Anal. Chem. 1992, 64, 134R-147R (F30) Vkw, C. M.; Chang, S. C.; Schardt, 6. C.; Weaver, M. J. J . phvs. Chem. 1991, 95, 7559-63. (F31) Cham. S. C.: Yau, S. L.; Scar&, 6. C.; Weaver, M. J. J . mys. Chem. ' lhl,95, 4787-94. (F32) Bard, A. J.; Fan, F.4.; Plerce, D. T.; Unwln, P. R.; Wiph. D. 0.; Zhou, F. Sclence 1991, 68-73. (F33) Bard, A. J.; Denauk, 0.;Friesner, R. A.; Domblaser, 6. C.; Tuckerman, L. S. Anal. Chem. 1991, 6 3 , 1282-88. (F34) Lee, C.; Kwak, J.; Anson. F. C. Anal. Chem. 1991, 63, 1301-4.
(F35) Scott, E. R.; White, H. S.; Phlpps, J. 6. J. Mmbrane S d . 1991, 58, 71-87. (F36) Yee, S.; Orianl, R. A.: Stratmann, M. J . Electrochem. Soc. 1991, 138, 55-61. (F37) Hunag. S.-M.; Yee, S.; Atanasoskl, R. T.; McMllen, C. S.; Orlenl, R. A,; Smyrl, W. H. J . Elecbpchem. Soc. 1991, 138, L63-4. (F38) Iseacs. H. S. J . Electrochem. Soc. 1991, 138, 722-8. (F39) Isaacs, H. S.; Davenport. A. J.; Shlpley, A. J . Electrod". Soc. 1991, 138, 390-3.
Planar Chromatographyt Joseph Sherma Department of Chemistry, Lafayette College, Easton, Pennsylvania 18042
INTRODUCTION This is a selective review of the literature of TLC and PC cited in Chemical Abstracts from Dec 11, 1989, to Nov 18, 1991. The literature search was augmented by consulting Analytical Abstracts, Chemml Titles, and Current Contents, and the following important journals publishing apers on TLC and PC were searched directly: Journal orChromatography (includin its bibliography issues), Journal of Chromatographic k i e n c e , Chromatographia, Analytical Chemistry, Journal of Liquid Chromatography,Journal of AOAC International, and Journal of Planar Chromatography. Approximately 3800 papers were published in the period under review on the theory, techniques, and applications of TLC. Only a small number of papers continue to report new research in PC. The review is limited to papers in journals easily accesible to US. scientists. This eliminates coverage of many pa ers in journals published in a foreign language, especially thinese, Japanese, Polish, and Russian. A paper by Ronald Majors ( I ) reported the results of a survey of 10 international TLC experts from academia, industry, and overnment concerning their opinions on the most im rtant a!vanm in the past decade and f u F e trends. The f o g + significant advances were cited: an mcreased variety of precoated h- erformance layers, including chiral phases and most of t e onded silica gel phases used in column HPLC; improved instrumentation for spotting, developing, and scanning layers and for applying detection reagents; development of optimization strategies and special techniques, such as scanning of radioactive zones and coupled TLC/MS. Predictions on the future outlook for TLC included the following: hard-layer, glass-backed plates will remain the standard format for the near term; improvements w i l l be made in instruments resulting in a higher degree of automation of procedures such as sample preparation and application, layer development, and scanning; uantitative TLC will become more useful and reliable and ess labor intensive; improved image analyzing systems will be developed, leading to accurate and more rapid analyses; coupled techniques, such as HPLC/HPTLC, HPTLC/MS, SFC/TLC, will be more widely used; and new and improved layers will be produced, including more specific surfacemodifiedphases. In addition, the article compared TLC and HPLC in terms of their relative advantages and disadvantages and discussed whether the techniques were competitive or complementary and the differences in the acceptance of TLC between America and Europe. Sherma also compared TLC and HPLC in another review article (2). Three symposia on planar chromatography were held in 1991: the Sixth International Symposium on Instrumental Planar Chromatography (Interlaken, Switzerland, in April); the Second Congress of Planar Chromatography of the Association de Reserche en Chromatographie Planaire (ARCP) (Chatenay-Malabry,France, in June); the National Sympo-
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Abbreviations used throughout the review are listed in Table 11. 134 R
Table I. Review Contents Introduction General Considerations Books, Reviews, History, and Student Experiments Theory and Fundamental Studies Chromatographic Systems (Mobile and Stationary 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 Pharmaceuticals, Drugs, and Alkaloids Purines, Pyrimidines, and Nucleic Acids Steroids Toxins Vitamins Miscellaneous Organic Compounds Inorganics and Organometallics
sium on Planar Chromato aphy-Modern Thin Layer Chromatography (Somerset, Nfin Sep). Topics featured at these symposia included overpressure and automated multiple development of layers, video recording techniques for image analysis, theoretical aspects and optimization, improved methods for quantitative TLC, and planar chromatography cou led with HPLC, SFC, and FTIR and mass spectrometry, antmany practical papers and posters were resented on applications of TLC. Two reports on the InterEken symposium were published (3,4). Despite the trend toward increasing use of modern TLC with expensive instrumentation, most notably in Western Europe, it is still undoubtedly true that most work with TLC is being done throughout the world with relatively inexpensive basic equipment and manual techniques. On the basis of the literature, it appears that TLC may be used and researched most widely in Eastern European, Asian, and Third-World countries; more than 50% of the papers published on TLC originate in these countries, with relatively few from American laboratories. Whether in ita instrumental or classical form, there is no doubt that TLC is an important analytical method that can serve as a valuable complement for HPLC and GC and that it deserves to be more greatly appreciatd and widely used in analytical laboratories throu hout the world. One of the reasons often cited for t i e relatively low use of TLC in the United States is the lack of training offered in American universities. Camag is attempting to fill this void
0003-2700/92/036~134R~10.00/0 0 1992 American Chemical Society
PLANAR CHROMATOGRAPHY ~orephs-0 received a B.S. in "istry from Upsala College, East Orange, NJ, in 1955 and a Ph.D. in analytical chemistry from Rudgers University in 1958. His thesis research in ion exchange chromatography was under the direction of the late Wm. Rieman 111. Dr. Sherma joined the facutty of LafayetteCollege in Sept 1958 and is presently John D. & Frances H. Larkin Professor
r
Table 11. Abbreviations
AMD
ir
and Head of the Department of Chemistry and is in charge of t h r e e courses in analytical Chemistry. Dr. Sherma independently i'v and wtth others has written or edited over 350 papers, chapters, books, and reviews covering chromatographic and analytical methods. His current research interests are in quantitative TLC, mainly applied to lipid analysis, pesticide residues, and food additives. He is editor for resldues and elements of the Joumal of AOAC International.
by continuing to offer periodic instrumental TLC training courses. Also mentioned is the lack of text books on modern TLC. To help overcome this shortage of information, Sherma and Fried edited a comprehensive Handbook of Thin Layer Chromatography (5),which contains 1047 pages and includes 13 chapters on the principles and practice of TLC and 18 chapters on applications written by over 40 expert authors from around the world. Chromatographic analysis plays a vital role in forensic science. A feature article in Analytical Chemistry (6) on analytical methods for detecting fraudulent documents contained a number of examples of the use of qualitative and quantitative TLC in this area. GENERAL CONSIDERATIONS A. Books,Reviews, History,and Student Experiments. A general book on TLC ( A I ) and books covering planar chromatography in the life sciences (A2) and detection reagents and methods (A3) were published. A new volume on carbohydrates in the CRC Handbook of Chromatography series was published in 1991; this handbook contains considerable TLC, HPLC, and GC data, as well as information on sample preparation and solute detection (A4). The following aspects of planar chromatography were reviewed: resolution and separation capacity (A5),Chromarod-Iatroscan TLC-FID (A6),enantiomeric separations ( A7, A8), comparisons of TLC and HPLC chiral separations (A9), applications to diagnosis of inborn errors of metabolism (AIO), pharmaceutical analysis (AII),clinical analysis (AIZ),analysis of chemical warfare agents (AI3),and industrialenvironmental monitoring (AI4). Other pertinent reviews are cited in the sections below. A book outlined the history of chromatography from its inception up to the development of TLC (AI5),and a paper presented the historic development of paper chromatography from the work of Runge up to the contribution of Tswett (A16). Student laboratory experiments were devised to illustrate the use of TLC for comparing the intermolecular forces of common organic functional groups (AI 7); microscale synthesis of a dipeptide from its component amino acids and its analysis by chiral-phase TLC (AI8);conical TLC (AI9);analysis of amino acids by PC and TLC (AZO);analysis of unknowns by melting points and TLC in combination (A21);TLC determination of caffeine, saccharin, and sodium benzoate in beverages (A22);and isolation and characterization of brain lipids by SPE and TLC (A23). B. Theory and Fundamental Studies. Theoretical aspects of TLC were reviewed (BI). The effect of the external pressure on the efficiency of off- and on-line OPLC (BZ)and prediction of R, values for disubstituted benzene derivatives (B3)were studied. A 25 factorial design was used to evaluate the effect of C1 through C5normal alcohols on the PC separation of selected amino acids (EM).The adsorption properties of aromatic acids and aldehydes on Florisil and silica gel were compared using TLC (B5). Zone shape in column-planar separations was found to be dictated by a combination of nonselective bulk transport forces, such as elution, solvent evaporation,and capillarity, as well as the selective forces of classic TLC (B6). A computer study was made on some
automated multiple development C2 alkyl-bonded silica gel CI2 alkyl-bonded silica gel cl8 alkyl-bonded silica gel charged couple device CCD chemical ionization mass spectrometry CIMS coefficient of variation cv EDTA ethylenediaminetetraaceticacid ELISA enzyme-linked immunosorbent assay FABMS fast atom bombardment mass spectrometry FID flame ionization detector Fourier transform infrared FTIR GC gas chromatography (gas-liquid chromatography) gel permeation chromatography GPC H E W height equivalent to a theoretical plate HPLC high-performance (column) liquid chromatography HPTLC high-performance thin-layer chromatography LSIMS liquid secondary ion mass spectrometry MSPD matrix solid-phase dispersion NMR nuclear magnetic resonance NP normal phase OPLC overpressured (or overpressure) layer chromatography PAH polynuclear aromatic hydrocarbon paper chromatography PC preparative layer chromatography PLC PMA phosphomolybdic acid PTFE poly(tetrafluoroethy1ene) PTH phenylthiohydantoin distance of zone migration/distance from origin to Rf solvent front RIA radioimmunoassay log (1 - R /R,) Rln RP reversed (or reverse) phase RPC rotation planar chromatography SBD ammonium 7-fluorobenzo-Zoxa-1,3-diazole-4sulfonate SERS surface-enhanced resonance Raman spectroscopy supercritical fluid chromatography SFC SFE supercritical fluid extraction SIMS secondary ion mass spectrometry SPE solid-phase extraction thin-layer chromatography TLC time of flight TOF uv ultraviolet visible vis c 2 c 1 2 cl8
performance indices used in TLC (B7). A general approach to R, adjustment in TLC was proposed (B8). Activation of adsorbent layers by anhydrous organic solvent (B9),applicability of a simple optical sensor for examining preadsorption phenomena of solvent vapor (BIO), and an absolute method for determination of the molecular weight distribution of oligomers based on HPTLC and high-resolution NMR spectrometry (BII) were studied. A model of the TLC process that takes into account molecular association in ternary mobile phases was discussed (BIZ). The thermodynamic theory of adsorption TLC was used to describe the retention and separation of aliphatic amino alcohols (BI3). The Snyder-Soczewinski approach was used to interpret the retention behavior of a series of @-carbolines in NP-HPTLC in relation to different solute, solvent, and stationary-phase characteristics (BI4). TLC retentions on silica gel of 20 (E)- and (2)-oxazolones and related cinnamates were studied as a function of mobile-phase effects or Hammett constants (BI5). The effect of surface-phase composition on retention mechanism in adsorption TLC with binary mobile phases was studied (BI6). A parameter was established for predicting retention in NP- or RP-TLC with binary solvent mixtures (BI7). The electrostatic interactions operating in NP- and RP-HPTLC systems used for steroid separations were analyzed by studying the relationship between log (1/R ) values and the molecular polarizabilities of the solvents ($18). The interactions between silica gel and alkali- and alkaline-earth-metal ions were studied in aqueous solutions by OPLC (B19). Three polarity-indicating dyes were employed to characterize the mobile-phasestationary-phase interface of TLC plates (BZO). Structure-retention relationships were examined for di- and trisubstituted derivatives of biuret on CISlayers (B21). Exact equations were proposed ANALYTICAL CHEMISTRY, VOL. 64, NO. 12, JUNE 15, 1992
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for calculating the R, values on silica gel of 9 isosorbide esters from their structures (B22). RP-TLC was used to study a new retention model for R, prediction with methanol-water mobile phases (B23),the retention behavior of 26 triphenylmethane derivatives based on R , values (B24),salting-in and salting-out effects on the retention of 15 dansylated amino acids in aqueous acid mobile phases (B25),the interactions of chlorhexadine with 42 nonionic surfactants in charge-transfer systems (B26),and dependence of the silanophyl effect on the chemical structure of peptides and on the type of mobile phase (B27). The separation mechanism for 20 tris(/.?-diketonato)complexes of transition metals on polyacrylonitrile was investigated (B28). The retention characteristics of 2 pyrolyzed and 2 chemically modified sllicas were explored in ads0 tive and RP-TLC using pesticides as solutes (B29). Force%low development in an overpressured developing chamber was used to determine the porosity, permeability, and apparent particle size of commercial TLC plates and Empore sheets (B30). The predictive abilities of some parameters describing the effect of the structure of metal chelates on their pro erties in RP-TLC were compared (B31). Data were evaluat.ec?(l332), and an equilibrium simulation model was constructed (B33) for the silica gel TLC of metal chelates. Steric effects that occur when aliphatic alcohol mobile phases are used in the adsorption TLC of alkanediols were studied (B34). Snyder’s generalized equation was applied to the characterization of ternary mobile-phase systems used to separate isomeric naphthalene derivatives (B35). Graphical methods were investigated for determining the resolution ran e for PAHs in TLC systems containing binary and ternary mogile phases (B36). Correlations between solvent ranking of 1-and 2-D solvent systems were based on either the solvent ranks or the actual values of the separation functions (B37). Published TLC optimization procedures were reviewed (B38).The following studies of optimization were reported: computer-assisted methods for mobile-phase or mobile-phase and impregnate ion concentrations for optimal separation of 12 PTH-amino acids (B39);computer-assisted optimization of multicomponent selectivity using a matrix-design statistical technique (B40);a comprehensive approach for mobile-phase optimization in pesticide separations, including an optimum graphics system, multifactor optimization system, and computer simulation-diagram optimization (B41);comparison of selectivity for various silica-diluent modifer systems (B42); retention versus eluent composition relationships (B43, B44); a computer program for selecting the optimal eluent composition for a given set of solutes from a database (B45);computer-assisted selection of an optimum stepwise gradient program (B46);optimization of a three-component mobile phase for separation of dolaproine isomers (B47);optimization of displacement development (B48); aphic presentation of binary mobile-phase optimization 6 4 9 ) ; an optimization function and its application to multidimensional TLC of amino acids (B50);optimization of mobile phases for separation of 1-(2-pyrimidy1)-3-methyl-5-pyrazolone derivatives (B51,B52);development of a personal-computer solvent optimization program, OPTISOLV, based on the PRISM model (B53);a new approach based on the dependence of the r e d u d HETP on the reduced flow rate at the end of development (B54). The following aspects of relations between TLC and HPLC were investigated: computer-assisted transposition of conditions in HPTLC to HPLC for separation of pesticides (B55); prediction of HPLC retention behavior of benzodiazepine derivatives by TLC (B56);theoretical considerations of the use of TLC as a pilot technique for HPLC (B57). TLC was used to determine the lipophilicity of triazine and cyano henyl derivatives (B58),cortisone derivatives (B59), 17 a n i n e and phenol derivatives (B60), xanthine and adenosine derivatives (B61, B62), and nonionic surfactants (B63). C. Chromatographic Systems (Mobile and Stationary Phases). Silica el continues to be the most widely used layer material for TL8,but increased application of bonded-phase layers has occurred over the past 2 years. A new class of flexible, sorbent-impregnated,binderless PTFE sheets (Empore) has been available for several years, but these layers have not yet found wide favor. Reviews were published on sorbents and precoated layers for NP, partition, RP, and ion-exchange TLC (C1);hydrophilic modified silica gels with bonded amino,
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cyano, or diol grou s (C2);and precoated plates with concentrating zones (&). Dip washin with methanol was found to be the most effective methJfor precleaning TLC plates ( ~ 4 )studies . were made on variation of the stationary phase in order to gain selectivity advan es (C5) and the effect of silica support surface pH on the P-TLC retention of amino acids (C6). Properties and application of the followin layers were reported: se aration of bryostatins 1 and 2 (87)and pharmaceuticals rC8) on multimodal plates with adjacent silica gel and Cu layers, a newly synthesized su port named -aminoplast” for the separation of amino acids (891,HP latea prepared from 3-pm silica gel (CIO),calcium sulfate & I ) , orous glass sheets (C12),and diol-bonded silica el (C13). h e t a l ions were separated on chitin, chitosan, ancftheir derivatives (C14), on a newly synthesized carbamide-formaldehyde polymer (C15),and on mixed stationary phases containing two or more kinds of functional groups in a controlled proportion (C16). Organic electrolytes were separated in ion-pair RP-HPTLC systems (C17). Layers impregnated with silver were used to separate triglycerides according to the number of double bonds (a dynamic impre ation process produced plates with superior reproducibEy) (C18),with Fe(II1) for separation of carboxy- and hydrox benzene derivatives (C19),and with salicylic, syringic, andro-phthalic acids for metal ions (C20). Optical and structural isomers were separated by develo ment with 8-cyclodextrin solutions as mobile phases (C21, &2). The following studies of enantiomer separations were reported use of chiral mobile-phase additives in RP-TLC (C23);NP-TLC usin chiral ion interaction agents (C24); synthesis of a novel, c L a l selector for ligand exchange TLC (CW); preparation of modified amino-bonded plates (C26, C27);separation of amino alcohols using a chiral counterion in the mobile phase (C28). Empore TLC sheets were evaluated (C29, C30),compared with conventional glass-backed plates (C31),and applied in forced-flow planar chromatography ((232). D. Apparatus and Techniques. Reviews were written on instrumental TLC (DI), HPTLC in the clinical laboratory ( 0 2 ) ,principles and potential of gradient elution (03),industrial a plications of HPTLC ( 0 4 ) ,multidimensional and multimdal TLC (05-07), automation of TLC with a robot (D8), applications TLC-FID to the analysis of lipids and llutants (D9),and documentation of planar chromatograms y nondensitometric methods (010). The following apparatus were described an automated sample applicator for quantitative TLC (011);an automated “4 X 4” sample applicator for a plying four 10-pL spots to a 10- X 10-cm plate (012);a m d f i e d horizontal chamber for stepwise gradient elution (013);a m e r based on photodiode array detection and fiber optic bundles for measuring UV spectra of separated zones (014);a compact, portable laptop TLC scanner (D15);the Rotachrom RPC instrument (016). Descriptions of the following techni ues were ublished: planar chromatography with rectangdar packef capillary columns and electrochemical detection (D17);resonant twophoton ionization spectroscopic analysis of TLC zones using pulsed laser desorption/volatilization into a supersonic jet expansion of COzfor transport into a TOF mass spectrometer (018);use of subambient temperatures to improve resolution (019);silica gel TLC with ascending double un regnation of silicone oil in opposite directions (020); m&imodal 2-D procedures for separation of tax01 and related compounds (021);mobile-phase transfer from OPLC to medium pressure LC (022);computer-misted sample cleanup in LC from TLC data (023). Optimization procedure for displacement chromatography were outlined (0241, and separations by spacer-displacement and carrier spacer-displacementTLC were compared (025). Direct coupling of HPLC and TLC was described (026)and a plied for the identification of PAHs in marine sediment by gorescence excitation and emission spectroscop (027). Procedures for optimization of gradient AMD were JLcribed (OB), AMD was coupled online with HPLC (0291and , AMD was used to analyze extracts of Echinacea species (030). Overpressure derivitization is a new method that is carried out by pressing an absorbent polymeric pad, wetted with derivitization reagent, onto the TLC plate ( 0 3 1 , 0 3 2 ) . The direct use of Empore TLC sheets in OPLC (033) and the
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PLANAR CHROMATOGRAPHY
principles of long-distance OPLC, a new multilayer development techni ue (034), were described. Analytical OPLC, preparative 8PLC, and HPLC with the same phase system were compared for the separation of closely related terpenoids
(0355). Methods for the coupling of TLC and FTIR spectrometry were reported ( 0 3 6 , 0 3 7 ) . Methylbenactyzium bromide in human urine was determined by TLC and pyrolysis GC (038). SFE ( 0 3 9 ) and SFC ( 0 4 0 ) were coupled with TLC. E. Detection and Identification of Separated Zones. The following detection reagents were described cyclodextrins, Triton X-100, and n-alkanes used as fluorescence enhancers to lower the detection limit of SBD-glutathioneby a factor of 2 (El); oxalic acid, dithiooxamide, or dithizone followed by ninhydrin to detect amino acids with various specific colors to aid identification (E2);acetylacetone-formaldehyde for detecting amino acids as yellow spots under W light (E3);trifluoroacetic a n h y d r i d d u m iodide for nitrones and nitroxide radicals (E4) and nitroso compounds (E5); AsC13/HI04and UV light for plant growth regulators (E6); 2-(trichloromethy1)benzimidazole for o-phenylenediamine (237); mouse monoclonal antibody 2D4 for ganglio-series gangliosides (E8);Fast Black K salt for differentiation of aliphatic primary and secondary amines by color (E9). Nitrogen or helium flushing enhanced the detection of thiols after derivitization with SBD; the method was recommended for other fluorescent compounds on silica gel layers (ElO).The liquid crystal method, which involves mapping of the chromatogram by transferring organic substances from a TLC plate to a liquid crystal layer, was applied to chemical warfare agents and pesticides separated on carbon layers ( E l l ) . An ultrafast data acquisition system for UV detection of bidimensional chromatograms (E12) and back ound correction and normalization of W/vk reflection anfabsorption s ectra (E13) were described. Spectral reflectance and uorescence were measured on layers by use of fiber optics and a photodiode array (E14). Amorphous fumed silica was used to enhance by 200-400% the fluorescence signal of a mixture of PAH compounds separated by PC (E15). A supersonic jet fluorometric method was able to detect down to 10 ng of separated compound on flexible silica gel sheets (E16). Iron porphyrins from coal were separated by multi le, sequential development and characterized by MS an{ paramagnetically shifted proton NMR spectrometry (E17). A new method for preparing SERS-active layers involved deposition of silver colloidal spheres on HPTLC plates (El@,and these layers were used to identify highly fluorescent molecules such as acridine orange and 2-aminofluorene by in situ resonance Raman scattering spectrometry (E19). TLC coupled with FTIR spectrometry was reviewed (E20). An algorithm developed for TLC/FTIR identification of functional groups in organic compounds was described (E21), and the method was used to analyze coal extracts (E22). Near-IR spectrometry was shown to be an alternative for chemical reaction-based detection and identification methods in the TLC analysis of pharmaceutical compounds (E23). A eat amount of activity was noted in the combination of T f i with MS. TLC was combined with CIMS for anal ing mixtures of crude reaction products from a synthesis; g e c t transfer of silica gel TLC zones to a solid probe was made using a Carbowax 20M pellet (E24). The Wick-Stick technique was modified by introducing ammonium chloride as a volatile salt substrate for analysis of TLC spots by direct sample introduction MS (E%). Several specific interface devices for combining planar chromatography with MS were described (E26). Analysis of planar chromatograms by FAB and laser desorption MS was reviewed (E27). Laser MS was used to analyze compounds directly on thin layers ( E 2 8 4 3 0 ) . A sim 1, microtransfer technique was developed for indirect TL&FABMS and TLC/CIMS determination of carbohydrates (E31). Evaluation was reported of a TLC/FABMS interface used in conjunction with two FAB matrixes, glycerol and thiodiethanol, for drug metabolism studies (E32). Coupled TLC FABMS was applied to the analysis of porphyrins (E33) an ecdysteroids (E34) directly on layers. Pa ers on the following aspects of TLC combined with S dwere ublishd a general discussion of directly coupled TLC/SIM&EW; an optical CCD camera integrated with a SI mass spectrometer for imaging planar chromatograms
R
d
(E36);obtaining product ion mass spectral data of oligosaccharides by using both LSIMS with BIE-linked scanning and FAB ionization with collision-induced dissociation/tandem MS (E37); an optimized procedure for the analysis of nonvolatile compounds from the surface of silica gel HPTLC plates by LSIMS (E38); o timization of primary beam and ion extraction optics of a 81 mass spectrometer for use with TLC (E39); quantitative analysis by TLC/SIMS (E40); TLC LSIMS applied to investigations of drug metabolites (E41/;TLC/LSIMS determination of major bile salts in dog bile (E42). F. Quantitative Analysis. Scanning densitometry for quantification in planar chromatogra hy (FI, F2) and sourcea of error in densitometric evaluation &‘3)were reviewed. The Soczewinski equation was applied to the optimization of elution systems in spectrodensitometricTLC (F4). Reducing HP silica gel layer thickness from 0.2 to 0.1 mm yielded increased response in scanning densitometry for absorption and fluorescence modes (FS).Pharmaceutical applications of postchromato aphic derivitization in quantitative TLC were presented, anfthe importance of dipping rather than spraying of reagents was emphasized (F6). A quantitative TLC-enzyme immunostainin method was developed for determination of gangliosides an! sulfatide in human cerebrospinal fluid (F7). A video densitometer was developed, evaluated, and compared with a conventional scanning densitometer (F8). Phospholipids were quantified by in situ FT near-IR spectrometry (Fs).Photoacoustic spectroscopy by the rear-surface detection method was proposed for use with aluminum foil TLC plates (FIO),and the method was used to quantify p b levels of cobalt on silica gel plates treated with 1-&pyridylazo)-2-naphthol (FI1). Voltammetric electrochemical detection coupled with TLC was demonstrated for the in situ quantification of trace organic compounds (F12). Polarity dependent fluorescence of 8(pheny1amino)-1-naphthalenesulfonate was used to develop a quantitative evaluation method for nonfluorescent compounds on TLC plates (F13). Computer averaging of data was shown to improve uantification of circular thin-layer chromatograms (1714). omputer-controlled scanning procedures were described for plates on which spots from 1-D development are not in a straight line and for 2-D chromatograms (F15). A general rocedure for algebraic linearization of calibration curves h 1 6 ) and integration problems in quantitative TLC (F17)were illustrated. Computer programs were described that allow scanning and evaluation of 2-D chromatograms (F18). Method validation in the bioanalybical laboratory (F19) and in pharmaceutical analysis (F20) and chromatographic suitability tests (3’21) were described. G. Preparative Layer Chromatography and RadioThin-Layer chromatography. SFC was combined with TLC on a semipreparative scale by condensing the effluent from large-diameter SFC columns onto the origins of preparative layers (GI). A new solid-phase sample ap lication method and device enables sample application in tEe entire cross section of a preparative layer with the advantages of in situ sample concentration and cleanup and formation of an extremely sharp initial zone (G2). A new device was presented to provide suitable mobile-phase velocity for the circular development mode of classical PLC (G3). Mixtures of lipids and phospholipids were separated by centrifugallyaccelerated PLC at 300-500-mg levels (G4). Detection of radioactivity distribution on layers with positron-sensitive detectors, linear analyzer, and digital autoradiograph was reviewed (G5). The first 14Ccalibration plate is available for optimization and calibration of radio-TLC linear analyzers (G6). The design and performance of the Vanguard scanner, which is intended to replace positronsensitive proportional counters for the 1-D and 2-D measurement of more energetic @ and soft y emitters on pa er and thin-layer chromatograms (G7), and the AMBIS d k 2 imaging system for computer analysis of 1-D and 2-D layers and gels (G8, C9) were described. Band-broadening effects can be avoided and the sensitivity of film detection methods for weak j3 radiations highly increased by using polystyrenebased ion-exchange lanar chromatography instead of silica gel (GIO). A new gtector for 2-D radio-TLC, the di ital autoradiograph, was described and evaluated (G11). 148-labeled paracetamol metabolites and 8-blockerswere analyzed
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by OPLC with on-line detection using a radio-flow cell and off-line detection usin a linear analyzer and proton NMR spectrometry (G12). l1 labeled flumazenil and ita metabolitm were determined in animal and human blood by using TLC with an automatic linear scanner (G13). A radiochemical TLC assay was used to measure 3-hydroxy-3-methylglutaryl-CoA lyase activity in cell extracts (G14).
8
APPLICATIONS Unless otherwise stated, the references below involved TLC on precoated plates or sheeta. TLC plates are rarely handcoated today, unless a special layer is required that is not available commercially or the cost of precoated plates is a factor. TLC as ap lied to pesticide analysis will be reviewed by this author in e! t 1993 Applications Review issue of this journal. H. Acids and Phenols. The relationships between R, and k’values and methanol and acetonitrile concentration of the mobile hase for aromatic alkoxy and hydroxy acids were investigatecfin CI8systems (HI). Lactic acid enantiomers were separated on silica gel layers impregnated with Cu(I1) (H2). N-Methylaspartic acid isomers were resolved by ligand-exchange TLC on commercial chiral plates (H3). A TLC screening method allowed detection and quantification of hippuric, mandelic, and phenylglyoxylic acids in urine after occupational exposure to aromatic compounds, and results were compared with those by GC (H4). Cz (H5) and cyanobonded silica gel HP layers in combination with propyl (H6) ion-pair re ents were used to separate dihydroxybenzoic acids. H P X C with scannin densitometry was applied to the determination of 18-a-and l&@-glcyrrhetinic acids (H7) and usnic acid in the lichen Usnea &ida (H8).The lipophilicity of arylsulfonylalkanoic and arylsulfonylcycloalkanemboxylic acids was determined by measuring R, values on a polyamide layer (H9). Natural phenolic compounds were separated by AMDHPTLC on silica gel layers employing methanol, ethanol, ethyl acetate, dichloromethane, and hexane solvents and fluorescence detection (H10). Phenols were separated on layers of silica gel and silica gel mixed with an aqueous heteropoly compound and detected usjng Kodak CD-3 reagent (HII). New spray reagents, orthanihc acid, o-dianisidine, and their diazotized products, were used to visualize 22 phenols after TLC on silica gel G layers developed with chloroform-methan01 (982) mobile phase (H12). Pentachlorophenolwas quantified in wooden containers and urine of exposed workers by a densitometric HPTLC method having detection and quantification limits of 10 and 27 ng/ spot, respectively (H13). A TLC method was described for screening and quantification of pentachlorophenol in tallow at 0.5-20 ppm involving extraction, alumina column cleanup, development of a preadsorbent silica gel la er with hexaneacetone-methanol-acetic acid (35:10:5:0.1[ detection with silver nitrateammonia reagent, and densitometry (H14). I. Amino Acids, Peptides, and P + s i r ~ s TLC . separation of dansyl and dinitrophenyl derivatives of amino acids (11) and amino acid enantiomers (12) was reviewed. A discussion of clinical amino acid screening was presented, including R, values and minimum detectable levels for 60 compounds (13). Amino acid separations were studied in several planar chromatography systems: 24 acids on papers impregnated with Sn(1V)and Th(1V) phosphosilicate cation exchangers using water, alcohols, acids, acetone, benzene, ether, and phenol as developing solvents (14);15 acids with n-butanol-acetic acid combined with water, chloroform, or ethyl acetate as mobile phases on plain and nickel chlorideimpregnated silica gel plates (15);chitin layers with monocomponent and binary mobile phases (resulta were elaborated using widely-accepted TLC theories) (16);and 14 acids on mixed admrbenta containing silica gel and alumina or cellulose with benzene-containing mobile phases (17). Ninhydrin detection reagent was modified by addition of D-camphor (18)and various acids (19) for improved identification of amino acids. Sensitivity limits and color differentiation in amino acid detection were improved by spraying lavers with 1.3-indanedione or o-mercaptobenzoicacid prior td ninhydrin (110). TLC/densitometrv was used to determine 0.5 me/L of phenyldanine in bl& serum as an indicator of phenylketo138R
ANALYTICAL CHEMISTRY, VOL. 64, NO. 12, JUNE 15, 1992
nuria (11l ) ,reduced and oxidized glutathione after fluorescent labeling of the thiol group (1121,and physiological aromatic acids on cellulow with development by sodium sulfate solution to facilitate ninhydrin detection (113). A new chiral reagent, NSP-C1, was synthesized and used to derivatize amino acid diastereomers, and the resulting amides were resolved by TLC (114). TLC on Chiralplatm with acetonitrile-methanol-water (4l:l) mobile phase was used to evaluate reaction products in the synthesis of modified phenylalanine and tyrosine derivatives and for the determination of the amino acid configuration of synthetic peptide analogues prepared starting from the racemic aromatic amino acids (115). TLC of peptides and proteins was reviewed (116). Aspartame and ita precursor stereoisomers were separated on a Chiralplate and on a Chiralcel OD column (117). Protein and peptide hydrolyzate were analyzed by 2-D cellulose TLC and densitometry, and the method was applied to luteinizing hormone (118). J. Antibiotics. Separation of seven tetracyclines was carried out on silica gel impre ated with buffered EDTA, with dichloromethanemethanoKater (59356) as the mobile phase (J1). Monensin, lasalocid, and salinomycin were screened in animal tissues by TLC with bioautogra hy detection (J2). Residues of aminoglycosides, macrolifes, tetracyclines,and chloramphenicolwere screened in poultry meat by silica gel and cellulose TLC after extraction and CI8column cleanup (J3). Densitometry was used for the following quantitative determinations: nebramycin components at a level of 2.5-55 %/spot using a charring procedure (J4);gentamicin C residues in poultry meat at ppb levels by silica gel HPTLC with 20% KHzPO mobile phase and ninhydrin or fluorescamine detection tJ5);tetracyclines, with a sensitivity of 400-900 ng/ spot, by use of cyanophase HPTLC lates, methanol-acetonitrile-0.6 M aqueous oxalic acid mokle phase, and scannin at 254 nm (J6).Three polyether carboxylic antibiotica in lipi! extracts of Streptomyces h groscopicus were separated and determined by TLC on Zhromarods SI1 developed with chloroform-methanol-formic acid (97:40.6) and FI detection
(57). K. Bases and Amines. A total of 12 aromatic amines were
separated b TLC on silica el G impregnated with 1% of the surfactant &iton-X 100 Silica gel layers impregnated with picric acid and developed with cyclohexane-benzene mixtures were also used for the chromatography of aromatic amines (K2). (2,4-Dinitrophenyl)pyridiumchloride was synthesized and used as a colorimetric or fluorometric detection reagent for primary and secondary amines, amino acids, thiols, thiolactones, and carboxylic acids (K3). Methyl (trichloromethy1)acetimidate was used as a selective chromogenic reagent for detection of o-phenylenediamine and some of ita derivatives at 80-2000-ng levels (K4). Monoethanolamine and glycine betaine were quantified in plants by use of two ion-exc e columns and silica gel TLC (K5). Histamine was separate from polyamines and determined by spectrodensitometric TLC after dansyl derivativization (K6). L. Carbohydrates. TLC, GC, HPLC, and SFC of carbohydrates were reviewed (L1). Mucin-derived oligosaccharides were analyzed by medium-pressure GPC and TLC on aminopropyl-bonded silica developed with acetonitrile10 mM triethylamine acetate (3:2) (L2). Twenty carboh drates were separated on amino-bonded plates and detectedaith a sensitivity similar to that obtained with spray and dip reagenta by merely heating and inspection under UV light (L3, L4). Six of the eight possible sucrose monostearate structural positional isomers were separated by TLC and further differentiated by use of a specific chromogenic detection reagent (L5). Immobilization of a-amylase in paper improved the sensitivity of the silver nitrate detection method for high molecular weight oligosaccharides (L6).o-Toluidine was used as a reagent in HPTLC for detection and scanning at 295 nm of reducing sugars in rotein hydrolyzates with a sensitivity range of 50-100 pmof(L7). The following densitometric determinations were reported xylose, 3-O-methylglucose, and rhamnose in urine with arabinose as internal standard by scanning at 400 nm after ap-
(21).
9
PLANAR C H R O M A T W A P H Y
plication of aminobenzoic acid detection reagent in an automatic dipping chamber (the CV of the method was
