arnrned Thin-layer or Improved okus A. d e Zeeuw Laboratory for Pharmaceu tical and Analytical Chemistry, State University, Antonius Deusinglaan 2, Groningen, The ~ e t h e r ~ a ~ ~ s
To obtain full benefit of the influence of vapor in TLC a new dewelopment chamber has been designed. This chamber provides full vapor control over the entire late. ~ p t i ~conditions ~ m can be established by vapor ~ r o g r a m m i n gand the migration rate of each individual spot can be guided. Thus, more efficient separations are obtained, particularly of chemically related substances which are inadequately separated with the classical TLC t chniques. The properties of the v a ~ o r - p r o g ~ a m m i nchamber g are discussed and results are shown in separations of dyes and sulfonam ides.
RECENT work ( I , 2) on the influence of solvent vapor in thin layer chromatography has shown that if development of the plate is done in unsaturated chambers then separations with multicomponent solvents are more efficient than in saturated chambers. 'The improvements are caused by a concentration gradient of adsorbed vapor on the dry part of the plate, with the gradient mainly dependent on the rate of evaporation of the solvent components and on their affinity for the adsorbent used. It will be clear that little or no control can be exercised on the extent to which the gradient develops on the plate after development is started. With highly polar solvent components the gradient may become too steep on the lower parts of the plate, with the upper parts showing no gradient at all because maximum vapor adsorption will take place here. With less polar solvent components the gradient may be too flat or will not reach its maximum because of fast development. Furthermore, differences in evaporation velocity between the solvent components may cause failures in the desired gradient. Thus, although unsaturated chambers may well yield improved separations the conditions are not necessarily optimum for every case. Therefore, we have searched for development techniques providing a more efficient control of the vapor processes during development. This has led to the apparatus described below, which allows full vapor programming over the entire plate, thus making it possible to affect the migration of each individual spot. APPARATUS
Description. The vapor-programming chamber (Figure 1), VP-chamber (patents applied for, soon obtainable from C. Desaga, Heidelberg), consists of three parts, all of chromium plated brass. The solvent reservoir, A , is a rectangular tank 20 x 1 x 2 cm. The ground plate, B, 20 X 20 X 1 cm, is fitted with a solvent reservoir holder and a tube to pass warm water, D , insulated from the ground plate by 2 mm of asbestos and 4 fixation clamps, E. The inner part of the ground plate contains a tube system for water-thermostating, with the inlet and outlet visible at F. The trough chamber, C, contains 21 troughs, Ld. 6 rnm and depth 12 mm. The partition walls have a thickness of 2 mm, the
(I) R. A. de Zeeuw, J. Chromatogr., 32, 43 (1968). (2) R.A. de Zeeuw, ANAL.CHEM., 40,915 (1968). 28 34
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side walls are 5 mm. The chamber has been made of welded flat oval tubes, with the tops sawed off. A space of about 1 mm is needed between the trough chamber and the solvent reservoir to prevent disappearance of solvent by capillary action between the walls. Working Procedure. The samples are spotted on normal 20- x 20-cm TLC plates, 2-2.5 cm from the bottom edge of the plate and at least 1 cm apart. The side edges and the bottom edge are stripped 0.5 cm wide. The troughs are filled with a series of liquids of appropriate compositions to give suitable vapors. Most often the liquids show increasing polarity from bottom to top. This can be done, for example, by using mixtures of two or more solvent components of different polarities, with the mixtures having an increased proportion of the more polar components. The empty solvent reservoir is placed in the holder and fitted with a folded strip of filter paper, 18.5 X 2 cm, with I crn folded over the inner wall. The side walls of the chamber are fitted with small strips, about 0.5 mm thick. The TLC plate is then placed on the chamber, the adsorbent downward, facing the troughs, the stripped side edges resting on the side strips, and the bottom edge just covering the solvent reservoir. The side strips should prevent the adsorbent layer from touching the walls and the thickness should be adapted lo that of the layer. The solvent reservoir is then filled with about 25 ml of the appropriate solvent, which is subsequently sucked up by the filter paper strip and thus brought in contact with the plate. The solvent reservoir and the filter paper are pressed smoothly to the plate by means of two springy metal strips underneath the reservoir. This ensures good contact between the plate and the paper strip. Figure 2 shows the VP-chamber at work, with development in process horizontally. The use of the VP-chamber permits vapor adsorption by the adsorbent from the underlying troughs. Thus, by filling the troughs with suitable liquid mixtures the vapor conditions can be programmed over the entire plate and optimal vapor gradients can be obtained because every desired polarity can be applied to the various parts of the plate via the vapor phase. Materials. All parts of the VP-chamber are of chromium plated brass to ensure sufficient thermoconductivity. The troughs are made of flat-Qval tubes, ranging from 6 mm to 17 mm in diameter with walls 1 mm thick. The tops are sawed off and a trough has a depth of about 12 mm. The troughs are kept together by side walls of 5 rnm and by welds along the partition walls. The thickness of the TLC glass plates may vary between 1 and 4 mm. Sagging plates or sheets or insufficiently flat plates can not be used. Teflon (DuPont) or gasket plate are suitable materials for the side strips. Teflon is inert to most organic solvents but is somewhat slippery to use. Both are obtainable in several thicknesses and have good flexibility. Thickness of the Side Strips. The thickness of the side strips plays an important role in the applicability of a certain
Figure 1. The vapor-programming chamber in parts A , solvent reservoir; B, ground plate; C,trough chamber. The ground plate is equipped with a warm water tube (D),fixation clamps (E), and an internal tube system for water-thermostating, with the inlet and outlet visible at F
Figure 2. The vapor-programming chamber at work The solvent makes the adsorbent transparent in relation to the underlying troughs and the filter paper strip becomes visible
gradient. It will be clear that because of the differences in liquid compositions in the troughs, strong vapor diffusion will occur if the space between the trough walls and the adsorbent is too large. These diffusions may well cause anomalies in the direction of solvent flow making the separation worthless. On the other hand the strips can not be too thin because the adsorbent must not touch the walls. If so, development stops immediately and the spots readily diffuse along the walls. It should be noted that most adsorbents swell more or less when wetted by the solvent. The rate of swelling is dependent on the polarity of the solvent and the thickness of the strips should be adapted to this phenomenon. In our experiments with layers of 0.25 mm good results were obtained with strips of 0.3-0.5 mm for solvents like hexane, benzene, chloroform, and ether, 0.5-0.8 mm for acetone, ethylacetate, and lower alcohols, whereas ammonia-containing solvents needed strips o f 1 mm. Saturation. To be sure that reproducible quantities of vapor are available for adsorption, it proved to be necessary to equilibrate the plate over the filled troughs for 10 minutes after it was fixed in position. During this period the small volumes over the troughs become almost saturated, resulting in a fixed amount of vapor available for adsorption. Not until after this saturation period is the solvent reservoir filled and development started. The duration of the saturation period was experimentally established by comparing the reproducibility of the separations. If highly volatile components are used, the saturation period can be reduced to 5 minutes. Temperature. It will be clear that constancy of room temperature, within =k 1 "C, is required to obtain reproducible separations. However, in vapor-programmed TLC it was
found that with higher room temperatures-over 22 "Cpoorer separations were obtained in comparison with results at lower temperatures because the migration rates of the spots decreased markedly at higher temperatures. Presumably this is caused by too much adsorption of vapor which is then followed by condensation on the plate. Thus, solvent transport from the solvent reservoir will be diminished and, consequently, the migration rate of the spots will decrease. Hence, cooling of the VP-chamber is necessary in those cases and we therefore have equipped the ground plate with an internal tube system to be linked with a water bath thermostat having a lower temperature than the ambient room temperature. The temperature of the cooling system, if kept constant, thus enables good control of the vapor adsorption rate. In our investigations at average room temperatures of 21 rt 1 "C optimal separations wereo btained with cooling temperatures between 18-20 "C. Continuous Development. Very careful development is needed to separate closely related substances of almost identical polarity. In those cases a solvent with low polarity and a flat vapor gradient, especially on the lower parts of the plate, should be used. However, this implies a slow migration of the substances, with the spots still migrating at the lower parts of the plate when the solvent front has already reached the top of the plate. To overcome this difficulty the VPchamber can be used for continuous development. The top end of the plate (1 cm) is not accompanied by an underlying trough, but lies 0.5 cm over a warm water tube. The solvent reaching the top will evaporate, thus allowing continuous solvent transport over the plate. With solvents of low volatility the warm water tube should be kept at about 50 "C. With highly volatile solvents, warming is not necessary for evaporation is fast enough. VOL. 40, NO. 14, DECEMBER 1968
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When publishing chromatographic data obtained with continuous development, the length of a run can no longer be stated. In our opinion the development time should be given or a colored reference substance should be used, stating the required distance to be moved by this reference. The optimal development time or the optimal distance to be migrated by the reference substance can be established when searching for optimal vapor gradients. Furthermore the term R, value requires a special definition for continuous development. We prefer to define the Rf values for continuous development as the distance moved by the substance divided by the distance of the starting point to the top of the plate. The Construction of the Vapor Gradients. In vaporprogrammed TLC one should use a running solvent of rather low polarity, giving the substances under investigation a small migration rate. The solvent may be single or multicomponent. The troughs underneath the starting pointsi.e. the first two troughs-are filled with liquids having the same composition as the running solvent. Trough 3 is then filled with a liquid having a slightly higher polarity than the running solvent, trough 4 is filled with again a slightly higher polar liquid than in trough 3, etc. This can be done for example by using mixtures of two solvents, one polar, one nonpolar, with the mixtures having an increased percentage of the polar component. Thus, a stepwise vapor gradient would be obtained which can be compared to the continuous gradient in the unsaturated N-chamber. However, one severe difficulty arises. Migration of the upper parts of a solute spot will be accelerated sooner than the lower parts and tailing will consequently result. Fortunately, this can be avoided by interspersing troughs with liquids of low polarity between the troughs containing the more polar mixtures, thus having a decelerating effect on the migration of the spots, particularly on the upper parts. If, for example, separation is required of chemically related sulfonamides ether can be used as the running solvent. With ether the substances should have R, values between 0.05 and 0.25. Troughs 1 and 2, the former underneath the starting points, are also filled with ether. Trough 3 is then filled with ether-methanol 80:20, the methanol having an accelerating effect, troughs 4 and 5 are filled with ether (deceleration), trough 6 with ether-methanol SO :50, troughs 7 and 8 with ether, trough 9 with ether-methanol 20:80, etc. It can be seen that the polarity difference between the accelerating and decelerating troughs increases when going upward but this must be done to avoid tailing. In some instances it may be necessary to use three or more deceleration troughs after one acceleration trough. We do not know how the process of deceleration and acceleration works in actuality ; however, with the process compact spots can be obtained, without decreasing the improved separation efficiency. We presume the improvements not only to be caused by the acceleration forces but also by a more or less selective deceleration, with the less polar substances decelerated to a higher extent than the more polar substances. It should be observed that the accelerating-decelerating stepwise gradient obtained in the VP-chamber is basically different from what is called gradient elution. The latter has a pushing effect on the spots, suitable for separations of substances showing great polarity differences. In the VPchamber a pulling gradient is obtained which is more suitable for separations of closely related substances with small polarity variations. e
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EXPERIMENTAL Solvents and Standards. All solvent components were reagent grade (E. Merck). Solvent compositions are given by volume. Solutions of color dyes in benzene, 0 . 4 x wjv and solutions of sulfonamides, 0.1% wjv in acetone, were used as test substances. All substances were 99.0% chromatographically pure. In case of sulfonamides, a reference substance (R), 4-nitroaniline, was run on each chromatogram. TLC Apparatus and Procedures. Silica gel GF 254 (E. Merck) was used as adsorbent, 30 grams/60 ml distilled water to prepare 5 plates, 20 x 20 cm, for a layer thickness of 0.25 mm. After spreading (Desaga apparatus) the plates were air dried for 15 minutes, heated for 30 minutes at 110 "C in an oven with a fan, then cooled and stored in a desiccator. Samples, 0.003-0.005 ml, were applied with 0.01-ml micropipets (Desaga) with the aid of a hot air blazer, 2-2.5 cm from the bottom edge of the plate, and 1.5-2 cm apart. The side edges and the bottom edge were stripped 0.5 cm wide. After spotting, the plates were left in the ambient atmosphere for at least 15 minutes to ensure water adsorption in equilibrium with the ambient relative humidity. The vapor-programming chamber was used for development, whereas normal tank chambers (N-chambers), 21 X 21 x 9 cm, were used as controls. In the latter the bottom edge of the plate was not stripped. The troughs of the VPchamber were filled with about 5 ml each of the appropriate liquid mixture, then the plate was fixed in position. After a 10 minutes saturation period, the solvent reservoir was filled with 25 ml of solvent. N-chambers contained 100 ml of solvent and were saturated with solvent vapor by lining the inner walls with filter paper. After 45 minutes the plate was introduced and development started. The solvent was allowed to run 17 cm over the starting points. In the VPchamber continuous development was applied, with the development time given in minutes. The composition of the liquids in the troughs is given in the photographic illustrations. All experiments were made at 21 =t 1 "C and a relative humidity of 26-30z (color dyes) or 33-35z (sulfonamides). Within these ranges, reproducibility of the chromatograms was obtained. Detection was carried out under UV light, 254 nm (Camag Universal lamp), followed by photography under two such lamps on Agfacolor CT 18 Diapositive film with an Asahi Pentax type SV camera with 49 mm UV ghostless filter, exposure 15 seconds, distance 70 cm, aperture 5.6. RESULTS AND DISCUSSION The separation of butter yellow, sudanred G, indophenol, and 4-nitroaniline in N-chambers with benzene as solvent is far from optimal as can be seen in Figure 3A. The spreading over the plate is poor and, furthermore, sudanred G and 4-nitroaniline do not separate. Changing the solvent to benzenechloroform 80 :20 does not improve the spreading or the separation significantly (Figure 38). The spots run higher but the spreading remains almost the same. Sudanred G and 4-nitroaniline are separated but the latter now coincides with indophenol. The use of an unsaturated chamber would not provide better results, for the gradient is too flat for benzene-chloroform, and with more polar components the gradient. becomes too steep. With the aid of the vapor-programming chamber however, a much better separation is obtained (Figure 3C). Benzene is used as the solvent and decelerating liquid in combination with a simple benzene-chloroform vapor gradient. The four substances are clearly separated and the spots are not enlarged as compared to those obtained in the N-chambers. Furthermore, the entire plate is used to spread the spots. If necessary, the mutual distances between the spots can be VOb. 40, NO. 14, DECEMBER 1968
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changed by applying slightly different vapor gradients-i.e., if a fifth component would be present between sudanred G and 4.-nitroaniline, the migration rates of the three spots can be altered to obtain optimal separation and spreading. The time required for the separation in the VP-chamber is longer than in the N-chamber because of continuous development, but this is partially compensated for by the fact that less time is needed for the saturation of the VP-chamber. Similar improvements could be obtained in the separation of a selection of sulfonamides : sulfaguanidine, sulfisomidine, sulfathiazole, sulfacetamide, sulfadimidine, sulfapyridine, sulfisoxazole, and sulfanilamide. These substances are poorly separated in N-chambers with ether-methanol 90 :10 as solvent (Figure 44. Changing the solvent ratio to ether-methanol 80:20 results in higher R, values but the separation becomes worse (Figure 4B). With the VPchamber all substances are clearly separated with the separation suitable for identification purposes. Ether-methanol 95 :5 is used as solvent in combination with an ether-methanol vapor gradient and ether as decelerating liquid. The separation is shown in Figure 4C. It can be seen that now 70 of the plate is used to spread the spots. The over all time required for the separation in the VP-chamber is less than in the N-chamber because of the short saturation period.
The reproducibility of the separations in the VP-chamber is good. It will be obvious, however, that the factors involved in the reproducibility such as room temperature, cooling temperature, relative humidity, saturation, thickness of the strips and the layers, must be kept constant. Small variations in these parameters will have great influence on the vapor gradient and subsequently on the separation. Thus, it becomes clear that vapor-programmed TLC offers new possibilities in the identification of closely related substances which are often inadequately separated by classical TEC techniques. Furthermore vapor-programmed development can also be valuable in preparative TLC, for the improved separations will allow more efficient isolations. ACKNOWLEDGMENT
The author is grateful to J. Wijsbeek for skillful assistance, to J. S. Faber and D. A. Doornbos for discussions, and to A. Oosterhoff and G. P. Rijskamp for constructing the VPchamber. RECEIVED for review July 15, 1968. Accepted August 16, 1968. Taken in part from a lecture presented to the Gordon Research Conference on Separation and Purification, New London, N. H., August 7-11, 1967.
2,6-Dichloroquinone4-Chloroimide as a Reagent for Amines and Aromatic Hydrocarbons on Thin-Layer Chromatograms Joseph H. Ross Indiana Uniaersity, South Bend, Ind. 46615 The range of utility of 2,6-dichloroquinone 4-chloroimide as a spray reagent for organic compounds on silica gel TLC plates was surveyed. Avariety of intense colors was produced with primary, secondary, and tertiary aromatic amines, carbazoles, primary and secondary aliphatic amines, aromatic hydrocarbons, and an enol (2,4-pentanedione). Amides, amine oxides, and other oxidized nitrogen compounds gave weak or negative tests, as did aromatic compounds with electron attracting groups. The relationship of amine structure to color formation and the mechanism of reaction are discussed. The effects of some chromatographic variables on color formation with the reagent are reported. RF values with neutral solvents a r e given.
It was reported that the color produced with the amines persisted after spraying with borax solution, but that ammonia was not used because it darkened the silica gel background ( 4 ) . 2,6-Dibromoquinone-4-chloroimide was investigated as a spray reagent for a variety of ring-substituted primary aromatic amines on silica gel (7); the colors changed, but did not disappear, upon treatment with ammonia. In a comparison of dichloroquinone and dibromoquinone chloroimides as reagents for substituted 3,4-methylenedioxybenzenes, the dichloro compound was reported to be superior (8). Some other types of compounds which have been detected by the quinone chloroimide reagents are pyridoxine (9 and page 545 of ref. 3), indole (lo), hydrazones ( I I ) , sulfur ~ , ~ - D I C H L Q R O ~ U I(N,2,6-trichloro-l,4N ~ N E - ~ - ~ ~and ~O ~ Ocompounds ~ ~ ~ ~(12-14). ~ Dichloroquinone chloroimide has now been found to give benzoquinone-4-imine) and 2,6-dibromoquinone-4-chloroimide, referred to as Gibbs ( I ) reagents, have been used as spot test and chromatographic spray reagents for phenols and cer(4) A. Seher, Fette, Seven, iind Anstrichm., 61, 345 (1959). tain other types of compounds. Dichloroquinone chloroi( 5 ) R. F. van der Heide, J . Chromatogr., 24,239 (1966). (6) V. A,nger and S. Ofri, Mikrochim. Acta, 1964, 109. mide has been recommended as a specific reagent for phenols (7) L. Fishbein, J. Chromatogr., 27, 368 (1967). (2, 3), on the basis that the color reaction with phenols occurs (8) L. Fishbein, ibid., 22,480 (1966). after treatment of spot paper with ammonia, which is re(9) K. Randerath, “Thin-Layer Chromatography,” Academic Press, ported to cause the color produced by aromatic amines to New York, N. Y.,1965, p 157. (10) L. J. King, D. V. Parke, and R. T. Williams, Biochem. J., 98, disappear. The reagent has also been used for detection 266 (1966). of antioxidants, including phenolic types and a few primary (11) V. Anger and S. Ofri, 2.Anal. Chem., 204,263 (1964). and secondary amines, on silica gel chromatographic plates (12) M. T. Cuzzoni and T. P. Lissi, Farmaco, Ed. Sei., 19, 771 (1964); Chem. Abstr., 62, 3408 (1965). (13) M. T. Cuzzoni and T. P. Lissi, ibid.,. 981;. Chem. Abstr., 62, (1) H. D. Gibbs, 9.Bid. Chem., 72, 649 (1927). . ?671 (1965). (2) F. FeigI and E. Jungreis, ANAL.CHEU.,31, 2099 (1959). (14) S. Kamiya, Bcinseki Kagaku, 8, 596 (1959); ibid., 58, 9612 (3) F. Feigl. “Spot Tests in Organic Analysis,” 6th ed., Ekevier, (1963). New York, N. Y . , 1960, p 138. 21 38
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(4-6).
