Planar chromatography.

s and the ICI Web of Science from November 1, 1999 to November 1, 2001. The literature search was augmented by consulting Analytical Abstracts, and the following 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. Because of a prescribed limitation of 200 references, coverage is more restricted than in my previous biennial reviews of planar chromatography dating back to 1970. Only the sections formerly under the General Considerations heading are included in this review, while sections on the TLC of individual compound classes, formerly included under the Applications heading, are omitted. Applications to specific compounds of many types are mentioned throughout the review, particularly in the section on Quantitative Analysis. Publications in the past two years on the history, theory, methodology, instrumentation, and applications of TLC continued at a high level, with over 2000 articles being found that are within the scope of this review. 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. The attempt was made to cite only important publications representative of the current practice and significant advances in the field. 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 papers written in Chinese. Chemical Abstracts citations are given for cited references not published in English. Most TLC papers originated from laboratories outside of the United States, especially Europe and Asia, but were published in English. Five publications in the review period addressed the state of the art in TLC. Sherma (1) wrote an encyclopedia article that described all important techniques and equipment in contemporary TLC, ranging from simple, inexpensive qualitative screening TLC to highly efficient, instrumental, quantitative HPTLC: sample preparation, stationary phases, mobile phases, application of standards and samples, chromatogram development, zone detection, documentation of chromatograms, zone identification, quantitative analysis, TLC-spectrometry, TLC-HPLC, preparativelayer chromatography (PLC), thin-layer radiochromatography, and rod TLC with flame ionization detection. Poole (2) reviewed planar chromatography at the turn of the century, including approaches to kinetic optimization and increased zone capacity (forced-flow development, electroosmotic flow, unidimensional multiple development, two-dimensional development) and on-line coupling of TLC and HPLC. The complementary features of columns and layers and advantages of TLC (evaluation of the whole sample, simultaneous sample cleanup and separation, screening in surveillance programs, multiple applications in the pharmaceutical industry, use of layers as a substrate for spectrometry) were discussed. Poole and Dias (3) described an approach to method development in TLC including definition of the problem and sample information, layer and mobile-phase selection, and mobilephase optimization. Freemantle (4) reported on the increasing use of TLC as a tool for combinatorial synthesis, with the layer acting as a large number of reaction vessels and providing a means of purifying the products. Parallel syntheses are carried out in a series of spots on the origin line of a silica gel layer, with microwave irradiation used to accelerate the reactions. The plate is then developed to separate and identify the reaction products. DerMarderosian (5) explained that TLC is widely used in natural product chemistry as the fastest and simplest procedure for separating and identifying plant and other natural product substances. Several color atlases exist that help to rapidly identify herbal substances. The use of TLC as a yes-or-no method often precludes the need for other, more expensive chromatography methods because seeing no zones on a layer after a rapid separation experiment indicates a low or nonexistent level of the analyte. The symposium Planar Chromatography 2000 was held on June 24-26 in Lillafured, Hungary, organized by the International Society for Planar Separations in honor of Professor Rudolf Kaiser, a leading pioneer and expert in TLC, on the occasion of his 70th birthday. This meeting was reported on by Davies (6), who noted that a recurring theme throughout the program was the increasing use of planar techniques in the biosciences. A symposium was held at the 115th AOAC International Annual Meeting, September 9-13, Kansas City, MO (Abstracts 901-906) on HPTLC for the Anal. Chem. 2002, 74, 2653-2662 10.1021/ac011764f CCC: $22.00 © 2002 American Chemical Society Analytical Chemistry, Vol. 74, No. 12, June 15, 2002 2653 Published on Web 01/29/2002 analysis of botanicals. This is becoming a very important application area for stability testing, quality assessment, fingerprint identification (7), and product surveys of herbal medicinal products as safety and regulatory concerns grow. Method development courses were offered periodically in Wilmington, 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. A cumulative compilation of abstracts from volumes 51-82 (May 1983 through March 1999) can be purchased from Camag on a CD-ROM that is searchable by key word (author name, substance, technique, reagent, etc.). In addition to a review of the literature and descriptions of new products, issues of the Camag CBS contain a section on applications, e.g., determination of drugs in the Czech Republic, pharmacological and pharmacodynamic testing of veterinary drugs, quality control of herbal medicines in Korea by HPTLC, separation of calystegines by HPTLC with automated multiple development (HPTLC-AMD), and HPTLC determination of antioxidant potency in the latest issue, no. 87, September 2001. A large number of applications are listed and can be requested on the Camag website . Diverse information on TLC methods and products is available on-line by entering the phrase “thin layer chromatography” or “TLC” on a website search engine such as or . HISTORY, STUDENT EXPERIMENTS, BOOKS, AND REVIEWS The history of the development of TLC and HPTLC (A1) and the production and application of ion-exchange resin papers (A2) was discussed. Laboratory experiments involving TLC for high school and college students were devised to illustrate separation of polycyclic aromatic hydrocarbons (PAHs) in environmental samples by twodimensional (2-D) TLC (A3), determination of lipophilicity of sulfonamide antibacterial drugs by reversed-phase (RP)-TLC (A4), combination of computational investigations and TLC in the undergraduate organic chemistry laboratory (A5), complete analysis of a biologically active tetrapeptide utilizing TLC and mass spectrometry (MS) (A6), identification of pharmaceuticals via computer-aided TLC (A7), the advantages of TLC in studying the effects of changing conditions on the final products of organic reactions (A8), and separation and identification of monoand disaccharides (A9) An updated and expanded third edition of the Handbook of Thin Layer Chromatography, edited by Sherma and Fried, is in preparation and will be published by Marcel Dekker, Inc. in late 2002 or early 2003. A general book chapter on TLC (A10) and a chapter on bioanalysis (A11) were published. A chromatography encyclopedia (A12) had TLC articles on detection, multidimensional development, densitometry, mobile-phase optimization, overpressured layer chromatography (OPLC), theory, quantitative structure-retention relationships, TLC-MS, immunostaining detection, sandwich chambers, and layer materials. Three special issues of the Journal of Thin Layer Chromatography & Related Technologies on TLC were edited by Sherma and Fried (vol. 22, no. 1 and no. 10, 1999, and vol. 24, no. 10, 2001). Special sections of the Journal of AOAC International were edited by Sherma on TLC-densitometry (vol. 83, no. 6, 2000) and by Renger on TLC in pharmaceutical analysis (vol. 84, no. 4, 2001). As mentioned above, comprehensive review of TLC applications to specific compound classes is not possible in this article because of space restrictions. A search of the literature showed that, as in the recent past, the most active area of publication of new methodology was in pharmaceutical and drug analysis. The Encyclopedia of Chromatography (A12) had articles on TLC analysis of amino acids, mycotoxins, plant toxins, antibiotics, carbohydrates, ceramides, coumarins, lipids, lipophilic vitamins, pesticides, pharmaceuticals, phenols, plant extracts, steroids, taxoids, pigments, and dyes. Applications review articles were published on TLC analysis of the following compounds: amino acids, derivatives, and enantiomers using impregnated layers (A13); sphingolipids (A14); enantiomers of chiral drugs (A15, A16); pesticides (A17); fumonisins (A18); and impurities in drugs (A19). The TLC analysis of numerous compound types in food and agricultural samples (A20, A21), pharmaceutical and forensic samples (A22), and clinical samples (A23) was reviewed. Other pertinent reviews are cited in the sections below. THEORY AND FUNDAMENTAL STUDIES The following are a selection of papers reporting theoretical and fundamental TLC studies that were chosen to illustrate some active research areas. The determination of lipophilicity by reversed-phase TLC as an alternative to the shake flask 1-octanol/water partition coefficient method has been studied for many types of compounds. As an example, the lipophilicity of some metallic complexes of diclofenac with potential antiinflammatory activity was determined by RP-TLC on octadecyl (C-18) and cyano bonded silica gel plates with water-methanol mixtures as mobile phases (B1). Mobile-phase optimization studies were carried out by numerical taxonomy techniques for 1,4-benzodiazepine mixtures (B2), by use of a mixture-design approach with solvation-parameter model for ternary mobile phases in RP-TLC (B3), by use of information theory and numerical taxonomy methods for flavonoids in plant extracts (B4), and by use of desirability functions and designs according to the PRISMA method (B5). Studies were made relating solute retention behavior to temperature (for macrocycles by RP-TLC) (B6), mobile-phase composition (for hydrocarbons and quinones) (B7), and solute structure (for photosystem II inhibitors) (B8). Rm values of hydrocarbons and their quinones were found to be most accurately predicted using chromatographic data-topological index dependence (B9). The role of lateral analyte-analyte interactions in the process of TLC band formation was elaborated (B10). AMD involves separation using repeated incremental developments of increasing length with a mobile-phase gradient of decreasing strength carried out in a computer-controlled instrument. The method, which can achieve a zone capacity of at least 30-40, is being applied increasingly for resolution of complex mixtures but deserves still more use based on its capabilities. Optimization of the solvent combination, development time, number of development steps, drying time between each run, and preconditioning parameters of the silica gel plates was described for the AMD-TLC separation of calystegines, a class of nortropane antibiotics, and of precursors of their biosynthesis (B11). 2654 Analytical Chemistry, Vol. 74, No. 12, June 15, 2002 CHROMATOGRAPHIC SYSTEMS (STATIONARY AND MOBILE PHASES) The following studies of layer materials were carried out: thermoanalytical study of silica gel chemically modified with amino, mercapto, octyl (C-8), and C-18 groups (C1); the influence of electric fields on surface interactions of silica gel and alumina adsorbents (C2); determination of the extent of surface coverage of C-18, octadecyl, cyano, and diol chemically bonded phases by Raman spectrometry (C3); and integration of impedance sensors in a cellulose layer for detection of zones (C4). The great majority of TLC analyses are carried out using commercial, precoated normal-phase (NP) silica gel TLC layers. The following are examples of publications reporting the use of a variety of other plates: laned, preadsorbent HP silica gel for densitometric quantification of lipids and phospholipids in infected snails (C5); HP silica gel coded to meet good laboratory practice (GLP) standards for quantitative assay of the active ingredient dimenhydrinate in motion sickness tablets (C6); C-18 bonded silica gel for quantification of the sunscreen octocrylene in lotions (C7); cellulose for separation and identification of metal-peptidoglycan monomer complexes (C8); amino and diol bonded silica gel for analysis of biogenic amines, alkaloids, and their derivatives (C9); and chitin for separation of phenol and its derivatives (C10). The separation of many metal ions was studied on layers composed of mixed titanium and silicon oxides (C11); titanium(IV) silicate ion exchanger (C12); bismuth silicate ion exchanger (C13); stannic arsenate or molybdosilicate mixed with silica gel, alumina, or cellulose (C14); and silica-zirconium tungstophosphate (C15). The following analyses on impregnated layers were reported: fatty acid methyl esters on silver-loaded silica gel (C16); antibiotics on hydrocarbon-impregnated silica gel HPTLC plates (C17); peptides on alumina impregnated with paraffin oil (C18); and 3d metal ions on silica gel impregnated with silicone fluid DC 200, triaryl phosphate, and tri-n-butyl phosphate (C19). The conditions used for modifying silica gel with metal salts were optimized (C20), and dynamic and static modification of silica gel and C-3 bonded silica gel stationary phases with surfactants (C21) were