(93Bi Llealor. D.. Townshend. A.. Anal. Chim. Acta,’39,235 (1967). (94B) hfichalski, E., Gelowa, H., Chem. Anal. (Warsaw), 12, 147 (1967). (95B) hlikhailova, V., Evtimova, B., Bonchev, P., Mzkrochzm. Acta, 1968, 922. (96B) Mottola, H., Freiser, H., ANAL. CHCM., 40, 1266 (1968). (9713) hlottola, H., Haro, &I.,Freiser, H., abzd., p 1263. (98B) hlunnely, T., ibid., p 1494. (99B) Nedved, J., Chem. Listy, 6 2 , 598 (1968). (100B) Ochiai, E., Coord. Chem. Rev., 3, 49 (1968). (101B) Oiwa, K., Kimura, T., Alakino, H., Kinoshita, Y., Bunsekz Kagaku 17, 805 (1968). (102B) Ottaway, J., Fuller, C., Sllan, J., Analyst, 94, 522 (1969). (103B) Pardue, H., Deming, S., ANAL. CHEY.,41, 986 (1969). (104B) Parker, R., Pardue, H., Willis, B., ibzd., 42, 56 (1970). (105B) Pausch, J., Margerum, D., ibid., 41, 226 (1969). (106B) Peshkova, V., Astakhova, E., Dolmanova. I.. Savostina., V.., Acta Chim. Hung., 53, 121 (1967). (107B) Peshkova, V., Dolmanova, I., Bogdanovich, I., Voronina, R., Poddubienko, V., Ushakova, N., Opred. Mikroprimesei, 1, 13 (1968) (cf. Ref. Zh. Khim., 1969, Abstr. No. 3G145). >
>
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1108B) Pooa. G.. Costache. D..’ Rev. Roum. ~‘hik., 12, 963 (1967). (109B) Zbid., 13,573 (1968). (llOB) PoDa, G., Costache, D., An. Univ. Bucarestl. Ber.’ Stiint Natur. Chim.. 17. (111B) Proskuryakova, G., Zh. Anal. Khim., 22,802 (1967). (112B) Rechnitz, G., ANAL.CHEY.,40, 455R (1968). (113B) Rechnitz, G., Accounts Chem. Res., 3, 69 (1970). (114B) Rechnitz. G.. Lin., Z.., ANAL. C H ~ M40, . , 696 (1968). (115B) Rodriguez, P., Pardue, H., ibid., 41, 1369 (1969). (116B) Zbid., p 1376. (117B) Rodziewicz, W., Kwiatkowska, I., Kwiatkowski, E., Chem. Anal. (Warsaw), 14, 55 (1969). (118B) Roginskii, S., Aliev, R., Berman, A., Lokteva, N., Semensko, E., Yanovskii, AI., Dokl. Akad. Nauk. SSSR, 176, 1114 (1967). (119B) Roy, R Al-Jallo, H., ANAL. CHEW,40, 1725 (1968). (120B) Sajo, I., Talanta, 15, 578 (1968). (121B) Schenk, G., Bazzelle, W., ANAL. CHEM.,40, 162 (1968). (122B) Sharipov, R., Songina, O., Zh. Anal. Khim., 21, 800 (1966). (123B) Srinivasan, K., Rechnitz, G., ANAL.CHEY.,40, 1818 (1968). (124B) Zbid., p 1955. (125B) Stewart, J., Lum, P., Amer. Lab., p 91 (November, 1969). ~
(126B) Sykes, A,, Advan. Znorg. Chem. Radiochem.. 10. 153 11967). (127B) Tanaka, AI., Funahashi, S., Shirai, K., Anal. Chim. Acta, 39, 437 (1967). (128B) Tikhonova. L.. Yatsimirskii. Zh. ‘ A n d . Khim.. 23: 1413 11968). (129B) Tockstein,‘ A., Tocksteinova, D., Collect. Czech. Chem. Commun., 34, 1625 ( 1969). (130B) Toren, E., Davis, J., Anal. Lett., 1, 289 (1968). (131B) Toren, E., Eggert, A,, Sherry, A,, Hicks, G., Clin. Chem., 15,811 (1969). (132B) Toren, E., Gnuse, I f . , Anal. Lett., 1, 295 (1968). (133B) Toropova, V., Anisimova, L., Pavlichenko, L., Bankovskii, Y., Zh. Anal. Khzm., 24, 1031 (1969). (134B) Toropova, V., Tamarchenko, L., ibid., 22, 576 (1967). (135B) Tsyganok, L., Chuiko, I’.,Reznik, B., Zav. Lab., 33, 5 (1967). (136B) Weichselbaum, T., Hagerty, J., Mark, H., ANAL.CHmf., 41, 848 (1969). (137B) Weisz, H., Klockow, D., Ludwig, H., Talanta, 16, 921 (1969). (138B) Yachiyo, K., Namiki, AI., Goto, H., Talanta, 13, 1561 (1966). (139B) Yatsimirskii, K., Kalinina, Y., Zh. Anal. Khim., 24, 390 (1969). (140B) Yoshino, Y., Takeuchi, T., Kinoshita, H., Uchida, S., Bull. Chem. SOC. Jap., 41, 763 (1968). (141B) Zeltmann, E., Diss. Abstr. I?, 28, 1815 (1967) (Univ. Microfilms, Ann Arbor, AIich., Order No. 67-13,842).
Chromatography Gunter Zweig and Richard 6. Moore, Life Sciences Division, Syracuse University Research Corporation, Syracuse, N. Y., and Joseph Sherma, Chemistry Department, lafayette College, Easton, Pa.
T
is based upon a selection of important papers published in 1968 and 1969 i n widely accessible journals in the fields of liquid column chromatography (excluding ion exchange), paper chromatography and thin-layer chromatography. The last review article which appeared in this journal in April 1968, covered the litera.ture through December 1967. The amount of published material has again increased compared to the previous period, with about 4000 items pertaining to these areas of chromatography having appeared in Chemical Abstracts during 1968 and 1969. Reflecting this increased activity, the Journal of Chromatography is now published fortnightly, and Chromatographia, an international journal on chromatography and related techniques, began publication in 1968. During the past two years, great activity and major advances were made in the methods, materials, and commercial equipment for liquid column chromatography in order to improve its efficiency and utility, especially for nonvolatile and heat-labile substances which cannot be analyzed by gas chromatography. Increased use is being made of small diameter columns packed with small diameter particles in combination with HIS REVIEW
fast flow rates for the rapid separation and analysis of multicomponent mixtures, especially those of biological interest. Development of universal and selective liquid detectors, hopefully comparable to those available for gas chromatography, continues. The greater importance of liquid chromatography wasindicatedby the change in title of the Journal of Gas Chromatography to the Journal of Chromatographic Science in 1969. The occurrence of two sessions on liquid chromatography a t the 1968 Eastern Analytical Symposium, and the presentation of many papers on liquid chromatography at the Fifth International Symposium on Advances in Chromatography in Las Vegas in January 1969, also gave evidence to the increasing interest in this field. Paper chromatography, much like gas chromatography, is now a standard, developed technique in which only minor advancements and refinements occurred. The use of loaded and modified papers of many types is now widespread. 4ttempts continued t o improve both quantitative paper- and thin-layer chromatography, neither of which is sufficiently accurate or reliable. Much research effort has been directed toward developing fast, selective
separations of all types of compounds by thin-layer chromatography. I n quest of increased resolution, gradient solvent, gradient layer, and vapor programming methods were developed, each a t the expense of the inherent simplicity which is such a n important advantage of normal thin-layer chromatography. Use of commercial preformed layers on either glass plates, plastic, or aluminum sheets has become widespread. The thrust toward a generalization of the theory of separations and the development of a field of “separation science” continues. Two sessions on separation science were held a t the 1969 Eastern Analytical Symposium. Courses are being taught in several colleges throughout the country and the first text book (85)on the subject has been published. We are pleased to record with coiigratulations that Morton Beroza and Julian F. Johnson were the winners of the 1969 and 1970 ACS Awards in Chromatography and Electrophoresis sponsored by Lab-Line Instruments Inc. GENERAL CONSIDERATIONS AND THEORY
New books have appeared introducing chromatography (2, 44),and short bi-
ANALYTICAL CHEMISTRY, VOL. 42, NO. 5, APRIL 1970
349 R
ographies of Runge, Goppelsroeder, and Reed have been published (184). Simple, reliable procedures for the demonstration of all varieties of liquid chromatography, suitable for students and teachers, are achieved with chloroplast pigments (421, 487). Lederer (256) proposes that R, values should be published with only two decimal places along with the solvent composition; other experimental conditions may be stated as a guide, although it is not possible to describe all pertinent, factors accurately. General chromatographic theory continues t'o be developed. Fundamental equations are derived for calculating the extent of separation in binary elution systems (365). General nonequilibrium theory of chromatography is developed for complex laminar flow transport (153) leading to an effective diffusion coefficient describing zone dispersion. This theory is applied (375) to a 2-channel flow model having a stepwise velocity exchange between channels. Chromatographic peaks are characterized as to plate height, shape, and contamination by computer methods (151); and such moments analysis is used for the discernment of low resolution, overlapping peaks (150). Desorption-mass transfer coefficients in chromatographic processes have been determined (378). Affinities of some basic solutes in adsorption Chromatography on silica gel are related to their ionization constant's (405). The relationship between molecular structure and chromatographic parameters continues to be studied (350). Guidelines for systematically optimizing chromatographic operat'ing conditions based upon simulation studies of the behavior of two model polar systems include: small saniple loading, finelydivided supports, development with fast-moving, selective solvents, and migration distances of one-quarter or onehalf the length of the support for single development (435). Thin layer and column separations of phenolic oligomers were improved by pretreatment of the absorbent with solvent vapors (452). The factors to be considered in order to improve the reproducibility of column and thin-layer results are discussed (479). The shape of the adsorption isotherm of water vapor affects the structure and activity of silica gel (342). Two studies (141, 284) of the reliability of various methods for determining the areas of chromatographic curves were made, and calibration factors for the simplified quantitative evaluation of chromatograms have been specified (49)* COLUMN ADSORPTION AND PARTITION CHROMATOGRAPHY
Theory and Fundamental Studies. The theory and experimental techniques of liquid column chromatog350 R
raphy have been reviewed (194, 277), and the theory of partition chromatography has been discussed in a series of papers (447). Equations were derived for calculating optimum conditions for minimum analysis time (177). Lloyd Snyder has studied the theory of optimized solvent programming (401, 403), the efficiency of columns connected in series (249),and the role of dispersion interactions in adsorption chromatography (899). The efficiency of presently attainable columns is given by (400) N Q 2
=
0.34PO.3t0.7dP-Q.3
when NQ2 is the number of effective plates, P is the pressure, t is the time, and d, is the particle size. One study (270) indicates an advantage when the column temperature is controlled, while another (397) finds little advantage in the use of positive and negative temperature programming. Band broadening in different types of packed columns (160) and in the detector and the detector-column joint (159) has been determined. The classical Van Deemter equation must be expanded for application to fast liquid chromatography (469). Computers can be used on-line to acquire chromatographic data and to process the data to evaluate column parameters (466). Knox and coworkers have examined the effect of the column to particle diameter ratio on the dispersion of unsorbed solutes (230), derived general equations for several optimized parameters (231),and compared gas and liquid chromatography on identical columns in terms of reduced plate heights and reduced mobile phase velocities (232). Techniques and Apparatus. A new gradient elution chromatography (36) has dual columns, a 9-chamber solvent programmer, and UV and differential refractometer detectors in series. A splitter inlet system (896),an automatic apparatus for loading a number of samples a t preselected times (435), and other devices (119, 236) for automating column chromatography have been described. Nucleic acids have been fractionated on polylysine-kieselguhr columns (15, 16) and organic acids on urea-Celite columns (62). Rare earths are separated by extraction chromatography on silica gel-HDEHP columns developed with aqueous HC1 (187) and free porphyrins on tributyl phosphate columns developed with a p H gradient of Sorensen buffers (302). Several promising new stationary phases were developed. Brush packings for gas and liquid chromatography consist of a liquid phase which is chemically attached to a silica support (161). Alkyl groups have been anchored to Kieselguhr (418), and various silicones to supports containing free silanol groups (15). Controlled
ANALYTICAL CHEMISTRY, VOL. 42, NO. 5, APRIL 1970
surface porosity column packings as developed by Kirkland yield efficient, fast separations (223-225). Computers may be used to select optimum systems (111) and elution gradients (157). Simultaneous temperature and solvent gradients allow the separation of diasterioisomer salt derivatives (137). New packing methods which minimize particle segregation and uneven compaction produce stable columns with plate heights of the order of 0.1 mm (391). A complete separation of the C1 to C4olefins and paraffins in 19 see was obtained on an Aerogel column, consisting of a short glass tube of 0.016-inch diameter loosely packed with highly dispersed silica brush packing (216). Eluent flow may be accelerated by centrifugation (286). An alumina column with continuously increasing activity is prepared by injecting an appropriate quantity of water with an air flow (189). Problems involved in the application of liquid chromatography to process control have been considered (82). Column and thin-layer chromatography in succession have been used to analyze urinary aldosterone (54). A new fraction collector for large-volume separations consists of a Beckman Model 132 collector with a modified timing system (166).
Detection and Quantitative Analysis. General discussions and evaluations of various liquid detectors (64, 200), and the practical aspects of on-line processing of chromatographic data (167) have appeared. A variety of automatic detectors have been described: a high performance ultraviolet photometric detector (222), a sensitive recording differential refractometer (273), a low volume conductivity recorder (352), flame ionization detectors (55, 271), a thermal conductivity detector with an Araldite-coated thermistor (324), thermal detector with strong ion exchange resins as the detector adsorbent (314). A modified microadsorption detector eliminated negative peaks by continuously adding an adsorbent slurry into the detector cell ($97). An apparatus for the automatic detection of basic compounds in the eluates of CM-cellulose columns is based on the continuous extraction of bromocresol green complexes of the basic compounds in the eluate into chloroform for colorimetric monitoring (283). -4microthermocouple measures the heat of adsorption of individual bands along the axis of a column (290). T-labeled substances in eluents are determined by Cherenkov counting in a liquid scintillation counter with no quench corrections necessary (279). Complex mixtures separated by drycolumn chromatography are analyzed by breaking the column into segments which are inserted directly into a mass
spectrometer (25). A computer program is described (109) for the identification and quantitative analysis of protein amino acids from the output of the integrator attached to an automatic amino acid analyzer. Use of peak heights for the quantitation of amino acids is also accurate and reliable (508). COLUMN GEL CHROMATOGRAPHY
Review articles on t'he history and development (296, 436), theory and mechanics (8, 255), and methods and results of gel chromatography (89, 242, 475) have appeared. Theory and Fundamental Studies. The separation mechanism of gel permeation chromatography was investigated (68) by static experiments. The solute molecules are excluded from part of the inner volume of the gel particles which are entirely available to the solvent molecules; the excluded volume increases with an increase in the molecular size of the solute, and a linear relation exists between the logarithm of the molecular size and the excluded volume; the excluded volume is independent of solute concentration. h d sorption effect's between the solute and the gel matrix are observed in some systems. Other studies of the following have been reported : dependence of separation efficiency on experimental conditions (185), theoretical analysis of zone dispersion (32),correction of instrument spreading by using Fourier analysis (442), effect of flow rate on gel permeation chromat'ography (482), theoretical study (132) of optimum conditions for highest resolution and speed in gel filtration and gel permeation (these conditions are long, narrow columns with fine particles and high pressure drops, temperature and solvents chosen to minimize viscosity, and relatively small pores but large total pow volume), efficiency of gel permeation columns as a function of the pore size of the gel and the elution velocity (292), effect of the crosslinking density of poly(viny1 acetate) gels on separation efficiency (67), relation between the molecular sizes of' acids and alcohols and their elution volumes in gel permeation Chromatography (57), equilibrium theory for the gel chromatography of branched and linear polymer chains ( 5 2 ) , relation between pore size distribution and polymer separation in gel permeation chromatography (51), stochastic niodel for gel permeation chromatography a t high flow rates (50), Gibbs-Donnan effects in chromatography on a Sephadex G-25 column ( S l l ) , atypical gel chroniatography in the system Sephadex LH-20-isopropanol ( S I @ )retention , and separat,ionprocesses operative in columns of porous silica beads (26). Gel permeation and con-
ventional solution chromatography have been compared as methods for determining molecular weight distribution of polymers (380). The gel filtration behavior of inorganic saks has been studied (309,310,372). Techniques a n d Apparatus. Peak broadening is reduced significantly by t h e use of a microrefractometer detection cell and t h e elimination of all excess tubing, including the heat exchanger, in a commercial gel permeation chromatograph (33). An empirical calibration technique for gel (351) permeation chromatography makes it unnecessary to use narrow molecular weight fractions in the determination of the molecular weight distribution of high polymers on a routine basis. Various packings were experimentally evaluated as stationary phases for gel chromatography: porous silica beads ($GO), hydroxyalkoxypropyl derivatives of Sephadex in lipophilic solvents (A$?), oil-insoluble copolymer gels (288), porous glass treated with hexamethyldisilazane to eliminate adsorption (66), 30-300 p beads of cellulose (QO), and surface-bonded silicones (13). The chromatographic properties of Sephadex G-15 are improved by boiling the gel in HC1 for 2 hours prior to packing the column (138). Comparisons of silica gels with different pore structures (162) for the fractionation of polystyrenes and of various gels for the chromatography of polymeric hydrated Fe(II1) (267) have been made. Good results are obtained on a column packed with mixed spherical particles of uniform size but different porosity (291). -1tandem column containing a bottom layer of Xmberlite XE-64 ionexchange resin, an intermediate layer of Bio-Gel P-60, and a top layer of Bio-Gel P-300 was used to purify beef heart cytochrome C (408). A cationexchange column connected in series with a Sephadex G-10 column allows the quantitative determination of anionic detergents (306). The usual liquid detectors are used for gel chromatography. X hydrogen flame detector with a rotating platinum gauze disk iiistead of a moving wire has been designed for gel permeation chromatography (323). Infrared detection of the effluent (66) and direct ultraviolet scanning of the column (45) can be used. Paper chromatographic solvents were used for the partition chromatography of nucleosides and nucleotides on gel columns (247). -1 recycling system with continuous ultraviolet recording of the effluent or a capillary column with a moving wire flame ionization detector was used for the separation of CI9 and CZIsteroids on lipophilic Sephadex (315). Gel filtration can be scaled up for the
purification and preparation of compounds of biological interest (60). It can be advantageous to calculate molecular weight averages from the area under a trace of a gel permeation chromatogram rather than by the conventional count height technique (182). PAPER CHROMATOGRAPHY
X review of current and future trends in paper chromatography (471), a book introducing chromatography on impregnated glass fiber (155), and discussions (149, 158, 407) of the factors which influence the reproducibility of Rf values have appeared. The ascending frontal paper chromatography of cations a t low concentration is explained by an ion exchange retention while deviations a t higher concentrations are explained by a second linear adsorptive mechanism (234, 235). Methods for measuring and controlling the moisture content of paper are presented (43'7). Relations between R , values and molecular structure were determined for two homologous series of pyridine derivatives (404). X twoparameter formula permits R, values in a solvent system to be predicted if the values in a second system are already known (333). Papers have been impregnated with titanic tungstate for the separation of metal ions (363), 2% KC1 for the radial separation of Cu(1) and Cu(I1) (%I), di-n-butyl phosphate for the reversed-phase separation of alkaline earths (79), and p H 8.6 Tris-maleate buffer for the separation of organic iodine compounds (360). Glass fiber sheets impregnated with silver nitrate provide separations of fatty acid methyl esters (142). Diethylaminoethyl cellulose anion-exchange paper was used for the separation of Hg(I1) from many other metals (385) and the separation of nucleoside phosphates (298). Retention of h m ( I I I ) , Ce(III), and La(II1) in reversed phase chromatography is higher on paper treated with a mixture of thenoyltrifluoroacetone (HTTX) and tri-n-octylphosphate (TOP) than on paper treated with either one alone (80). The three metals are separated with 0.05J1 HCI on paper impregnated with 0.05M TOP. Serum 0.15-11 HTT.1 and body fluid proteins were determined by radial chromatography on nitrocellulose membranes (348), and the mechanism of adsorption of the prcteins to the membranes was elucidated (349). Hydrofluoric acid-hydrochloric acid solvents (especially ethan01-40% HF6N HCI, 75:5:20 v/v) (346) and benzyl alcohol solvents (e.g., benzyl alcoholdioxane-8N HC1, 2 : 2 : 1 v/v) (345) give excellent separations of many metal ions on M7hatman Xo. 1 paper.
ANALYTICAL CHEMISTRY, VOL. 42,
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NO. 5, APRIL 1970
351 R
Commercial silica gel loaded papers developed with partition solvents in one or two dimensions provide separations of polar substances such as amino acids and sugars (472). The basic principles of radial chromatography are discussed in terms of dynamic sorption equations (358). The construction and use of a sandwich developing chamber with low solvent requirement for paper and thin-layer radial chromatography is detailed (91). Multiple ascending development with different solvents (four times with methyl ethyl ketone-pyridine-wateracet'ic acid, 70: 15: 15:2, and three times with butanol-acetic acid-water, 4: 1: 1) separates the free amino acids in human blood serum (81). Partition, adsorption, and ion-exchange paper chromatography were performed a t -30 "C by using low freezing solvent mixtures (322), and a new temperature gradient chamber for paper or thinlayer Chromatography producing a gradient of 2'/cm was described and applied to the separation of Xi(I1) and Mn(I1) on Whatman KO.4 paper with butanol saturated with 4N HC1 as the solvent (266). X a n y ions with normally equal R , values were separated by paper strip chromatography in a cylindrical jar around which was wound a copper wire carrying a current to produce a magnetic field (390). Kew detection techniques were as follows: sodium tetraphenylborate-bromophenol blue test for potassium ions (2%); 1% azulene in sulfuric acid as a spray for aromatic aldehydes, furfural derivatives, sugars, dinitrophenyl-hydrazones and azines ( l o g ) , and a series of spray reagents in sequence providing a combination of specific reactions for the staining of peptide maps on Whatman No. 31111 paper (106). Autoradiography continues to be used to locate radioactive spots (360). Major sources of error in quantitative paper chromatography arise from hand spotting small initial spots (machine spotting reduces this error) and the variation in properties of different pieces of paper (corrected by using standard and test solutions on each sheet). Under normal conditions, no further error is produced during the migration of the solute zones (110). Fluoromet'ry was employed for the determination of polynuclear hydrocarbon air pollutants eluted from glass fiber chromatograms (107). Polyaminocarboxylic acids are sprayed after separation with the Folin-Ciocalteau reagent andn determined by densitometry a t 6000 A (251). Polythene-backed paper facilitates the uniform distribution of locat'ing reagents and produces a n increase in the spot color intensity (287). A cooperative, inter-laboratory study (280) showed an error of over i: 10% for the paperchromatographic analysis of mixtures 352 R
of pure amino acids and even greater scattering for hydrolyzates; ion-exchange analysis was more reliable. Silicic acid-glass fiber paper is superior to thin-layer plates for the bioautography of antibiotics (460). The presence of glass fiber in the vials does not cause quenching during the measurement of excised radioactive spots by liquid scintillation counting (142). Proper performance of drying and equilibration procedures are critical for reproducible counting of tritium on paper chromatograms (389). Activation analysis of paper chromatograms, especially involving activation after solvent development, has been reviewed (48). Automated methods have been described for starting the run a t a preselected time (229) and for detection and termination of a descending mobile phase (40). The following automated paper chromatography methods have been reviewed (37): preparation, development, and drying of the paper, direct scanning of the paper, computational procedures and results. THIN-LAYER CHROMATOGRAPHY
An annotated bibliography for the years 1964-68 (181) and reviews of terminology (419) and newer techniques (303, 30.4, 929) (including salting out, exclusion-diffusion, ion exchange, double irrigation, three-directional chromatoelectrophoresis, etc.) have appeared. A theoretical treatment (402) comparing four thin-layer techniques suggests the following order of decreasing general gradient-layer TLC performance : (best), gradient elution TLC, polyzonal TLC and vapor-programmed TLC (worst); none of these techniques can compete with optimized stepwise or gradient elution column liquid adsorption chromatography in terms of speed or resolution and none ever exceeds normal (single solvent) TLC with respect to maximum resolution. -1 review of the application of TLC to analytical toxicology has been published (499)' n'umerous fundamental investigations have been made: the behavior and influence of volatile polar solvent components on several adsorbents in various tyges of developing chambers (456); the preparation and usefulness of commercial precoated plates for standardizing TLC ( 1 G 4 ) ; the role of the solvent vapor (92, 93) (it is concluded that unsaturated chambers can give better separations than saturated chambers with the same reproducibility, and that separations can be improved by vapor not originating from the developing solvent); the influence of humidity variations in the silica gel TLC of hypnotics (94) (Efvalues initially increase with increasing humidity, then
ANALYTICAL CHEMISTRY, VOL. 42, NO, 5, APRIL 1970
decrease a t higher humidity; constant humidity is essential for reproducible TLC); the apparent influence of alumina layer thickness on the R, values of dyes which is found to be attributable rather to the degree of vapor-phase saturation (207); the relationships between the flow rate of one-component (461) and mixed (462) solvents and the viscosity of the solvent and the surface area of the silica layer (4G2) (flow rate decreases with increasing solvent velocity or surface area) ; the dependence of R j values on the structure of the adsorbent with one-component (464) and mixed (4G5) solvents; the influence of changes in the composition of twocomponent solvents during chamber saturation and development, and the influence of chamber saturation on R j values (971); the hydrogen-ion concentration gradients for'med on alginic acid and CM-cellulose layers during development with acids, buffers, and salts (69). The influence of humidity on many different solvent-sorbent systems was studied and the following conclusions reached (370): for oxides and magnesia silicate, increasing humidity results in higher R j values with nonpolar solvents; with polar solvents and these sorbents, humidity has no effect; polyamide, carbon and graphite are not affected by humidity; for cellulose, increasing humidity results in lower R j values. Sorbents can be ordered as follows: silica gel (most adsorptive), magnesium silicate, alumina and kieselguhr; an elutropic series of solvents for silica gel and alumina a t 35y0 relative humidity is hexane, cyclohexane, carbon tetrachloride, benzene, chloroform, ether, et'hyl acetate, and acetone. The separation of isomeric mono- and disubstituted peroxybenzoic acids on polyamide and cellulose layers resulted from hydrogen bonding between bhe solutes and absorbents (219). The free energy of phosphate groups has been calculated from the relations between R , values on cellulose layers and the structural units of condensed phosphates (205). Theoretical studies of a molecular model of adsorption chromatography (40s)have been made. Chromatographic Systems. Many different types of layers have been employed: acetylated cellulose for polycyclic aromatic hydrocarbon air pollutants ( % I ) , silica gel impregnated with silicone for the reversed phase separation of antibiotics (30) and aliphatic polycarboxylic .acids (427), silica gel hipregnated with an electron acceptor (pj:romellitic dianhydride) for the separation of polycyclic hydrocarbons (389); polytetrafluorethylene for the separation of metal ions and organic compounds (364), polyethylenimiiie-cellulose for the two-dimensional separation of nucleic acid components (856), silica gels with different pore sizes (163) for
the fractionation of polystyrenes (both adsorption-partition and exclusion mechanisms were observed), mixed silica gel-cellulose for metals (319) and for the direct analysis of crude plant extracts (441), sintered glass for the separation of dyes and alkaloids ( & I ) , talc for free and esterified porphyrins (478), silver nitrate impregnated silica gel (17, 489, cellulose impregnated with Primene JM-T hydrochloride (a liquid anion exchanger) for the reversed phase separation of metal ions (145, 146), polyamide and cellulose-polyamide for nitrophenols (143) and organophosphorus pesticides (199), zirconium hypophosphate for metal ions (237), magnesium hydroxide for polynuclear hydrocarbons (221), magnesium oxide for plant constituents (217), alginic acid for metal ions (70, 71), silica gel containing 3y0 KOH for fatty acids ( I O S ) , kieselguhr impregnated with paraffin oil for the reversed phase separation of sterols (88),silica gel pretreated with 0.2X NaH2P04 for the two-dimensional separation of fucose and acetylhexosamines (197), silaniaed silica gel for fatty acids (191), silica gel containing 5% ammonium dodecamolybdate or Zn2 Fe(CN)6 for alkali metal ions and containing 5y0 Co2[Fe(CN)6] for di- and trivalent cations (262), silica gel impregnated with sodium acetate, NaH2P04 or Na2HP04 for carbohydrates (254), magnesol (magnesium acid silicate) for flavonoid compounds (252), graphitized carbon black for biphenyl and terphenyls (SIO), and Sephadex for food dyes (326). Comprehensive studies (336, 337) of thin layers of many types on flexible plastic supports have been made. Commercial preformed layers were compared for separations of watersoluble vitamins (356) and chloroplast pigments (387). Quartz and silica gel layers have been compared (5). Polyacrylonitrile, polyacrylamide, and poly(N-acetylacrylamide) were investigated as new thin-layer adsorbents and compared to polyamide (188). Silica gel and alumina layers prepared using an agar-agar binder (272) exhibited improved compactness and mechanical resistance compared to conventionally prepared layers. The preparation and properties of many different kinds of coated glass tubes have been described (259). A statistical analysis was made of the results obtained by TLC of a simple dye mixture on commercial precoated tubes and foils (213). Molten salts can be used as eluents for the TLC of inorganic ions on silica gel ( 1 ) . Homogeneous, aaeotropic mixtures, which are stable over a wide range of temperature and pressure, have been used as solvents for the TLC of psychopharmaceuticals (364). Solubilization thin layer chroma-
tography was originally defined (386) as a procedure fortheseparationof waterinsoluble organic nonelectrolytes by development on layers of strong-acid or strong-base polystyrene ion exchange resins with mixed aqueous organic solvents. The term has since been used (206, 317) to denote a method for separating metal ions by differences in solubility in the developing solvents (e.g., methanol, ethanol, acetone, dioxane-HN03, ethanol-HX03) of the compounds which are formed by the metals with a precipitant (e.g., NazHAs04 or a polysulfide polymer) included in the thin layer (silica gel or silca gel-cellulose). Techniques and Apparatus. Devices for the automatic application of samples as spots (28),streaks (414) and strips (459) have been described. A modified wire loop (369) which has the form of a surface capillary is more reliable for the routine, manual application of aqueous solutions than conventional circular wire loops. A capillary funnel can be used to evaporate solvents of extracts and for directly transferring the residues to the layer (98). Spots from one chromatogram can be dry-transferred to a fresh layer by applying the removed spot t o an adsorbent-free area on the second plate and pressing on the plate with a spatula (429). X thermomicro procedure has been described (411, 412) for separating volatile substances from solid materials and applying the substances directly to a TLC plate; the organic or inorganic mixture is heated in a glass cartridge or ampule with a conical or capillary tip, and the volatilized substances are deposited as a spot on the plate located at the tip. Small amounts of volatile substance can be concentrated by gas liquid chromatography prior t o application on the layer (245). Substances can bechanged toderivatives [e.g., trimethylsilylated carbohydrates, (258)] or degraded [e.g., alkaloids, with UV light, (9)] prior to chromatography to facilitate detection, identification, or analysis. I n addition t o normal ascending development, circular development has been used with layers on glass plates (172) and plastic sheets (480). Twodimensional development separates pesticide residues from sample interferences (220). One-dimensional multiple development with the same solvent (285) or different solvents (275) can improve resolution. Horizontal development with the solvent fed to one edge of the plate by a capillary allows adsorbents without binders to be used (203). Detection and resolution of trace components can be increased by heating the plate during development so that the solvent evaporates from the plate and the concentration increases in the center of the zone (445). A general survey of stationary phase
gradients has appeared (312), and methods for preparing pH-gradient layers have been described (415). A new horizontal chamber for preconditioning the layer with certain vapors in order to produce gradients has been described (128). A similar chamber (95) designed by de Zeeua for his “vapor-programmed TLC” provides vapor control over the entire plate leading (it is claimed) to more efficient separations; Snyder’s theoretical treatment (402) does not indicate the superiority of this technique. Continuous elution is achieved by using a device for warming the top of a TLC plate so that the solvent evaporates (444). Continuous elution and collection of the spots are achieved if a glass capillary is used as a support for the adsorbent layer (451). Ultrathin liquid films (73) and vacuum evaporated thin films (1 p) of metal oxides (72) have provided some very rapid separations. Micro TLC was performed on adsorbent placed in a uniform scratch ( 20.2 mm wide and 0.1 mm deep) on a glass slide (104); the detection reagent is applied as a smear and spots are observed under a microscope. Microscale two-dimensional chromatography was performed on 5 X 5 cm. plates (458). Glass threads (6 p thick) were coated with silica gel and used for the separation of very small samples (289). Plates coated with a small strip of strong-acid ion exchanger in cellulose a t the origin while the rest of the plate is covered with pure cellulose can be used to analyze the amino acids in urine without desalting the sample (241). Plates with a rounded groove to provide a thicker layer of adsorbent a t the origin also allow a sample clean-up process to be carried out as part of the separation (63). Methods for standardizing adsorbents include the determination of R f values of various dyes on prepared plates (154, and the measurement of the viscosity of the adsorbent suspension used for making the plates (463). For preparative TLC, a sample applicator has been designed ( 7 ) , and a sample-loading method has been used in which a narrow (3-mm) channel of adsorbent is removed from the layer. The sample is mixed with the adsorbent, and the mixtures is placed into the channel (470). Preparative chromatographic development can be by continuous descending elution in a tank with an upper solvent reservoir (196). A large-scale 40-cm vapor-programming chamber for preparative TLC has been described (96). Solid silica gel rylinders (813) have been prepared in cellophane tubes for preparative purposes. Detection and Identification of Solutes. Silica gel impurities can interfere in the qualitative identification of organic compounds eluted from T L C
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plates (409). The following chemical methods have been described for making separated spots visible: Tinopal optical brightness as fluorescent contrast indicators for lipids (347), nitroso-R salt for cobalt(II1) amine complexes (156), tetracyanoethylene for aromatic hydrocarbons and substituted aromatics (269), and iodine vapors for free and N-protected peptides (228). The mechanism of detecting various organic substances with fuchsine dyes was studied (394). Seven chromogenic reagents for detecting primary, secondary, and tertiary amines were systematically evaluated (24). Detection and documentation methods for polyamide TLC were reviewed (484). Instrumental methods which have been employed for characterization and identification include: direct spectrophotometry and fluorometry for amino acids, peptides, and nucleic acids (328); mass spectrometry (363) for phenol and aniline derivatives (202) ; fluorescence and phosphorescence for steroids after treatment with sulfuric acid (75); and differential thermal analysis for dicarboxylic acids (274). Free fatty acids have been identified by their spot shapes (105). Separated spots were eluted from the support into KBr powder on the TLC plate with 100-150 p1 of a dry, nonpolar solvent; the KBr powder was added to a KBr microdisk and pelletized for infrared spectroscopy (84). Fractions can be collected directly on KBr for subsequent IR spectral measurement (196). An experiment suitable for students illustrating the identification of separated dyes by reflectance spectroscopy has been published (122). Quantitative Analysis. A book on quantitative thin-layer and paper chromatography has been published (384). A detailed, three-part study of quantitative TLC procedures has appeared (383), as has a review of ultramicro quantitation by TLC (226). Swinny filter holders have been adapted for the collection and elution of samples from TLC plates (307). Visual comparison is still used for quantitative estimation (106, 284). A quantitative method based on the effect of the amount of sample on the increase in H E T P value has been described (443). Reflectance densitometry has been employed for the analysis of amino acids (183), metal ions (123, 473), and pesticides (29). Elution-spectrophotometric (195) and in situ (62) direct densitometric (113, 168, 337) methods continue to be widely used for quantitative analysis. A spot removal method has been compared with densitometry for the determination of metal ions (147). Two-dimensional scanning with a point of monochromatic light followed by off-line data processing by computer
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was used for zones on microscope slides (136). A novel densitometer was used to evaluate the negatives from photographs of chromatograms (53). Bandtype thin-layer chromatograms were evaluated in situ (227). The reproducibility of several colorimetric procedures has been determined (192). Other quantitative methods include: direct fluorometric scanning (204, 330), time-of-flight mass spectrometry (426), ultramicro carbon analysis (440),X-ray emission spectrometry ( 2 6 4 , and ultraviolet remission (201, 377). Radioactive Substances. Sealed disposable plastic bags have been used in place of conventional chambers for the T L C of radioactive substances (153). A review of instruments and procedures for carbon-14 and tritium radioassay by thin-layer and gas chromatography has appeared (398). Solid (359) and liquid (198) scintillation radioassay methods have been discussed. A device for measuring tritium has been described (367). Neutron activation analysis was used to determine selenomethionine (22) and scintillation autography for pyridoxal-l*C and -aH metabolites (65). APPLICATIONS
Acids and Related Substances. A variety of carboxylic acid and several dinitrophenylhydrazones have been separated by thin-layer chromatography (TLC) ( l o o ) , aliphatic polycarboxylic acids by reversed TLC on silica gel impregnated with silicone (427) or treated with acetic acid prior to solvent development (294). Quantitative estimation of free fatty acids has been based on the linear relationship between concentration and spot size after TLC (106) and identification attempted by characteristic spot shape; differential thermal analysis (DTA) has been used for the identification of dicarboxylic acids after TLC on silica gel (274). TLC (27) and two-dimensional paper chromatography (68) of 2,4dinitrophenylhydrazones of keto acid standards and those isolated from urine and sweat have been described; a new spray reagent for a-hydroxy and keto acids separated by paper and thin-layer chromatography is an alcoholic solution of cerric ammonium nitrate (438). Fourteen urinary acids are separated and quantitatively estimated by silica gel column chromatography (321); maleic and fumaric acids are resolved by gel filtration on Sephadex G-10 ( 4 1 ) ; aminoalkylphosphonates have been studied by column chromatography (366). Alkaloids. Opium alkaloids have been separated on silica gel by onedimensional (416) and circular TLC (174); opium and opium preparations have been assayed by column partition
ANALYTICAL CHEMISTRY, VOL. 42, NO. 5, APRIL 1970
chromatography (395). Belladonna alkaloids may be determined by a combination of liquid-liquid extraction and column chromatography on acid siliceous earth (38). LSD and other indole alkaloids are identified after ultraviolet degradation, by TLC on silica gel G (9). Reproducibility for the determination of digitoxin, morphine, and capsaicine has been verified by TLC-colorimetry (191). Amino Acids. An evaluation was made of recent improvements in automatic column chromatographic amino acid analysis which separates asparagine from glutamine without adversely affecting the resolution of other amino acids (535). Another automated method used the dinitrophenpl and dinitrophenylene derivatives to effect separation of tyrosine and lysine (248). Gel filtration on Sephadex G-10 separated phenylalanine, tyrosine, and tryptophan from each other and other amino acids (240). Thin-layer chromatography still remains a popular method of separation of amino acids and peptides. Twentyfive nickel-amino acid complexes were separated on silica gel G plates ( 2 1 4 , while polyamide thin layer plates were used to determine Rf values of eighteen N-benzyloxycarbonyl amino acids and three esters in five solvent systems (467). Cellulose MN-300 thin-layer plates were used for two-dimensional separation of sixty-three amino acids and related compounds (178). The 5-nitro2-pyridyl and 3-nitro-2-pyridyl derivatives of fourteen amino acids were used to determine Rf values in six solvent systems (56). All dansyl derivatives of amino acids were separated by two 2-dimensional chromatographic developments combined with thin-layer electrophoresis (305). Sugars and amino acids were quantitated after two-dimensional paper chromatography with a ninhydrin cadmium acetate reagent (211). The quantitation of amino acids with 1,3-dimethylalloxan had several advantages over ninhydrin, although it did not react with proline or hydroxyproline (367). A mixture of twenty amino acids was separated by circular chromatography after preseparation into neutral, acidic, and basic groups (170). Selenomethionine from animal liver or muscle is determined by paper chromatography followed by neutron activation analysis of the excised spot corresponding to methionine (22). Antibiotics. More than 300 antibacterial preparations were divided into five groups based on chromatography (34). Classification was based on Rf values in different solvent systems. Reversed phase thin-layer chromatography on silica gel G impregnated with silicone DC 200 was used to develop R , values for the sodium, potas-
sium salts, or free acids of twelve penicillins (30). The R, values for these compounds increased in proportion to the acetone concentration in the mobile phase. The use of silicic acid-glass fiber sheets in bioautography assays of gentamicin decreases the amount of antibiotic required, and larger zones of inhibition are obtained. Development time is twice as fast as with thin-layer plates (460). Infrared analysis followed by thin-layer chromatography was utilized to detect degradation products formed a t room temperature in pharmaceuticals containing benzoylperoxide (148). Bases and Amines. This is not a well defined class and the reader should refer to other sections in this article, such as Amino Acids, illkaloids, Drugs, and Miscellaneous. Separation of a new hallucinogen, 4methyl - 4,2 - dimethoxy - CY - methylphenethylamine, is separated by a variety of thin-layer methods from related drugs such as amphetamine, methamphetamine, mescaline, bufotenine, and dimethyltryptamine (129). The application of several reagents useful for detection of imidazoles, indoles, and phenols is applied to detection of histamine and biogenic monoamines following thin-layer chromatography (14). A sensitive method for the quantitative determination of spermine, putrescine, and spermidine following separation on Whatman cellulose CC41 has been described. The amines are visualized with ninhydrin. Identification of seminal fluid by thin-layer chromatography is based on detection of choline and spermine (190). The minimum amount of semen detectable is 1 pl. Thujols and thujolamines have been separated on silica gel G plates and the compounds visualized with saturated phosphomolybdic acid in ethanol (21). Thin-layer chromatography of N , N disubstituted hydroxylamines and stable nitroxides on silica Gel G was examined (474). Spots were detected with 1% potassium permanganate or exposure to iodine vapor. Seven chromogenic reagents for detecting primary, secondary, and terteriary amines on thin-layer Chromatograms have been systematically evaluated (24). Of this group, salicylaldehyde was useful only for detecting primary amines while pdimethylaminobenzaldehyde detected most aromatic amines and a few of the aliphatic amines examined. Bile Acids. Deoxycholic acid and chenodeoxycholic acid have been separated by T L C on silica gel G with isooctane-ethyl acetate-acetic acid and quantified a f t e r elution from the thin-layer plates (419). Bile acids from rat were separated with two solvent systems, chloroform-methanol-acetic acid and benzene-isopropanol-acetic acid also on silica gel G (392). The use
of alkaline solvents was recommended to improve the staining technique with iodine and molybdophosphoric acid (426). Chromatography of conjugated bile acids was carried out on glass fiber paper impregnated with silica gel (12.4). Carbohydrates. The application of thin-layer chromatography t o problems of carbohydrate chemistry, particularly to the separation of monoand disaccharides was the subject of a review (374). A rapid method of identification of urinary sugars employs polycarbonate chromatographic sheets (334). Another method for carbohydrates in biological fluids uses monodimensional separation of sugars by thin-layer chromatography (253). Visualization of sugars on circular thinlayer chromatograms is achieved with an anisaldehyde-sulfuric acid spray reagent (173). A method for detecting reducing and nonreducing sugars in micro amounts uses an aminoguanidinesulfuric acid reagent (278). This method is specific for fucose. Fucose and acetylhexoseamines are also separated by two-dimensional chromatography on thin-layer plates pretreated with 0.2M mono- basic sodium phosphate (197). Galactose, glucose, mannose, and other sugars were separated by this method. Di-, tri-, and tetrasaccharides were separated from their component monosaccharides using silica gel G thin-layer plates impregnated with 0.02M boric acid and double development in one direction (209). Fructose, glucose, and galactose were partitioned by development with the first solvent. A new group of chromogenic reagents for location of 2-deoxy sugars was introduced (373). These reagents are fluorescent and will detect 0.5-5 pg of material depending upon the particular sugar and pH. A combination of fractional precipitation and gel permeation chromatography is claimed to be a more definite analytical tool than either method alone for the molecular weight determination of cellulose esters (39). Trimethylsilylated carbohydrates, such as TMScY,P-D-glucose, methyl ThIS-a and p-Dglucopyranoside, have been examined qualitatively by TLC using silica gel (258).
Carbonyls. 2,4 - Dinitrophenylhydrazone derivatives of aliphatic and aromatic aldehydes were separated on thin-layers of silica gel G, Carbowax 4000, and a small amount of Tinopal WG (36); silica gel SG 41 containing 5% powdered Sn has also been recommended (&V). Aromatic aldehydes have been converted to their respective isonicotinoyl hydrazones and resolved by circular TLC (175). Conjugated aromatic and heterocyclic aldehydes after separation by paper chromatography or TLC on silica gel were detected with 1%azulene (108).
Dyes. Common dyes used in biological staining have been studied by T L C and the impurities detected; this technique is claimed to be much faster than paper chromatography (101). Dyes have been used to standardize the water content in silicic acid used for TLC by a comparison of the R f values (154). Azo derivatives of chromotropic acid have been separated by paper chromatography (393). Similarly, azo dyes used as fluorometric chelating agents have been separated on a Sephadex column (19). Food dyes have been studied by TLC coated with Sephadex G-25 (326). Enzymes, Proteins, and Peptides. The most significant advance in separation of enzymes is by affinity chromatography (77). An inhibitor is covalently bonded to Sepharose. The enzyme which reacts with the inhibitor is selectively separated from other proteins. Affinity chromatography can be used to separate active from inactive staphylococcal nucleases. h single step 4000-fold purification of avidin from egg white was achieved with a biocytinSepharose column (76). Micromolecular interactions were assessed by equilibrium molecular sieving. The binding of an antibody fragment (MW 50,000) to its antigen (R'IW 150,000) was successfully studied (420). An apparatus combining diffusion and filtration of macromolecules through an agar gel plate was described (276). Bacterial impurities in a column packing may produce significant alterations to the substances being separated. A novel fraction collector permits the collection of samples under sterile and apyrogenic conditions (343). Immunochromatographic techniques combine thin-layer and immunodiffusion methods (238) and are designed to correlate precipitin and chromatographic patterns of protein fractions. High resolution techniques of peptide mapping use cellulose and polyamide thin-layer plates. The number of peptides detected from G actin increased 30% over paper methods previously used (130). Serum and body fluid proteins can be separated by chromatography and electrophoresis on nitrocellulose membrane filters (348); however some adsorption of the proteins to the filter due to electrostatic charge does occur (349). Fats and Lipids. h review of separation of lipids, fatty acids, glycerides, and other compounds of biological origin by thin-layer and paper chromatography has appeared (455). Two methods for the microdetermination of brain lipids (168) or blood serum (453) by densitometry following thin-layer chromatography are available. Good separation of phospholipids is achieved on silica gel Hbarium carbonate thin-layer plates (410). Phospholipids from human ser-
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um may be quantitatively estimated after separation into four main classes (476). Estimation of trisaturated glycerides in fats and mixtures of peanut oils is accomplished by argentation thinlayer chromatography (20). Quantitative analysis of radioactive fatty acids is performed by argentation chromatography of their methyl esters on glass fiber sheets (14%’). Quantitative densitometric estimation of glycolipids and phospholipids can be determined by spraying thin-layer plates with 3% copper acetate in 8% phosphoric acid (113). Methyl esters of branchedchain or straight-chain fatty acids may be chromatographed in the presence of urea and the resulting fractions quantitated by gas chromatography (62). Fractionation of neutral lipids and phospholipids by thin-layer chromatography was compared with silicic acid column chromatography (218). There was close agreement between values obtained by the two methods. Thinlayer chromatographic behavior of isomeric long-chain aliphatic compounds was examined using five chromatographic adsorbents and five developing solvents (297). Hormones and Steroids. Paper chromatography of steroids is reviewed (126) including methods and equipment. Another review (266) deals with thin-layer chromatography of sterols and steroids. An improved solvent system consisting of 20% acetone-isopropyl ether resolved four adrenal steroids (263). The precise location of steroids on thin-layer plates is determined by the use of marker dyes (449). This location procedure was demonstrated with urinary steroids. Iodine is used as a nondestructive location agent for several steroids on thinlayer plates; however, three other steroids appeared to be permanently modified (417). Rapid thin-layer separation of 17-keto steroids from urine followed enzymatic hydrolysis of the sample (87). Instant thin-layer chromatography (ITLC) and thin-layer chromatography (TLC) of steroids were compared (127). Steroids were visualized under ultraviolet light with Rhodamine B. No significant difference in chromatographic media was noted. Thirty-seven 1-oxygenated steroids were chromatographed in various solvent systems (376). The 1,11diketones were strongly polar compounds. Migration distances and separation factors were given for ten closely related steroids on silica gel plates (481). Fifteen isomeric triterpenes were separated as their acetate or benzoate derivatives on Arasil B thin-layer plates (116). R j values for these compounds in two solvent systems are given. A group of tetracyclic triterpenes, the cucurbitacins, which are of interest for their antitumor activity were separated 356R
on kiesel gel thin-layer plates (486). Several methods of visualization were used; however, a bioassay was the most sensitive method of detecting activity. Hydrocarbons. Because of the increasing concern of the existence of polynuclear hydrocarbons in polluted air, a variety of novel chromatographic methods have been developed. Thin-layer chromatography on acetylated cellulose powder plates was used to separate 3,Cbenzopyrene, 3,4benzofluoranthene, l12-benzoanthracene, and l12-benzopyrene (381); magnesium hydroxide has been shown to be superior to aluminum oxide as adsorbent for TLC (221); glass fiber paper impregnated with silica gel has been recommended for the separation of phenalen-1-one and 7H-benz (de)anthracen-7-one and visualized with fumes of trifluoroacetic acid (107). The incorporation of an electron acceptor, pyromellitic anhydride, into silica gel thin-layer plates improves the chromatographic separation of polynuclear hydrocarbons (389). Thin-layer chromatography of biphenyl and terphenyl isomers on graphitized carbon black was studied; the resolved compounds were detected by sublimation onto paper impregnated with tetracyanoquinodimethane, forming intensely colored *-complexes (320). Wax mixtures have been analyzed by TLC (362) and tall oil by gel permeation chromatography on Riobead S gel (cross linked nonionic polystyrene) (69). Naphthenes and paraffins have been separated by gas-liquid chromatography on a sodium hydroxide-treated molecular sieve (46). Monomers and Polymers. (This section does not cover Proteins and Peptides, and the reader is referred to t h a t section.) Polysulfones, carboxyterminated polybutadienes, and polystyrenes have been fractionated by gel permeation chromatography (380); poly(viny1 chloride) adsorption onto CaCO3 particles was studied by a similar technique (112). Copolymers of styrene with methyl methacrylate and methyl acrylate were studied for heterogeneity by TLC on silica gel G (216). Polystyrenes with molecular weights ranging from 5000 to 870,000 have been separated by TLC on silica gel with different pore sizes (163). Poly(oxyethylene) glycols have been determined by. reversed-phase partition chromatography with butanol adsorbed on silicone-treated Celite as stationary support and 10% NaCl as mobile phase (239). Vinyl monomers based on undecanoic acid have been studied by TLC on silica gel G or by reversed phase TLC on silicone-impregnated silica gel (246). Natural Products. Flavones and flavonols have been separated by column chromatography on Sephadex LH-20 (212). Flavonoids have been resolved by TLC on Magnesol (magne-
ANALYTICAL CHEMISTRY, VOL. 42, NO. 5, APRIL 1970
sium acid silicate) and visualized by ultraviolet scanning before and after spraying with 20% Na&03 (262). Thin-layer chromatographic patterns of extraneural gangliosides are complex and only serve as a guide to distinguish them from brain gangliosides (362). Scilla glycosides, separated by TLC, have been determined quantitatively by direct densitometry, fluorimetry, and UV transmission using Kieselgel G with fluorescein for TLC (2&). Pesticides. Two reviews have appeared describing the qualitative and quantitative analyses of pesticides by thin-layer chromatography (193) and the chromatography of alkylating agents (116). Two-dimensional TLC on alumina-coated plates (220) and silica gel G (6) will resolve 11 different organochlorine pesticides. This method is also suitable for samples which have not been thoroughly cleaned up prior to chromatography (220). Pesticide mobility has been determined on thin layers of soils (186) and alumina (484). High-speed liquid-liquid chromatography in less than 60 sec has been reported (223). Infrared spectroscopy in combination with TLC has been successfully applied for the identification of D D T and related compounds (4). Organochlorine insecticides may be removed quantitatively from thin-layer plates with hot benzene (301). By the incorporation of rancid vegetable oil in the adsorbent coating of thin layers, the interference of fatty impurities during the TLC of chlorinated pesticides is suppressed, presumably by the reaction with hydrogen peroxide from the rancid oil (296). Thin-layer chromatography of organophosphorus insecticides has been accomplished on silica gel G (260), polyamides (199,468), AlzOa and magnesium silicate (131). Organophosphate insecticides from river waters have been determined by TLC and gel chromatography (Sephadex LH-20) and gasliquid chromatography (12). Paper chromatography (117) and thin-layer chromatography have been used to identify a wide variety of organophosphorus insecticides from biological materials (118). For the detection of organophosphorus insecticides on TLC plates, the following systems have been used, based on the inhibition of cholinesterase by these insecticides: Indophenyl acetate(substrate) and bee brain cholinesterase (477); B-bromoindoxylacetate and beef liver homogenate (282). Semiquantitative analysis of dimethoate residues in foods has been accomplished by TLC (3). High speed liquid-liquid chromatography with controlled-surface porosity-supports (b,P’-oxydipropionitrile) and hexane as carrier will resolve E P N insecticide (224). Substituted urea herbicides have been resolved by liquid-liquid chromatography and detected a t a sensitivity of
10 ng by ultraviolet photometry (222). Two-dimensional T L C has been applied to the separation of uracil herbicides (457). Triazine herbicides were determined quantitatively by reflectance and absorption spectrophotometry after T L C (121). Hydriodic acid has been used as a detection reagent for several classes of pesticides on T L C plates: carbamates, thiocarbamates, substituted ureas, and nitrogen-containing organophosphorus insecticides (11). The fungicide Botran (2,6-dichloro4-iiitraniline) and its metabolites are visualized on silica gel thin-plates by spraying with an electron acceptor such as chloranil or 2,4,7-trinitro-9-fluorenone with a sensitivity of about 5 pg; the same method is applicable to other substituted anilines and phenols (202). Pharmaceuticals. (see also Antibiotics). A collaborative study for reserpine in tablet formulations is based on the chromatographic elution from a Celite column followed by spectrophotometric analysis (23). Common barbiturates have been analyzed by TLC on cellulose plates using a sodium sulfate-mercurous nitrate color reagent a t a sensitivity of about 0.4 pg (78). Other drugs which have been analyzed by thin-layer chromatography include imidazoline derivatives (134), appetite depressants, e.g. Preludin, Ritulin (325), anticonvulsant drugs (341), and acetanilide-based analgesics (135). Phenols. Two-dimensional T L C of plant phenols has been described and the subsequent i n situ fluorometric analysis; the adsorbent was cellulose powder-silica gel ($04). Isomeric monosubstituted phenols have been separated by paper chromatography and visualized with ceric ammonium nitrate reagent (15 2 ) . An exhaustive study on the separation of phenols and phenol carboxylic acids by TLC on cellulose has been reported (99). Nitrophenols have been separated on polyamidetreated thin-layers (143) or on silica gel G-amylopectin (430). Phenolic and heterocyclic fluorescent compounds have been studied by gel filtration chromatography on Sephadex G-15 (86). Pigments. Although most papers which were reviewed deal with naturally occurring pigments, one paper dealt with the isolation and classification of pigments from liquid and dry inks by chromatography and their identification by infrared spectroscopy (281) . A detailed study on comparative chromatography of the chloroplast pigments is recommended reading to choose between numerous techniques such as column with various adsorbents, paper, and thin-layer (482). Thin-layer chromatography is the preferred technique for chloroplast pigments (382), chlorophyll and carotenoids from marine algae (210) and seawater (126). Free
porphyrin and esters from urine have been determined by T L C on talc as adsorbent (478). Free porphyrins have also been separated by extraction chromatography on columns of tributyl phosphate (302). Porphyrin esters have been purified by gel filtration on Sephadex LH-20 (18). Free bile pigments have been separated by ascending T L C on polyamide gel, while the esters have been partially resolved on silica gel G thin-layer plates (338).
Purines, Pyrimidines, RNA, and DNA. A variety of supports have been used to separate purines and pyrimidines. Column packings for liquid chromatography include Sephadex G-25, G-15, and G-10 (139), and Dowex I (261) and Biogel P-4 (847). Thin-layer supports include Whatman CC41 Chromedia (257), polyethylenimine-Cellulose MN300 (355), and cellulose (61). A review dealing with the use of cellulose for the separation of nucleotides and their derivatives has been published (327). Separation of nucleoside phosphates on diethylaminoethyl cellulose paper has been successfully accomplished ($98). Substances labeled with 32P in effluents from liquid chromatography columns can be detected by Cherenkov counting (279). Chemical quenching is eliminated by Cherenkov counting and no quench corrections are necessary. Mixtures of RNA and DNA have been separated by polylysine kieselguhr chromatography (15,16). This method gives partial separation of different genetic markers on the Escherichia coli B chromosome, rat liver and calf thymus DNA in a highly polymerized form has been purified with agarosegel filtration (868). Cytoplasmic and mitochondrial transfer RNA's show chromatographic differences which can be demonstrated by reversed phase chromatography (43, 361). Toxins. Several improved chromatographic procedures for the analysis of aflatoxins have been described. Thin-layer plates coated with silica gel and used within 2 hours after activation a t 110 "C gave improved resolution for aflatoxins BI, B2, G1, and G2 (388). The addition of water to the solvent system also improved the T L C resolution of the toxins (423). Aflatoxin Bl in cottonseed meals was concentrated by column chromatography on Celite, followed by T L C (454). A new detecting reagent for aflatoxins was 2,4-dinitrophenylhydrazine in H2S04 and ethanol and a sensitivity of 0.5 pg was claimed (74). Vitamins. Methods which can be used to successfully extract and chromatograph the members of the vitamin A and vitamin D families have been reviewed (85). Separation methods for the tocopherols in animal tissues were also the subject of a recent
review (31). An improved method for the quantitative determination of atocopherol and coenzyme Q in rat liver involves the saponification of tissue lipids, thin-layer chromatography, extraction of the coenzyme Q and a-tocopherol from the T L C plates, and colorimetric assay of each compound (10). Chromatography of vitamin A compounds on multibore silicic acid columns employing gradient elution gave good resolution of the applied compounds (486). Microchromatographic determination of vitamin C in fruit and vegetable extracts was achieved on paper strips (140). Reversed phase chromatography was used to separate vitamins D Pand DI and related compounds (102). This system separates ergosterol from 7-dehydrocholesterol and cholesterol. Good separation and recovery of ergocalciferol from related compounds was achieved on alumina GF thin-layer plates (344). Separation of an organocobalt derivative of vitamin B12 by cellulose thin-layer chromatography results in faster and better resolution than paper methods (114). R, values in the thin-layer system are very sensitive to changes in the organic ligand. Circular thin-layer chromatography has been used for the semiquantitative estimation of water soluble vitamins (172). Silica gel was the chromatographic adsorbent. Miscellaneous Applications. Substituted indanols have bnen resolved by reversed-phase T L C on cellulose impregnated with ethyl oleate (144). N ,N-Dibutylacetamide and N ,N-dibutylbutyramide have been resolved by TLC on silica gel and detected with a successive spray of dinitrophenylhydrazine, followed by iodine (448); .V-substituted maleimides and N-(chloropheny1)succinimides have been separated on silica gel thin-layers (97). Polynitro aromatic compounds have been determined quantitatively by T L C on silica gel G and spectrophotometric analysis after elution of the resolved spots (195). Anionic detergents have been analyzed by liquid chromatography on Sephadex G-10 followed by a cation exchange column for the determination in equivalents per gram (306). Urea, thiourea, dicyanodiamide, biuret, and nitroguanidine have been separated by automatic liquid chromatography and detected by thermal energy (314). Qualitative identification of the reaction products following hydrogenation of quinoline has been accomplished by liquid-solid column chromatography (4011'
Simple indole compounds have been chromatographed on silica gel thinlayers containing a fluorescent indicator; detection was by Ehrlich reagent (p-dimethylaminobenzaldehyde) (340). a-Tocopheryl acetate in feed supple-
ANALYTICAL CHEMISTRY, VOL. 42. NO, 5, APRIL 1970
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ments can be separated from naturally occurring tocopherols by T L C and saponification, followed by a final separation by thin-layer chromatography on silica gel GF2s4; the final product is eluted and determined colorimetrically (300). Commercial food emulsifiers (Span) can be identified by T L C (244). Organosilicon compounds have been studied by T L C (120, 446) and silicon polymers by paper chromatography (293). A simplified paper chromatographic method was described for mass screening of blood and urine samples of newborn infants for the detection of inborn errors of metabolism (424). Inorganic Ions. Thin-layer chromatography was t h e most widely reported technique for the separation of inorganic cations, anions, and metal complexes. EDTA complexes were separated on silica gel H plates (428, 450). Other metal complexes which were separated by TLC included ethylenediamine acetic acid, dimethylethylenediamine, and diethylethylenediamine (428). The cis- and trans-isomers of Co ethylenediamine were separated on thinlayer microcrystalline cellulose (179) and silica gel (156). T L C and column chromatography of anions on microcrystalline cellulose was reported (180). Alkali metal polyiodides have been separated by TLC on silica gel (208). Circular T L C has been used for the separation of Co, Cu, and Ni ( l 7 6 ) , noble metals (169), and a number of anions (171). Sc, Y, the rare earths, Th, and U(1V) have been resolved on thin-layers of cellulose (318) and silica gel-cellulose (319). Boron as boric acid was determined in caviar by T L C (47). A new thin-layer adsorbent for the separation of metal ions is Primene (246). JM-T (long-chain amine) Other adsorbents for T L C of inorganic ions are zirconium hypophosphate (237), and alginic acid (70, 71). Silica gel H R containing 5y0 ammonium dodecamolybdate has been recommended for the separation of alkali metal ions; cobalt ferrocyanide-silica gel HR is suitable for di-and trivalent cations (262). Precipitation thin-layer chromatography has been applied for the separation of Ni, Cd, Cu, Pb, Fe(III), and Bi by the incorporation of monobasic sodium arsenate into the silica gel adsorbent (206). -4 similar principle was applied by impregnating a polysulfide polymer into the silica gelcellulose adsorbent (317 ) . Quantitative estimation by reflectance spectroscopy after TLC has been reported for Cu, Ni, and Zn (123). Paper chromatography has been applied for the separation of Cu(1) and reflectance densitometry Cu(I1) (%?I), of Fe, Mn, Zn, anti Cu in plant material (473),and fifty-two cations on paper im358 R
pregnated with titanic tungstate (353). Several solvent systems have been studied for the paper chromatography of inorganic ions, HF-HC1 (346) and benzyl alcohol-HC1 (345). Reversedphase chromatography on paper treated with a mixture of thenoylfluoroacetone and tri-n-octylphosphate has been shown to separate Am(III), Ce(III), and La(II1) with dilute HC1 as mobile solvent (80); with di-n-butyl phosphate as stationary phase, the alkaline earth ions could be separated with HC1 (79). Paper chromatography at low temperatures of -30 “ C was studied for Bi(II1) (322) and temperature gradient for Ni(I1) and Mn(I1) (266). -4new detecting reagent for potassium was reported (233); activation analysis of paper chromatograms containing various metal ions and anions has been reviewed (48). Gel filtration of anions (309, 310), extraction chromatography of rare earth ions on silica gel columns (187), and automatic ion-exchange-extraction chromatography of cations from biological materials (368) have been reported. Sulfite in biological fluids was reacted with 14C-Ilr-ethylmaleimide and the resultant adduct purified by paper chromatography and electrophoresis (299). LITERATURE CITED
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Electron Microscopy R. M . Fisher and A. Szirmae, Edgar C. Bain laboratory for Fundamental Research, United States Steel Corporation, Research Center, Monroeville, Pa., and I, H . McAlear, Catholic University, Washington, D. C.
T
HE ELECTRON MICROSCOPE is almost universally used in support of research into the relations between structure and properties of nonbiological materials and between morphology and function in the case of biological materials so that literally thousands of papers relating to electron microscopy (EM) appear each year. For example, during the current review period 1968-69, more than 200 papers involving EM instrumentation or utilization appeared in just four leading national and international journals which deal largely with experimental physics. The overburdened general electron microscopist trying to keep track of this flood of literature may be disappointed (or relieved) to learn that this somewhat superficial coverage is intended for beginning students or managers with some responsibility for electron microscopy rather than practicing specialists. Most of the aspects of EM discussed in the following paragraphs were already of some importance two years ago and were included in the 1968-69 review. For this reason the reader may find it helpful to refer to that article for additional information on some topics as well as for more general comments on electron microscopy which served to introduce the various sections of the previous review.
362 R
LITERATURE OF ELECTRON MICROSCOPY
As in 1968, major contributions to the literature of electron microscopy are listed separately with the references under the categories of journals, conference proceedings, and new books. This list is the result of a diligent, but by no means exhaustive, check of our own libraries and the publishers’ indices. We apologize to authors and to our readers for the inevitable but nevertheless regrettable omissions. The journals cited are those containing a considerable amount of material of interest to most microscopists, but a substantial fraction of the important papers on electron optics, instrumentation image theory, and techniques appear in the various national journals on “Applied Physics.” Specific applications of electron microscopy will, of course, be found in the various chemical, biological, solid state physics, and metallurgical journals. The Proceedings of EM meetings are commonly in the form of two-page extended abstracts and largely serve as advance notices of research to be published in detail elsewhere. With the exception of the list of journals, references given previously are not repeated here but it must be pointed out that the 1968 book list includes some of the most
ANALYTICAL CHEMISTRY, VOL. 42, NO. 5, APRIL 1970
important titles in the available literature of electron microscopy. Specific references are excluded from this review to avoid questions of credit and priority, especially as we believe few readers would rely on this source for such information. Where further details of a topic are required, we urge our readers to scan the conference proceedings and meeting reports to identify individuals active in the various fields. INSTRUMENTATION
Commercial Instruments and Accessories. The manufacture (and sale) of electron microscopes is a n extremely competitive business so t h a t new or improved models now appear on the market quite regularly. The usual target dates for unveiling new models are t h e occasion of the EMSA, European Regional, or International meetings although the actual availability of the new models frequently lags far behind. It does not seem appropriate to make specific references to the various commercial instruments. It is also rather futile as the readers can glean more detailed and more current information by scanning the advertisements in the scientific journals. Furthermore, a call or card to any manufacturer or his
