acid 0.005 mo1 /1 Titrisol ampoule 6595 Rhodizonicacid disodium salt GR 5 g 804 L(+)Tartaric acid GR 250 g, 1 K, K, 50 K 629 Rubeanie acid GR 10g, 100 g 808240 Tetracyanoethylene for synthesis 10 g 1512 Silver nitrateGR 25 g, I00 g, 250 g, 1 K 171 Tetrafluoroboric acid 9990 Silver nirrate solution 0.1 mo1/1TitrisuP 1 ampoule (fluoroboricacid) about 35% 21/2 L, 60 K 6261 Sodium GR 250 g, 1 K 8126 1,2,5,8-Tetrahydroxyanthraquinone 6268 Sodium acetate anhydrous GR 250 g, 1 K, 5 K, 50 K (quinalizarin) GR 50 g 6688 Sodium azide extra pure 100 g, 250 g, 1 K, .50 K 8103 Tetrazolium blue for microscopy 1 g, g 6391 Sodium carbonate 10-hydrare tryst. GR 1 K, 5 K, 50 K 8180 2-Thioba rbiruric acid reagent for sorbic acid 25 g 6392 Sodium carbonate anhydrous GR 500 g, 1 K, 5 K, 50 K 8167 Thymol tryst. extra pure 100 g, 1 K 6404 Sodium chloride crysr. GR 500 g, 1 K, 5 K, 50 K 8176 Thymol blue indicator 5 g, 25 g 6448 tri-Sodium citrare 2-hydrate GR 500 g, 1 K, 5 K, 50 K 7815 Tin (11) chloride GR 100 g, 250 g, 1 K 6336 Sodium dichromate CR 250 g, 1 K 7811 Tin(IV) chloride fuming GR 100 ml 6346 Sodium dihydrogen phosph are 1-hydrate GR 500 g, 1 K 1307 Titan yellow GR 50 g 6507 Sodium dithionite (LAB) 500 g, 71/2 K 9613 4-Toluenesulfonic acid (monohydrate) GR 100 g, 500 g 6329 Sodium hydrogen carbonate GR 500 g, 1 K, 5 K, 50 K 1273 Toluidine blue 0 for microscopy 50 g 6587 1/15 M di-Sodium hydrogen phosphate solution 1 L 807 Trichloroacetic acid GR 100 g, 250 g, 1K 6498 Sodium hydroxide pellets GR 500 g, 1 K, 5 K, 50 K 808260 Tritiuoroaceticacid for synthesis 25 ml, 100 ml, 1 L .5588 Sodium hydroxide solution 10% (1.11) GR l L 2635 2,4,6-Trimerhylpyridine GR 50 ml, 250 ml 5591 Sodium hydroxide solution min. 27% (1,30) GR 21/2 L, 60 K 8380 2,3,5-Triphenyttetrazohum chloride 10 g, 100 g 9961 0.01 N Sodium hydroxide solution Titrisoll' ampoule 583 Tungstophosphoric acid GR 100 g, 250 g

112 113 8473 Uranyl acetate GR 25 g, 100 g, 500 g 8487 Urea GR 500 g, 1 K, 5 K, 50 K 8510 Vanillin 100 g, 1 K 8696 Xanthydro] ahn. 10% in methanol 10 ml 8789 Zinc powder GR 500 g, 1 K 8802 Zine acetate GR 250 g, 1 K 8816 Zinc chloride dry GR 250 g, 1 K 8917 Zirconium(IV) oxidechloride GR 25 g, 100 g