Vapor-Programmed Preparative Thin-Layer Chromatography, a Technique with Improved Resolving Power Rokus A. d e Zeeuw and J a a p Wijsbeek Laboratory for Pharmaceutical and Analytical Chemistry, State Unioersit Antoniur Deusinglaan 2, Groningen, The Netherlands Further investigations on t h e properties a n d analytical applications of vapor-programmed TLC have shown t h a t significant separation improvements c a n b e obtained by using t h i s technique in preparative TLC. A large-scale 40-cm vapor-programming c h a m b e r for preparative purposes is described a n d t h e working procedure discussed. Results a r e shown in s e p a r a tions of d y e s and closely related sulfonamides. The much better separations obtained with t h i s technique indicate t h a t vapor-programmed preparative TLC may b e successful in analyses impossible to conduct with conventional TLC methods.
PREPARATIVE THIN-LAYER CHROMATOGRAPHY is widely applied to orovide adeauate sevaration of lamer auantities of materia.I, usually on the milligram-gram scale. The technique is simple; the resolving power is, in general, comparable to .. , ILL m . me ~ ~ . . .-, ~~...L~ that of analytical microgram ~ range; m u buustances are easily detected, by fluorescent methods or spray reagents. Various attempts have been made to use conventional liquid column chromatography for preparative separations after having defermined the best adsorbent and the best solvent system by preliminary checks of smaller quantities on thin layers (1-5). This procedure, however, was often inadequate, particularly with multicomponent solvents. Recent studies on the role of solvent vaoor in TLC have now revealed that with these solvent types the separation conditions on columns are markedly different from those in TLC, so that often a TLC separation cannot be translated directly onto a column (6, 7). To date, preparative TLC is usually carried out on adsorbent layers 0.5, 1, or 2 mm thick, spread on 20 X 20 cm, or 20 X 40 cm glass plates, or for very large quantities 20 X 100 cm (8). The usual equipment can be used for the preparation of the layers, although small modifications may he desirable in some instances. The sample is applied in a uniform line o r band 15 to 25 mm from the bottom edge - of the date. chis band should be as narrow as possible, because the band \width after separatic)n is highly dependent on the width of the initial band at th e start.
Figure 1. Preparative chamber, 40 X 20 cm
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limitations and sometimes its resolving power leaves something to be desired. Two major difficulties of preparative work may be mentioned: Often two substances, just separated by analytical TLC, :annot be separated on a preparative scale as a consequence ,f the higher loadings and the larger initial band width which cannot always be avoided. If an impurity or a decomposition product is to be separated and isolated from a parent substance, rather high quantities (1) J. M. Miller and J. G. Kirchner, ANAL.CHEM., 23, 428 (1951). (2) J. M. Miller and J. G. Kirchner, ibid., 24, 1480 (1952). (3) E. Stahl, Arch. Pham., 292, 411 (1959). (4) H. Dahn and H. Fuchs, Helu. Chim. Aero, 45, 261 (1962). (5) B. Loev and K. M. Snader, C/rem. Ind. (London), 1965, 15. (6) R. A. de Zeeuw, ANAL.CHEM.,40,915 (1968). (7) J. H. van Dijk and W. J. Mijs, 2.Anal. Chem., 236,419 (1968). (8) H. Halpaap, Chem.-lng.-Tech., 35,488 (1963). 90
vapor-programming
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sample be applied.~ Accordingly, ~ need to ~ - the band ~ of the parent compound may become so broad that it overlaps the small band of the impurity or decomposition product, preventing adequate isolation.
We therefore investigated whether vapor-programmed TLC, which was highly effective in TLC of smaller quantities of material (9, IO), would also be able to increase the resolving power in preparative TLC. By means of this technique optimal vapor conditions can be established all over the plate, making it possible to affect and guide the migration of the individual substances. In this work the technique was applied in separations of dyes and several impurities and in separations of three closely related sulfonamides frequently used in pharmaceutical preparations. APPAR.4TUS Description and Materials. Although the previously described 20-cm VP chamber (9) can be used for preparative work, the need to handle larger quantities of material has led to the design of a 40-cm preparative VP chamber (also obtainable from C. Desaga, Heidelberg), for the use with 20 x 40 cm TLC plates. The chamber is shown in Figure 1. The dimensions are: solvent reservoir 40 X 1 X 2 cm, ground plate 40 X 20 X 1 cm, trough chamber 40 X 17.5 X 1.7 cm with 21 troughs, i.d. 6 nun, depth 12 mm, partition walls 2 mm, and side walls 5 mm. The trough chamber is made of rolled brass, with the troughs directly milled out. The top surface of the trough chamber is also milled to obtain a completely flat surface. After milling, the trough chamber and the other brass parts are nickel- and chromium-plated. Brass was chosen, to ensure sufficient thermoconductivity. 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 these parts. Working Procedure. Samples are applied on normal 20 X 40 cm TLC plates as a narrow band, 2.5 cm from the bottom edge of the plate and at least 2.5 cm from the side edges. TLC plates should preferably be of glass with a thickness of 4 mm to avoid sagging during development. The side edges and the bottom edge of the adsorbent are stripped 5 mm wide. The troughs of the VP chamber are filled with liquids of appropriate composition to give optimal vapor (9) R. A. de Zeeuw, ANAL.CHFM., 40,2134 (1968). (10) R. A. de Zeeuw, J. Pharm. Pharmacal., 20,54S (1968).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 1. JANUARY 1970
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Table I. Trough NO. (bottom to top) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
I BQ B B-C (90 B B B B-C (60 B B B B B C B B B
0
I1 111 B C-A B C-M-A (50 40 10) B-C (20 80) C-A B C-A B C-A B C-M-A (20 70 10) C-A C B C-A B C-A B Ac-M-A (20 70 10) B C-A B C-A B C-A B Ac-M-A (20 70 10) B C-A B C-A B C-A B Ac-M-A (20 70 10) B C-A B C-A B C-A 25% ammonia, Ac = acetone, C-A = chloroform, saturated with 25% ammonia.
+ +
+
+ 10)
+ +
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+ +
+ +
B
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B = benzene, C = chloroform, M Liquid compositions given by volume.
Compositions of Vapor Programs Vapor program
=
methanol, A
=
programs. Searching for such optimal programs should be done in the same way as searching for an optimal solvent in conventional TLC. The principles of compounding the vapor programs are identical to those described for analytical VP-TLC and combinations of acclerating and decelerating troughs should be used (9). The layer thickness of the adsorbent should be in the order of 1 mm when spread. Thicker layers may be used and have a greater capacity, but they do not always give as good resolution (11). It is very important to apply the sample in as narrow a band as possible, since broader bands have a distinctly negative effect on the resolution. Hence, manual application should be avoided and a suitable automatic mechanical applicator should be used. In our investigations the Desaga Autoliner (12) yielded excellent results. Further details of the working procedure are identical to those for the 20-cm VP chamber, except that the filter paper strip is now 37 X 2 cm and the solvent reservoir is usually filled with SO ml of solvent. For extra long development additional solvent supply during the run is necessary. Thickness of Side Strips. The applicability of a certain vapor program is dependent on two factors: The distance between the adsorbent and the trough walls must be very small to keep vapor diffusions between the troughs as small as possible; this is very important for successful use of the VP chamber. However, the adsorbent must not touch the trough walls during development; most adsorbents swell more or less, the rate of swelling being dependent on the polarity of the solvent and the vapors used. The thickness of the side strips should thus be adapted to these requirements. In our experiments with 1-mm layers when spread, good results were obtained with 1-mm strips for nonpolar liquids like hexane, benzene, 1.3-mm strips for medium polar liquids like chloroform, ether, and acetone, and 1.5-mm strips for strongly polar liquids like ethanol, methanol, acetic acid, and ammonia. Teflon (Du Pont) is a suitably inert material for the side strips and available in several thicknesses. Saturation. To be sure that reproducible quantities of vapor are always available, the dry plates are equilibrated over the filled troughs for at least 10 minutes prior to development. After this period, in which the space over the troughs becomes saturated, the solvent reservoir is filled and development started.
Temperature. Vapor-programmed TLC requires constancy of room temperature within i l "C, so that reproducible results can be obtained. Cooling of the ground plate to 2" to 3 "C below room temperature is, in general, required to avoid too much vapor condensation on the adsorbent layer. The cooling temperature must also be kept constant. Furthermore, the VP chamber should be protected from draft, air circulation caused by air conditioning, etc. The conditions in the VP chamber are easily destroyed by such air streams due to temperature changes. To overcome this problem-work was done in a fully air-conditioned and climatized room-we used a 20 X 2.5 X 60 cm Plexiglas hood, placed over the VP chamber during development. The hood is open at the back and gives suitable protection. For the 20-cm VP chamber a 20 X 25 X 35 cm hood may be used. Continuous Development. Separation of closely related substances by VP-TLC generally requires continuous development (9). Moreover, the broader bands in preparative work require even longer developments than in qualitative work before a separation is obtained (13). Construction of Vapor Program. The same principles as in qualitative VP-TLC can be applied to preparative work. However, because the sample is now applied as a band rather than a spot narrower than the band, the acceleration of the migration of the band must be carefully controlled to suppress tailing. This should be done by using small concentrations of the more polar liquid in the lower troughs and a sufficient number of decelerating troughs, filled with a liquid of a distinctly lower polarity than in the adjacent accelerating troughs. EXPERIMENTAL
Solvents and Standards. All solvent components were reagent grade (E. Merck). Solvent compositions are given by volume. The Desaga test mixture (Stahl) was used in the investigations of dyes, consisting of a solution of butter yellow, Sudan red G , and indophenol, 0.15 % wjv in benzene. In the investigations of sulfonamides a mixture of three sulfapyrimidines was used-a solution of sulfadiazine, sulfamerazine, and sulfamethazine, each 0.2 % wjv in acetone. The latter substances were according to the trisulfapyrimidine mixture, United States Pharmacopeia XVII.
(11) F. J. Ritter and G. M. Meyer, Nature, 193, 941 (1962). (12) E. Stahl and E. Dumont, J. Chromatog., 39, 157 (1969).
(13) R. D. Bennett and E. Heftmann, J. Chromatog., 12,245(1963). ANALYTICAL CHEMISTRY, VOL. 42, NO. 1, JANUARY 1970
91
Figure 3. Separation of sulfonamides ohtained with &" . . . . . . . * I -n"e"+erl , I . . . ,,.dopment, conventional develvp.m....L, and with development in V:P chamher
Figure 2. Separation of color dyes with conventional development anid development in VP chamber
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A. Conventional developnlent in saturated chamber, 40 solvent chloroform-methanol-25% ammonia (50 lo), temperature 20.9 "C, rel. humidity 29%, saturation 60 min, development 67 min B. As under A, development repeated twice C. VP chamber, solvent chloroform-methanol-25 Z 1 1_9 -, vel. ammonia (75 20 5), t-mn _ _ _ _ _2 r .... hiimirlitv 27Z, saturation 10 min, development 180 min, strips 1.5 mm, cooling 19 "C, Vapor._ Program. I11-(see Table .. .. . . ." I) . . 1 = sultadlazme, z = sulkamerazuIe, 3 = sulfametnazme. Plates. Silica gel PF 254 366, layer thickness 1 mm when spread. Load 30 mg
A. Conventional development in saturated chamber, solvent benzene, temp. 21.0 "C, rel. humidity 27%, saturation60 min, develoimerit 48 min B. VP chamber, solveint benzene, temp. 21.2 "C, rel. hnmirlitv -. 2747. .atwntio 10 min, development 125 min, ___.._____i ,",._._.__in strips 1.3 mm, cooling 19 "C. Vapor Program I (see Table I) C. As under B, but with solvent benzene-chloroform I ,~" , *",.
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E"\ V...". D,m".nm rr \,aT . M . I\ 1 = indophenol, 2 = Sudan red G, 3 = butter yellow, 4 to 10 = impurities. 366, layer thickness 1 mm Plates. Silica gel PF 254 when spread. Load 30 mg ,C"
92
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ILL Appararus ana rroceonres. xiica gel rr LJ'I t JOO (E. Merck) was used as adsorbent, 70 grams per 175 ml of demineralized water to prepare two 20 X 40 cm plates, layer thickness 1mm when spread. After spreading (Shandon apparatus) the plates were air-dried for 3 hours, heated for 45 minutes at 110 "C in an oven with a fan, then cooled and stored in a desiccator (Desaga). Samples (20 ml of the test mixture or 5 ml of the sulfonamide solution) were applied with the Desaga Autoliner (Stahl) 2.5 cm from the bottom edge of the plates, in streaks of approximately 32 cm. The side edges and the hottom edge of the plate were stripped 0.5 cm wide. The 40-cm preparative vapor-programming chamber was used for development, whereas normal 50 X 25 X 20 cm preparative tank chambers (Shandon) were used as controls. In the latter the bottom edge of the plate is not stripped. The troughs of the VP chamber were filled with about 10 ml each of the appropriate liquid mixture; then the plate was fixed in position. After a 10-minute saturation period the solvent reservoir was filled with 50 ml of solvent. Tank chambers contained 300 ml of solvent and were saturated with solvent vapor by lining the inner walls with filter paper. After 60 minutes the plate was introduced and development started, The solvent was allowed to run 17 cm over the starting line. In the VP chamber continuous development
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compositions of the liquids in the troughs are listed in Table I. All experiments were done at 21 + 1 "C and a relative humidity of 26 to 30z. Within these ranges reproducibility of the chromatograms was observed. Detection was carried out under ultraviolet light of 254 nm (Camag Universal lamp with 25 X 7.5 cm filter), followed by photography under two such lamps on Agfacolor C T 18 Diapositive film with an Asahi Pentax type SV camera with 49-mm ultraviolet .. ghostless filter, exposure 2 seconds, distance 70 cm, aperture 5.6. ~~
RESULTS AND DISCUSSION The separation of the test mixture with benzene as solvent is incomplete (Figure 2, A). Because of the higher loadings in comparison to analytical TLC, indophenol and Sudan red G show overlapping. Furthermore, an orange impurity can he observed (marked 4, 5 ) between butter yellow and Sudan red G. The use of a more polar solvent such as benzene-chloroform mixtures does not improve the separation. The spots run higher hut Sudan red G and indophenol remain coincided. Unsaturated chambers were also unsatisfactory in this case.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 1, JANUARY 1970
Repeated development (8) has also been applied, but a suitable result could not be obtained, because butter yellow and indophenol showed the phenomenon of band splitting. By means of the preparative VP chamber and using Vapor Program I (Table I) separation of the three dyes could be significantly improved (Figure 2, B). Butter yellow, Sudan red G, and indophenol are clearly separated. Moreover, an impurity (6) could be detected just in front of the Sudan red band, whereas the orange impurity of Figure 2, A , appeared to consist of two components, a yellow and a red (upper band). These color differences cannot be seen in Figure 2, B, however. Experiments with the same substances, but with benzene50) and Vapor Program 11, revealed that chloroform (50 the test mixture also contained a number of impurities with a lower migration rate than indophenol (Figure 2, C ) . The concentrations of these impurities are rather low, however (0.1 to 0.5 but the above results make clear that the preparative VP technique is highly effective in the separation of these products, which are often overlapped by the main products. Accordingly, the better separations will allow easy isolation. When comparing the development times in conventional TLC and in the VP chamber the question may arise whether the separation improvements in the latter are not caused merely by the prolonged time of development. We therefore investigated continuous development in a conventional chamber, fdlowing the procedure of Bennett and Heftmann (13). With benzene as solvent and a development time of 125 minutes the bands of Sudan red G and indophenol could be separated, but then impurity 6 did not separate from Sudan red G, butter yellow ran into the solvent front, and impurities 4 and 5, remaining coincided, did the same. Continuous development for 125 minutes with benzene-chloroform (50 50) as solvent left Sudan red G and indophenol coincided and revealed only two impurity bands behind indophenol. Thus, continuous development compared unfavorably with VP chromatography. A strikingly improved resolving power was also obtained in the vapor-programmed separation of the sulfonamides. The
+
z),
+
substances under investigation are very closely related: sulfadiazine, NI-2-pyrimidinylsulfanilamide;sulfamerazine, Nl-(4-methyl-2-pyrimidinyl)sulfanilamide;and sulfamethazine, Nl-(4,6-dimethyl-2-pyrimidinyl)sulfanilamide,In analytical TLC a reasonable separation of the components can be obtained, but in preparative TLC the bands overlap as a consequence of the larger quantities of material. This is demonstrated in Figure 3, A , in which 10 mg of each substance was applied and developed with chloroform-methanol-25 40 10). Repeated development was unammonia (50 successful, as can be concluded from Figure 3, B, and the same result was obtained by conventional continuous development (13) for 180 minutes. In unsaturated chambers a somewhat better resolution was obtained because sulfamethazine could be separated from the other two, which remained coincided. With the preparative VP chamber, however, all substances were clearly separated, using chloroform-methanol-25 ammonia (75 20 5 ) as solvent, together with Vapor Program I11 of Table I. The result is given in Figure 3, C. Rechromatography showed the separated and isolated substances to be chromatographically pure to at least 99.5 %. The above experiments clearly indicate that vapor-programmed TLC promises new possibilities in the preparative field because of its higher resolving power in comparison to conventional preparative TLC. In particular this will be useful in the separation of closely related substances and in separations and isolations of small quantities of impurities or decomposition products, which are often overlapped by the parent component.
+ +
+
+
ACKNOWLEDGMENT
The authors are grateful to A. Oosterhoff and G. P. Rijskamp for constructing the VP chamber. RECEIVED for review September 8, 1969. Accepted October 29, 1969.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 1, JANUARY 1970
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