Anal. Chem. 1998, 70, 7R-26R
Planar Chromatography Joseph Sherma
Department of Chemistry, Lafayette College, Easton, Pennsylvania 18042 Review Contents General Considerations History, Books, Reviews, and Student Experiments Theory and Fundamental Studies Chromatographic Systems (Stationary and Mobile Phases) Apparatus and Techniques Detection and Identification of Separated Zones Quantitative Analysis Preparative-Layer Chromatography and Radio-Thin-Layer Chromatography Applications Acids and Phenols Amino Acids, Peptides, and Proteins Antibiotics Bases and Amines Carbohydrates Dyes and Pigments Hydrocarbons Lipids Pesticides Pharmaceuticals, Drugs, and Alkaloids Purines, Pyrimidines, and Nucleic Acids Steroids Surfactants and Detergents Toxins Vitamins Miscellaneous Organic Compounds Inorganics and Metal Organics Literature Cited
8R 8R 9R 9R 10R 11R 12R 12R 12R 13R 13R 13R 13R 13R 14R 14R 14R 15R 16R 18R 18R 18R 19R 19R 19R 20R 21R
This is a selective review of the literature of thin-layer chromatography (TLC) in Chemical Abstracts from November 1, 1995 to November 1, 1997. The literature search was augmented by consulting Analytical Abstracts, Chemical Titles, and Current Contents, and the following important journals publishing papers on TLC were searched directly: Journal of Chromatography (parts A and B and the bibliography issues), Journal of Chromatographic Science, Chromatographia, Analytical Chemistry, Journal of Liquid Chromatography & Related Technologies, Journal of AOAC International, Journal of Planar Chromatography - Modern TLC, and Acta Chromatographica. Publications in the past two years on the theory, techniques, and applications of TLC continued at a high level. Only a very small number of papers reported new research in paper chromatography, the other main classification of planar chromatography, but none of these was considered to be important enough to be included in this review. A computer-based search of Chemical Abstracts found that 1650 publications on TLC were abstracted in the review period. The attempt was made to cite only the most important publications describing significant advances in theoretiS0003-2700(98)00002-X CCC: $15.00 Published on Web 03/21/1998
© 1998 American Chemical Society
cal studies, methodology, instrumentation, and applications in this review. The review is mostly limited to journals easily accessible to U.S. scientists. This eliminates coverage of many papers in foreign language journals, most notably Chinese papers, which account for ∼15% of the TLC literature. Abstracts citations are given for references from the more obscure journals and papers not published in English. Although TLC was applied during the past two years in many fields, the greatest number of papers were published on the analysis of pharmaceuticals and drugs. Highperformance thin-layer chromatography (HPTLC) is finding increasing use relative to conventional TLC because of its lower detection limits, improved separations, and more accurate and precise quantification. Most TLC papers originated from laboratories outside of the United States, especially Europe and Asia. Several important articles were published in 1996 or 1997 that reviewed the latest trends and future prospects in TLC. Weins and Hauck (1) noted that the greatest number of published TLC papers from 1990 to 1996 were distributed in chemical synthesis monitoring and pharmaceutical, medical, clinical, food, environmental, toxicological, and biological-biochemical analysis, and they discussed advances in methodology and applications in these areas. Jaenchen (2) emphasized the advantages of the off-line character of TLC with storage of sample and standard fractions and simultaneous analysis of multiple samples on a single plate, and he discussed automation of the various steps in the procedure. A trend in the past two years has been the increased use of image analysis, which Jaenchen (2) states will not equal the specificity or, in many cases, the accuracy of densitometric evaluation but may be adequate in many analyses. Cserhati and Forgacs (3) reviewed trends in sample preparation and application, TLC sorbents, mobile-phase optimization and plate development, solute detection and identification, TLC as a pilot method for HPLC, determination of molecular parameters such as lipophilicity, and quantification and validation. Predictions were made for increased application of solid-phase extraction, increased emphasis on separations of enantiomers, development of sorbents for separation of bioactive macromolecules (peptides, proteins), description of more efficient optimization methods for preselection of layer and mobile-phase components, greater use of combined TLCspectroscopy methods, and growing demand for validation of quantitative results (e.g., pharmaceutical compendial and regulatory methods) in the future. The analysis of impurities and stability assessment in pharmaceutical laboratories were mentioned by Poudrier (4) as important applications of TLC. She also discussed the need to establish the robustness of TLC methods and, as part of the process, mobile-phase optimization by trialand-error, simplex, mixture design, and window diagram methods. In a review by Borman (5) of a symposium on the future of analytical chemistry at Pittcon ’97, M. Bonner Denton is quoted Analytical Chemistry, Vol. 70, No. 12, June 15, 1998 7R
as suggesting that thin-layer chromatography may be undergoing a rebirth. Denton cited a micronebulizer device for reproducible, repetitive application of up to 100 0.5-µL samples onto a plate and use of optical spectroscopy to analyze the developed spots with detection limits comparable to LC/MS, and he reported the detection of 3-5 pg of aflatoxin in peanut butter samples by the HPTLC method (see ref U4 below). Kalasz et al. (6) predicted future advances in the areas of mobile-phase optimization, twodimensional separations, and spectrometric and biological detection methods and greater use of displacement and forced flow development. The 10th anniversary meeting of the U.K. TLC Forum was held on June 3-5, 1996, at the University of Surrey. The lectures presented at this meeting, which were reviewed by Wilson (7), covered the following topics: approaches to increasing the separation power of TLC, state of the art in scanning densitometry, special techniques for forced-flow planar chromatography, advances in radioactivity detectors, argentation chromatography separation of isomers of capsaicin and triglycerides, isolation and identification of pharmacologically active natural products, the utility of TLC in a busy clinical laboratory for analyzing drugs of abuse, aflatoxin determinations, improved separations on C-18 and DIOL layers compared to silica gel, and identification of substances by HPTLC coupled with in situ spectrometry (MS/MS, NMR, IR, Raman). The Ninth International Symposium on Instrumental Planar Chromatography was held in Interlaken, Switzerland, April 9-11, 1997. The lectures presented at this symposium, which like those listed above illustrate many of the important current research areas in techniques and applications, were reviewed by Davis (8). The topics of these lectures included the following: comparison of TLC with other analytical methods with emphasis on their complementary nature; sample preparation and application; coupled methods (HPTLC-FT-IR, HPTLC with automated multiple development (AMD)-Raman spectrometry, HPLC/TLC); method development; validation of quantitative TLC using a digital video camera; in situ radioisotope measurement; documentation of chromatograms; theoretical thermodynamic and practical experimental approaches to TLC optimization; and numerous applications in food and cosmetic, environmental and toxicological, and pharmaceutical analyses. The special importance of TLC in the latter area is indicated by the offering of a session on applications of high-performance TLC in the pharmaceutical industry at the Eastern Analytical Symposium in November 1996, and the recent increasing acceptance of modern TLC methods by the European Pharmacopoeia and other legislative bodies (8). The next regular International Planar Chromatography Symposium is scheduled to be held in Interlaken in May 1999, having as a theme the complementary aspects of TLC and HPLC (9). A TLC symposium is also planned for May 1998 in Hungary in celebration of the 60th anniversary of the birth of TLC (8). Method development courses and instrumental TLC workshops were offered periodically in Wrightsville Beach, NC, by Camag. A bibliography service (CBS) is offered by Camag to keep subscribers informed about publications involving TLC. This service is available free of charge by mail from Camag or on-line at the Merck website . A cumulative compilation of abstracts from May 1993 through March 1997 can be purchased from Camag 8R
Analytical Chemistry, Vol. 70, No. 12, June 15, 1998
on a CD-ROM that is searchable by key word (author name, substance, technique, reagent, etc,). The Merck site also contains a glossary of TLC terms and much other information on TLC, as does the Camag site 440 nm (J2). Quinolonic antibiotics such as oxolinic acid were determined in fish feed and fish meat with 10 ppb sensitivity by extraction with acetonitrile-aqueous KCl (1%) + KOH (0.02M), SPE cleanup, separation on silica gel layers impregnated with K2HPO4, detection with sulfuric acid-HCl reagent, and fluorescence scanning at 320 nm (J3). Virginiamycin antibiotics were extracted from fermentation broths, purified by preparative HPLC, and analyzed by silica gel HPTLC with chloroform-methanol (92:8) mobile phase and reflectance scanning at 235 nm (J4). TLC on a mixed layer composed of silica gel, aluminum hydroxide, and magnesium hydroxide enabled better identification and more rapid and efficient separations of cephalosporins compared to plain silica gel (J5). Bases and Amines. Chromatographic behavior studies were carried out for 26 primary aromatic amines on iron(III) tungstophosphate layers with respect to the influences of the exchanger concentration in the layer and the acidity and salt concentration of the mobile phase, and many useful separations were achieved (K1). Detection amines at 1-ng levels was based on their inhibition of oxydoreductases, and the change in enzyme activity was proportional to the amount of analyzed amine (K2) and sulfonamides on normal and reversed phases in order to determine hydrophobicity parameters (K3). The presence of carcinogenic amines in textiles arising from azo dyes was determined by HPTLC with AMD and densitometry (K4). Catecholamines (epinephrine, norephinephrine, dopamine) were quantified in rat plasma by a method involving deproteinization, adsorption on acidic alumina, acetylation, extraction of the acetyl derivatives on a C-18 minicolumn, HPTLC separation, and densitometry at 415 nm using isoprenaline as internal standard (K5). The retention behavior of three series of aromatic amides was studied on silica gel layers using eight binary nonaqueous mobile phases, and results were presented in terms of the nature of the solute and mobile phase (K6). A simple method for detection of prohibited azo dyes was based on TLC analysis of their amine cleavage products; characteristic colors from specific diazotization and coupling reactions and Rf values were used to identify the amines (K7). Carbohydrates. The retention behavior of sugars and sugar alcohols was examined on silica gel, Florisil, alumina, and aminobonded silica layers using various aqueous and nonaqueous mobile phases; the proton acceptor solvents dioxane and THF were found to be very suitable for sugar separations (L1). Combination of two one-dimensional TLC systems based on Analytical Chemistry, Vol. 70, No. 12, June 15, 1998
13R
propanol and butanol improved the separation of oligosaccharides in urine screening analyses (L2). Reducing and nonreducing sugars could be differentiated by two novel methods employing Somogyi, Nelson, and bicinchoninate coloring spray reagents; 100 µg of glucose could be detected as a red-violet spot but nonreducing sugars such as sucrose and trehalose were not detectable (L3). Highly selective sugar separations were demonstrated on diol layers using AMD with acetonitrile-water and acetonitrileacetone-water gradients (L4). HPTLC-AMD with acetonitrileacetone-water gradients was also used for carbohydrate monitoring in the quality control of commercial beer production (L5). Sucrose and fructan oligosaccharides were quantified in enzyme and physiological studies by silica gel TLC using propanol-ethyl acetate-water (45:35:20) mobile phase, detection with ureaphosphoric acid reagent, and reflection densitometry of the resulting blue spots (L6). A TLC scanning method for determination of lactose in fecal samples was found to be more accurate for evaluation of lactone intolerance compared with the traditional anthrone method (L7). Fructose, glucose, and sucrose were simultaneously quantified in beverages such as sodas and iced teas by direct spotting of diluted samples on preadsorbent HPTLC silica gel 60 plates impregnated with sodium bisulfite and pH 4.8 citrate buffer, triple development with acetonitrile-water (85:15), zone detection with R-naphthol reagent, and reflectance scanning of the blue-purple zones at 515 nm (L8). Similar TLC methods, with the added use of ethyl acetate-acetic acid-methanol-water (60:15:15:10) mobile phase, were employed to analyze sugars in the hemolymph and digestive gland-gonad complex of Biomphalaria glabrata and Helisoma trivolvis snails maintained on restricted diets (L9). Dyes and Pigments. The HPTLC analysis of basic and cationic dyes (M1) and planar chromatographic analysis of leather dyes (M2) were reviewed. TLC analysis of reactive dyes released from wool fibers by alkaline hydrolysis digestion was found to provide important forensic information (M3). Quality control procedures for the TLC analysis of writing inks were described for use by document examiners and forensic chemists (M4). TLC was used to characterize commercial Coomassie brilliant blue R-250 after its purification (M5). Surface-enhanced resonance Raman spectrometry was used for selective detection of structurally similar aminotriphenylmethane dyes separated by TLC; spectra were recorded with a multichannel micro-Raman spectrometer after applying colloidal silver solution to the analyte spots (M6). HPTLC methods using silica gel preadsorbent plates and scanning densitometry with SPE for sample preparation were described for the quantification of lycopene and permitted synthetic food dyes in alcoholic and nonalcoholic beverages (M7). The TLC behavior of several crown ether derivatives of mesotetraphenylporphyrin were compared on silica gel, alumina, and cellulose layers with different organic mobile phases; every pair of isomers could be resolved using these sorbents (M8). TLC on silica with petroleum ether-acetone-diethylamine (10:4:1) and detection under 365-nm UV light was applied to the determination of carotenoids (β-carotene, lutein) in wood (M9). The following TLC studies of flavonoids were published: effect of different fluorescence intensifiers on the fluorodensitometric determination of flavones and flavonols (the most improvement was achieved by use of silicone oil or paraffin for lipophilic flanonoids and poly14R
Analytical Chemistry, Vol. 70, No. 12, June 15, 1998
(ethylene glycol) 4000 for lipophilic aglycons as well as glycosides) (M10); identification of the flavonoids and alkaloids from Peruvian Uncaria tomentosa bark in quality control (M11); systematic optimization of the mobile phase for separation of flavonoids extracted from two Mediterranean plants (M12); classification of species and hybrids of Crataegus (hawthorne) by means of characteristic flavonoid patterns produced by 2-fold development of silica gel with ethyl acetate-acetic acid-water followed by ethyl acetate-MEK-formic acid-acetic acid-water (M13); complexation of flavonoids with cyclodextrins and cyclodextrin derivatives examined by TLC on cellulose (M14); separation of some flavonoids and coumarins on diol-modified silica gel with various organic modifiers in heptane as the mobile phase (M15); and phytochemical characterization of Epilobium angustifolium and differentiation from other Epilobium species based on flavonoid patterns on silica gel and polyamide HPTLC layers and HPLC with photodiode array detection (M16). Anthocyanins were determined in Malva silvestris L. by HPTLC with reflectance densitometry at 530 nm; the method was compared with gradient elution HPLC-DAD, and HPTLC was found to be more sensitive and to give comparable quantitative results (M17). Hydrocarbons. RPTLC separations of hydroxy derivatives of polycyclic aromatic hydrocarbons were described with acetonitrile-water and methanol-water as mobile phases using a DS sandwich chamber (TLC) and in an overpressured chamber with visualization in 254- and 365-nm UV light and by reaction with fast blue B salt; off-line MS of scraped and extracted zones was used to confirm compounds in samples of airborne particulate matter (N1). TLC on sorbent rods with flame ionization detection (Iatroscan TLC-FID) is applied most widely for the analysis of hydrocarbons and lipids. The following examples illustrate hydrocarbon determinations, and two applications and a review of TLC-FID are included in the next section on lipids. Four oil and condensate samples were analyzed by TLC-FID, and quantitative data were found to be more reliable for low-molecularweight compounds than that obtained by HPLC and conventional TLC (N2). Coal-tar pitch was characterized by an improved method with a total analysis time of
