Streak Reagents for Chromatography ARTHTJH L. L E K O S E S ~ , RICHARD T. RIORAVEK, A K D JACK IC. C-IRLTON2 Coates Chenticul Laboratories, Louisiana S t a t e Cniversity, Baton Rouge, La. Thirteen reagents are presented which have been developed for use in the detection of colorless zones on a silicic acid column. Some of the tests are n e w and some are known tests which have heen adapted to the chromatographic technique. The compounds studied w-ere dissolved in benzene, adsorbed on silicic acid, and developed with benzene. The preparation and stability of reagents are discussed; the compounds detected, the reagent used for the detection, and color of reaction product are tabulated.
I
S THE original work by LeRosen and conorkers ( 7 ) sixtecn
reagents were reported which had been found useful in the detection of colorless zones on chromatographic columns. The preparation, stability, and perceptible reaction characteristics of those reagents were discussed and the compounds for which they could be used were listed The present work is a by-product of studies made on adsorbent specificity and steric factors in polysubstituted conipounds. As in the original paper, some of the tests are new and some have been adapted from known tests to the detection of colorless zones on a chromatographic column It was not the purpose of this investigation to establish limits of identification or reaction kinetics, or to make extensive interference studies. However, it is believed that the tests presented here will be of considerable value in qualitative analysis, where chromatographic adsorption is used as a means of separating organic mixtures. The organic compounds used in this work were prepared as 0.01 -11solutions in benzene and adsorbed on Merck reagent grade silicic acid employing benzene as developer I n Table I is found a list of the compounds studied, the reagents effective in their detection, and the color of the reaction product. PREPARATION AND USE OF REAGEhTS
In the follouing section are listed the reagents studied, their preparation, and pertinent remarks concerning stability and reaction characteristics.
1. Gold Iodide Complex. This reagent is prepared by adding 2 drops of a 2 .If (approximately) solution of gold chloride to a solution containing 0.5 gram of potassium iodide dissolved in 25 ml. of water. This reagent streaks brown to black against a pink background when amines and sulfides are present on the column. The appearance of the characteristic bron-n-black reaction product with sulfides is enhanced by exhaling gently on the column a few seconds after streaking. The reagent should be prepared fresh about once a month. 2. Ceric Nitrate (3). This reagent is prepared by dissolving 90 grams of ceric ammonium nitrate in 225 ml. of warm 2 S nitric acid. The reagent streaks brown to black against a yellow background in the presence of phenols and aromatic amines. Substituted phenols react slightly or not a t all. 3. o-SafranineBromine. The column is first streaked with a solution containing 3 mg. of safranine dissolved in 25 ml. of mater. The column is then overstreaked n i t h a 0.1 M solution of potassium bromide to n,hich has been added 4 or 5 drops of bromine. Overstreaking with bromine solution bleaches the red streak of the safranine, but when di- and triaryl amines, unsaturated compounds, and sulfones are present the red color of the safranine is quickly regenerated in the zone. Unsaturated compounds of low molecular weight are not easily detected with this reagent. 4. Vanillin in Sulfuric Acid (9). This reagent is prepared as a 1% solution of vanillin in concentrated sulfuric acid. Cnsuhstituted phenols give pink or red zones against a colorless background using this reagent. Some suhstituted phenols give weak positive reactions. Although the reagent takes on a dark coloration after standing for some time, it will give characteristic reactions with phenols several months after preparation. 1
2
Decewl. Present address, University of Arkansas, Fayetteville, Ark.
5 . Bismuth Iodide Complex. Potassium iodide, 1 Jf, is added to 1 11.1’ bismuth nitrate until the precipitate formed is just dissolved. This reagent is used to test for amines. Xliphatic amines give a reddish-orange zone against a yellow-orange background. Aromatic amines react similarly but not as vividly. The reagent should be prepared fresh once a month. 6. Vanadate-8-Quinolinol (1). The colurmi is first streaked Jyith a solution of ammonium vanadate containing 200 to 300 mg. of vanadium per liter, then overstreaked with a 2.5% solution of 8-quinolinol in 6% acetic acid, Alcohols containing four carbon atoms or more react to give orange-brown zones against a dull green background. The reaction is faster with alcohols of higher molecular weight. 7 . Chloranil in Dioxane (6). This reagent is prepared as a saturated solution of chloranil in dioxane. When streaked on aliphatic amines a green zone develops against an orange background. Ammatic amines give weak tests and the zone, instead of being bright green in appearance, is dull green to black. 8. Ammonium Chloride-Zinc Dust (8). Ammonium chloride (0.5 gram) and zinc dust (0.5 gram) in 10 ml. of 50% ethyl alcohol are heated to boiling. This solution, when streaked on a column as a warm slurry, gives a yellow zone after a short period of time when nitro compounds are present. 9. Stokes Reagent (11). Ferrous sulfate (1.5 grams) and tartaric acid (1 gram) are dissolved in 50 ml. of water. At the time of use, 1.0 ml. of the reagent is placed in a test tube and 1 M ammonium hydroxide is added dropwise until the precipitate that first forms just dissolves. Then approximately 1.0 ml. of 50% potassium hydroxide is added. This mixture is shaken and streaked on the column. Nitro compounds readily give a red zone on a greenish background. On standing, the Thole column soon turns red. 10. Mercury Iodocyanide Reagent (10). The reagent is prepared by dissolving 1.6 grams of mercuric cyanide in 25 ml. of water to which is added 25 ml. of a solution containing 0.8 gram of iodine and 1.6 grams of potassium iodide. This solution, when streaked on a column containing organic acids, develops a red color along the edge of the streak a t the site of the zone. After sufficient time a red zone against a vhite background is formed at the site of the adsorbed acid. 11. Formaldehyde in Sulfuric Acid ( 4 ) . To 10 ml. of concentrated sulfuric acid 0.2 nil. of 37% formaldehyde is added. This solution is streaked on a column to give colored zones in the presence of adsorbed aromatic hydrocarbons. In general, noncondensed rings are denoted by red zones: condensed nuclei are indicated by green or blue-green zones. Aromatic ethers, thiophene, and a few miscellaneous compounds give color reactions with this reagent. Because benzene creates an interference, these compounds Kere dissolved in heptane and developed with that solvent. This reagent should be used Kith adequate ventilation, because formaldehyde is a strong lung irritant. I t should be prepared fresh daily 12. Doctor Test ( 8 ) . A plumbite solution is prepared by addin 1.25 grams of litharge (PbO) to a solution of 7.5 grams of s o 8 u m hydroxide to 40 ml. of water. This solution when streaked on a column gives yellow zones in the presence of mercaptans (thiols), thiophenol, and hydrogen sulfide. If a saturated solution of sulfur in n-heptane, benzene, or other nonpolar solvent (the solvent is usually the same as the developer used) is streaked over the plumbite solution, the yellow zone turns gray and finally black. The sequence of reagents may he interchanged with similar results, as the saturated sulfur solution will also give yellow zones in the presence of the compounds mentioned above which, again, turn black on overstreaking with plumbite solution. 13. Antimony Pentachloride in Carbon Tetrachloride (6). The reagent is prepared by adding 7 . 5 ml. of antimony penta-
1335
ANALYTICAL CHEMISTRY
1336
Reagent 1. Gold iodide complex
2. Ceric nitrate.
3.
+Safraninebromine
4. Vanillin in sulfuric acid 5.
Bismuth iodide complex
6 . Vanadate-
8-quinolinol
-
I .
Chlor ani1 in dioxane
E. Ammonium chloride-zinc dust 9. Stokes reagent 10. hfercury iodocyanide reagent 11. Formaldehyde in
sulfuric acid
Table I. Compounds Detected Ethyl sulfide Isoamyl sulfide Benzyl sulfide Butylamine Amylamine Decylamine Hexadecylamine Diamylamine Aniline Ethylaniline Diethylaniline Phenol Resorcinol 2-Saphthol o-Toluidine o-Chlorophenol o-Cresol Ethylaniline Diethylaniline 1,3-Pentadiene Styrene Phenol Resorcinol 2-Xaphthol Catechol Butylamine Amylamine Decylamine Hexadecylamine Diamylamine Diethylaniline Butyl alcohol Amyl alcohol Hexyl alcohol Myristyl alcohol Butylamine Amylamine Decylamine Hexadecylamine Diamylamine Aniline Ethylaniline Nitrobenzene Nitropropane m-Dinitrobenzene Xitrobenzene Xitropropane m-Dinitrobenzene Acetic acid n-Heptylic acid Lauric acid Benzoic acid Benzene Toluene o-, m-. p-Xylene E thylbenzene Cumene
Compounds, Reagents, and Color Developed Color Compounds Developed Detected Reagent Brown-black Mesitylene 11. Formaldehyde in Dark brown Triethylbenzene sulfuric acid Brown Hexaethylbenzene (Contd.) Black Diphenylmethane Black Triphenylmethane Black Dibenzyl Black Stilbene Black Kaphthalene Brown-black, slow Diphenyl Brown, faint Tetralin Black Anthracene Brown Phenanthrene Brown Fluorene 1,3&TriphenylBrown Brown benzene BIown Thiophene Brown Carbazole Red Anisole Red Phenyl ether Red Phenol Red 2-Saphthol Red Benzyl alcohol Red Benzyl mercaptan Blue-black Chlorobenzene Red Methyl-2-naphOrange thyl ketone Orange Thiophenol 12. Doctor test Orange Benzyl mercaptan Orange Hydrogen sulfide Orange Benzene 13. Antimony pentaOrange chloride in carbon Naphthalene Pink Anthracene tetrachloride Red Diphenyl Brown Ethylbenzene Orange-brown 1,3,5-TriphenylGreen benzene Green Triphenylmethane Green Dibenzyl Green Stilbene Green Thiophene Green-black Phenanthrene Green-black Hexaethylbenzene Tellow Tetralin Yellow Carbazole Yellow Phenol Red 2-Xaphthol Red Benzyl alcohol Red Methyl-2-naphRed thyl ketone Aniline Red Red hiethylaniline Dimethylaniline Red ;inisole Red Phenyl ether Red Thiophenol Red Benzyl mercaptan Rec!-tan Ethyl disulfide Red
chloride to 20 ml. of carbon tetrachloride. This solution when streaked on the column gives positive tests in the presence of aromatic hydrocarbons. In general, nonfused aromatic nuclei give yellow zones: condensed nuclei shift the color of the zone toward the violet end of the spectrum. h few other aromatic compounds also give positive tests. This reagent should be handled with caution, because of the extreme volatility of the antimony pentachloride, and should be well atoppered when not in use t o prevent the hydrolysis of antimony pentachloride. ACKNOWLEDGMENT
The author8 wish to express their appreciation to the Office of S a v a l Research for financial assistance provided them under Contract x7onr-356, T. 0. I\-. LITER4TURE CITED
(1) Buscarons, F., Marin, J. L., and Claver, J., Anal. Chim. Acta. 3, 310 (1949).
Color Developed Tan Orange-red Yellow Red Red Dark red Brown Green Blue-green Red-brown Yellow-green Green Green Blue Red-violet Green Red-violet Violet Red-violet Brown Red Orange Red Orange Black Black Black Tellow Gray Blue-green Red-blue, fade Light orange Green Yellow Yellow Green Yellow Blue Yellow Orange Green Green Brown Red-orange Yellow Green Tan Orange Light orange Light orange Yellow Orange Light red, fade
(2) Cheronis, N. D., and Entrikin, J. B., “Semimicro Qualitative Organic Analysis.” p. 141, Yew York, Thomas Y. Crowell Co.. 1947. (3) Duke, F. R., and Smith, G. F., IND. ESG. CHEW,~ I L ED., . 12, 201 (1940). (4) Ficklen, J. B., “Manual of Industrial Health Hazards,’’ p. 43. Lancaster, Pa., Science Free Press Printing Co., 1940. (5) Frehden, O., and Goldschmidt, L., Mikrochim. Acta, 1, 33s (1937). (6) Hilpert, S.,and Wolf, L., Ber., 46, 2215 (1913). (7) LeRosen, 4.L., Monaghan, P. H., Rivet, C. A., Smith, E. D.. and Suter, H. A., ANAL.CHEM.,22, 809 (1950). (8) Shriner, R. L., and Fuson, R. C., “Systematic Identification of Organic Compounds,” p. 149, Sew York, John Wiley & Sons. 1942. (9) Takahashi, T., Untersuch, S a h r . u . Genussm.. 27, 820 (1915). (10) Welcher. F.. “Chemical Solutions,’’ p. 221, New York, D. Van Kostrand Co., 1942. (11) Ibid.,p. 333. RECEIVED for review April 10, 1952.
Accepted May 23, 1952
