Planar Chromatography - Analytical Chemistry (ACS Publications)

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Anal. Chem. 2002, 74, 2653-2662

Planar Chromatography Joseph Sherma

Department of Chemistry, Lafayette, Collegte, Easton, Pennsylvania 18042 Review Contents History, Student Experiments, Books, and Reviews 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 Thin-Layer Radiochromatography Literature Cited

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This is reviews covers the literature of thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) found by computer-assisted searching in Chemical Abstracts 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. 10.1021/ac011764f CCC: $22.00 Published on Web 01/29/2002

© 2002 American Chemical Society

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 Analytical Chemistry, Vol. 74, No. 12, June 15, 2002 2653

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 mono- and 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-densitom2654

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etry (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).

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 compared. Mobile phases that were evaluated included tributyl phosphate for metal separations on stannic arsenate layers (C22), buffered solutions containing cationic surfactants for phenol separations (C23), micellar mobile phases for amino acid separations on alumina and Li+-impregnated alumina (C24), surfactant-modified mobile phases for metal ion separations on silica gel (C25), and di-2-ethylhexyl orthophosphoric acid for separation of quinolines (ion-association TLC) (C26). TLC is being used to an increasing degree for separation of enantiomeric compounds, especially compounds of pharmaceutical interest, on chiral layers, layers impregnated with a chiral selector, or layers developed with chiral mobile phases. The use of cyclodextrins for resolution of enantiomers by TLC was reviewed (C27), and the following papers are examples of TLC separations of enantiomers: aminoglutethimide, acetyl aminoglutethimide, and dansyl aminoglutethimide with β-cyclodextrin and derivatives as mobile-phase additives (C28); dansyl amino acids with β-cyclodextrin mobile phases (C29); 1,4-dihydropyridine derivatives on a chiral layer of the ligand-exchange type (C30); dansyl-DL-amino acids on silica gel impregnated with vancomycin (C31); and (()-

ephedrine and atropine on silica gel impregnated with pure L-tartaric acid and L-histidine, respectively (C32). Paper was used as a layer with low adsorptive capacity (preadsorption layer) in TLC on rectangular and circular plates (C33). Multicolumn plates for TLC were designed as a parallel array of adsorbent strips 3 mm wide and tested successfully for capillary flow and forced-flow TLC (C34). APPARATUS AND TECHNIQUES Sample preparation methods for TLC are generally similar to those for gas chromatography (GC) and HPLC except that cruder samples with minimal purification can often be analyzed because of the single use of disposable plates. Sample cleanup and separation are often accomplished during the same run, and irreversibly sorbed impurities remaining at the origin cause no problems as they would in HPLC. Traditional and modern sample preparation methods were reviewed, including sampling, drying, and grinding the sample, dissolving the sample, steam distillation/ solvent extraction, Soxhlet extraction, sonication, accelerated solvent extraction, pressurized solvent extraction, microwaveassisted extraction, supercritical fluid extraction (SFE), and solidphase microextraction (D1). Solid-phase extraction (SPE) is especially widely used for extraction and cleanup of analytes prior to TLC. As examples, a screening assay involving SPE and HPTLC with quantification by densitometry was described for the nicotine metabolite cotinine in urine (D2), and RP-SPE with zonal silica gel TLC was used to determine paclitaxel and 10-deacetylbaccatin III in some Taxus species (D3). There are a variety of and manual and automatic devices available commercially for sample application; automated application instruments are usually recommended for maximum accuracy and precision in quantitative analysis. A new manual multiapplicator was described (D4) for simultaneous deposition of up to 15 samples successively on several plates of different sizes. It was shown to provide results equivalent to those obtained with a more expensive commercial autosampler for qualitative and quantitative analysis. Different types of filter paper were used as concentrating zones to facilitate application of initial zones onto plates (D5). The influence of a perpendicular homogeneous electric field on the TLC separation by horizontal development of PAHs and quinoline derivatives was studied on silica gel, alumina, cellulose, and polyamide layers (D6). TLC served as a tool for reaction optimization in microwave-assisted synthesis of N′-substituted arylpiperazines directly on a plate (D7). Single-development, unidimensional capillary flow ascending development in a mobile-phase vapor-equilibrated, large-volume glass chamber continues to be the main technique reported for TLC and HPTLC, with use of other techniques, such as multiple, 2-D, and forced-flow development, to improve resolution of complex mixtures in certain analyses. For example, 2-D TLC was used for a multiresidue screen of cardiotoxins in gastrointestinal contents (D8) and for the simultaneous separation of lysophospholipids from the total lipid fraction of crude biological samples (D9). Double development on a plate with preadsorbent zone, one for cleanup and the other for analysis, followed by fluorescence densitometry was used for determination of the antibiotics flumequine and doxycycline in milk (D10). Analytical Chemistry, Vol. 74, No. 12, June 15, 2002

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Practical aspects of mucopolysaccharidosis screening of urine samples by TLC, including scored layers, sequential development, a modified development chamber with spouts to add and remove mobile phases without opening the cover, and careful choice of standards, were described (D11). A novel biomolecular chemical screening approach for discovery of biologically active secondary metabolites combined RP-TLC of microbial extracts with binding studies toward DNA (D12). Special modes of development such as unidimensional multiple development (UMD), stepwise gradient development, and AMD were used for the micropreparative separation on silica gel and alumina of tertiary and quaternary alkaloids from roots (D13). Phenol derivatives from Oleum thymi were quantified by TLC with gradient elution and densitometry (D14). Displacement TLC, involving development of a spotted, dry layer with a mixture of carrier and displacer solvents, was described and compared to the usual elution development technique for one- and twodimensional separations (D15). A quick, economical, and reliable method for screening tuberculosis pharmaceuticals in the laboratory or field is based on use of a portable kit involving grinding of samples, development on a plastic-backed silica gel sheet with mobile phase, and application of detection reagents in polyethylene bags, or detection under UV light; sample zones are compared to high and low reference standards developed on the same layer to determine whether the drug content is within the specification range (D16). Forced-flow development techniques (rotational planar chromatography, RPC; OPLC; electroosmotic flow) were reviewed (D17). In OPLC, the plate is covered by a sheet of flexible material and subjected to a high external pressure, and the mobile phase is pumped through the stationary phase (layer) as in HPLC. A separate review on OPLC was written stressing the characteristics that make it a bridge between TLC and HPLC (D18). Some technical problems associated with construction of chambers for temperature-controlled TLC were elaborated (D19). Cholesterol and bile acids were separated using thermostated TLC on water-wettable C-18 plates in vapor-saturated chambers at temperatures ranging from 5 to 60 °C (D20). Chiral ligand-exchange separation of enantiomers over the past 30 years was reviewed (D21). Enantiomers and racemic compounds were resolved by inclusion TLC, in which derivatized and underivatized cyclodextrins are used as mobile phases on achiral microcrystalline cellulose triacetate plates (D22). Amino acids derivatized with 1-fluoro-2,4-dinitrophenyl-5-1-alanine amide were enantiomerically resolved on an RP-TLC plate without using impregnation or a chiral mobile phase (D23). The quality of paper and color stability were found to be important parameters affecting the permanence and durability of chromatograms produced by a charge-coupled device (CCD) camera or densitometer and ink-jet printer system for archiving purposes (D24). Multimodal (or multidimensional) separations are achieved by combining TLC with another complementary chromatographic technique. Published examples of multimodal methods include the following: analysis of pesticides in foods and surface waters and wash additives in sewage plants by HPLC coupled on-line with TLC through the DuoChrom interface (D25); separation of some flavonoids using an isocratic RP-HPLC gradient development 2656

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NP-TLC off-line coupled system (column fractions were collected, evaporated, and applied to the plate) (D26); separation of watersoluble choline metabolites by 2-D high-voltage electrophoresis and TLC (D27); 2-D capillary electrophoresis-TLC separations of amino acid enantiomers using electrospray, electrofilament mode transfer to the layer (D28); and rod-TLC with desorption by a pyrolysis unit interface directly into a GC/MS instrument for marine lipid class and molecular species analysis (D29). TLC on sorbent-coated quartz TLC rods (Chromarods) with flame ionization detection (Iatroscan TLC-FID) is a quite widely used technique, mostly for the analysis of samples such as bitumen (D30), petroleum, and oils (D31) for hydrocarbons and of marine samples (D32) and rice (D33) for lipids. A novel recent application of rod TLC-FID is the detection of paralytic shellfish poisoning toxins in plant and animal tissues (D34). DETECTION AND IDENTIFICATION OF SEPARATED ZONES Detection in TLC is based on natural color, fluorescence, or UV absorption (fluorescence quenching on phosphor-impregnated layers) of separated zones or on the use of various universal or selective chemical or biological detection reagents. An advantage of TLC lies in the ability to use a number of detection methods and reagents in sequence on a single layer to increase the amount of information obtained. Identification is tentatively based on the correspondence of Rf values and detection characteristics between sample and standard zones but must be confirmed by other evidence, such as off- or on-line coupling of TLC with spectrometric methods. An instrument has been available for many years from Camag for standardized application of detection reagents by the dipping technique with defined vertical speed and immersion time. A new instrument, the ChromaJet DS 20, is now available from Desaga for automated application of reagents by spraying (E1). The following new postchromatographic derivatization reagents for TLC zone detection were reported: diphenylamine-anilinephosphoric acid for glycoconjugates (E2); chloranilic acid for cimetidine and famitodine (E3); 4-chloro-5,7-dinitrobenzofurazan and 7-chloro-4,6-dinitrobenzofuroxan for amino compounds (E4); 3,5-dinitrosalicylic acid-ninhydrin for amino acids (distinguishable colors were formed for different acids) (E5); resorcinol and thiourea (modified Roe-Papadopoulos method) for fructose and fructosyl derivatives (E6); π-acceptor reagents for common drugs, with formation of characteristic colors (E7); 0.01% 9-formylacridine in dichloromethane for metal cations (E8); manganese chloridesulfuric acid for lipids (quantification by scanning densitometry) (E9); and oxolin-1,2,3,4-tetrahydro-1,4-dioxo-2,2,3,3-tetrahydroxynaphthalene for biogenic amines (E10). Visual detection of quinones (E11), cyanobacterial hepatotoxins (E12), and higher fatty acids (E13) was studied by comparison of 11, 17, and 18 different reagents, respectively. Berberine-impregnated plates were used successfully for fluorescence detection and densitometric quantification of a broad variety of alkanes (E14). Simple heating of a silica gel plate containing fluorescent indicator provided reagent-free detection of creatine as a fluorescencequenched zone; this thermochemical activation method, which did not a produce a fluorescent zone as previously reported, was the basis for quantification of creatine in nutrition supplements by densitometry (E15).

Reviews were written regarding protocols for the TLC-immunostaining detection of glycosphingolipids with monoclonal antibodies on HPTLC plates (E16) and TLC-blotting detection using poly(vinylidene difluoride) membrane (Far-Eastern blotting) (E17). Free cortisol in urine and feces of guinea pigs was determined by TLC with a rapid (1-2 h), improved competitive protein-binding assay involving the use of dexamethasone as a marker for cortisol zone localization on silica gel 60 sheets and buffer instead of organic solvents to recover cortisol (E18). Screening for acetylcholinesterase inhibitors from Amaryllidaceae at 10-200-ng levels was carried out by silica gel TLC in combination with bioactivity staining using Ellmam’s reagent (E19). A new bioassay was developed combining the simplicity of direct bioautography with the improved resolution of 2-D TLC; mixtures of structurally diverse antifungal agents were tested to discover new natural products with activity against agriculturally important fungal pathogens (E20). Monesin, narasin, lasalocid, salinomycin, and maduramicin were detected and identified in feed at 0.5-2 ppm by silica gel TLC with ethyl acetate-toluene-2-propanol (17.5:2: 0.5) mobile phase and bioautography using Bacillus subtilis culture medium (E21). Novel software was introduced for improved compound identification by library searching of UV spectra based on corrected Rf values, in situ UV spectral correlation, and spectrum maximum wavelength comparison (E22). The application of TLC combined with Fourier transform infrared spectrometry (TLC-FT-IR) was reported for the following analyses: ozone-induced phospholipid hydroperoxides in epithelial cells on silica gel HPTLC plates (E23); color pigments of Trichoderma harzianum by RP-TLC-FT-IR (E24); impurities in flurazepam on specialized plates containing 50% magnesium tungstate (E25); and color pigments of chestnut sawdust by offand on-line TLC-FT-IR and TLC-densitometry (E26). Studies of combined TLC-surface-enhanced Raman spectrometry (SERS) were carried out on silica gel 60F TLC, HPTLC, and Raman TLC plates using traditional capillary and overpressured developments; spectra were enhanced using a silver sol prepared according to the modified Lee-Meisel procedure (E27). Although not an application aimed at compound identification, Raman spectrometry has proven invaluable to study bonded stationaryphase stability; the extent of hydrolytic cleavage of C-8 ligands from the surface of bonded layers was quantified for TLC with mobile-phase buffers of pH 1-10 (E28). The coupling of fluorescence line-narrowing spectrometry (FLNS) with TLC and other liquid chromatography techniques was reviewed (E29). FLNS coupled with TLC on poly(ethylenimine)-cellulose sheets and a 32P-post-labeling technique was shown to provide low-picomole detection of benzo-R-pyrene (BAP) tetrols and a BAP-DNA adduct (E30). Research involving the combination of TLC with MS was a very active area in the past two years. Important publications on TLC/MS include the following: a review of methods for structure elucidation of saponins (E31); direct microextraction and analysis of rough-type lipopolysaccharides by TLC/matrix-assisted laser desorption/ionization (MALDI)-MS (E32); analysis of cationic pesticides by TLC/MALDI-MS (E33); a critical, general review of TLC/MS, including MALDI, surface-assisted laser desorption (SALDI), and the TLC-electrospray interface, with some specula-

tions as to future prospects (E34); analysis of the pharmaceutical compound UK-224,671 and related substances by TLC/MALDItime-of-flight (TOF)-MS (E35); direct MALDI-TOF-MS of glycolipids on thin-layer plates and polymer transfer membranes (E36); separation and detection of carcinogen-adducted oligonucleotides by TLC/MALDI-MS (E37); application of TLC/ MALDI-TOF-MS to identification of unknown mixtures produced in an organic synthetic process (E38); quantification of caffeine by off-line TLC/MS (E39); and 2-D TLC separation and MS identification of anthraquinones isolated from the fungus Dermocybe sanguinea (E40). A new TLC/MALDI-MS direct coupling method that recovers ∼100% of the analyte was described. The method makes use of a hybrid plate in which a silica gel layer and a MALDI layer are configured adjacently on a common backing. After TLC separation, the plate is rotated 90°, and the separated analyte zones are eluted from the silica gel layer to the MALDI layer via the capillary action of the latter. Low-femtomole detection limits were demonstrated for small cyclic peptides (E41). QUANTITATIVE ANALYSIS Although the majority of planar chromatography analyses are carried out on a qualitative or semiquantitative (visual comparison) basis, modern computer-controlled slit-scanning densitometers that mechanically scan sample and standard chromatograms in tracks on the layer allow selective, sensitive, accurate, and precise quantitative analyses to be carried out by HPTLC. Reports of the use of electronic scanning or image analysis with a video densitometer (CCD camera) based on total illumination of the plate with the light source continued to increase during the review period, and it is becoming especially important in the qualitative (fingerprint) and quantitative analysis of herbal or botanical medicines and food supplements. Validation of results obtained by TLC is increasingly addressed in the literature because of the quality demands imposed on analyses, such as those performed under pharmaceutical compendial, regulatory, and GLP/GMP standards. TLC is considered by some analysts to be the most reliable and informative separation method because samples, standards, and control samples are developed at the same time on a single layer with the same mobile phase; samples are not eluted as in column procedures; and the chromatograms that are produced simultaneously but independently are stored in separate tracks on the layer and can be evaluated and validated in a variety of ways after the chromatographic procedure. A comparative study of TLC-densitometry and HPLC for the determination of biogenic amines in fish and fishery products found that TLC-densitometry was faster and less expensive, allowed simultaneous screening of several samples at one time, and required less sophisticated instrumentation and technical skill (F1). Another important advantage of TLC-densitometry is that it is often possible to investigate dirty samples without timeconsuming pretreatments because the layer is used only once, as opposed to sequential analysis with HPLC that requires purified samples so that contaminants do not build up and destroy column performance. Although most reported quantitative analyses are carried out with commercial slit-scanning densitometers (e.g., Camag, Desaga, or Shimadzu), other scanners have been described in the literature. A flatbed scanner with 12-bit intensity resolution and Analytical Chemistry, Vol. 74, No. 12, June 15, 2002

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600 dpi minimum linear resolution along with Igor Pro and a fast PC were purchased for under $2500 and used as a densitometer for natural or stained colored TLC zones (F2). A fiber optical HPTLC scanner was described that took measurements with a set of 50 fibers at a distance of 400-500 µm above the plate, had spatial resolution of 160 µm, and scanned 450 spectra simultaneously in the range of 198-610 nm in less than 2 min (F3). Neutral lipids were scanned with a laser-excited fluorescence scanner after separation by double development on silica gel and detection with rhodamine 6G spray reagent (F4). Scanning laser densitometry was also used to examine the levels of platelet membrane phospholipids, after separation by 2-D TLC (F5). A simplified densitometer consisting of a UV light-emitting diode for fluorescence excitation at 370 nm, photodiode with peak sensitivity of 440 nm and a 418-nm cutoff filter for detection of fluorescence intensity, operational amplifier integrated circuit, analog-to-digital converter, and portable PC with software for data recording was developed for the inexpensive determination of aflatoxins at 1-ng levels by TLC (F6). A scientifically operated charge-coupled device detector and HPTLC plates were utilized for analysis of tetracycline pharmaceutical products in the fluorescence mode with detection limits of 0.1-0.5 ng and recovery of >85% (F7) and of famotidine, acetaminophen, caffeine, and aspirin at nanogram levels by fluorescence quenching (F8). The basic principles of image processing in HPTLC were described in terms of the signal, noise, pixel operations, histograms, deconvolution operations, and morphological operations; definitions of scanning densitometry and 2-D digitized chromatograms were given in terms of vector or matrix algebra (F9). The influence of instrumental settings (aperture and number of accumulated frames) on the quality of captured images (background response, baseline noise, sensitivity, reproducibility) was studied for the Camag video densitometer in the fluorescence quenching mode by measurements of the model compound atrazine in the UV spectral region on TLC and HPTLC plates containing a fluorescent indicator or phosphor (F-plates) (F10). Slit-scanning densitometry with a commercial instrument and video densitometry with a conventional flatbed scanner and commercial software were compared for linearity, precision, and detection limits in the quantification of some test dyes. Video densitometry was found to have equivalent precision, a broader linear range, and better sensitivity in this study (F11). However, the opinion of most TLC practitioners appears to be that the sensitivity, resolution, selectivity, and applicability to absorption and fluorescence measurements of video densitometers do not match slit-scanning instruments at this time. It is likely that advances in camera technology and software developments will lead to the eventual replacement of slit scanning densitometry by image analyzers. The following TLC-densitometry calibration studies were carried out: application of a new fuzzy regression algorithm that was claimed to be the most suitable regression method in quantitative TLC (F12); calibration based on weighted regression functions (F13); and comparison of linear and nonlinear calibration for silica gel HPTLC analysis of dansyl derivatives of polyamines utilizing a fiber-optic fluorescence detector (F14) and for spec2658

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trodensitometric stability-indicating silica gel TLC determination of ranitidine hydrochloride (F15). Validation is a critical part of quantitative TLC method development, and virtually all publications include some amount of quality assurance data. For example, a method was developed for determination of loratadine in pharmaceutical preparations involving dilution of syrups or extraction of the analyte from tablets with 96% ethanol, TLC on silica gel plates with chloroform-ethyl acetate-acetone (5:7:7) mobile phase, and absorbance-reflectance scanning at 250 nm; it was demonstrated that the selectivity, precision, and accuracy of the method were suitable for its use in pharmaceutical industry quality control laboratories (F16). An OPLC method for determination of aflatoxins in wheat was validated in terms of specificity (retention factor, resolution, asymmetry factors), linearity, accuracy, precision, detection limit, and quantification limit; robustness was also tested and found to be critically dependent on whether a TLC or HPTLC plate was used (F17). An extremely important paper (F18) discussed practical approaches for validation of pharmaceutical TLC analytical procedures within the guidelines of the International Conference on Harmonization (ICH). Basic acceptance criteria for evaluation of validation experiments based on practical experience are proposed for all levels at which TLC is used, i.e., qualitative identity testing, active ingredient assays, semiquantitative limit tests, and quantitative determination of impurities. The validation parameters considered were specificity, detection limit, linearity and range, precision (repeatability, intermediate precision), accuracy, and quantification limit. In addition, selected parameters for robustness testing of given procedures and quality assurance of quantitative TLC testing by control charts were described. The Eurachem/Citac Guide on measurement of uncertainty establishes general rules for evaluating and expressing measurement uncertainty across a broad spectrum of measurements and presents a special calculation procedure called the “error-budget approach”. This model was tested using the quantitative HPTLC determination of sodium glutamate in a food product as an example (F19). After dividing the procedure into stages and evaluating each one, it was found that the total uncertainty estimated by the error-budget method was less than one-fifth of the value obtained from a validation study. It was concluded that the contribution from sources selected and processed according to the Citac guide is so small that it can be neglected. The following recent publications illustrate the wide variety of analytes and sample types to which TLC-densitometry has been applied: TLC-fluorescence densitometry of Ginko biloba terpenes by postcolumn thermochemical derivatization and quality survey of commercial ginko products (F20); quantitative determination of pesticides in soil by TLC-video densitometry (F21); hydrocarbon types in petroleum and coal-derived products by UV densitometry on berberine-impregnated silica gel plates (F22); amino acids from sanguine plasma using dual-wavelength flyingspot scanning (F23); caffeic acid in Dipsacaceae family plants by gradient elution TLC-densitometry (F24); gardenia yellow in food by RP-TLC-densitometry using crocetin as an indicator (F25); phospholipids in a pharmaceutical drug by scanning and video densitometry (F26); malic and lactic acids in wines by silica gel TLC with detection using bromophenol blue reagent and zigzag

scanning at 430 nm in order to monitor malolactic fermentation (F27); determination of the phospholipid/lipophilic compound ratio in liposomes by double development separation and scanning after detection by charring plates with specific visualization reagents (F28); flavonoids in Vaccinium leaves by silica gel TLC with gradient multiple development and videodensitometry (F29); vitamin C and dipyrone in bulk and pharmaceutical dosage forms by silica gel TLC using a water-methanol (95:5) mobile phase followed by direct scanning at 260 nm (F30); cortisol and cortisone in human urine by fluorescence densitometry after derivatization with isonicotinic acid hydrazide (F31); lutein and β-carotene in Cerithidia californica snails infected with two species of larval trematodes by C-18 RP-HPTLC and visible-mode scanning (F32); and carbohydrates in Biomphalaria glabrata snails maintained on a high-fat diet by TLC on laned, preadsorbent silica gel HPTLC plates, detection with R-naphthol reagent, and visible-mode scanning (F33). PREPARATIVE-LAYER CHROMATOGRAPHY AND THIN-LAYER RADIOCHROMATOGRAPHY The various roles of planar chromatography in medicinal and aromatic plant research for separation, identification, quantification, purification, isolation, and screening of compounds were described in detail in a valuable paper (G1). Special attention is paid to analytical, micropreparative, and preparative forced-flow techniques, e.g., OPLC and RPC. The special features of these methods are compared in tables, and purification and isolation procedures using forced-flow techniques are shown in flowcharts. Applications of PLC to substances of different classes are presented. Construction and use of a low-cost mechanical applicator for PLC were described (G2). Procedures were presented for optimization of systems for zonal micropreparative and preparative chromatography on silica gel in horizontal sandwich chambers of an alkaloid extract from Fumaria officinalis; adsorbent containing the separated fractions was scraped from the layers, transferred to small columns, and eluted, and the eluates were analyzed (G3). Zonal micropreparative silica gel TLC with gradient elution involving increasing methanol concentrations in pH 6.0 aqueous acetate buffer was used to isolate milligram quantities of the alkaloids sanguinarine and chelerithrine from the roots of Chelidonium majus L (G4). The isolation of the taxoids paclitaxel and cephalomannine from yew tissues was performed by stepwise gradient elution alumina column chromatography followed by zonal TLC on silica gel with dichloromethane-dioxane-acetonemethanol mobile phase and TLC on silanized silica gel with methanol-water (G5). Kava lactones were separated by RPC on silica gel with mixtures of hexane-ethyl acetate or hexane-dioxane as mobile phases (G6). Polydispersed polysiloxanes (1-2 g) were separated into narrow molecular weight fractions in 30-60 min using the Cyclograph centrifugal chromatography system (G7). The histamine H2-receptor antagonists cimetidine, famotidine, nizatidine, and ranitidine hydrochloride were isolated by PLC on silica gel 60F layers with ethyl acetate-acetone-water (5:4:1) mobile phase, extracted from the adsorbent, and identified by UV spectrometry (G8). Monoacylglycerols derived from butter oil by fungal degradation were separated on silica gel-F PLC plates with hexane-diethyl ether-formic acid (80:20:2) mobile phase; lipid

bands were detected under UV light or with iodine vapor and scraped and eluted with hexane-2-propanol (3:2), and recovered lipids were derivatized to trimethylsilyl ethers and analyzed by GC/MS (G9). The principal methods used in radio-TLC (also termed thinlayer radiochromatography) for detecting and quantifying radioactive zones in 1-D and 2-D chromatograms are autoradiography, zonal analysis (scraping followed by scintillation counting), and direct measurement using radiation detectors. Autoradiography and the related technique fluorography were reviewed in a book chapter (G10). OPLC-digital autoradiography (DAR) and HPLC with a radiation detector were applied to the qualitative and quantitative determination of drug metabolites after extraction from different biological samples (G11). A new type of radiodetector for direct digital autoradiography of TLC plates was evaluated in a study of lipid and steroid metabolism; the detector, which is based on the concept of parallel avalanche detection, enabled detection of zone radio signals of 2.0 dpm and quantitative measurement in the range of 5-50 dpm for 14C (G12). A new radiolabeling method to determine the lipoxygenase and hydroperoxide lyase specificity in olive fruit pulp employed incubation of enzyme preparations from the pulp with radiolabeled linoleate, followed by fractionation of the resulting lipidic products, pretreated with 2,4-dinitrophenylhydrazine, on TLC plates coated with poly(ethylene glycol) 400 (G13). A simple TLC method was described for simultaneous determination of taxol and its metabolites in microsomal samples in order to examine the inhibitory effect of NK-104, a potent inhibitor of HMG-CoA reductase, on taxol metabolism; after incubation of 14C-taxol with human liver microsomes, the supernatants were developed on silica gel using a mobile phase consisting of toluene-acetone-formic acid (60:39:1) and quantified with a bioimaging analyzer (G14). When using radio-TLC for purity testing of 99Tcm-tetrofosmin injections with instant thinlayer chromatography-silica gel (ITLC-SG) sheets and dichloromethane-acetone (65:35) mobile phase, it was found that the following precautions were necessary to eliminate artifacts: the sample spot applied should be in the 10-20-µL range, the initial zone should not be force-dried with air, and the sheets should not be dried before use (G15). Joseph Sherma received a B.S. in chemistry from Upsala College, East Orange, NJ. in 1955 and a Ph.D. in analytical chemistry from Rutgers University in 1958. He joined the faculty of Lafayette College in 1958. He is currently John D. and Frances H. Larkin Professor Emeritus of Chemistry and continues to supervise undergraduate students in analytical method development and interdisciplinary analytical chemistry-biology research. Dr. Sherma independently and with others has written or edited over 525 papers, chapters, books, and reviews covering chromatographic and analytical methods. He has been editor for residues and trace elements of the journal of AOAC International for 20 years and is on the editorial boards of the Journal of Planar Chromatography-Modern TLC, Acta Chromatographica, Journal of Environmental Science and Health, Part B, and Journal of Liquid Chromatography & Related Technologies.

LITERATURE CITED (1) Sherma, J. Thin Layer Chromatography. In Encyclopedia of Chromatography; Meyers, R. A., Ed.; John Wiley & Sons Ltd.: Chichester, U.K., 2000; Vol. 13, pp 11485-11498. (2) Poole, C. F. J. Chromatogr., A 1999, 856, 399-427. (3) Poole, C. F.; Dias, N. C. J. Chromatogr., A 2000, 892, 123-142. (4) Freemantle, M. Chem. Eng. News 2000, (March 13), 9. (5) DerMarderosian, A. Chem. Eng. News 2000, (May 22), 6. (6) Davies, I. J. Planar Chromatogr.-Mod. TLC 2000, 13, 244-247.

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(7) Rajani, M.; Ravishankara, M. N.; Shrivastava, N.; Padh, H. J. Planar Chromatogr.-Mod. TLC 2001, 14, 34-41. HISTORY, STUDENT EXPERIMENTS, BOOKS, AND REVIEWS (A1) Ettre, L. S.; Kalasz, H. LC-GC North Am. 2001, 19, 712-714, 716, 718, 720-721. (A2) Lederer, M. Chem. Intell. 1999, 5, 32-34. (A3) Crisp, G. T.; Williamson, N. M. J. Chem. Educ. 1999, 76, 1691-1692. (A4) Fontes, P. M. A. J. Chem. Educ. 2001, 78, 533-534. (A5) Hessley, R. K. J. Chem. Educ. 2000, 77, 202-205. (A6) LeFevre, J. W.; Dodsworth, D. W. J. Chem. Educ. 2000, 77, 503-504. (A7) Macherone, A. J., Jr.; Siek, T. J. J. Chem. Educ. 2000, 77, 366367. (A8) Papageorgiou, G. Educ. Chem. 1999, 36, 132-134. (A9) Wagner, G.; Sommer, K. Naturwiss. Unterr. Chem. 2001, 12, 29-32; Chem. Abstr. 2001, 134, 340081z. (A10) Touchstone, J. C. Handb. Anal. Tech. 2001, 1, 327-344. (A11) Wiltshire, H. In Principles and Practices of Bioanalysis; Venn, R. F., Ed.; Taylor & Francis Ltd.: London, UK., 2000; pp149159. (A12) Cazes, J., Ed. Encyclopedia of Chromatography; Marcel Dekker, Inc.: New York, 2001. (A13) Bhushan, R.; Martens, J. Biomed. Chromatogr. 2001, 15, 155165. (A14) Van Echten-Deckert, G. Methods Enzymol. 2000, 312, 6479. (A15) Subert, J.; Slais, K. Pharmazie 2001, 56, 355-360. (A16) Aboul-Enein, H. Y.; El-Awady, M. I.; Heard, C. M.; Nicholls, P. J. Biomed. Chromatogr. 1999, 13, 531-537. (A17) Sherma, J. J. AOAC Int. 2001, 84, 993-999. (A18) Meredith, F. I. Methods Enzymol 2000, 311, 361-373. (A19) Ferenczi-Fodor, K.; Vegh, Z. Prog. Pharm. Biomed. Anal. 2000, 4, 146-182. (A20) Sherma, J. J. Chromatogr., A 2000, 880, 129-147. (A21) De Brabander, H. F.; de Wasch, K. In Residue Analysis in Food: Principles and applications; O’Keeffe, M., Ed.; Harwood Academic Publishers: Amsterdam, 2000; pp 145-175. (A22) Kalasz, H.; Bathori, M. Handb. Anal. Sep. 2000, 1, 439-501. (A23) Jain, R.; Sherma, J. Planar Chromatography in Clinical Chemistry. In Encyclopedia of Chromatography; Meyers, R. A.. Ed.; John Wiley & Sons Ltd.: Chichester, U.K., 2000; Vol. 2, pp 1583-1603. THEORY AND FUNDAMENTAL STUDIES (B1) Sarbu, C.; Demertzis, M. A.; Kovala-Demertzi, D. Acta Chromatogr. 2000, 10, 222-229. (B2) Cimpoiu, C.; Hodisan, T. J. Pharm. Biomed. Anal. 1999, 21, 895-900. (B3) Dias, N. C.; Poole, C. F. J. Planar Chromatogr.-Mod. TLC 2000, 13, 337-347. (B4) Medic-Saric, M.; Stanic, G.; Bosnjak, I. Pharmazie 2001, 56, 156-159. (B5) Pelander, A.; Summanen, J.; Yrjonen, T.; Haario, H.; Ojanpera, I.; Vuorela, H. J. Planar Chromatogr.-Mod. TLC 1999, 12, 365372. (B6) Zarzycki, P. K.; Nowakowska, J.; Chmielewska, A.; Wierzbowska, M.; Lamparczyk, H. J. Chromatogr., A 1997, 787, 227233. (B7) Maciejewicz, W.; Soczewinski, E. Chromatographia 2000, 51, 473-477. (B8) Qin-Sun, W.; Ling, Z.; Hua-Zheng, Y.; Hua-Yin, L. J. Planar Chromatogr.-Mod. TLC 1999, 12, 301-305. (B9) Pyka, A. J. Planar Chromatogr.-Mod. TLC 1999, 12, 293-297. (B10) Prus, W.; Kaczmarski, K.; Tyrpien, K.; Borys, M.; Kowalska, T. J. Liq. Chromatogr. Relat. Technol. 2001, 24, 1381-1396. (B11) Scholl, Y., Asano, N.; Drager, B. J. Chromatogr., A 2001, 928, 217-224. CHROMATOGRAPHIC SYSTEMS (STATIONARY AND MOBILE PHASES) (C1) Dranca, I.; Coman, V.; Constantinescu, R.; Dogar, F.; Marutoiu, C.; Lupascu, T. J. Planar Chromatogr.-Mod. TLC 2000, 13, 48-51. (C2) Malinowska, I. J. Planar Chromatogr.-Mod. TLC 2000, 13, 4-8. (C3) Kowalska, T.; Kowalik, G.; Daniel, P. Acta Chromatogr. 2000, 10, 85-96. (C4) Wu, J.; Sansen, W. Analyst (Cambridge, U.K.) 2000, 125, 1375-1377. (C5) Cline, D. J.; Fried, B.; Sherma, J. Acta Chromatogr. 2000, 10, 183-189. (C6) DiGregorio, D.; Westgate, E.; Sherma, J. Acta Chromatogr. 2000, 10, 190-194. (C7) Fisher, J.; Sherma, J. J. Planar Chromatogr.-Mod. TLC 2000, 13, 388-390. (C8) Sesartic, Lj.; Hadzija, O.; Brajenovic, N. J. Liq. Chromatogr. Relat. Technol. 2001, 24, 1511-1514. 2660

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(C9) Baranowska, I.; Zydron, M. J. Planar Chromatogr.-Mod. TLC 2000, 13, 301-306. (C10) Malinowska, I.; Rozylo, J. K.; Jamrozek-Manko, A. J. Planar Chromatogr.-Mod. TLC 2000, 13, 37-41. (C11) Ghoulipour, V.; Husain, S. W. Ann. Chim. (Rome, Italy) 2001, 91, 111-116. (C12) Ghoulipour, V.; Husain, S. W. J. Planar Chromatogr.-Mod. TLC 2000, 13, 354-358. (C13) Ghoulipour, V.; Husain, S. W. Anal. Sci. 2000, 16, 1079-1081. (C14) Mohammad, A.; Yousuf, R.; Hamid, Y. Acta Chromatogr. 2001, 11, 171-182. (C15) Mohammad, A.; Iraqi, E. Indian J. Chem. Technol. 2000, 7, 223-226. (C16) Flieger, J.; Szumilo, H. J. Planar Chromatogr.-Mod. TLC 2000, 13, 426-431. (C17) Dhanesar, S. C. J. Planar Chromatogr.-Mod. TLC 1999, 12, 280-287. (C18) Cserhati, T.; Forgacs, E.; Deyl, Z.; Miksik, I.; Eckhardt, A. J. Chromatogr., A 2001, 910, 137-145. (C19) Sharma, S. D.; Sharma, S. C.; Sharma, C. J. Planar Chromatogr.Mod. TLC 2001, 14, 16-20. (C20) Shtykov, S. N.; Sumina, G.; Smushkina, E. V.; Tyurina, N. V. J. Planar Chromatogr.-Mod. TLC 2000, 13, 182-186. (C21) Flieger, J.; Szumilo, H.; Gielzak-Kocwin, K. J. Liq. Chromatogr. Relat. Technol. 1999, 22, 2879-2894. (C22) Mohammad, A.; Iraqi, E.; Khan, I. A. Chromatography 2000, 21, 29-36. (C23) Mohammad, A.; Anwar, S.; Iraqi, E.; Khan, I. A. N. Acta Chromatogr. 2000, 10, 195-212. (C24) Mohammad, A.; Agrawal, V. J. Planar Chromatogr.-Mod. TLC 2000, 13, 365-374. (C25) Mohammad, A.; Agrawal, V. J. Planar Chromatogr.-Mod. TLC 2000, 13, 210-216. (C26) Soczewinski, E.; Wojciak-Kosior, M. J. Planar Chromatogr.-Mod. TLC 2001, 14, 28-33. (C27) Schneiderman, E.; Stalcup, A. M. J. Chromatogr., B 2000, 745, 83-102. (C28) Aboul-Enein, H. Y.; El-Awady, M. I.; Heard, C. M. J. Liq. Chromatogr. Relat. Technol. 2000, 23, 2715-2726. (C29) LeFevre, J. W.; Rogers, E. D.; Pico, L. L.; Botting, C. L. Chromatographia 2000, 52, 648-652. (C30) Mielcarek, J. Drug Dev. Ind. Pharm. 2001, 27, 175-179. (C31) Bhushan, R.; Thiong’o, G. T. J. Planar Chromatogr.-Mod. TLC 2000, 13, 33-36. (C32) Bhushan, R.; Martens, J.; Arora, M. Biomed. Chromatogr. 2001, 15, 151-154. (C33) Berezkin, V. G.; Markov, A. B. J. Planar Chromatogr.-Mod. TLC 2000, 13, 470-472. (C34) Berezkin, V. G.; Mardanov, R. G.; Markov, A. B. Ind. Lab. (Diagn. Mater.) 2000, 66, 366-368. APPARATUS AND TECHNIQUES (D1) Namiesnik, J.; Gorecki, T. J. Planar Chromatogr.-Mod. TLC 2000, 13, 404-413. (D2) Bazylak, G.; Brozik, H.; Sabanty, W. Pol. J. Environ. Stud. 2000, 9, 113-123. (D3) Glowniak, K.; Wawrzynowicz, T.; Hajnos, M.; Mroczek, T. J. Planar Chromatogr.-Mod. TLC 1999, 12, 328-335. (D4) Maboundou, C. W.; Grosse, P.-Y.; Delvordre, P.; Vermerie, N. J. Planar Chromatogr.-Mod. TLC 1999, 12, 373-377. (D5) Berezkin, V. G.; Malinowska, I.; Rozylo, J. K.; Makarov, A. B.; Mardanov, R. G. J. Planar Chromatogr.-Mod. TLC 2000, 13, 82-87. (D6) Malinowska, I. J. Planar Chromatogr.-Mod. TLC 1999, 12, 408-415. (D7) Williams, L. Chem. Commun. (Cambridge) 2000, (6), 435436. (D8) Holstege, D. M.; Francis, T.; Puschner, B.; Booth, M. C.; Galey, F. D. J. Agric. Food Chem. 2000, 48, 60-64. (D9) Yokoyama, K.; Shimizu, F.; Setaka, M. J. Lipid Res. 2000, 41, 142-147. (D10) Choma, I.; Grenda, D.; Malinowska, I.; Suprynowicz, Z. J. Chromatogr., B: Biomed. Sci. Appl. 1999, 734, 7-14. (D11) Lee, B. Y.; Yanamandra, K.; Thurmon, T. F. LC-GC North Am. 2001, 19, 422-424, 426. (D12) Maul, C.; Sattler, I.; Zerlin, M.; Hinze, C.; Koch, C.; Maier, A.; Grabley, S.; Thiericke, R. J. Antibiot. 1999, 52, 1124-1134. (D13) Waksmundzka-Hajnos, M.; Gadzikowska, M.; Golkiewicz, W. J. Planar Chromatogr.-Mod. TLC 2000, 13, 205-209. (D14) Bazylko, A.; Strzelecka, H. Chromatographia 2000, 52, 112114. (D15) Bariska, J.; Csermely, T.; Furst, S.; Kalasz, H.; Bathori, M. J. Liq. Chromatogr. Relat. Technol. 2000, 23, 531-549. (D16) Kenyon, A. S.; Layloff, T.; Sherma, J. J. Liq. Chromatogr. Relat. Technol. 2001, 24, 1479-1490. (D17) Nurok, D. Anal. Chem. 2000, 72, 634A-641A. (D18) Nyiredy, S. TrAC, Trends Anal. Chem. 2001, 20, 91-101. (D19) Zarzycki, P. K. J. Planar Chromatogr.-Mod. TLC 2001, 14, 6365. (D20) Zarzycki, P. K.; Wierbowska, M.; Lamparczyk, H. J. Chromatogr., A 1999, 857, 255-262. (D21) Davankov, V. A. Enantiomer 2000, 5, 209-223.

(D22) Lepri, L.; Del Bubba, M.; Cincinelli, A.; Boddi, L. J. Planar Chromatogr.-Mod. TLC 2000, 13, 384-387. (D23) Nagata, Y.; Iida, T.; Sakai, M. J. Mol. Catal. B: Enzym. 2001, 12, 105-108. (D24) Vovk, I.; Muck, T.; Novak, G. J. Planar Chromatogr.-Mod. TLC 2000, 13, 276-280. (D25) Morlock, G. E. CLB Chem. Labor Biotech. 1999, 50, 410412; Chem. Abstr. 2000, 132, 87350e. (D26) Hawryl, M. A.; Soczewinski, E. Chromatographia 2000, 52, 175-178. (D27) Utal, A. K.; Coleman, P. D. J. Microcolumn Sep. 2000, 12, 419-428. (D28) DeVault, G. L.; Sepaniak, M. J. Bitumen 1998, 60, 133-135. (D29) Hudson, E. D.; Helleur, R. J.; Parrish, C. C. J. Chromatogr. Sci. 2001, 39, 146-152. (D30) Masson, J.-F.; Price, T.; Collins, P. Energy Fuels 2001, 15, 955-960. (D31) Schwarz, G.; Ecker, A. Pet. Coal 1999, 41, 185-188. (D32) Striby, L.; Lafont, R.; Goutx, M. J. Chromatogr., A 1999, 849, 371-380. (D33) Nishiba, Y.; Sato, T.; Suda, I. Cereal Chem. 2000, 77, 223229. (D34) Indrasena, W. M.; Ackman, R. G.; Gill, T. A. J. Chromatogr., A 1999, 855, 657-668. DETECTION AND IDENTIFICATION OF SEPARATED ZONES (E1) Hahn-Dienstrop, E.; Koch, A.; Muller, M. Chromatographia 2000, 51 (Suppl.), S302-S304. (E2) Anderson, K.; Li, S.-C.; Li, Y.-T. Anal. Biochem. 2000, 287, 337-339. (E3) Chukwurah, B. K. C.; Ajali, U. Boll. Chim. Farm. 2000, 139, 260-262. (E4) Evgen′ev, M. I.; Evgen’ev, I. I.; Levinson, F. S. J. Planar Chromatogr.-Mod. TLC 2000, 13, 199-204. (E5) Laskar, S.; Sinhababu, A.; Hazra, K. M. J. Indian Chem. Soc. 2001, 78, 49-50. (E6) Muro, A. C.; Rodriguez, E.; Abate, C. M.; Sineriz, F. Folia Microbiol. (Prague) 1999, 44, 647-649. (E7) Ogoda Onah, J. Acta Pharm. (Zagreb) 1999, 49, 217-220. (E8) Patrut, A.; Marutoiu, C.; Marutoiu, O.-F.; Moise, M. I. J. Planar Chromatogr.-Mod. TLC 2000, 13, 151-153. (E9) Thanh, N. T. K.; Stevenson, G.; Obatomi, D.; Bach, P. J. Planar Chromatogr.-Mod. TLC 2000, 13, 375-381. (E10) Zenkova, E. A.; Degterev, E. V. Pharm. Chem. J. 2000, 34, 91-94. (E11) Kocjan, B. J. Planar Chromatogr.-Mod. TLC 2000, 13, 396397. (E12) Pelander, A.; Ojanpera, I.; Lahti, K.; Niinivaara, K.; Vuori, E. Water Res. 2000, 34, 2643-2652. (E13) Wardas, W.; Pyka, A. J. Planar Chromatogr.-Mod. TLC 2001, 14, 8-15. (E14) Cossio, F. P.; Arrieta, A.; Cebolla, V. L.; Membrado, L.; Domingo, M. P.; Henrion, P.; Vela, J. Anal. Chem. 2000, 72, 1759-1766. (E15) Wagner, S. D.; Kaufer, S. W.; Sherma, J. J. Liq. Chromatogr. Relat. Technol. 2001, 24, 2525-2530. (E16) Ishikawa, D.; Taki, T. Methods Enzymol. 2000, 31, 157-159. (E17) Ishikawa, D.; Taki, T. Methods Enzymol. 2000, 31, 145-157. (E18) Fenske, M. Chromatographia 1999, 50, 428-432. (E19) Rhee, I. K.; van de Meent, M.; Ingkaninan, K.; Verpoorte, R. J. Chromatogr., A 2001, 915, 217-223. (E20) Wedge, D. E.; Nagle, D. G. J. Nat. Prod. 2000, 63, 10501054. (E21) Michard, J.; Joubert, M. Ann. Falsif. Expert. Chim. Toxicol. 2000, 93, 83-93; Chem. Abstr. 2001, 134, 294720g. (E22) Pelander, A.; Ojanpera, I.; Sistonen, J.; Sunila, P. J. Liq. Chromatogr. Relat. Technol. 2001, 24, 1425-1434. (E23) Hemmingsen, A.; Allen, J. T.; Zhang, S.; Mortensen. J.; Spiteri, M. A. Free Radical Res. 1999, 31, 437-448. (E24) Kiss, G. C.; Forgacs, E.; Cserhati, T.; Vizcaino, J. A. J. Chromatogr., A 2000, 896, 61-68. (E25) Stahlmann, S.; Herkert, T.; Roseler, C.; Rager, I.; Kovar, K.-A. Eur. J. Pharm. Sci. 2001, 12, 461-469. (E26) Cserhati, T.; Forgacs, E.; Morais, M. H.; Ramos, A. C. J. Liq. Chromatogr. Relat. Technol. 2001, 24, 1435-1445. (E27) Horvath, E.; Katy, Gy.; Tyihak, E.; Kristof, J.; Redey, A. Chromatographia 2000, 51 (Suppl.), S297-S302. (E28) Kowalik, G.; Kowalska, T. J. Planar Chromatogr.-Mod. TLC 2000, 13, 348-353. (E29) Gooijer, C.; Kok, S. J. Chem. Anal. (N. Y.) 2000, 156, 307331. (E30) Kok, S. J.; Evertsen, R.; Velthorst, N. H.; Brinkman, U. A.Th.; Gooijer, C. Anal. Chim. Acta 2000, 405, 1-7. (E31) Schopke, T. Proc. Phytochem. Soc. Eur. 2000, 45, 95-106. (E32) Therisod, H.; Labas, V.; Caroff, M. Anal. Chem. 2001, 73, 3804-3807. (E33) Vermillion-Salsbury, R. L.; Hoops, A. A.; Guseev, A. I.; Hercules, D. M. Int. J. Environ. Anal. Chem. 1999, 73, 179-190. (E34) Wilson, I. D. J. Chromatogr., A 1999, 856, 429-442. (E35) Crecelius, A.; Clench, M. R.; Richards, D. S.; Mather, J.; Parr, V. J. Planar Chromatogr.-Mod. TLC 2000, 13, 76-81.

(E36) Guittard, J.; Hronowski, X. L.; Costello, C. E. Rapid Commun. Mass Spectrom. 1999, 13, 1838-1849. (E37) Isbell, D. T.; Guseev, A. I.; Taraneko, N. I.; Chen, C. H.; Hercules, D. M. J. Mass Spectrom. 1999, 34, 774-776. (E38) Matsumoto, K.; Habaue, S.; Ajiro, H.; Okamoto, Y. J. Mass Spectrom. Soc. Jpn. 1999, 47, 274-280. (E39) Prosek, M.; Golc-Wondra, A.; Vovk, I.; Andrensek, S. J. Planar Chromatogr.-Mod. TLC 2000, 13, 452-456. (E40) Raisanen, R.; Bjork, H.; Hynninen, P. H. Z. Naturforsch., C: J. Biosci. 2000, 55, 195-202. (E41) Mehl, J. T.; Hercules, D. M. Anal. Chem. 2000, 72, 68-73. QUANTITATIVE ANALYSIS (F1) Shakila, R. J.; Vasundhara, T. S.; Kumudavally, K. V. Food Chem. 2001, 75, 255-259. (F2) Johnson, M. E. J. Chem. Educ. 2000, 77, 368-372. (F3) Spangenberg, B.; Klein, K.-F. J. Chromatogr., A 2000, 898, 265-269. (F4) Kishimoto, K.; Urade, R.; Ogawa, T.; Moriyama, T. Biochem. Biophys. Res. Commun. 2001, 28, 657-662. (F5) Soares, J. C.; Dippold, C. S.; Wells, K. F.; Houck, P.; Mallinger, A. G. Psychiatry Res. 1999, 86, 107-112. (F6) Stroka, J.; Anklam, E. J. Chromatogr., A 2000, 904, 263-268. (F7) Liang, Y.; Simon, R. E.; Denton, M. B. Analyst (Cambridge, U.K.) 1999, 124, 1577-1582. (F8) Simon, R. E.; Walton, L. K.; Liang, Y.; Denton, M. B. Analyst (Cambridge, U.K.) 2001, 126, 446-450. (F9) Ebel, S.; Henkel, T. J. Planar Chromatogr.-Mod. TLC 2000, 13, 248-253. (F10) Petrovic, M.; Kastelan-Macan, M.; Ivankovic, D.; Matecic, S. J. AOAC Int. 2000, 83, 1457-1462. (F11) Mustoe, S. P.; McCrossen, S. D. Chromatographia 2001, 53 (Suppl.), S474-S477. (F12) Sarbu, C. J. AOAC Int. 2000, 83, 1463-1467. (F13) Sarbu, C.; Cabzac, S. J. Liq. Chromatogr. Relat. Technol. 2000, 23, 273-280. (F14) Linares, R. M.; Ayala, J. H.; Alfonso, A. M.; Gonzalez, V. J. Liq. Chromatogr. Relat. Technol. 2000, 23, 2653-2668. (F15) El-Bayoumi, A. E.-A.; El-Shanawany, A.; El-Sadek, M. E.; Abd El-Sattar, A. J. Pharm. Biomed. Anal. 1999, 21, 867-873. (F16) Indrayanto, G.; Darmawan, L.; Widjaja, S.; Noorrizka, G. J. Planar Chromatogr.-Mod. TLC 1999, 12, 261-264. (F17) Papp, E.; Farkas, A.; Otta, K. H.; Mincsovics, E. J. Planar Chromatogr.-Mod. TLC 2000, 13, 328-332. (F18) Ferenczi-Fodor, K.; Vegh, Z.; Nagy-Turak, A.; Renger, B.; Zeller, M. J. AOAC Int. 2001, 84, 1265-1276. (F19) Prosek, M.; Golc-Wondra, A.; Vovk, I. J. Planar Chromatogr.Mod. TLC 2001, 14, 100-108. (F20) Peishan, X.; Yuzhen, Y.; Haoquan, Q.; Qiaoling, L. J. AOAC Int. 2001, 84, 1232-1241. (F21) Petrovic, M.; Babic, S.; Kastelan-Macan, M. Croat. Chem. Acta 2000, 73, 197-207. (F22) Cebolla, V. L.; Membrado, L.; Vela, J.; Garriga, R.; Henrion, P.; Domingo, M. P.; Gonzalez, P. J. AOAC Int. 2000, 83, 14741479. (F23) F23) Simon, G.; Liana, G.; Letitia, G. J. Pharm. Biomed. Anal. 2001, 26, 681-685. (F24) Kowalczyk, A.; Matysik, G.; Rzadkowska-Bodalska, H.; Cisowski, W. J. Planar Chromatogr.-Mod. TLC 2001, 14, 175177. (F25) Ozeki, N.; Oka, H.; Ito, Y.; Ueno, E.; Goto, T.; Hayashi, T.; Itakura, Y.; Ito, T.; Maruyama, T.; Turuta, M.; Miyazawa, T.; Matsumoto, H. J. Liq. Chromatogr. Relat. Technol. 2001, 24, 2849-2860. (F26) Essig, S.; Kovar, K.-A. J. AOAC Int. 2001, 84, 1283-1286. (F27) Boido, E.; Dellacassa, E.; Carrau, F.; Moyna, P. J. Planar Chromatogr.-Mod. TLC 2001, 14, 175-177. (F28) Rodriguez, S.; Cesio, M. V.; Heinzen, H.; Moyna, P. Lipids 2000, 35, 1033-1036. (F29) Smolarz, H. D.; Matysik, G.; Wojciak-Kosior, M. J. Planar Chromatogr.-Mod. TLC 2000, 13, 101-105. (F30) Aburjai, T.; Amro, B. I.; Aideh, K.; Abuirjeie, M.; Al-Khalil, S. Pharmazie 2000, 55, 751-754. (F31) Fenske, M. Chromatographia 2000, 52, 810-814. (F32) Marsit, C. J.; Fried, B.; Sherma, J. J. Parasitol. 2000, 86, 635636. (F33) Kim, Y.; Fried, B.; Sherma, J. J. Planar Chromatogr.-Mod. TLC 2001, 14, 61-63. PREPARATIVE LAYER CHROMATOGRAPHY AND THIN-LAYER RADIOHROMATOGRAPHY (G1) Nyiredy, S. J. AOAC Int. 2001, 84, 1219-1231. (G2) Fisher, T. L.; Gilman, C. P. J. Chem. Educ. 2001, 78, 367. (G3) Jozwiak, G.; Wawrzynowicz, T.; Waksmundzka-Hajnos, M. J. Planar Chromatogr.-Mod. TLC 2000, 13, 447-451. (G4) Golkiewicz, W.; Blazewicz, A.; Jozwiak, G. J. Planar Chromatogr.-Mod. TLC 2001, 14, 95-99. (G5) Hajnos, M. L.; Glowniak, K.; Waksmundzka-Hajnos, M.; Kogut, P. J. Planar Chromatogr.-Mod. TLC 2001, 14, 119-125. (G6) Segiet-Kujawa, E.; Gorecki, P. Herba Pol. 1999, 45, 186-191.

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(G7) Gupta, S. K.; Sargent, J. R.; Weber, W. P. Anal. Chem. 2001, 73, 3781-3783. (G8) Gyeresi, A.; Gergely, M.; Vamos, J. J. Planar Chromatogr.-Mod. TLC 2000, 13, 296-300. (G9) Liu, Q.-T.; Kinderlerer, J. L. J. Chromatogr., A 1999, 855, 617624. (G10) Quemeneur, E. In Nucleic Acid Protocols Handbook; Rapley, R., Ed.; Humana Press Inc.: Totowa, NJ, 2000; pp 169-173. (G11) Kiss, B. D.; Mincsovics, E.; Nemes, K. B.; Klebovich, I. J. Planar Chromatogr.-Mod. TLC 2000, 13, 257-260.

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(G12) Vingler, P.; Gerst, C.; Boyera, N.; Galey, I.; Christelle, C.; Bernard, B. A.; Dzido, T.; Tardieu, F.; Hennion, C.; Filtuth, H.; Charpak, G. J. Planar Chromatogr.-Mod. TLC 1999, 12, 244-254. (G13) Salas, J. J. Grasas Aceites (Sevilla) 2000, 51, 168-172. (G14) Fujino, H.; Yamada, I.; Shimada, S.; Yoneda, M. J. Chromatogr., B: Biomed. Sci. Appl. 2001, 757, 143-150. (G15) Graham, D.; Millar, A. M. Nucl. Med. Commun. 1999, 20, 439-444.

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