Stanley Samuels N e w York University Medical Center Deparlment of Psychiatry and Neurology N e w York City
Micro-Thin-Layer Chromatography in Qualitative Organic Analysis
Like paper chromatography, thin-layer chromatography (TLC) has a number of advantages that make it well suited to the teaching laboratory. I t is a widely used modern research technique appropriate to the qualitative and quantitative analysis of complex mixtures. Its versatility permits it to he used to illustrate a wide range of chromatographic concepts and chemical reactions. I t is particularly economical--of time, money, and materials-making it suitable for almost any budget. The general usefulness of TLC in the organic chemistrv lab has been renorted and its value in aualitative organic analysis suggested ( 1 ) . A number of features make TLC idcally suitable for application to systematic analytic schemes: (1) Migratory methods permit solubility characterination of a compound. (2) Spray reagents utilize functional group reactions. (3) Corrosive reagents can be used with TLC that cannot be used on paper chromatograms. (4) Many mixtures can he resolved and the components simultaneously analyzed. (5) Derivatives can be prepared and also chromatographed. (0) Unknowns can be identified by co-chromatography with suitable test compounds and cochromatography of the derivatives. (7) Reactions can
he performed with quantities of material too small for test-tube analyses. (8) Preparative scale TLC (on thick layers) can be used to purify an unknown for instrumental analysis. General Methods
Layers are prepared on microscope slides (2) by spraying a suspension of 10 g cellulose (MachereyNagel MN-300, Brinlcmann Instrument Co., Westbury, New Yorlc) in 70 ml acetone/water (1 :3). This is sufficient for 54 slides. An artist's mouth-type atomizer or an aerosol paint sprayer is conveniently used. The use of cellulose layers permits full utilization of the vast paper chromatography literature although silica gel and Kieselguhr are also used, especially for the corrosive reagents. Glass plates (3 in. X 3 in., Arthur H. Thomas #8274) can also be used, for two-dimensional applications. The layers are dried a t 70-100" with a hotplate or oven and stored in a clean place. The silica gel layers should be activated at about 110" for 30 min and then stored in a desiccator. Different amounts of the unknown are spotted on a series of slides using a nichrome loop or a fine capillary. The slides are used in testing the suitability of various
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detection reagents, which are used to observe the migration of the substance in the different test solvents. I n addition, the reagents give information on the chemical reactivity of the compound (Table 1). I n addition to the many reference texts on paper chromatography (5-8) and TLC (9, lo), many spot test (11) and functional group reactions (18) can be used for the detection of a refractory unknown and for later functional group analysis. Once a suitable detection reagent has been found and the required amount of the unknown determined, solubility characterization can begin. Table 1.
Detection Reagents
Method
Ref.
Iodine Vapor Ultraviolet light (long wave length) Alkaline permanganate Bramcresol green AICls CHCls ChlonneBenzidine
,+
Fnnctional group or class
(3) Unsat~wation,,lipids,sugars (3) Steroids, punnes, pyrimidines (3) Oxidizable groups (3) Acids and bases (4) Aromaticity (3) Amide or amine nitroeen
Solubility Classification
Suitable portions of the sample are spotted 1 cm in from one end of a series of coated microscope slides. Solvent testing can be carried out in parafilm covered 150-ml beakers, in Coplin staining jars, or (most conveniently) in 2-02 screw cap jars (A. H. Thomas 6284B). Two-dimensional chromatography on the 3 in. X 3 in. plates is best run in 16-oz screw cap jars (A. H. Thomas 6284F). A test solvent is added at the bottom of the jar to a depth of about 0.5 cm and the microslide is placed in it. The solvent is allowed to rise at least 2 in. before the slide is removed, dried, and treated with the detection reagent. The test solvents are of two types: the classification solvents of Shriner and Fuson (I$), and the elutropic series compiled by Randerath (10) (see Table 2). The two-solvent series is used to permit utilization of the analytic scheme of Shriner and Fuson and at the same time to find suitable solvents for subsequent TLC analysis of the unknown. I n series B (Table 2), if the compound remains a t or near the origin it is classified as insoluble, and if it remains at or near the front it is termed soluble. The solvents of series A are of decreasing hydrophilic properties. A compound which remains at the origin in one solvent and migrates to the front in the next may be suitably chromatographed in a mixture of the two or in another solvent (10) of intermediate polarity. Problems of streaking or doublespotting can frequently be controlled by the addition of a volatile acid, base, or buffer.
Functional Group Analysis
As already indicated there is a considerable literature on chromatographic spray reagents: functional group methods adapted as spray or dip procedures. Fusion techniques and elemental analysis have not yet been applied, but there has probably not been an urgent need for them. I t is not possible to give a comprehensive list of functional group spray reagents; in addition to the reference texts already cited, a very large multivolume bibliography has been published (13) and the literature is still expanding (14). Nevertheless, some suggestion of the use of the technique can be given (Table 3). I t should be pointed out that the methods are not necessarily specific, and standards should be run alongside. An Illustrative Specific Application of TLC Analysis
A patient with Refsum's Syndrome, a rare metabolic disorder (167,was found to have a peculiar substance in his urine. Following hydrolysis with glncuronidase, and solvent extraction of the free steroid fraction, a material was present on the papcr chromatogram which gave a pink spot with 10% sodium carbonate. Because of the possible relevancy of this substance to the disease and the very small amount present in the urine, a large number of the steroid papcr chromatograms were processed. The spot areas were eluted with methanol and concentrated for analysis by the technique described here. The material gave a permanent reaction with sodium carbonate, a transient reversible pink spot with ammonia vapor and a transient irreversible reaction with 5y0 sodium hydroxide. The reaction with ammonia was used for detection in establishing solubility characteristics and for finding a suitable TLC solvcnt. By means of the Shriner and Fnson solvent series, the substance was classified as group A2, suggesting that it might be a phenol. Testing with the elutropic solvent series showed it to be insoluble in water but very soluble in methanol. It was found to have an R, of about 0.5 in 30% (V/V) aqueous methanol. Reaction with sodium carbonate and heat followed by observation under long wavelength ultraviolet light revealed eleven components. The reactions with ammonia and sodium hydroxide suggested a substituted naphthoquinone (5); however, testing with potassium permanganate and reduced phosphomolyhdatc showed that it was neither a hydroquinone nor a quinone. I t was shown by reaction with powdered aluminum chloride and chloroform overspray that the substance was aromatic. Negative Table 3.
Selected Functional Group Spray Reactions
Functional Table 2.
(A) Classification solvent (after 10) Water Methanol Chloroform/methanol(95:5) Chloroform/metbanol (99: 1) Chloroform Toluene Petroleum ether
146
/
-
P"m," =-'.
Test Solvent Series
Journal of Chemkol Education
(B) Elutropio series (after 18) Water 5% NaHCOa 5% NaOH 10% HCl ...
...
Unsaturation Aldehvde Carb&vlic acid Amide Phosphate Phenol Amino Quinone Sulfhydryl Guanidino
R!=aa-nt -v-m-.."
Bromine Silver oxide Nit+-biuretrdimetbvlelvoxime . -. Chlorine-beneidine Ammonium molybdate Diazo-o-nitroaniline ~~-~ Ninhydrin 1% reduced ~bos~homolybdate Nitro russid; &~yrfroxyquinoline ~
~
~~~
Pd
(5) (3)
(id '(si
(3)
(Ri
?si
reaction with Ehrlich's reagent (Dimethyl-amino benzaldehyde) precluded an indole ring, and a negative reaction with the chlorine-benzidine reagent suggested the absence of nitrogen. The peculiar reactivity of the substance made it necessary to establish clearly whether or not it was a phenol, and additional spray reagents were developed. (See Table 4.) Table 4.
New Snrav Reaoents for Phenols
Reawnt
Ref.
Color*
Saturated alcoholic FeClr 25% HNOa; heat; 10% NalCOs; heat 1% Anisaldehyde in 10yo alcohol~o NaOH. heat ~iazo-D-Aitroaniline:5% NNaCOs
...
(4) (4)
Purple Orange Pink-Brown
(5)*
Red
Different phenols may give other colors. Because of the color wit,h NalCO. alone, NsHCOa was substituted in this reagent.
It may be pointed out that 1% alcoholic ferric ehloride (3) gave no reaction. It was observed that a loss of the pink color following a 5% sodium hydroxide spray accompanied the drying of the reagent and that the color loss was almost instantaneous following a spray with 50% sodium hydroxide. The data suggested a phenolic compound with an oxonium group and a labile struct,ure-possibly a lactone. A large amount of the methanol eluate was streaked across a cellulose thin-layer plate 8 in. X 8 in. and was chromatographed in 30% methanol. I t was detected with ammonia vapor and marked off. Following evaporation of the ammonia and loss of the pink color, the cellulose band was scraped from the plate and eluted with methanol. Evaporation revealed a few grains of a white amorphous powder which melted above 250". The methanol solution showed a sharp peak in the ultraviolet at 234 mfi. Comparison of the melting point data and chemical properties with compounds in the literature suggested phenolphthalein, which is a bis-phenolic lactone and melts a t 261". Its pink color with alkali is well known, and in the presence of excess alkali the colorless trisodium salt is formed. Since the patient was receiving no medication and endogenous synthesis was most unlikely, it was difficult to accept this for the identity of the unknown. However, a phenolphthalein standard gave the same color reactions as the unknown and the same R,values in 30% methanol and two other solvents (n-butanol saturated with 4M ammonia; and 10% sodium carbonate). The ultraviolet spectrum also matched exactly. Interrogation of the clinical staff did not reveal the source of this con~pound,which is a common ingredient of many laxatives. Careful questioning of the patient's mother disclosed that she had been supplying him with laxative tablets in the hospital. While the investment of time and effort revealed nothing new about the patient's disorder, it was most informative regarding the pitfalls of clinical biochemistry and the usefulness of this analytic procedure as a research tool. Conclusion
Chromatography on microplates is now finding increasing use as a microchemical technique because of its
1 inch
I
Two-dimenriond lhin-layer chromatography of omino acid.: Cellvlare NHI = 411. layer on 3-in. X 3-in. plate. Solvent 1: n - p r 0 p a n o l / 3 ~ ~ Solvent 2: n-prapanol/methyiethylketonn/25% formic odd = 5/3/2, 5mp moles of each omino acid (Colbiochem Standard Solution AA-5). detected with ninhydrin: 1, leucine; 2, iroleucine; 3, phenylolanine; 4, voline; 5, methionine; 6, tyrosine; 7, proline; 8, threonine; 9, danine; 10, rerine; 11, hirtidine; !2, glycine; 13, lyrine; 14, arginine; 15, glutomote; 16, asportote; 17,cyrteine.
great speed, high sensitivity, good resolution, and low cost. Bi-dimensional separation of the protein amino acids has been achieved in 30 minutes on 3 in. X 3 in. plates using a solvent system that requires three days with large filter paper sheets (l7),and more than 10 times as much material (see the Figure). The technique offers extensive possibilities in the teaching laboratory and in t,he research lab as well. Acknowledgment
The support and encouragement of Doctors William D. Drucker and William I
