(1 162) (1163) (1164) (1165) (1166) (1167) (1168) (1169) (1170) (1171) (1172) (1173) (1174) (1175) (1176) (1177) (1178) (1179) (1180) (1181)
I . S., lzv. Akad. Nauk SSSR, Ser. Khim., 1972, 1955. Vodenicharov. R., Kolusheva, A,, Farmatsiya, (Sofia), 22, 1 (1972). Vodzinskii. Y . V., Skvortsov, N. P., Korshunov. I . A.. Eiektrokhim., 9, 469 (1973). Voicu, V., Calusaru. A,, Talanta, 20, 659 (1973). Volke. J.. Naarova. M., Collect. Czech. Chem. Commun., 37,3361 (1972). Volke, J.. Skala, V., J. Electroanab Chem., 36, 383 (1972) Volke, J.. Volkova, V., Collect. Czech. Chem. Commun., 37,3361 (1972). Volova. I . G., Kuznetsova. M . A,, Brainina, K. Z., Sb. Nauch. Tr. Khim., Sverdiovsk, lnst. Nar. Khoz., 1971, 15. Vondrak. D., Barbosa, A. C.. Sb. Vys. Sk. Chem.-Technoi. Praze, Anal. Chem., HE, 73 (1972). Vorobev. N. E., Dzyuba. N. P., Farm. Zh., 26, 57 (1971). Wachholz. E., Pfeifer. S..Pharmazie, 27, 43 (1972). Wagenknecht, J. H., J. Electrochem. SOC.,119, 1494 (1972). Wagenknecht. J. H., J. Org. Chem.. 37, 1513 (1972). Walter. M.. Diss. Abstr. B , 31, 7154 (1971). Warren, L. E., Homer, S. M., Hatfield, W. E., J . Amer. Chem. SOC., 94, 6392 (1972). Wawzonek. S., Su, T. Y., J. Electrochem. SOC.,120, 745 (1973). Webb, J., ibid.. 119(2). 59c (1972). Webb. J. W., Janik, B.. Elving, P. J., J . Amer. Chem. SOC.,95, 991 (1973). Weitzman, P. D. J., Kennedy, I. R., Caldwell, R. A.. FEBSLett., 17, 241 (1971). Wenck, H., Schwabe. E., Schneider, F., Flohe. L.. Fresenius’ Z. Anal. Chem., 258, 267 (1972). Westmoreland. P. G., Day, R. A,, Underwood, A. L.. Anal. Chem., 44, 737 (1972).
(1182) Whitson, P. E., VanderBorn, H. W.. Evans, D. H., lbid., 45, 1298 (1973). (1183) Wilhelm, M., Federlin, P., Kern, J. M.. Bull. SOC.Chim. Fr., 1972, 1062. (1184) Williams, A. A., Timberlake, C. F., Tucknott, 0. G.,. Patterson, R. L. s., J. Scl. FoodAgr., 22, 431 (1971). (1185) Williams, A. I., Analyst, (London), 98, 233 (1973) (1186) lbid., p 1165. (1187) Williams, D. J.. Pearson, J. M.. Levy, M . , J. Amer. Chem. SOC.,93, 5483 (1971). (1188) Winecoff. W. F., I l l , O’Brien, F. L., Boykin, D. W., Jr., J. Org. Chem., 38, 1474 (1973). (1189) Woggon, H.. Sauberlich, H., Uhde. W. J., Fresenws’ Z. Anal. Chem., 260, 268 (1972). (1190) Yakhontov, L. N., Krasnokutskaya, D. M . , Akalaev, A. N., Palant, I. N., Vainshtein. Y. I., Khim. Geferotsikl. Soedin., 7, 789 (1971). (1191) Yamashita. K.. Imai, H., Rev. Poiarog., 18, 10 (1972). (1192) Yamshchikov, A. V., Lemin, N. N., Zh. Org. Khim., 8, 1267 (1972). (1193) Yanovskaya, L. A,, Dombrovsky, V. A,, Chizhov. 0. S.,Zolotarev, B. M., Subbotin, 0. A,, Kucherov, V. F., Tetrahedron, 28, 1565 (1972). (1194) Yanovskaya, L. A,, Umirzakov, B., Kucherov, V. F., Izv. Akad. Nauk SSSR, Ser. Khim., 1972, 823. (1195) Yarmukhametrova, D. K., “Mater. Nauch. Konf., Inst. Org. Fiz. Khim., Akad. Nauk SSSR,” Kazan, USSR, 1970 (Publ. 1971). (1196) Yoshida. T., Nippon Kagaku Kaishi, 1973, 1706. 111971 lbid... rD 1710. (1198) Yurko, D. G., Solomko, Z. F., Gergel. L. G., Bozhanova, N. Y., Dyshlevoi, V. P., Braichenko, V. T., Zh. Obshch. Khim., 42, 1081 (1972). (1193) Yusupova, N. K., Lankina, T. A,, Yamatova, R. G., Uzb. Khim. ’ Zh., 17, 21 (1973). (1200) Zahradnik, R., Rejholec, V.. Hobza, P.. I
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Carsky, P., Hafner, K., Collect. Czech. Chem. Commun., 37,1983 (1972). Zaitsev. P. M.. Grechishkina. V. N., Chasovskikh, L. S., Zaitseva. 2. V., Zh. Obshch. Khim., 42, 1084 (1972). Zaitseva, 2. V., Zaitsev, P. M.. Lubyanitskii, I . Y., Shuster. Y. A , , Tr. Gos. Nauch.-lssled. Proekt. lnst. Azotn. Prom. Prod. Org. Sin., 1972, 78. Zakharchenko, I . P.. Antropov, L. I., Vestn. Kiev. Politckh. lnst., Ser. Khim. Mashinostr. Tekhnol., 1971, 6. Zakharin, L. I., Kyskin. V. I., lzv. Akad. Nauk SSSR,Ser. Khim., 1971,2052. lbid., 1972, 214. Zakharkin, L. I., Orlova, L. V., Denisovich, L. I., Zh. Obshch. Khim., 42, 2217 (1972). Zakharov, V. A.. Bessarabova, I . M . , Barikov, V. G., Treshetkina, T. I., Elektrokhim., 9, 58 (1973). Zaporozhets, E. V . , Avrutskaya, I. A,, Babievskii. K. K., Belikov, V. M., Fioshin, M. Y., ibid., 8, 1243 (1972). Zaporozhets. E. V., Avrutskaya. I. A,, Fioshin, M . Y., Babievskii, K. K., Belikov. V. M., ibid., 9, 270 (1973). Zecchini, S., Pilloni, G., J. Electroanal. Chem., 36, 117 (1972). Zezula. I., Markusova, K.. Collect, Czech. Chem. Commun., 37, 1081 (1972). Zhantalai, B. P., Siisarenko, V. P., Zavod. Lab., 39, 6 (1973). ibid., p 143. Zhustareva. S. S.. Krunchak. V. G., Lomova, M. A,, Mikhailova, V. p., Khvorostin, Y. S.. Tr. Vses. Nauch.-lssled. lnst. Tsellyul.-Bum. Prom., No. 60, 170 11972) Zimiia; G. A,, Sedavkina. V. A,. lssied. Obi. Geterotsikl. Soedin., 1971, 99. Zuman, P., Ed.. “Progress in Polarography, Vol. 3, Interscience, New York, 1972. Zverina, V., Remes, M.. Kroupa, J., Zagner, Z., Matika, M., Collect. Ozech. Chem. Commun.. 37,839 (1972).
Chromatography Gunter Zweig Criteria and Evaluation Division, U.S. Environmental Protection Agency, Washington, D.C. 20460
Joseph Sherma Chemistry Department, Latayette College, Easton, Pa. 78042
In the period since our preparation of the last Chromatography review for this Fundamental Reviews issue, chromatography has remained a very widely used analytical technique, with over 8000 publications on all aspects of chromatography appearing in the world literature. This review covers the literature of liquid column chromatography, paper chromatography, and thin layer chromatography (excluding ion exchange, which is reviewed separately elsewhere in this issue). The authors have attempted to select the most important publications abstracted in Chemical Abstracts between December 20, 1971, and December 3, 1973, and when possible, papers were selected which were published in journals readily accessible to American scientists. In order to conserve space and prevent possible overlap with coverage in the Analytical Applications Reviews published by this Journal in alternate years, the extensive second section on “Applications of Chromatography’’ ar-
ranged according to compound types, which has traditionally appeared in this review, has been eliminated. Some selected applications of the various chromatographic methods are still mentioned below, however, under the appropriate headings. During the past two years, the American Chemical Society sponsored short courses on modern liquid chromatography and maintaining and troubleshooting chromatographic systems. The Chemical Rubber Co. two-volume “Handbook of Chromatography,’’ edited by Zweig and Sherma, and a book presenting a unified introduction to separation science by Karger, Snyder. and Horvath (Wiley-Interscience) were published, and a new supplement to the Journal Separation Science entitled Separation and Purification Methods (Dekker) appeared. Preston Technical Abstracts Co. introduced a service providing regular abstracts of the LC literature, and Sadtler offered an audio-visual teaching program on LC. An Institute for A N A L Y T I C A L C H E M I S T R Y , VOL. 46,
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chromatography training began in early 1973 in Bad Durkheim, West Germany, under the direction of R. E. Kaiser. The following symposia were published: ACS Symposium on Chromatography and Computers, held in September, 1971 in Washington, D.C., published in the December, 1971, and January, 1972, issues of Journal of Chromatographic Science; the First International Symposium on Computer Chromatography and Associated Techniques, held in Mainz, Germany, March 2 and 3, 1972, published in Chromatographia, volume 5 (2/3), 1972; the Camag Symposia on Advanced TLC held in Cherry Hill, N.J., April 26-28, 1971, and New York City, November 28-30, 1972, were published in Journal of Chromatography, volume 63 ( l ) ,1971, and volume 82 ( l ) , 1973, respectively; the Chromatography Anniversary Symposium, held a t the ACS National Meeting in New York City in August, 1972, published in the February, April, and June 1973 issues of Journal of Chromatographic Science; the Eighth International Symposium on Advances in Chromatography, held in Toronto, April 16-19, 1973, published in the April, May, and June 1973 issues of Analytical Chemistry; the Fourth Tswett Memorial Symposium, held in Milan, Italy, June 19-21, 1972, published in Journal of Chromatography, volume 77 (l),1973, and the November 22, 1972, issue of the Journal of Chromatography [volume 73 (2)] was dedicated as the Tswett Centenary Issue; the Fifth Symposium on Quantitative Flat-Bed Chromatography, held in Carlsbad, Czechoslovakia, September 11-13, 1972, published in Journal of Chromatography, volume 78 (l), 1973; and the International Symposium on Column LC, held in Interlaken, May 2-4, 1973, published in Journal of Chromatography, volume 83, 1973. The ACS Award in Chromatography, sponsored by Supelco, Inc. went to Albert Zlatkis of the University of Houston for 1973 and to L. B. Rogers of Purdue University for 1974.
LIQUID COLUMN CHROMATOGRAPHY General Considerations and Theory On the occasion of the centennial of the birth of M. Tswett, the inventor of chromatography, several papers traced the history of chromatography and Tswett's life and contributions (147A, 210A, 309A, 449A, 511A). A book by Munier on the principles of chromatography (375A), a practical supplement to pharmacopoeias covering the chromatography of drugs (501A), and a handbook on sorbents and carriers in Russian (331A) were published. Reviews appeared on general aspects of various chromatographic techniques (4A, 8A, 106A); preconcentration in analysis (238A);empirical rules determining retention behavior and column selection (438A); determination of thermodynamic and kinetic parameters by chromatography (272A, 412A); support materials for chromatography (374A); phase selectivity in gas chromatography and LC (273A); the importance of molecular diffusion (573A); zone spreading theories (296A); principles of chromatographic detection (274A, 414A); the chromatographic analysis of anthocyanins and flavones (430A), deoxyribonucleic acids (451A), chloroplast pigments (51I A ) , drugs in body fluids (259A), food products (63A), plant hormones (344A), and lipids (114A); and the continuous chromatographic refining of hydrocarbons ( 2 0 A ) . Column and thin layer chromatography were compared in detail ( 7 A ) .An undergraduate experiment demonstrates the column and thin layer separation of the product mixture from the nitration of phenol (446A), and a simple liquid column apparatus for teaching follows the elution of electrolytes by continuous monitoring of the conductivity of the effluent (275A). General chromatographic theory was developed and fundamental studies were carried out as follows. The errors in perpendicular allocation of overlapping peaks depend on peak shape (287A).A simple least squares analysis of chromatographic tracings is particularly applicable to the repetitive analysis of mixtures (103A). A modified random walk method provides plate height values which agree with those obtained from nonequilibrium theory (118A). Automated numerical analysis using the first de74R
rivative of the chromatogram rather than the total concentration curve is more sensitive for detecting the presence of peaks (117A). Expressions for the first, second, and third moments in nonlinear chromatography were derived and tested by computer simulation (67A-69A). Mathematical analysis was made of the single and dual reference peak normalization techniques (94A).Time normalization can be used in liquid chromatography to improve resolution without sacrificin analysis time by optimization of parameters such as cofumn length, carrier velocity, support size, and capacity ratio (193A). The dynamics of chromatographic zone expansion were studied with (556A) and without (555A) calculation of the velocity profile. Random errors in chromatography were discussed in terms of the combination of the arithmetic mean value and standard deviations of two data series (256A). Basic guidelines were outlined for effective use of correlation techniques in continuous chromatographic analysis (IOA). Optimum separations of multicomponent ion mixtures may be determined by solving algorithms, either graphically or by using computers (354A).The material balance concept of the chromatographic process, describing concentration as a function of location and time, has been considered as concerns automated liquid chromatography (228A). A simplified form of Wilson's equation for excess free energy is accurate for 11 common chromatographic systems (235A). Gravitational and hydrodynamic factors affect the transformation of veinlike chromatographic zones (270A).The effects of various sources of error on the measurement of peak maxima retention times were studied (179A). A theoretical derivation of the frequency response in nonequilibrium chromatography was given (170A). A limiting material transport function for a component in a model chromatographic system was derived and characterized by the capacity ratio, theoretical plate height, and sample load (229A). A logarithmic normal distribution curve approximately describes asymmetric chromatographic peaks (479A). Mathematical analysis of displacive chromatography in the presence of a rapid chemical reaction revealed that diffusion caused an asymmetrical front of finite length and form which depended upon various factors (381A). The optimum phase-volume ratio for most chromatography and countercurrent systems was found to be the lowest value of V practicable ( V = Vmot,~l/Vstatlonary) (364A). Reliable retention data can be obtained by frontal chromatography, but without following the breakthrough process in detail (455A). The blurring of bands in the case of random, slightly curved isotherms was studied (605A).The elution of a band a t an alternating average flow rate was analyzed theoretically considering a linear isotherm and kinetic-inhibition and longitudinal-diffusion effects (606A).An equation describing separation criteria for a multicomponent mixture was suggested for taking into account the quality and time of the separation and by choosing separation conditions (593A). Discrepancies between the Purnell equation and Glueckauf chart used in determining the minimum length column required to achieve a desired level of separation were found to arise because the Glueckauf approach does not account for column dead space (524A).An expression has been calculated for the resolving power of nonuniform chromatographic systems which yield non-Gaussian peaks (571A). Relations between peak positions and shapes and column characteristics were studied with a dynamic model of linear chromatography with three dimensionless parameters-namely, retention, axial dispersion, and mass transfer (557A).The peak shapes from and efficiency of preparative chromatographic columns under moderate overloaded conditions could be approximated by Gaussian curves and theoretical plate calculations assuming a linear sorption isotherm (560A). Liquid distribution in packed columns has been studied ( S A , 505A). Chromatographic solvents were classified according to the nature of their hydrogen bonding, acidic, and basic groups, and solvents were chosen for separations of lipids and other water insoluble substances (441A). Preferred liquid phases were selected using factor analysis (569A). The main sources of error in chromatographic data acquisition systems were evaluated (180A, 181A). Full areas of off-chart peaks were calculated using data obtained with the aid of integrators (442A).A new digital comput-
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Gunter Zweig is chief, chemistry branch, Criteria and Evaluation Div , Office of Pesticide Programs, EPA Previously, he was a senior scientist and director of the Life Sciences Div , Syracuse University Research Corp , 196573 Dr Zweig received his BS in chemistry and PhD in biochemistry from the University of Maryland in 1944 and 1952, respectively From 1951 to 1953 he was associated with R J Block at the Boyce Thompson Institute. Yonkers, N Y , and he coauthored with Block and R Le Strange, one of the first books on paper chromatography, ' Paper Chromatography, A Laboratory Manual" (Academic Press, 1952) In subsequent years, Dr Zweig, R J Block, and E L Durrum published two editions of "Paper Chromatography and Paper Electrophoresis " A new book on paper chromatography was published in 1971 by Academic Press and coauthored with Joseph Sherma From 1953-57 Dr Zweig worked as biochemist at the C F Kettering Foundation. Yellow Springs. Ohio, on problems related to photosynthesis and developed techniques of autoradiography and paper chromatography In 1957 he moved to the University of California where he headed up the Pesticide Residue Research Laboratory (later Agricultural Toxicology and Residue Research Laboratory) on the Davis Campus From 1963-64 Dr Zweig was awarded a Rothschild Fellowship and spent the year at the Weizmann Institute in Israel During the past 10 years, he has been active in the fields of analytical techniques and mode-of-action studies of pesticides, particularly herbicides, and has been editor of a seven-volume treatise on 'Analytical Methods of Pesticides, Plant Growth Regulators, and Food Additives" (1963-73, Academic Press) Vols V I and VI1 appeared in 1972/73 with Joseph Sherma as coauthor Dr Zweig has published over 70 articles in his specialty fields and is on the Editorial Boards of Separation Science, "Handbook of Analytical Toxicology," and Critical Reviews /n Analyfical Chemistry He is coeditor of the "Handbook of Chromatography " Dr Zweig is a member of ACS, AAAS (Fellow), New York Academy of Sciences, Sigma Xi, and the American Institute of Chemists
Joseph Sherrna received a BS in chemistry from Upsala College, N J , in 1955 and the PhD in analytical chemistry from Rutgers, the State University of New Jersey in 1958 under the direction of the late William Rieman I l l His thesis research involved development of the technique of solubilization chromatography Professor Sherma joined the faculty of Lafayette College in 1958 and is presently in charge of two courses in analytical chemistry At Lafayette he has continued research in chromatography and has worked for nine summers in this field with Harold Strain, James Fritz, Joseph Touchstone, and Gunter Zweig More than 90 publications and lectures concerned with column, paper, and thin-layer chromatography of metals plant pigments, and other organic compounds, the electrophoresis of metal ions and organic compounds, and the chromatographic analysis of pesticides have resulted from this research Professor Sherma and Gunter Zweig have authored books on paper chromatography (1971) and the analysis of pesticides (1972 and 1973), all three published by Academic Press A two-volume "Handbook of Chromatography' by these authors was published by Chemical Rubber Co in 1972 They have also prepared the last two biennial reviews of chromatography for Analytfcal Chemistry Professor Sherma spent the period from February-July 1972 on sabbatical leave at the EPA Perrine Primate Laboratory, Perrine. Fla , doing research on pesticide residue analysis in collaboration with T M Shafik In 1968 he presented an invited lecture on chromatography at Lafayette in conjunction with receipt of a Jones Award for Superior Teaching, and he received a second Jones Teaching Award ($1,000) in the fall of 1972 Professor Sherma is a member of the ACS, Sigma Xi, and phi Lambda Upsilon and is a Life Fellow of the American Institute of Chemists
ing integrator system automates up to four chromatographs simultaneously (217A). Chromatography systems incorporating the Muelheim computer (462A) and a programmable-gain amplifier for chromatograph-computer coupling (519A) were described. Iterative curve fitting of an eight-parameter function to chromatographic peak profiles by nonlinear residual least squares ( 9 I A ) and digitization errors in the measurement of statistical moments of peaks (9OA) were reported. Recent development trends in data processing in the chromatography laboratory were discussed and a flexible digital integration system and apparatus described and compared with other on- and offline systems (123A). Miscellaneous techniques reported include intervent dilution chromatography for the separation of strongly interacting macromolecules in a dynamic environment in which the association constant is continuously varied (278A), zone-melting chromatography of metal chelates (337A), countercurrent chromatography using a vertical helical tube in a centrifugal field and two liquid phases but no solid phase (242A), bubble column chromatography to study vapor-liquid equilibriums in general multicomponent systems (407A), field flow fractionation for separations of macromolecules (194A), precipitation chromatography (269A), and pulsed hydrodynamic circulation chromatography (97A). Adsorption a n d Partition LC Theory a n d Fundamental Studies. The molecular basis of selectivity in adsorption and molecular sieve chromatography was studied (284A).L. R. Snyder studied the ability of 46 nondonor solvent systems to create differences in the relative migration rates for 30 different nondonor solutes on columns of water-deactivated alumina (494A). The optimum temperature for activation of A1203 for maximum chromatographic activity was found to be 400" for 5 hr (498A). Silica gels were characterized by their pore systems and activities (202A). Calculated retention volumes in several model gradient elution adsorption systems were in close agreement with experimental values (416A). The selectivity of an immobile complexforming phase depended on the concentration of complexing agent (483A). Adsorption on graphite fiber surfac-
es was studied by continuous flow frontal analysis (482A). Practical guidelines for selecting optimum solvent systems in adsorptive LC were discussed in detail; these include the use of the Hildebrandt scale, binary solvent system graphs, and columns with surface activity limited to a single monoenergetic level where reactive hydroxyls are permanently bound (597A). A drop in efficiency by a factor of 50 was observed when metal tubes were used in lace of glass tubes for the chromatography of dyes on 60 silica gel (550A).The surface behavior of aluminum oxides was studied and reproducibility and data were predicted by measurement of the zone length of azobenzene on highly active and gradually deactivated sorbent (143A).Activity scales for adsorbents in column and TLC were established and correlated so that experimental conditions may be determined to give the same activity in the two methods for a given adsorbent (174A). Standardized adsorbents for use in column, dry-column, high pressure, and layer chromatography were described (584A).A series of five papers was written on the theory of chromatography of rigid rodlike macromolecules on hydroxylapatite columns (271A). Extraction chromatography in inorganic analysis has been reviewed (347A). The interfacial tension between the mobile and stationary phases in liquid-liquid chromatography reflects the partition properties of the two phases and can be a criterion for choice of the system to be used (I46A).The effect of adsorption phenomena on the identification of chromatographic zones in partition chromatography (34A) and the effect of the support on separation efficiency of columns in reversed phase partition chromatography were studied (421A). Chromatographic Systems. Column adsorption separations were carried out on the following column packings: single- and double-stranded RNA (257A, 352A) and polypeptides and proteins (36A) in hydroxylapatite, lysozymelike enzymes on chitin .coated cellulose (236A, 237A), proteins on calcium-deficient hydroxylapatite (247A), corrinoids on the nonpolar adsorbent Amberlite XAD-2 (559A), tRNA on benzoylated diethylamino ethyl (BD)-cellulose (572A, 604A), nucleic acid components on synthesized dihydroxyboryl-substituted polymers (463A, 464A), aromatic hydrocarbons on Porapak T (353A), chlorinated
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pesticides on precisely deactivated alumina and silica gel (358A), metal ions on chitin and chitosan (377A), lipids on lipophilic-hydrophobic cellulose and polysaccharide derivatives (4?7A), leaf nucleotides and nucleosides (197A) and cytokinins (40A) on poly(vinylpyrro1idinone) (Polyclar AT), DNA on basic polyamino acid kieselguhr (214A), racemic cyclic polyether on optically pure valine/ Celite (213A), racemic Troeger’s base on cellulose triacetate (216A), amino acid racemates on asymmetric polysulfamide (551A), rat bone marrow cells on glass beads eluted with EDTA (420A), copper on a-hydroxyoxime (166A), phenols on a macroporous polyacrylate resin (167A), metals on cadmium metal loaded columns (172A), phenols on Bentone-34 (286A), hormones on dextran-coated and uncoated charcoal (43A), mRNA on oligothymidylic acidcellulose (13A), microbial DNA strands on poly I-coated kieselguhr (314A), and proteins on (y-aminopropy1)triethoxysilane modified macroporous silica-aminosilochrom (1424). The preparation and properties of synthetic macroporous silica adsorbents (603A),powdered polyamide of required particle size (539A), vacuum-deposited metals on glass or plastic beads (392A), synthetic asymmetric polymers (345A), macroporous silochromes (169A), and alumina modified with alkali metal phosphates (378A) have been described. Applications of partition chromatography in inorganic analysis have been reviewed (33A, 514A). Supports reported for selective immobile liquid phases for metal ion separations include fluoroplast-4 (541A), Microthene-710 (531A),DMCS-treated Chromosorb W (477A), polytrifluorochoroethylene (Daiflon, Voltalef) (29A, 596A), Porapak Q (567A), and cellulose (47A). A scheme for the separation of 19 common cations into groups was devised using six columns in which organic solvents such as BusPo4 and trioctylamine were supported on Daiflon ( 2 A ) .Ionized organic compounds can be separated by ion-pair chromatography (140A).Foam chromatography is a method in which solid foams are supports for phases such as tributyl phosphate (54A), tri-n-octylamine (56A), or chloranil (55A). These columns have been used for rapid, high capacity metal-ion (54A-56A) and dye (332A) separations. Apparatus and Techni ues. Column packings may be graded according to particye size for sharp and reproducible separations by flotation in a stream of gas or liquid (529A).Vacuum packing was superior to conventional column packing for reversed phase chromatography columns (53A). Columns composed of tubing with necks containing filter plugs on both ends were designed for reproducible packing (138A). A simple column pressure applicator (&?A) and a solvent evaporator for collected fractions (157A) were described. Devices for effecting liquid gradients of various types were reported (80A, 250A, 393A, 432A, 566A). Columns packed with aluminosilicates and having coaxial, spiral deflection blades were used to separate hydrocarbon mixtures (132A), and aqueous polymer 2-phase extraction systems in which the two phases move toward each other in columns through alternating mixing and settling chambers, or the lower phase is stationary and the upper phase moves through the chambers, were used to separate nucleic acids and proteins (46A).Centrifugal column chromatography was reviewed (363A) and evaluated for the separation of steroid hormones on silica gel (590A), and an apparatus for centrifugal column preparation and development which can be placed directly in the cuvette of a centrifuge was patented (156A). Microsized glass “dry-column chromatography” columns were patented (27A), and preparative “dry-column chromatography” was carried out on water-deactivated silica gel packed in nylon casing. which was pierced at 1- to 2-cm intervals with disposable glass pipettes to remove samples for analysis (199A). Chromatin was fractionated on a hydroxylapatite column by elution with a thermal gradient (359A). Heftmann reported on the chromatofuge, a centrifugal apparatus for preparative, rapid, high-resolution radial-column chromatography (211A).Automatic fraction collectors (124A, 161A, 266A) and a paper tape control system for programming sample collection (348A) were described. An on-line reaction-colorimetric detection system in which all reactions are carried out in a gas-segmented liquid stream was used to detect carbonyl compounds separated by liquid-liquid chromatography 76R
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(119A). A commercial carbon analyzer consisting of an oven with combustion tube, absorption tubes, a specific IR detector for C02, and a recorder was adapted for monitoring separated sugars (589A).
Gel Chromatography This section will deal with column separations carried out in gels which provide separations by molecular size. The term gel permeation chromatography (GPC) is generally employed when the chromatographic system is composed of an organic solvent and a gel such as cross-linked styrene or porous silica beads, while gel filtration chromatography (GFC) designates an aqueous solvent with a dextran, acrylamide, or agarose gel. Reviews. Theoretical principles of GFC (322A), gel filtration in enzyme chemistry (57A, 298A), gel permeation in polymer fractionation and characterization (32A, 49A, 102A, 492A, 517A), gel filtration with Sephadex (121A), molecular sieve chromatography of proteins (220A), gel chromatography in organic media (178A), general aspects of GPC (72A, 99A, 212A, 361A, 405A), GFC of low molecular weight compounds (562A), and data interpretation techniques in GPC (398A) have been reviewed. A student experiment for determining the molecular weight distribution of polystyrene by GPC on porous silica gel was described (450A). Theory and Fundamental Studies. Dependence of the concentration effect in GPC on the solvent (267A), the effect of the pore size distribution of porous glasses on separation efficiency (IOOA, lOlA), the salt effect in GPC of partially charged polymers (104A), the theory of BakerWilliams precipitation chromatography (336A), theoretical models for peak migration in GPC (74A),a generalized method for correcting instrument spreading in GPC (86A), gel-solute-solvent interactions (96A), chromatography on a porous glass column at pressures up to 3500 kg/ cm2 (41A), molecular-statistical description of macromolecular separations (554A), undesirable peak broadening in GPC (239A, 240A), the magnitude of a finite equilibration effect (570A), the equilibrium theory of GPC (594A), the mechanism of GPC distribution coefficient (544A), the migration behavior of a solute introduced by frontal gel chromatography into a new solvent where it undergoes a kinetically controlled and irreversible isomerization (362A), adsorption effects with poly(viny1 acetate) gels (233A), interactions between proteins and gel matrixes (426A), a model for calculating the effect of concentration on the elution volume in GPC (443A), the effect of pore sizes and the chemical nature of silica gel surfaces on the GPC of polystyrene (19OA), and partitioning in gel chromatography (62A) have been investigated. Resolution per unit volume of GPC packing was increased by decreasing flow rate and sample and increasing column diameter; analysis time was optimized by decreasing column diameter and increasing column length and flow velocity (251A). Elution volumes of polystyrene increased with increased concentration and viscosity because of viscous fingering in columns of cross-linked polystyrene gels eluted with T H F (182A). Polystyrenes with long-chain branching could not be characterized using hydrodynamic volume as the size parameter (400A). Nonlinear fractionation effects in GPC increase when sample loading is increased, molecular weight distribution is narrower, and the column permeability mismatch with molecular size increases (397A).An increase in pore volume of macroporous glass beads increased the efficiency of separation of narrow polystyrene fractions (602A). Evaluation of six different GPC column configurations showed that the columns can be formed into compact shapes with little loss in column performance and that polymer retention volumes were not affected (577A). Equations were derived for direct determination of the instrumental spreading function parameters and their dependence on molecular weight (521A). Improved resolution and linearity were obtained in molecular weight distribution determinations when GPC columns were randomly arranged rather than when arranged in order of permeability levels; large errors were found when calibration curves were not obtained at the same relative concentration as samples (245A).GPC separations can be enhanced by choosing a solvent which can interact differ-
‘
entially with the compounds to be separated (83A). Complex mixtures are analyzed using GPC in combination with reversed phase partition LC (316A). In fast GPC, elution volumes were independent of flow rate and peak width volumes were low a t flow rates 535 ml/min. At high flow rates, the effect of viscous fingering decreased and loss of resolution was less than expected from the Van Deemter equation. Increased temperature decreased peak width and improved resolution (317 A ) . Efficiency increased with reduced column diameter and packing-particle size (395A). Studies of the following in GFC have been reported: determination of the particle size of biological particles with Sepharose 6B (175A), PMR spectra of salts adsorbed on Bio-Gel P-2 (409A), the concentration dependence of elution volumes for dextrans on Sephadex G-100 (380A),the chromatographic behavior of divalent metals on Sephadex G-10 (525A), correlation of differences in confirmation of nucleosides with their behavior on Sephadex LH-20 (518A),relation between adsorption values of aromatic organic compounds on LH720 and the delocalization energies of the compounds (513A), relation between structure and behavior of pyrimidine compounds on Sephadex G-10 (561A), fractionation of polyelectrolytes on Sephadex (507A), macromolecular changes resulting from changes in the environment of the solutes (14A), effects of sorption and molecular size of sugars, glycols, and derivatives of 2-imidazolidinone upon elution from Sephadex (349A), use of Sephadex G-25 to study rapid equilibria (325A),adsorption in a model dextran-hydroxypropyldextran aqueous two-phase system (304A), and hydrophobic retention forces on Sephadex LH-20 ( l 2 5 A ) . Column Packings. Macroporous silica gels have been used to separate polymers and proteins (285A), lipophilic Sephadex for mixtures of lipophilic and hydrophilic compounds (291A) and fatty acids (30A), Sephadex complexed with Cu for amino acids and peptides (294A), Sephadex G-25 for the size and ion exchange separation of indole derivatives (308A), trimethylsilyl derivatives of Sephadex for straight- and reversed-phase separations (523A), silica gel and porous glass for general GPC analyses (526A), styrene-DVB gel impregnated with chelating compounds for metal ions (536A), and acrylate beads esterified with Cz-6 poly01 for proteins (591A).The composition and properties of commercial GPC and GFC gels (113A), interactive gels with a pyridine type functional group (163A), and the basis of separations on various gels (162A) have been reviewed. Granular acrylamide-agarose mixed gels (54OA), vinyl acetate copolymer resin granules (371A), cross-linked copoly(viny1 alcohol-vinyl chloride and -vinylidene chloride) gels (372A),halo hydroxy alkoxy alkyl derivatives of Sephadex (488A), rehydratable agarose gel having hydrocolloid occlusions (428A),porous zincspinel-v-alumina gel (452A),N,N’-methylenediacrylamide copolymers for GFC (249A), a chiral derivative of Sephadex LH-20 ( 9 A ) , various dextran derivatives (343A), hydrophilic ethylene di(meth)acrylate copolymers (313A), and hydroxyalkyl or alkyl ethers of cellulose (137A) have been synthesized for gel chromatography. Apparatus and Techniques. A simplified swelling procedure for Sephadex G-200 involves addition of the gel to isotonic unbuffered saline and boiling for 2-3 min (122A). A Styragel column packing method providing 1100-1230 plates/ft used a liquid balancing mixture of two non-solvent components which did not swell the gel (116A). Enzymes are rapidly purified by gradient sievorptive chromatography, in which combined molecular sieve and adsorption effects are active (277A). Dye-complexed bacterial lipopolysaccharide was proposed as a void volume marker for permeation columns (206A). An internal support of 6-mm siliconized glass beads markedly improved flow rates obtainable with porous Sephadex gels without significant alteration of other properties (448A). The approach to construction of a gel chromatograph and commercial instruments and components were described (141A). A column for preparative scale separations is made up of units, each of which consists of a packed drum with a circular lid and base. Both the lid and base have a series of holes connected by pipes to a central pipe, by which the sample solution enters and leaves the drum and the eluent passes (139A). Alternate pumping recycle gel
permeation chromatography was described (44A).Molecular size distribution of a polymer was determined without viscous fingering by introducing a dilute 2olution onto the GPC column containing the same solvent, in a volume sufficient to produce a rise in polymer concentration in the eluate from 0 to that of the original solution (369A). Sectional acrylic tube columns allow use of different gels separated by a nylon fabric (246A). Nylon was analyzed by GPC using a mixture of solvents (241A). Differential GPC makes use of a reference and separation column for comparison of two polymer solutions; the solvent, a solution of a broad molecular weight distribution, is used in both columns, while the sample is injected in the separation column. The resulting chromatogram reflects only differences between the samples (98A). Simultaneous vertical gel chromatography and horizontal electrophoresis fractionated proteins and mucopolysaccharides (127A). GFC columns were developed while being vibrated (255A). An apparatus was described for automated GPC cleanup of fish and other tissue extracts prior to pesticide residue analysis (533A). IR (357A, 440A), atomic absorption (600A), thermal (599A), radiochemical (311A), UV (81A), and multiple (3A) detectors have been used for gel chromatography in addition to the common refractive index detector. GPC coupled with automatic viscometry characterizes homoand copolymers as to molecular weight distribution and intrinsic viscosity (192A). Computer techniques were developed for analysis of gel chromatograms and evaluation of molecular weight distributions (396A, 425A, 5OOA, 60lA). Various studies of universal calibration in GPC have been made (6A, 107A, 171A, 444A, 537A). A method for calibration and determination of Mark-Kuhn-Houwink constants for polydisperse polymers was proposed (31A). GPC calibration procedures were reviewed (115A), and a computer technique yielding from GPC data both differential and integral molecular weight distributions of macromolecules based on a universal calibration method (52A)was developed. AFFINITY CHROMATOGRAPHY During the past two years, affinity chromatography has become increasingly important for the purification of biological and biomedical substances. Affinity chromatography is a special type of adsorption chromatography in which the column packing has a specific attraction for the compound of interest. Column packings are composed of an insoluble matrix, usually a modified gel, to which one of an interacting pair of compounds ( e . g . , enzyme-inhibitor, hormone-carrier, or antigen-antibody pairs) is attached. The interacting component of the mixture is adsorbed on the column while all other substances will pass through, and it is later eluted by a solvent (buffer) having appropriate pH and or ionic strength or containing a competing material. he column is regenerated by washing with the original solvent. Reviews and General Studies. General reviews of the techniques, applications, merits, and limits of affinity chromatography for the separation of biological macromolecules have appeared (89A, 108A, 155A, 295A, 402A, 427A, 578A). Some solid supports include “spacer- or extension-molecules (arms)” for attachment of the immobile ligand, and preparation of such matrices was studied (386A). Preparation and evaluation of AMP- and NADSepharose (370A),N-hydroxysuccinimide ester derivatives of agarose for immobilization of proteins (11OA), glycopeptide-Sepharose (480A), antibody-coated columns for cell separations (574A), polysine-Sepharoses (576A), and cyclic AMP-Sepharose (530A) for affinity chromatography were described. Affinity chromatography has been performed on gels containing hydrophobic (222A, 481A) and mixed hydrophobic and ionic (595A) groups. Adsorbents and general procedures for purification of steroid-binding macromolecules (330A), phytohemagglutinin (38A), and acetylcholinesterase (439A) were described. In the latter paper, active site stoichiometry was determined by titration measurements. Ligands covalently bound to gels may be quantitatively determined by hydroxamate assay
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(586A) or dissolving of the matrix followed by direct spectral analysis (I49A). Column Packings and Applications. The following column materials and purifications have been reported: p-aminophenylmelibioside-succinoylaminoalkyl agarose for purification of a-galactosidases (346A), immobilized nucleotides for enzymes (327A-329A), N-acetylglucosamine-Sepharose derivative for wheat germ agglutinin (324A), trypsin immobilized on acyl azide derivatives of beaded agarose and porous glass for trypsin inhibitor of soybeans (320A), antibody-Sepharose for avian type C viral reverse transcriptase (328A), polyuridylic acid-Sepharose for mRNA (315A), immobilized ampicillin for penicillinase (3IOA), NADP-agarose hydrazide for glucose-6-phosphate dehydrogenase (303A), sulfanilamideSephadex for carbonic anhydrases (150A), N-methylacridinium derivative-CNBr Sepharose for acetylcholinesterase (135A), dodecylamine-agarose for plasma lipids (l26A), 2’-deoxyuridylic acid-Sepharose for thymidylate (II2A), insulin-agarose derivatives for insulin receptor of liver cell membranes (IOgA), haptoglobin-agarose for hemoglobin (289A), peptide-Sepharose for trypsin (265A), d-tubocurarine-Sepharose for acetylcholinesterase (254A), peptide-agarose for protocollagen proline hydroxylase (35A), aminoacyl-tRNA-bromoacetylamidobutyl-Sepharose for aminoacyl-tRNA synthetases (25A), UDP-agarose derivatives for galactosyltransferase (22A), 2-aminoethyl p-nitrophenyl methylphosphonate-Sepharose for acetylcholinesterase (IIA), vitamin BIZ-Sepharose for vitamin BIZ-binding proteins (5A), methotrexate-agarose for tetrahydrofolate dehydrogenase (87A), affinose-202 for acetylcholinesterase (84A), antibody-Sepharose for tryptophan-containing peptides (580A), serum albumin-Sepharose for ferriporphyrin c-peptide (575A), mitochondrial ATPase inhibitor protein-Sepharose 4B for mitochondrial ATPase (520A), NAD-porous glass for alcohol dehydrogenase (568A), modified GTP-Sepharose hydrazide for dihydroneopterin triphosphate synthestase (244A), c-aminocaproy1.D-tryptophan methyl ester-Sepharose 4B for stem bromelain (50A), penicillin-substituted Sepharose for penicillin-binding proteins (48A), concanavalin A-Sepharose for brain lysosomal aryl sulfatases (45A) and human a l antitrypsin (376A), antidinitrophenyl antibody column for trypsin complex (579A), L. culinaris phytohemagglutinin for glycoproteins (207A), oxidized glutathione derivativeagarose for glutathione reductase (203A), sucrose 1,4-lactone-Sepharose 4B for @-glucuronidase (204A), human IgG-CNBr Sepharose for human complement protein Clq (489A), hexanediamine-Sepharose 2B for cholinergic receptor protein (389A), AMP-Sepharose for enzymes (388A), antiinterferon globulin-Sepharose for mouse interferon (387A), lysine-Sepharose for preparing plasminogenfree fibrinogen (355A), propyllipoamide-glass for lipoamide dehydrogenase (476A), benzamidine-Agarose for bovine thrombin (456A), single-stranded DNA-Agarose for DNA-binding enzymes (454A), Butesin-Sepharose for plasminogen (607A), phosphocellulose for tyrosyl-tRNA synthetase (592A), inhibitor-glass for fungal P-galactosidase (587A), 0-glycosyl polyacrylamide gels for phytohemagglutinins (224A), fatty acid-Agarose for serum albumin (4IOA), cross-linked pectic acid for A . rziger endopolygalacturonase (429A), Cibacronblau-Sephadex for yeast pyruvate kinase (436A), organomercurial-Sepharose for histone fraction F3 (445A), antibody-Sepharose 2B for DNase II (447A), alginic acid-hydrazine Bio-Gel P-20 for alginases (154A), phenylalanine-Sepharose for tRNA ligase (I60A), SBI-CM-cellulose for kallikrein (I68A), amethopterin-Agarose for tetrahydrofolate dehydrogenase (I73A), and linoleyl aminoethyl agarose for soybean lipoxygenase (19IA). The Durification of ovine lutenizine hormone (183A). galactose-binding hemagglutinin (I 76A) and phytoagglutinins and phytotoxin (534A), 1251-labeled lysine-vasopressin ( I 6 5 ) , @-galactosidase (434A), methionyl-tRNA synthetase (433A), tannase (403A), bovine trypsin and thrombin (219A), brain tubulin (2I8A), anti-meningococcal antiserum (582A), mushroom-tyrosinase (198A), thermolysin and neutral metalloendopeptidases (399A), human @-fetoprotein (382A), tyrosine aminotransferase (366A), thiamine-binding protein (356A), acetylcholine receptor (457A), bovine pancreatic and hog spleen deoxy78R
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ribonucleases (453A), and neutral proteinase (499A) by affinity chromatography was reported. High Speed Liquid Chromatography Books and Reviews. The most actively researched area in the field of liqud column chromatography in the past biennium has been high speed (or high pressure, high resolution, high efficiency, or modern) adsorption, partition, and ion-exchange chromatography. A book by Perry on practical HSLC (408A) and one by Brown on biomedical applications (60A) were published. General reviews (129A, 184A, 196A), and reviews emphasizing sampling techniques (261A), optimization of separations (152A), column packings and instrumentation (260A), applications of reversed-phase LC on polymers coated or bonded to Zipax support (458A), and preparative LC (151A) appeared. Supercritical fluid chromatography, a procedure which is quite similar in many respects (including the apparatus which can be used) to HSLC, was reviewed (185A, 461A). Theory and Fundamental Studies. Peak retention, peak dispersion, and methods of improving performance were discussed and comparisons made among common column packings by using reduced plate height us. reduced velocity plots (292A). L. R. Snyder described a simple, rapid procedure requiring no calculations to evaluate the resolution required for a certain separation (495A) and the column length, pressure, and time required to achieve this level of resolution (496A). The influence of column parameters on peak broadening with brush type stationary phases was studied (20IA). Displacement peaks in liquid elution chromatography were studied using a model with computer simulation (497A). Expressions were derived for packed and capillary columns from which the optimum values of column parameters such as length, particle size, and flow velocity leading to the shortest separation times for a fixed pressure drop could be estimated. It was found that the maximum number of theoretical plates which can be generated per unit time is a strong function of the separation factor (493A). Structure-retention relations were studied for some aromatic hydrocarbons on columns of Zipax Permaphase ODS in HzO-methanol mixed solvents (490A). Incremental gradient elution is a HSLC procedure making use of a rational series of solvent mixtures (473A) such that a single solvent change between any consecutive member of the series will result in an approximately constant incremental change in the logarithm of the distribution coefficient of a solute. This solvent system can be used to resolve complex mixtures containing solutes of diverse polarities, thereby increasing the scope of HSLC in a manner similar to the use of programmed temperature GC. Factors affecting resolution and analysis time (475A) and an apparatus for use in this procedure (474A) have been described. The influence of coiling of HETP for AI columns packed with Corasil I supporting ODPN depended upon the column and coil radii (24A). Pressure induced changes in selectivity were noted for methyl and ethyl orange on silica gel (42A). The mechanism of reversed phase LC was studied on natural diamond having a surface similar to common inert bonded hydrocarbon phases (527A). The theory of laminar flow processes in open and packed columns was reviewed and applied to HSLC (120A). Columns packed with porous particles with diameters 0.6 gram li uid/gram support (144A). Different Brockmann activityqevels can be generated reversibly and reproducibly in situ by changing the water content of the organic mobile phase in alumina columns (145-4). The preparation and/or evaluation of the following HSLC column packings was described: bonded monomolecular polymer films on silicic supports (12A),ESi-N= bonded phases on silica gel (64A), a pellicular packing prepared by fusing molecular sieves as 2- to 4-pm layers onto hard core glass beads (360A), a pellicular packing prepared by reacting silica or alumina gel with an organosilane containing a C12-24 aliphatic hydrocarbon radical followed by reaction of the product with a (26-28 alcohol and halogenation under UV light (506A), solid dimeric Rh(T1) acetate deposited on Corasil I1 (365A), polar silicone-based chemically bonded phases (384A), polypeptide bound to resin coated glass beads, Porasil C, and Corasil I1 (195A), a pellicular polyamide (Pellidon) (422A), aluminum oxide type T (423A), and Zipax treated with hydrolyzed y-glycidoxypropyltrimethoxysilane and similar compounds (283A). The reactions of tri- and dimethylsilanes with Porasil porous silica and their role in tailoring chromatographic adsorbents were examined ( I 77A). Techniques a n d Apparatus. Developments in high pressure LC apparatus (35IA, 548A), selection of optimum instrument design criteria (598A), and the different models available from various suppliers of LC chromatographs (85A) have been discussed. Techniques were described for centrifugal packing of silica columns (431A), balanced density slurry packing of 5- and 10-g diameter porous particles (341A, 516A), in situ coating of supports with stationary liquids (282A),and uniformly filling a column under pressure with a suspension of the packing while solvent was passed under pressure simultaneously through an auxillary tube inserted into the column to the bottom (390A). A sampling system allows compressed samples to be transferred into a liquid chromatograph without previous pressure release (300A). A siphon-type flow meter (437A), a noise-free, starting point generator (553A), and an injection port and pressure-release device (79A) for HSLC and a sample injector for supercritical fluid chromatography (39A) were described. A modular design glass capillary column that can be operated a t 3000 psi (504A), a Teflon-lined steel column for use with corrosive liquids (391A), and column connection tubes that restrict the increase in band width (472A)were designed. Solvent pumps and delivery systems (37A, 253A, 262A, 334A, 335A) and automatic 2-chamber gradient-generating systems (71A, 95A) were described. A high pressure micro multiport switching valve allows many different operational modes and does not cause significant band broadening (232A). Small peaks covered completely by large peaks were discovered and determined by recycling fractions (233A).
d
A three-component, two-phase system in which the more polar phase was supported on silica gel provided partition separations of many compounds (215A). Differential chromatography was carried out on a chromatograph with dual, coupled parallel columns and separate eluate UV monitors (470A). Liquid chromatographs were designed (128A, 234A, 323A, 508A) and improved (418A), and modular systems were discussed ( 5 1 A ) .Preparative columns (17A, 585A), a production scale sequential chromatograph (21A),and apparatus for supercritical fluid chromatography (26A, 248A) were described. Computers were coupled to liquid chromatographs in order to analyze large amounts of data and identify chromatographic peaks (92A, 93A). The precision of an on-line coupled computer system was studied (SA). Detectors. The most popular LC detector at the present time is the UV-absorption detector followed by the differential refractive index detector. Much research, however, during the past two years was aimed a t improving the transport or moving wire detector and developing other detector types. Various LC detectors were reviewed and compared (299A, 394A). Double-beam, dual-wavelength UV (258A) and visible (564A) absorption detectors were developed, and the Beckman DU spectrophotometer was modified for use as a LC monitor (424A). Carbohydrates were reacted in the effluent with HzSO4 to produce a UV-absorbing chromophore (268A), and fatty acids were benzylated prior to separation to enhance UV detectability (415A). The use of refractometer detectors was reviewed (563A). The heat of adsorption detector was treated theoretically with the conclusion that it is not viable for LC (471A). Transport detectors consist of a moving wire or chain which picks up a fraction of column effluent and passes to an evaporator to remove the solvent, leaving the eluate as a residue. The residue is then transported either directly after pyrolysis or after pyrolysis followed by reduction (404A) into an FID. An improved FID and conveyor consisting of a steel spring with a twisted steel core moving horizontally around two pulleys provided a sensitivity of ca. 100 ng of lipids chromatographed through a silica gel column (510A). A burner provided with a source of alkali metal vapors (thermionic detector) increased the sensitivity and selectivity of detecting organophosphorus compounds ( 1 9 A ) . A detector increasing productivity by 10fold was developed (306A). A metal gauze rotating disk (134A); a rotating body with metal or quartz collecting surfaces impregnated with catalyst (133A); a wire coated with water glass, clay slurries, or saturated aqueous copper nitrate containing kaolin (419A); and spraying of the column effluent on a wire (543A) were suggested improvements in the transport system. Fluorescent solutes were detected at the low ng level using available commercial fluorimeters equipped with a small volume flow through cell (78A, 512A). New micro spectrofluorimetric detectors were developed (205A, 532A). A UV-detector and scanning fluorimeter were connected in series for analysis of complex mixtures (406A). Fluorigenic labeling techniques developed for TLC were applied to HSLC to increase detection sensitivity of nonfluorescent compounds (164A). Liquid scintillation counting of labeled compounds in effluents was reviewed (549A), a flow cell for measurement of weak 0-activity was designed (486A), heterogeneous and homogeneous counting compared (467A), and a radiochromatograph for simultaneous recording of light absorbance and radioactivity described (535A). Liquid chromatographs were coupled to mass spectrometers for pesticide (326A) and steroid (466A) analyses. Electron capture detection was used for LC effluents (383A). Electrochemical (253A, 288A, 333A, 522A) and conductometric (159A, 528A) detectors were described. Other reported detectors included capacitance (ZOOA, 41 7A, 552A) detectors, interferometers (18A, 208A), a universal mass detector based on the piezoelectric effect (468A), a rapid-scan spectrophotometer for continuous and instantaneous simultaneous recording of curves a t several fixed wavelengths (70A), and a flow through packed reactor for production of colored products of separated solutes (227A). Applications. The following compounds have been sep-
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arated and/or analyzed by high speed adsorption or partition LC: nitro- and chlorophenols (189A), oxindole alkaloids (252A), isomeric arene tricarbonylchromium complexes (188A), phenylthiohydantoin amino acids (187A), organoiron compounds (186A), steroids (82A, 158A, 230A, 305A, 321A, 367A), tRNA (435A), trisulfapyrimidines (413A), plasma oxypurines (411A), metal chelates (23IA), transplutonium elements (226A), phenolic acids (221A), ergot alkaloids (209A), substituted anilines and phenols (491A), pesticides (484A), aflatoxins (478A), DNPH carbonyl derivatives (401A), food constituents and additives (379A, 542A), compounds important in brewing (485A), amino acid metabolites (373A), bitter compounds (368A), vitamin A (547A), tocopherols (545A), complex biological mixtures (469A), isomers and complexes involved in vitamin BIZ synthesis (465A), nucleotides and nucleosides (59A, 61A, 65A, 460A), flavor mixtures (459A),purine and Strychnos alkaloids (588A), morphine and heroin (76A, 587A), a group of fat soluble vitamins (581A), ketazolam (565A),lipids (307A, 509A), acids in wines and fruit juices (502A),tricarboxylic acid cycle intermediates (503A),polycyclic aromatic hydrocarbons (243A, 264A, 290A, 515A, 546A, 558A), equine estrogens (66A), pharmaceutical compounds (16A), fluocinolone acetonide and its acetate (15A), artefacts in frying oils ( I A ) ,phenethylamines of forensic interest (77A), DNPH derivatives of aliphatic carboxylic compounds and glycols (73A), vitamin D (301A), methylprednisolone in milk (302A), glycosphinf,olipids (148A), prostaglandins (136A), lupulone and humulone (131A), lichin extracts ( I I I A ) , volatile N-nitroamines (105A), and sulfa drugs (297a). General applications (350A) and the theory and practice of high speed LC in criminalistics (75A)were reviewed. PLANAR CHROMATOGRAPHY General Considerations As was already observed in our recent reviews on chromatography, the trend in planar chromatography has been toward thin-layer chromatography, as judged by the research papers published during the past two years. Since paper and thin-layer chromatography are being considered under the heading of planar (or flat bed) chromatography, they will be treated in separate sections, but we anticipate that in future reviews the subject will be combined. The general section will deal with recent work applicable to paper and thin-layer chromatography. Books and Reviews. A teaching manual for paper and thin-layer chromatography and electrophoresis has been geared mostly to Shandon (England) equipment (11OB); however, the manual may be more generally applicable. An excellent book on pharmaceutical application of thinlayer and paper chromatography (266B) is being recommended as well as a review of paper and thin-layer chromatography (76B). The 1966-1969 bibliography of paper and thin-layer chromatography has been published (268B).A useful Czech-English-German-Russian vocabulary on chromatography will be helpful to readers of foreign literature on this subject (267B). Papers, ready-foruse plates, and flexible sheets for chromatography have been reviewed (265B). Theory and Fundamental Studies, Factors which affect the variability of Rf values in paper and thin-layer chromatography have been summarized (432B) and rebutted (248B). The relationship of concentration and spot area has been approached from a theoretical standpoint (293B), and factors affecting the accuracy of the determination of purity of radiolabeled pharmaceuticals have been discussed (290B).The resolution of spots by temperature gradient chromatography has been critically reviewed (153B).The effect of ionic equilibrium on the separation of ionizable substances (amino acid derivatives) has been studied on impregnated paper and cellulose powder thin-layers (309B). Similar studies have been conducted with amino acids and peptides (175B). Rhj functions have been studied for use in structural analysis of organic compounds (19B) and protein binding of corticosteroids and androgens (138B). A correlation has been found between structure of pterins and chromatographic behavior on paper and thin-layers (84B).A simple electro80R
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static model exists for the chromatographic behavior of primary metal dithizonates (21B). Apparatus and Techniques. Flat-bed and column chromatographic techniques are critically evaluated for qualitative and quantitative work (202B). The resolution by paper and thin-layer chromatography with temperature gradients follows an equation derived from open column chromatography (154B). A semi-automatic spot applicator for quantitative thinlayer and paper chromatography has been developed (395B),and the precision of different types of micropipets has been studied (104B). Phenols derivatized as p - ( N , N dimethy1amino)benzene-p’-azobenzoateshave been separated on paper and thin layers (71B). Detection and Identification. A simple glass sprayer for thin-layer and paper chromatograms has been developed (500B). Several novel detection systems for radiolabeled compounds on thin-layer and paper chromatograms have been described: spark-chamber radioscanner (433B); liquid scintillation (37B) for low-energy @-emitters; and low-temperature solid scintillation fluorography (372B). Bioautography for antibiotics separated by paper and thin-layer chromatography has been reviewed (27B). A specific method for the detection of threonine and allo-threonine on paper and thin-layer chromatograms has been developed (450B). Several new spray reagents for the following compounds have been described: peroxides (19OB);adenine-containing residues (252B). Quantitative Analysis. Quantitative flat-bed chromatography has been reviewed (269B), and an inexpensive color chart for recording colors of spots in quantitative work has been described (286B).A modification for densitometric estimation of R f values has been proposed (48B). The photographic quantitation of flat-bed chromatograms using various exposure times is described (358B). An automatic spotter for thin-layer and paper chromatograms assures quantitative delivery (42B). Applications. A number of studies have been reported on comparison between chromatography of various solutes by paper and thin-layer chromatography: thioureas, thiazoles, and thiazolines (480B); review of pterins (83B); penicillins (344B); pesticides (149B); dihydrochalcone sweeteners (I47B); separation of Tc(VI1) and M O W ) (336B); chlorite, hypochlorite, chlorate, and perchlorate ions (463B).In most cases, thin-layer chromatography was the preferred method. P a p e r Chromatography Reviews and Books. A book on the paper chromatography of antibiotics appeared in Russian (39B). The past, present, and future development of paper chromatography was reviewed (514B) and the conclusion reached that although there are still many applications for paper chromatography, most new developments take place in thinlayer chromatographic techniques. Theory and Fundamental Studies. Solubility products (I&) of metal ions were predicted by chromatography on stannic acetate-impregnated paper from the observed R, values (380B). Structural analyses of polyfunctional carboxylic acids have been studied by two-dimensional paper chromatography (247B). Structure and R f values of heterocyclic azo dyes have been correlated from paper chromatographic data (17B). The migration of dicarboxylic acids (Cz to C6) and lactic acid, as studied by paper chromatography, followed an empirical relationship based on the ratio of acid front to solvent front (313B, 324B). The separation of metal ions by paper chromatography was found to be temperature-dependent (2898);and reversedphase chromatography was applied to study the distribution coefficient of Zr and Hf (51lB).Amino acids in foods were studied and results compared by paper and ion-exchange chromatography (222B). Chromatographic Systems. A patent has been issued for sorptive sheets suitable for chromatograpic separations, manufactured from styrene-divinylbenzene copolymer and fibers made from cellulose, polypropylene, PVC, or glass fibers (IOOB). Quantitation of reducing carbohydrates following separation by ion-exchange paper chromatography, was accomplished by measuring the amount of tritium found in
NaB3H4-reduced sugars (75B). Resin-loaded papers have been found suitable for the separation of mercury and methyl mercury (246B). Platinum metals and gold have been analyzed qualitatively on paper impregnated with Amberlite LA-1 ion-exchange resin (374B). DEAE paper chromatography was used for the separation of intermediates of the pyrimidine bidsynthetic pathway (18IB). Partition and salting-out chromatography were combined to separate dansyl amino acid derivatives (311B) on finely textured paper (Whatman 20). Lanthanides were separated by circular chromatography on paper impre nated with oxine (318B). The separation of Ni(I1) and Tlh) was studied a t different temperatures on paper impregnated with different amounts of stannic phosphate (378B). Metal ions were separated on paper impregnated with titanium(1V) phosphate (379B) and stannous ferrocyanide ( B I B ) . Carotenoids were chromatographed on papers filled with silica gel and alumina (487B); ascorbic acid and related compounds were resolved by glass fiber paper chromatography (188B). The paper chromatography of metal halide and thiocyanate complexes using liquid anion exchangers has been reviewed (373B). A versatile solvent system for the paper chromatographic separation of inorganic cations consists of chloroform-acetone-isoamyl alcohol-llN HC1 (1:1:1:0.5) (369B). Alkali metal ions have been separated rapidly by centrifugal chromatography with a phenol solvent saturated with 2M HC1 (426B). Several different solvents are described as second solvent in two-dimensional chromatography of amino acids to yield maximum resolution ( I 62B). Apparatus a n d Techniques. Cu(I), Ag(I), and Au(1) ions have been investigated by paper chromatography as their thiourea complexes (276B);12 sugars have been separated as titanium color complexes (387B). Temperature-gradient chromatography on Whatman No. 3 was found to separate Ni(II), Co(II), and Mn(I1) ions, whereas multiple development for conventional paper chromatography had to be applied to achieve equal resolution (155B). Aromatic carboxylic acids were identified by comparing paper chromatograms before and after electrolytic reduction (120B). Guidelines were established for the successful paper chromatographic separation of amino and phenolic acids from biological fluids (397B). A simple Plexiglas apparatus has been described for washing of chromatography papers prior to development (140B). An improved method for the recovery of compounds from paper chromatograms has been devised (I07B). Conical paper chromatography appears to offer advantages over conventional methods (63B). A simple wooden device for holding paper in place for sample applications has been described (18B). Detection and Identification. The detection of weakly W-absorbing spots on paper chromatograms can be improved by placing the paper on top of a thin-layer chromatographic plate made of silica gel F254 (244B). Iodine vapor is useful as a general detection agent for most organic compounds on paper chromatograms (439B). Carbohydrates on paper chromatograms can be detected as fluorescent spots by using ethylenediamine sulfate as spray reagent (187B);citric and aconitic acid can be visualized with fluorescence using a spray of K&O3-acetic acid and UV-radiation at 366 nm (242B). L-Malate has been studied with optical rotary dispersion after elution from paper chromatograms (82B). Cyclic AMP has been determined by paper chromatography, followed by enzymic conversion to AMP, which in turn is re-chromatographed; final measurement is by counting of radioactivit y (339B). A detailed study on the identification of 160 phenols and derivatives in several solvents and using 15 color reagents has been reported (389B). Two-dimensional paper chromatography of plant polyphenols was greatly improved by pre-treating the paper with di-Na EDTA (377B). The fluorescence of keto steroids is greatly enhanced on cellulose (paper chromatography) when using the Zimmermann chromogenic sway, but only weak fluorescence is observed on TLC (61B).
A new spray reagent for Ce, Th, Fe, Mo, Cu, and Au cations is 3-phenyl-4,5,7-trihydroxycoumarin(88B). Neutron activation analysis has been further applied for the detection of S (384B) and metal ions (321B) on paper chromatograms. Quantitative Analysis. Quantitative analysis of sugars has been accomplished by paper chromatographic separation followed by atomic absorption spectrometry of reduced silver (167B) and double reflectance densitometry (292B). Alkali and alkali earth metal ions have been quantitatively determined electrolytically by clamping the developed paper chromatogram between a graphite and mercury electrode (67B). Paper chromatographic methods for the quantitative determination of common metal ions found in soil compare favorably with results obtained from spectrophotometric analyses (236B). The quantitative determination of micro-amounts of tin in fungicides has been reported (44B). Miscellaneous Applications. Dansylamino acids, especially dicarboxylics, cystine, and amides, have been separated by two-dimensional paper chromatography (310B); iodoamino acids have been resolved on paper with aqueous solvents (201B). Cyclic AMP-dependent protein kinase has been assayed by paper dhromatography following the phosphorylation of histone using s2P-ATP(258B). A quick paper chromatographic method for monosaccharides (less than one hour) is especially suitable for students (199B). Intermediates of D-glucuronic acid metabolism have been resolved by paper chromatography (375B). Oxidized pyridine nucleotides (NAD and NADP) have been separated by paper chromatography (400B). Norepinephrine and its five metabolites are separated on cellulose phosphate paper (256B). Isomeric thioureas, thiazoles, and thiazolines have been separated by descending paper chromatography (320B). Reversed-phase paper chromatography has been used to separate free sterolse.g., cholesterol and 7-dehydrocholesterol (297B). Phenols from ground water have been determined by paper chromatography and identified by the reaction with chlorine dioxide (464B). Azo dyes and pigments have been separated by paper chromatography and TLC (142B). An improved solvent for the paper chromatographic separation of polyphosphates consists of 50 g chloroacetic acid and isopropanol-acetone-water-0.25% EDTA-concd NH40H (250:350:300:100:15, V/V) (510B). Zinc dithiophosphates and dialkyl dithiophosphates have been separated by partition paper chromatography using paper impregnated with different nonpolar solvents (423B). Thiourea complexes of Zn, Cd, and Hg(I1) perchlorates, fluoroborates, and trifluoroacetates were repared and studied by paper chromatography using &3thiourea (357B). Li, K, and Na as their chlorides were resolved by paper chromatography (80B). The following inorganic mixtures were resolved by paper chromatography: As(III), Sb(III), Sn(IV), and Mo(V1) ( 6 B ) and arsenatearsenite (451B). Thin-Layer Chromatography Books a n d Reviews. A new book on thin-layer chromatography was published in Romanian (350B) and Randerath’s was translated into French (385B). A monograph for thin-layer chromatography for the analytical laboratory and instruction has been written (146B),and more recently a book on quantitative thin-layer chromatography (474B). Thin-layer chromatography literature abstracts covering the period 1968-71 have been published ( 4 1 0 B ) . Historical aspects and the future of development of paper chromatography have been covered in two reviews (22% 424B). Thin-layer chromatography in pharmacognosy has been reviewed (484B), as well as TLC on polyamide layers (501B)and TLC of alkaloids (485B). Theory a n d Fundamental Studies. A simple model has been proposed between electron donor solvent and silica gel in adsorption chromatography (436B). Advanced concepts in thin-layer chromatography have been discussed (435B). An adsorption isotherm has been derived and its application to TLC demonstrated (512B). The possibility is discussed of using information theory for characterizing TLC separations (281B). A N A L Y T I C A L C H E M I S T R Y , V O L . 46, NO. 5, A P R I L 1974
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The reproducibility of Rf-values in TLC has been reviewed (87B). The non-ideal shape of spots near the solvent front by TLC, so-called “hammer effect,” has been explained (77B).The spreading of zones or spots has been investigated on different adsorbents and attempts made to explain the phenomenon on a theoretical basis (814 325B, 348B, 366B). The influence of temperature and temperature-gradients on resolution of solutes by TLC has been studied (260B, 346B). The chromatographic behavior of amines on ion-exchange thin-layers has been compared with that in column chromatography and similar factors for affinity selectivity have been found (255B). The importance of ionic equilibrium during chromatography has been demonstrated on cellulose thin layers with amino acids (312B).I t has been shown that a relationship exists between molecular structure of neutral organophosphorus compounds and their adsorption affinity on silica gel (240B, 241B). An equation has been derived relating RM values with the composition of a two-component solvent, applicable to high-pressure liquid and thin-layer chromatography (337B). Different corrected R f values are obtained from TLC on silica gel layers with and without the addition of kieselguhr (92B). A simple model of adsorption chromatography has been demonstrated by TLC of solutes with two groups capable of adsorption on silica (157B). The effect of geometric arrangements of the atoms within the molecule (e.g.,&/trans isomerism) on the chromatographic behavior on thin layers has been demonstrated (356B). The correlation between the RM-values in adsorption and partition thin-layer chromatography has been shown to be linear (338B).The effect of vapor phase during TLC on separation efficiency has been thoroughly investigated (26B, 353B, 460B). The dependence has been shown to exist between HETP and amount of substance applied on thin layers with different adsorbents (354B). A detailed study has beep reported on the relationship between structure of coumarins and their chromatographic mobility on different thin layers (148B); similar studies have been performed with methyl-a-D-glucopyranosides (429B). An almost perfect relationship has been shown between RMvalues of penicillins and the logarithm of the partition coefficients (35B). Practical rules for optimization of TLC-systems are based on a relationship between RMvalues and loearithm of mole fraction of Dolar solvent (437B, 438B). Polyamide adsorbents used for TLC have been systematicallv studied. and it was Dossible to detect the mesence of a binder based on a thehetical equation (20B):The influence of adsorbents in thin-layer adsorption chromatography has been reviewed (470B). A mechanism has been proposed for the fractionation of polystyrene polymers on silica-gel thin layers (216B). Additional studies on relating migration on thin-layers to chemical structure have been reported for pesticides (218B), aromatic aldoximes (324B), and polyphosphates (475Bi. The role of statistics is cited in an exploratory program for TLC of organic sulfonates (16B). Sample Preparation. For specific compounds and substrates, the reader is referred to the section on Miscellaneous Applications. However, we want to cite one reference on the sample preparation of amino acids from urine prior to TLC (396B).The sample, after adjustment to pH 2, is chromatographed on ion-exchange resin (AG 50W-X8), the impurities are removed with water, the amino acids eluted with NHIOH, evaporated, and spotted on thinlayer for chromatography. Chromatographic Systems. A substrate tape suitable for fully automatic TLC has been patented (49B). A dispersion of powdered adsorbents mixed with a solution of resinous binder is suitable for aerosol dispension to prepare thin-layer plates (342B). Layer pre-conditioning techniques are described for adsorption TLC (200B). Another patented method for the preparation of thin-layers from an aqueous paste of adsorbent containing a binder, has been described and should become commercially available (105B). Sorbent gradients may find wider application than that reported for the separation of dyes (448B). A study has been made comparing the flow qualit y of thin-layer finished sheets, some of them for rapid Y
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flow (102B); a standardized method has been proposed (498B). Chromafol, a new material for flat-bed chromatography, consisting of divinylbenzene-ethyl-vinylbenzene-styrene copolymer in a cellulose binder, has been used for the separation of 2,4-dinitrobenzyl esters of fatty acids (99B). Silica gel particles ground in a ball mill to a suitably small size are used as adsorbent in TLC simplifying the microchromatography of lipids (455B). Pre-coated TLC plates have been made from silica geland alumina-sintered plates (329B, 331B) and contain no organic binder and thus are very stable and can be used repeatedly after washing (197B). These plates are prepared by mixing silica gel or alumina with lead silicate, soda lime, borosilicate glasses, coating the adsorbent on suitable plates, and heating a t 570-780” (344B). The sintered plates can also be fabricated from a luminescent glass and fluorescent pigment, aiding in the visualization of colorless compounds, like vitamins and alkaloids (333B). Scanning electron microscopic examination of the sintered plates revealed that the silica gel had not melted under the condition of heating (330B). Similarly, sintered rods containing silica gel and alumina as adsorbent have permitted the separation of cholesterol, triglyceride, and ester (332B). Glass fiber sheets have been used to separate cocaine and some metabolites (294B). Glass powder will serve as support for TLC of metal cations (137B). Binder-free cellulose powder TLC and electrophoresis will separate lower peptides and amino acids (316B). Ultrathin silica gel and alumina layers on glass have been investigated as TLC support (135B), and alumina has been used as adsorbent for the TLC of inorganic ions (249B).Laboratory-prepared a-Al203 (231B) and y-Al203, produced by anodization, have been tested as adsorbents for TLC (245B). Silica gel with different microporous structures has been compared for quantitative TLC (497B). Other applications for differently treated silica gel have been reported: cadmium acetate impregnated silica gel for TLC of aromatic amines (513B); silica gel modified with ammonium bisulfate for steroids and lipids (473B);oxalatesilica gel for TLC of 2-hydroxy fatty acids and acyl coenzyme A (486B). Silver nitrate-impregnated TLC has been reviewed (299B) and has been found to stabilize unsaturated compounds during TLC (152B); the technique has also been applied to the separation of polyphenols (467B). The preparation of polyamide plates has been patented (46IB, 477B). The procedure for preparing polyamide layers has been described (392B) and the parameters for porous structure influencing resolution have been studied (417B). A variety of adsorbents have been tested for different applications: Cu(II) modified cellulose layers for TLC of amino acids (213B);barium sulfate for the separation of metal-complex dyes (73B); magnesium hydroxide for the separation of chlorinated pesticides (303B);chitin adsorbent for the separation of nucleic acid derivatives (458B);molecular sieves (95B, 203B); vinyl polymer of the type poly(vinylpyrro1idone) for corticosteroids (15B, 298B). Other adsorbents for the application of specific separations have been described: China clay seems to have many advantages over silica gel (419B);sodium silicate as binder was disclosed in a patent (416B);poly(ethy1enimine)cellulose or PEI-cellulose for the separation of purine and pyrimidine nucleotides from nucleosides and free bases (390B);Dowex 50 ion-exchange resin for water-soluble antibiotics (347B); charcoal for steroids (370B); soil as a chromatographic medium was reviewed (405B).Theoretical and practical aspects of the stationary phase in TLC were reviewed (411B). A number of solvent systems have been described and their special applications: a new eluent for the separation of opium alkaloids (165B); a semi-solid solvent Solvid (471B);dimethylsulfoxide for inorganic ions (427B);a new solvent system for TLC of taurine-conjugated trihydroxy bile acids (236B);acetone-nitric acid as a solvent for inorganic ions (118B);solvent systems for basic dyes ( I I B ) ;a chromatographic system containing salting-out agents for separation of rare earths (14B). The distribution of solvent on thin-layers was studied
of free fatty acids and other lipids (22B); thermofractography by which the sample is heated over a linear temperature range and continuously collecting the condensates on thin-layer plates prior to chromatography (445B,446B); hot-plate chromatography tending to increase rate of evaporation of solvent during development (483B) ; temperature-gradient TLC (185B, 186B);and four-directional development of lipids (362B). A micro-method involving TLC is described to determine the melting point of small amounts of pure materials, which would not be possible by conventional methods (279B). Other novel techniques of TLC have been applied to (221B). specific separations, as for example: instant-TLC of carReady-for-use TLC plates were evaluated for the separation of radioiodides (33B)and heavy metal ions (371B); bofuran insecticide (56B); TLC-screening of urinary amino acids (232B); simplified TLC of DNP-leucine silica gel sheets, films and glass plates were compared for (398B);miniature TLC of phenylthiohydantoin derivathe separation of drugs of toxicological interest (409B). tives of amino acids (440B);sequential TLC of urinary Apparatus a n d Techniques. A number of advances in acidic glycosaminoglycans (192B);argentometric TLC of the automation of sample application, preparation of steryl acetates (193B);duo-zonal TLC of alcohols (488B); thin-layers, solvent development, elution of zones, and TLC of metal dithizonates (101B); and TLC of inorganic qualitative evaluation of TLC have been reported during ions as their diethylthiocarbamate derivatives (382B). the past two years. Detection a n d Identification. A review on qualitative An apparatus has been developed for applying thinTLC has been published (508B). A generalized technique layers on parallel plates for gradient TLC (502B)and for the visualization of organic compounds on thin-layers spreading thin-layers (490B).Several ingenious sample is the carbonization of TLC’s by means of a heating block application devices have been reported or patented-all (278B).A new detector system for chromatograms on adtending to automate TLC and produce more uniform chromatograms for quantitative analyses (209B, 21 7B, sorbent layers inside quartz or Pyrex tubes is based on zone-pyrolysis or combustion (304B,305B). 239B,302B,360B,418B). A promising approach to increasing sensitivity of thinA complete automatic chromatographic system for the layer chromatograms is by means of cylindrical support of analysis of street drugs, but applicable to other separaadsorbents or very thin active layers (367B).The influtions, consists of a “Mylar” chromatographic tape, coated ence of adsorbents on the in situ fluorometric evaluation with adsorbent, a solvent delivery system, spray delivery, of TLC has been studied (355B).Reflectance spectroscopy and densitometry ( 5 B ) . An automated thin-layer applicaA spectrophotometer of TLC’s has been reviewed (123B). tor has been devised for the microanalyses of serum lipids, for direct evaluation of TLC’s has been constructed eliminating deproteinization step (111B). Another system (447B). By treating developed TLC’s with HF, a number for automated TLC has been patented (50B) and is deof organic substances can be detected by infrared specscribed (51B). An apparatus for reversed-phase smalltroscopy (28B). In situ Raman spectroscopy has been utiscale TLC has been developed (85B). Rapid sample preplized for the detection of benzophenone derivatives (2B). aration and spotting technique (114B)and rapid TLC of A simple device is described for the introduction of sugars have been described (106B). thin-layer chromatographic samples into a mass specA sandwich-type thin-layer system has been patented trometer (326B).A polarographic electrode permits the (476B), and a simple adjustable holder for micro-TLC redirect determination of TLC’s without elution ( I 12B). ported (26IB).The movement of small TLC plates and X-ray fluorescence (I89B)and electrical conductivity dethe application of samples from a syringe are synchrotectors have been adapted to TLC (94B). nized, and the scanning device is modified ‘to hold small TLC’s can be recorded on transparent photocopying plates (28723).TLC and gas-liquid chromatography are paper after charring with KzCrz0, or sulfuric acid (43%). coupled using a coated glass tube, corresponding to the TLC plate, and a moving heating element which volatiliSeveral improvements for detection of radiolabled substances on TLC’s have been reported: a device for increaszes the separated compounds for GLC (259B). ing sensitivity (12B);computerized contour mapping of Developing chambers for TLC in a saturated atmotwo-dimensional radiochromatograms (415B); a new scansphere (69B)and rapid equilibration (422B)have been ner (306B);and automatic scraping followed by liquid described. A mechanical device provides a continuous scintillation radioassay (434B3. flow of solvent for development of TLC’s (173B).An apparatus has been patented allowing a constant rate of solNumerous reports have appeared on the detection of specific compounds or classes after chromatographic devent movement (168B). Apparatus for circular TLC velopment. Among the important published techniques (489B)and centrifugal radial chromatography have been are the following: mercurous nitrate spray reagent for a described (253B,254B). Thread chromatography utilizes variety of organic compounds (219B); Pinakryptol Yellow degreased yarn threads wound around a glass frame which on cellulose layers for organic anions (319B);bromine vais irrigated by suitable solvents (251B). An apparatus has been devised to perform simultaneous TLC and electropors for the detection of aromatic diamines (235B);1,1‘phoresis (491B). diazido-4,4’-disulfostilbenefor the detection of aromatic amines and phenolics (466B); Preparative thin-layer chromatography has been the diazotization with 2-naphsubject of several reports and reviews (172B,211B, 224B, tho1 for the detection of primary arylamines (210B). 2MB, 495B, 509B).Microcrystalline cellulose thin-layers Confirmatory tests for aflatoxins separated by TLC have been prepared by adding butanol to cellulose (277B). have been devised (282B,300B),and the fading of fluorescent spots could be prevented by shielding the thinThe elution of resolved compounds from thin-layer chromatograms has been accomplished by a computerlaver d a t e s with another glass d a t e during hieh-intensitv controlled scraper (116B, 117B);a simple device combinirGadikion (322B). ing scraping and vacuum aspiration (452B);a simple Dansyl-amino acids have been identified by TLC on scraping device fashioned from photographic film (462B); three adsorbents using a single solvent (86B);tryptophan or a micro-scale vacuum collector (66B). and other indoles have been visualized with a phosphoric A motorized spraying device permits the uniform appliacid smav and heat (43B):several rare amino acids have cation of detection reagents (444B);an apparatus has been ^detected with’ o-diacetylbenzene spray reagent been patented for the drying and visualization of TLC’s (233B). (352B).TLC’s were copied by treating them with iodine A modified p-anisidine technique has been reported for and contacting with a starch-coated polymer film (307B). the detection and permanent recording of free and combined fructose on paper chromatograms-the method can A number of novel techniques have been developed to improve resolution by TLC. Among these are functional be adapted to TLC (257B).Bile acids and lipids have been detected with dimethyl-POPOP (24B);phospholipchromatography in which the sample is chemically modigradient-thickness TLC fied prior to development (283B); ids on TLC have been analyzed by in situ X-ray emission
(119B)and the effect of ”3, HCl, or water in the laboratory atmosphere on TLC using Silufol thin-layers (see also above) (68B).Several solvent systems were investigated Refor TLC of cardiotonic aglycons and glycosides (46B). versed-phase extraction TLC (55B, 250B) and reversedphase TLC (54B,79B)were systematically investigated. A novel solvent system consisting of the addition of small amounts of NaCl to water was effective in the separation of some cephalosporins (59B).Three-dimensional TLC was used to evaluate spice oils and oleoresins (160B),and chromatography on cellulose strips on a slight incline was capable of resolving compounds with similar R f values
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spectrometry (65B) and with a spray of 8-anilinonaphquantitative technique involves semi-automatic streaking thalene-1-sulfonate(15IB, 182B). of solutes and analyses after elution of developed zones Organosulfur and thiazole compounds can be distin(93B). guished by two spray reagents (31B); thiols and other orMost of the refined methods which have been reported ganosulfur compounds are detected with a spray of are based on direct photometry of resolved spots or zones: PdClz-calcein, liberating fluorescent calcein during the spectrophotometric measurements with fiber optics process (132B).Phenolics are visualized on TLC’s with Nu (403B);linear densitometry (349B,479B);simultaneous cobaltinitrite (29B)and a reagent composed of NaN02transmittance and reflectance measurements (478B); Nu tungstate-trichloroacetic acid (30B).Indoles have flying-spot measurement in the ultraviolet region (158B, been characterized by i n situ fluorescence before and after 159B).Computer applications to quantitative photometric sulfuric acid-treatment (271B). methods of TLC are discussed (363B)and applied to the Organophosphorus pesticides have been detected on analysis of pesticide formulations (238B). TLC’s as fluorescent spots by treating the chromatograms An electrophoresis densitometer has been modified for with PdClz-calcein (34B);1,2-dichloro-4,5-dicyanobenzo- use in quantitative scanning of TLC plates (205B). The quinone (127B);8-hydroxyquinaldine (128B);acidlbase operating principles of a commercial densitometer, Vitatreatment (57B); or by natural fluorescence (274B). Electron TLD 100, are described (230B), and a correction detron acceptor reagents like 2,4,7-trinitro-9-fluoreronehave vice to account for light scattering has been developed been utilized for the detection of pesticides and photode(341B). A double-beam photometer is capable of measurcomposition products (270B,272B).Enzymic inhibition ing the concentration of light absorbing substances on techniques have been developed for the detection of 2,4Reflectance methods (1B, 212B) and TLC plates (505B). D-type herbicides (144B)and organophosphorus insectiother direct photometric techniques for quantitative TLC cides, the latter using indoxylacetate as substrate (45B). analysis have been studied in detail (183B).Direct quanThe movement of pesticides in soil has been studied by titative determination of TLC plates has been accomsoil-TLC (179B)employing an algae bioassay. plished by amperometric measurements (206B) and couIn vitro titration of unconjugated bilirubin has been acpled as chromatography (121B). complished after TLC on Sephadex thin-layers (48IB). Befow are listed some applications for quantitative Electron-acceptor reagents have been used for the detecanalyses of a variety of compounds and ions separated by tion of phenothiazine derivatives separated by TLC TLC: Fatty acids, sugars, steroids, prostaglandins after (115B). Penicillins have been detected with chloroplatinic the introduction of a fluorescent chromophore (431B); amino acids in biological fluids (4B); catecholamine meacid (361B),and the bioautographic detection of monensin and tylosin was improved by first spraying the TLC tabolites and 5hydroxyindoleacetic acid (53B);primary plates with tetrazolium salts (25B). and secondary amines as DANS amide derivatives (413B); Benzophenone derivatives are identified as their 2,4sugars (430B,504B); aflatoxins in cottonseed products (368B); radio-labeled phospholipids (503B); serum lipids dinitrophenylhydrazones (386B). Isoprenoid quinones after by charring technique (234B); serum lecithin (449B); neureversed-phase TLC are visualized if the Kieselguhr G tral lipids (394B)and serum lipids (139B). plates are impregnated with Rhodamine 6G or Nu fluorescein (393B). Other applications of quantitative TLC are: steroids as DANSYL, derivatives (351B);cholesterol by fluorimetry Molybdenum salts have been used for the detection of steroids after TLC (32%) and compared (425B);it was (295B); lipids by photodensitometry and an internal stanfound that the H4SiMo12040-reagent was most sensitive dard (506B); simultaneous determination of phospholipids, free fatty acids and cholesterol, triglycerides and es(425B). Trace steroids present in commercial prednisolone terified cholesterol (141B);gallic acid (122%); polychloriproducts were identified by TLC and mass spectrometry nated biphenyls (PCB’s) (60B);alkaloids (280B):ergot al(74B).Exposure of thin-layer plates to HC1 vapors at 4” kaloids (220B); primary and secondary amines in blood and 40” distinguishes 3-hydroxy-4-ene from 3-hydroxy-5ene steroids (204B). and urine (296B);drug mixtures by scanning of remission Combination TLC and Weisz ring oven technique will peaks (406B);3-indoleacetic acid and serotonin (166B); identify a number of metal and alkali metal ions (169B, tocopherols (IOB); isomeric nitrophenols by polarography 208B, 288B).Heavy metal ions, like Cd, Zn, Cu, Ag, Hg, (161B); Mobam insecticide by reflectance measurements and Fe, have been detected on thin-layer plates by a ure(130B); porphyrins (96B); alkene and hydroxyalkane sultransition metals as thio-6-diketonate comfonates (7B); ase inhibition technique (143B).Thirty-five metal ions have been detected with 4,6-dihyroxy-3’,4’-dimethoxyau- plexes (301B),and Zr(1V)-Hf(1V) mixtures (335B). rone on paper chromatograms, but the technique should Miscellaneous Applications. This section deals with selected applications of TLC, which in the authors’ opinbe applicable to TLC (89B).Metallic tetramethylenediion are significant and may not be covered by Reviews thiocarbamates have been determined by a combination dealing with specific classes of compounds. This section, of TLC and infrared spectroscopy (275B). therefore, is not intended to give the reader a comprehenQuantitative Analysis. This subject has been reviewed sive survey of the literature, which would be beyond the (171B,226B). Errors in quantitative analysis by TLC have scope and space of this article. been discussed (328B).Variations between individual opQuantum yield determination of 3H-labeled tetraphenerators and application techniques are compared (47B). ylbicyclohexenol has been accomplished by TLC and raThe distribution of substances on thin-layers is an impordioassay (98B).TLC in air pollution research has been retant factor in quantitative analyses (414B). A simple inexviewed (404B). pensive applicator for TLC spotting will assure good reAmino acids, amines, and peptides have been analyzed producibility (402B).A critical study has been prepared by TLC: diphenylindonyl-substituted thiohydantoin deon light absorption methods for i n situ quantitative TLC rivatives of amino acids (198B);phenylthiohydantoin deanalysis (126B). Calibration curves from direct fluorimerivatives (194B, 195B); amino acid hydantoins (454B). try or photometry of TLC’s are transferable if internal Amino acid sequence of peptides has been determined by standards are used (227B).Recent advances in photoTLC on ion-exchange resin-coated chromatoplates (399B). densitometric evaluations of TLC’s are reviewed and a Dansyl-leucine and isoleucine have been resolved by TLC newly developed instrument is described (364B).The (108B), while TLC was utilized to separate the diastetheory of quantitative analysis on TLC’s by absorption and fluorescent densitometry has been presented (156B). reoisomers of di- and tripeptides (191B). Peptides with N-terminal histidine have been separated by TLC and Another theoretical study deals with fluorescence techcharacterized by o-phthalaldehyde-induced fluorescence nique (365B).The utility of planimetric determinations (103B). Two-dimensional TLC on cellulose layers comhas been investigated using the TLC system of organic bined with electrophoresis has been applied to the separaacids (229B).Transmittance densitometry is more sensition of synthetic peptides (315B).Primary and secondary tive than reflectance due to the bathochromic shift of 1amines have been chromatographed on paper and thincarvone studied as model compound ( 9 B ) . layers as p - (N,N-dimethylamino) -p’-azobenzamides Detailed experimental procedures are iven for the approximate quantitation of metal ions y TLC (421B). (72B). Aflatoxins in food (8B) and aflatoxins MI and Mz in Quantitative TLC on different adsorbents (499B)and spemilk (223B)have been determined by TLC. Ochratoxins cifically silica gel (472B)has been studied. A simple 84R
A N A L Y T I C A L CHEMISTRY, V O L . 46, N O . 5 , A P R I L 1974
A and B and their esters have been analyzed in barley by TLC (323B). W-Methyltetrahydrofolate-homocysteine transmethylase activity has been assayed by TLC techniques (97B). Nucleic acid enzyme reactions of intact bacterial cells have been assayed by determining reaction products by TLC (228B).Two-dimensional TLC has also been applied to the separation of nucleosides (62B), methylated bases of ribonucleic acid (317 B ) ; acid-soluble nucleosides and bases by one-dimensional TLC (345B);quantitative mapping of oligonucleotides (308B). C1- to Cs-alcohols have been separated by TL,C as 3,5dinitrobenzoates (64B). Mono- and oligosaccharides have been resolved by TLC on cellulose thin-layers (443B).Organic acids of the citric acid cycle have been studied by TLC on silica gel thin-layers, to which MgS04 and NaZC03 have been added (23B). Fatty acids (C1 to C12) have been separated by TLC as corresponding p-nitrobenzyl and p-bromophenacyl esters (91B). Thin-layer chromatography of lipids has been reviewed (420B).Lipid classes have been separated by TLC (134B, 237B, 343B). Bile acids have been separated from neutral lipids by TLC (150B). Cis- and trans isomers of 1,2-benzylidene glyceryl esters have been separated by TLC, which could not be achieved by column or gas chromatography (207B). Triglycerides have been determined on TLC plates coated with silica gel containing ammonium sulfate and rendering the plates fluorescent upon heating and exposing to UV radiation (482B).Mammalian neutral lipids have been separated on glass-fiber paper impregnated with silica gel (359B). Phospholipids have been determined quantitatively by TLC and color development with a modified sulfomolybdic acid reagent (40B). Thin-layer chromatography has been widely used for the detection, assay, and screening of commonly abused drugs (52B, 70B, 113B, 184B, 391B, 401B). Thin-layer chromatography and gas chromatography as aids in forensic medicine have been reviewed (428B), as well as TLC in toxicology (133B). Alkaloid drugs have been assayed and stability has been tested by a TLC-technique (407B). Biological amines in human urine have been determined quantitatively by TLC and fluorescence measurements (457B). Catechol amines and related products have been analyzed by circular TLC as an aid in the diagnosis of neural crest tumors (215B). Over 106 slightly volatile substances of toxicological significance have been studied by TLC (3B).Broadspectrum tetracycline antibiotics have been separated by TLC on EDTA-impregnated cellulose layers (243B). The continued progress in the analysis of pesticides by TLC has resulted in a number of important papers during the past two years. The detection of pesticides on thinlayer chromatograms by enzyme inhibition techniques has been reviewed (291B). The classification of pesticides has been accomplished by the determination of mobilities (453B). Chlorinated pesticides have been analyzed by micro-thin TLC (492B);DDT and related compounds on aluminum oxide chromatoplates (36B);benzene hexachloride by micro-thin TLC (493B); semi-quantitative determination of toxaphene (469B). Tetra- and octachlorodibenzo-p-dioxins, toxic impurities in a number of chlorinated pesticide products, have been separated from PCB’s and chlorinated pesticides by TLC (507B). PolychlorinatLITERATURE CITED Liquid Column Chromatography Aitzetmueller, K , J. Chromafogr, 1973, 83,461-9. Akaza. Ikuko; Tajima, Takatoshi: Kiba, Toshivasu. Bull. Chem. SOC. Jap. 1973, 46(4)., 1199-204. Albaugh. E. W.; Talarico. P. C.. J. Chromatogr. 1972, 74(2), 233-53. Ali, Syed L., Pharm. Ztg. 1973, 118(29). 1139-46. Allen, Robert H.; Mehlman, Carol S., J. Biol. Chem. 1973, 248(10), 3660-80. Ambler. M. R . . J. Polvm. Sci.. Po/vm. Chem. Ed. 1 9 7 3 , 1 1 ( 1 ) , i g l - 2 0 1 . . Amos, R . ; Perry, S. G.. J. Chrornatogr. 1973,83. 254-54. Anderson, D . M. W.; Dea, I . C. M.,
ed biphenyls have been separated from organochlorine pesticides by TLC on alumina plates (388B). Several organophosphorus insecticides have been analyzed by TLC: trichlorphon and dichlorvos by enzymatic detection (109B); azinphosmethyl (l25B) and other organothiophosphorus pesticides by in situ fluorimetry (124B). The separation and identification of carbamate insecticides has been reported on polyamide thin-layers ( l 9 6 B ) . Pesticide mobility in soils has been studied using silty clay loam thin-layer plates ( I 77B, 176%). N-Methyl carbamate insecticides have been separated on thin-layers as fluorescent “dansyl” derivatives ( 229B). Atrazine and linuron residues in environmental samples have been determined by TLC (376B) and carbamate and urea herbicides as fluorescent dansylated derivatives (131B).Acidic herbicides were converted to corresponding nitro derivatives and resolved by TLC ( I 6 3 B ) . Benomyl and other systemic fungicides have been separated and determined by TLC (494B, 496B). A fluorometric method for the TLC determination of thiabendazole derivatives has been developed (327B). Organomercury compounds have been detected on thinlayer chromatograms by the urease inhibition technique (145B). Ethylenethiourea (ETU) has been separated from Zn and Mn ethylenebisdithiocarbamates by TLC 138B). Chlorophyll a and b in plant extracts have been determined by TLC on sucrose thin-layers (262B); algal pigments were separated on sucrose- and silica gel thin-layers (412B). Water-soluble dyes have been separated on polyamide thin-layers (459B);two-dimensional TLC was used for porphyrin methyl esters (408B); pteridines have been determined quantitatively by direct fluorometry on thinlayers (I80B). Preparative TLC was applied to the separation and isolation of isomeric chalcone-flavanone mixtures
(MB). TLC was capable of separating structurally similar estrogenic steroids (78B),and testosterone has been resolved from hydrotestosterone by TLC on silica gel thin layers (264B). Miscellaneous organic compounds have been studied by TLC: resolution of mixtures of isomeric mono- and diarylthioureas, thiazoles, and thiazolines (273B);isomeric cresols and phenols (468B); polyphenols on silver nitrateimpregnated plates (465B); alkyl-, benzo-, and naphthaquinones as mono- and dinitrophenylhydrazones (214B); phthalic esters (41B). Polymers have been separated by TLC: polyethylene fractionation by thin-layer gel premeation (330B); methacrylates (284B);and dextrans (13B). Advances have been reported in the application of TLC to cation and anion inorganic analyses: semi-quantitative determination of 31 cations ( I 7 4 B ) ; cation analysis on microscope slides coated with cellulose powder (58B);separation of Al, Ti, In, Fe, and Ga and detection by alizarin (441B);separation of Cu, Pb, Bi, Cd, Hg, As, Sb, and Sn (442B). Metal chelates of nitrilotriacetic acid and EDTA have been separated by TLC (383B);quaternary ammonium cations of cations (285B). Sulfide, sulfite, sulfate, and thiosulfate ions have been determined by TLC ( I 70B). Hydrogen peroxide has been identified ( I 6 4 B ) . Racemic [C0(en)3]~’ is resolved by TLC on d- quartz plates ( 176B).
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