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Continuous flow thin-layer chromatography
NOTES
Continuous
595
flow thin-layer
chromatography
The desire to combine the advantages of a continuously running solvent as found in descending paper chromatography with the well-known advantages of the thin-layer chromatographic technique (TLC) has lecl to the development ‘of many ingenious assemblies for continuously fiowing thin-layer chromatographyl-a. The use of the term “continuous” in thin-layer chromatography has become somewhat confused in the literature, possibly because of semantics involved in translations. Several worker& 3~~9 11 use the terms “continuous development” or “continuous flow” when referring to a continuously flowing solvent acting in a manner similar to that of descending paper chromatography. Other authorsGJ3 use the term “throughflow” chromatography and refer to the “overflow” of solvent. The term “continuous chromatography” is reserved by some workers 10 to describe t!rose processes wherein both the solvent flow is continuous and the sample is continually fed to the system, In our work we will use the terms “continuous flow” or “continuous development’ in the sense of a continuously flowing solvent with a single application of sample We have devised two methods which are as effective as any reported and arr simple to assemble. The materials and dimensions described are offered as a .guidt since the individual worker may have other equipment which can be readily adaptec’ to the same principles, The first method was based on the continuously flowing ascending papel chromatographic technique described by FISCHBACHAND LEVINE’ and is similar tc ones described recently by BOBRITTI and TRUTER~.In this method a 15 x 20 cm TLC plate, prepared by the rod and tape procedure of LEES AND DEMURIA~, is placed in 2 cylindrical jar (diam. 17.5 cm, height 23 cm) and supported from below so tha about 2 cm of the coated plate extends above the top of the jar. Solvent is placed in the jar so that about I cm of the lower edge of the plate is contacted by the solvent the samples previously having been applied about 3 cm from the bottom edge of the plate. While carefully holding the coated plate vertically in the jar, two plain glas plates are placed on top of the jar leaving a narrow slot through which the TLC platl extends. These cover plates are then slid against the coated plate, taking care not tc break the coating. The openings on either side of the coated plate are sealed wit1 adhesive tape, thus leaving about z cm or more of the coated plate exposed to thm atmosphere. Two plates can be developed simultaneously if they are placed back-ta back. To reduce the traveling distance of the solvent, spots may be placed furthe from the bottom edge and more solvent added to the jar. Other methods that hav been used to reduce the solvent traveling distance were to employ shallower solven jars or to place the solvent in a small trough which could be raised or lowered 1:’ regulate the solvent traveling distance. The shorter this distance the greater th flow rate. The second method devised by us was a horizontal one in which a TLC plate i used as the lid (coated sicle down) of a shallow glass tray serving as the solvent chair ber. The plates (IS x 20 cm) were coated with silica gel or other adsorb,ent by th B rod and tape method described previously. Some of the coating of the TLC plate j 1; scraped from three edges of the plate so as to restrict the solvent flow to one directio (Fig. I). Method I3 gives a somewhat better vapor seal than method A, but in eithe case solvent vapor leakage is not too great and saturation conditions are easil J. Clwomatog.,
18 (1965)
5g5-5q
NOTES
596
maintained because of the close proximity of the solvent layer and the TLC plate. Samples were applied about 2 cm in from the edge of the coating on the side opposite the unscraped end of the plate (cf. Fig. I), The developing chamber is simply a-glass photographic tray approximately18 x 13 x 3 cm, the edges of which have been ground flat so’ as to form a tight seal with a plate glass lid. These trays are usually made of soft glass and are easily ground down with a Carborundum paste. h
“LONG”PLATE
-METWOD
A
,b
“LONG”PLATE-METHOD b.
B ,b
tj “SWORT” PLATE-
METHOD
A
“SWORT” PLATE-
METHOD
B
Fig. I. Preparing horizontal thin-layer plates. (a) Silica gel costing: (b) area. from which silica gel is removed; (c) area of silica gel contacted by solvent wick; (cl) section of TLC plate contacted by edge of solvent tray; (x) sample application points,
Larger trays have also been used with correspondingly larger TLC plates. About ml of the developing solvent is placed in the tray and a rolled paper wick either 15 cm wide for the “short” plates or IO cm wide for the “long” plates is placed in the proper end of the chamber (Fig. 2). The paper wick is made by loosely rolling a IO cm long piece of chromatographic grade paper such as Whatman No. 3MM of the proper width into a roll approximately 2 cm in diameter. A piece of heavy glass rod (12 mm diam,) is inserted into the center of the roll to anchor the wick in place, The upper side of the rolled wick should contact the silica gel layer just before the point where the samples are added and care must be taken so as not to have the roll so bulky that it touches the applied samples or so tightly rolled that it does not make good contact. Cellulose sponge can be cut to form a wick and works quite well for ma.ny solvents except that unwanted materials were found to be extracted from the sponge itself in several cases. Once the TLC plate is prepared and the samples applied to it, the plate is inverted over the chamber so as to serve as a lid; the unscraped edge of the plate is exposed to the atmosphere for at least 2 cm. 200
J. Chvomafog.,
I8 (1965) 595-595
NOTES
Fig. 2. Apparatus
597
for horizontal
continuous
flow thin-layer
chromatography,
.
:
Continuous development by the horizontal or ascending methods depends on the constant evaporation of solvent as it reaches the exposed end of the coating layer. The rate of the solvent migration through the coating varies inversely with the distance to be traveled and directly with the volatility of the solvents used. Short plates, while permitting a large number of solvent passes, restrict the actual distance in which the separation is to be achieved. The vertical and later the horizontal methods were first applied to the separation of cholesterol and desmosterol. More recently the separation of these compounds as the alcohols has been described by WOLFMAN AND SAC~S~~ using a reversed-phase TLC technique. Fig. 3 shows the secaration obtained for these compounds as the acetates* when a silica gel G plate was developed in the horizontal manner for one hour using benzene-hexane (I : 3). This time is equivalent to about three “passes”; one “pass” being the time required for the solvent front to reach the far edge of the plate. The spots were visualized by spraying the plate with concentrated sulfuric acid, heating, and then observing the plate under white or long-wave ultraviolet light. Fig. 4 illustrates the separation of cholesterol and desmosterol, as the free alcohols, by the horizontal technique using a silver nitrate impregnated silica gel G plate. The developing solvent was benzene-ethyl acetate (95 :5) and the separation took about 6 h. Sulfuric acid was used to visualize the spots. AVIGAN et ~1.~4 used silver nitrate impregnated plates for separating these compounds, but found it necessary to use the acetate forms. We found that by dipping a previously prepared silica gel G plate in a 5 o/osolution of silver nitrate in acetonitrile, fvllowed by air drying to remove the acetonitrile, we were able to obtain much less background discoloration than when the method of preparation described by AWGAN was used. * Snnlplcs of Ohc alcohol Chas. Pfizer 13 Co., Inc.
and acetate
forms wwe lcinilly’ suppliecl by Dr. S.
I<. FIGDOR of
J. Clwomutog., 18 (1965)
595-598
NOTES
598
Fig. 3, Scparai& mosterol acetate
‘of’ oholestorol atid desmosterol acetates. CA = cholesterol (impure); MA = mixture of the two acetates. Photographed
Fig. 4. Separation of cholesterol and desmosterol. C = cholesterol ; D &I = mixture of the two alcohols. Rhotographed by white light.
The techniques described, can be adapted to and, besides affording good separations, have the pensiveness. These methods have been applied to clesmosterol and can be applied to other difficultly We wish to acknowledge
=
acetate: DA = desby ultraviolet light.
desmosterol
(impure);
many shapes and sizes of vessels, attributes of simplicity and inexthe separation of cholesterol and separable compounds.
the assistance of Mr. I. CI-IMTJRA in preparing
CIwomatogva$hy Laboratory, Medical Research Laboratories, Clzas. Pfa’zev & Co., Inc., Groton, Coma. (U.S.A .)
Fig.
I.
T. M. LEES M. J. -LYNCH F. R. MOSWER
I J.M. BOBBITT, TJain-Layer CJtromatograpJt_l,, Reinhold, New York, 1963. z L. G~LDEL, W. ZIMMERMANN AND D. LOMMER,~. PJqsiol.Chem., 333 (1963) 35.,. 3 R. BENNETT AND E. HEFTMANN, J. CJzromatog., 12 (x963) 245. 4 M. ANWAR, J. Chsm. Educ., 40 (1963) 29. 5 ill.BRENNER AND A. NIEDERWIESER, Evperientia, 17 (IgGI) 237. 6 N. Z~LLNER AND G. WOLFRAM, Klilz. Wochscltr., 40 (1962) 1098. 7 HI. ~ISCI-IBACH AND J. LEVINE, .%iClZCe, 121 (1955) 602. 8 T. LEES AND DXMURIh, J. CitYOmatOg., 8 (1962) 108. g E.TRUTER, J. Cltromatog., 14 (1964) 57. IO S. TURINh, v. MARJANOVIC-MRhJOVhN hND &r. OBRADOVIC, A?zaZ.CJJenz., 36 (1964) 1905. I I I<. RANDER~TW, TJain-Layer Chromatography, Academic Press, New York, 1963. 12 L. WOLPMAN AND 33. ShcEIS, J. Lipid. Res.. 5 (IgG4) 127. 13 Thin-Layer CJtvomatogvapJty wills Adsovbents, Woelm Publication AL IO, M. Woclm, Rschwege, W. Germany, 1963. 14 J, AVIGAN, DEWITT S. GOODMAN AND D. STEINBERG, _I..i;ifik$ Res., 4 (1963) 100. First received June 3rd, 1964 Modified November myth, 1964 J, Cilzvomatog..
I 8 (1965) 595-598
Continuous
595
flow thin-layer
chromatography
The desire to combine the advantages of a continuously running solvent as found in descending paper chromatography with the well-known advantages of the thin-layer chromatographic technique (TLC) has lecl to the development ‘of many ingenious assemblies for continuously fiowing thin-layer chromatographyl-a. The use of the term “continuous” in thin-layer chromatography has become somewhat confused in the literature, possibly because of semantics involved in translations. Several worker& 3~~9 11 use the terms “continuous development” or “continuous flow” when referring to a continuously flowing solvent acting in a manner similar to that of descending paper chromatography. Other authorsGJ3 use the term “throughflow” chromatography and refer to the “overflow” of solvent. The term “continuous chromatography” is reserved by some workers 10 to describe t!rose processes wherein both the solvent flow is continuous and the sample is continually fed to the system, In our work we will use the terms “continuous flow” or “continuous development’ in the sense of a continuously flowing solvent with a single application of sample We have devised two methods which are as effective as any reported and arr simple to assemble. The materials and dimensions described are offered as a .guidt since the individual worker may have other equipment which can be readily adaptec’ to the same principles, The first method was based on the continuously flowing ascending papel chromatographic technique described by FISCHBACHAND LEVINE’ and is similar tc ones described recently by BOBRITTI and TRUTER~.In this method a 15 x 20 cm TLC plate, prepared by the rod and tape procedure of LEES AND DEMURIA~, is placed in 2 cylindrical jar (diam. 17.5 cm, height 23 cm) and supported from below so tha about 2 cm of the coated plate extends above the top of the jar. Solvent is placed in the jar so that about I cm of the lower edge of the plate is contacted by the solvent the samples previously having been applied about 3 cm from the bottom edge of the plate. While carefully holding the coated plate vertically in the jar, two plain glas plates are placed on top of the jar leaving a narrow slot through which the TLC platl extends. These cover plates are then slid against the coated plate, taking care not tc break the coating. The openings on either side of the coated plate are sealed wit1 adhesive tape, thus leaving about z cm or more of the coated plate exposed to thm atmosphere. Two plates can be developed simultaneously if they are placed back-ta back. To reduce the traveling distance of the solvent, spots may be placed furthe from the bottom edge and more solvent added to the jar. Other methods that hav been used to reduce the solvent traveling distance were to employ shallower solven jars or to place the solvent in a small trough which could be raised or lowered 1:’ regulate the solvent traveling distance. The shorter this distance the greater th flow rate. The second method devised by us was a horizontal one in which a TLC plate i used as the lid (coated sicle down) of a shallow glass tray serving as the solvent chair ber. The plates (IS x 20 cm) were coated with silica gel or other adsorb,ent by th B rod and tape method described previously. Some of the coating of the TLC plate j 1; scraped from three edges of the plate so as to restrict the solvent flow to one directio (Fig. I). Method I3 gives a somewhat better vapor seal than method A, but in eithe case solvent vapor leakage is not too great and saturation conditions are easil J. Clwomatog.,
18 (1965)
5g5-5q
NOTES
596
maintained because of the close proximity of the solvent layer and the TLC plate. Samples were applied about 2 cm in from the edge of the coating on the side opposite the unscraped end of the plate (cf. Fig. I), The developing chamber is simply a-glass photographic tray approximately18 x 13 x 3 cm, the edges of which have been ground flat so’ as to form a tight seal with a plate glass lid. These trays are usually made of soft glass and are easily ground down with a Carborundum paste. h
“LONG”PLATE
-METWOD
A
,b
“LONG”PLATE-METHOD b.
B ,b
tj “SWORT” PLATE-
METHOD
A
“SWORT” PLATE-
METHOD
B
Fig. I. Preparing horizontal thin-layer plates. (a) Silica gel costing: (b) area. from which silica gel is removed; (c) area of silica gel contacted by solvent wick; (cl) section of TLC plate contacted by edge of solvent tray; (x) sample application points,
Larger trays have also been used with correspondingly larger TLC plates. About ml of the developing solvent is placed in the tray and a rolled paper wick either 15 cm wide for the “short” plates or IO cm wide for the “long” plates is placed in the proper end of the chamber (Fig. 2). The paper wick is made by loosely rolling a IO cm long piece of chromatographic grade paper such as Whatman No. 3MM of the proper width into a roll approximately 2 cm in diameter. A piece of heavy glass rod (12 mm diam,) is inserted into the center of the roll to anchor the wick in place, The upper side of the rolled wick should contact the silica gel layer just before the point where the samples are added and care must be taken so as not to have the roll so bulky that it touches the applied samples or so tightly rolled that it does not make good contact. Cellulose sponge can be cut to form a wick and works quite well for ma.ny solvents except that unwanted materials were found to be extracted from the sponge itself in several cases. Once the TLC plate is prepared and the samples applied to it, the plate is inverted over the chamber so as to serve as a lid; the unscraped edge of the plate is exposed to the atmosphere for at least 2 cm. 200
J. Chvomafog.,
I8 (1965) 595-595
NOTES
Fig. 2. Apparatus
597
for horizontal
continuous
flow thin-layer
chromatography,
.
:
Continuous development by the horizontal or ascending methods depends on the constant evaporation of solvent as it reaches the exposed end of the coating layer. The rate of the solvent migration through the coating varies inversely with the distance to be traveled and directly with the volatility of the solvents used. Short plates, while permitting a large number of solvent passes, restrict the actual distance in which the separation is to be achieved. The vertical and later the horizontal methods were first applied to the separation of cholesterol and desmosterol. More recently the separation of these compounds as the alcohols has been described by WOLFMAN AND SAC~S~~ using a reversed-phase TLC technique. Fig. 3 shows the secaration obtained for these compounds as the acetates* when a silica gel G plate was developed in the horizontal manner for one hour using benzene-hexane (I : 3). This time is equivalent to about three “passes”; one “pass” being the time required for the solvent front to reach the far edge of the plate. The spots were visualized by spraying the plate with concentrated sulfuric acid, heating, and then observing the plate under white or long-wave ultraviolet light. Fig. 4 illustrates the separation of cholesterol and desmosterol, as the free alcohols, by the horizontal technique using a silver nitrate impregnated silica gel G plate. The developing solvent was benzene-ethyl acetate (95 :5) and the separation took about 6 h. Sulfuric acid was used to visualize the spots. AVIGAN et ~1.~4 used silver nitrate impregnated plates for separating these compounds, but found it necessary to use the acetate forms. We found that by dipping a previously prepared silica gel G plate in a 5 o/osolution of silver nitrate in acetonitrile, fvllowed by air drying to remove the acetonitrile, we were able to obtain much less background discoloration than when the method of preparation described by AWGAN was used. * Snnlplcs of Ohc alcohol Chas. Pfizer 13 Co., Inc.
and acetate
forms wwe lcinilly’ suppliecl by Dr. S.
I<. FIGDOR of
J. Clwomutog., 18 (1965)
595-598
NOTES
598
Fig. 3, Scparai& mosterol acetate
‘of’ oholestorol atid desmosterol acetates. CA = cholesterol (impure); MA = mixture of the two acetates. Photographed
Fig. 4. Separation of cholesterol and desmosterol. C = cholesterol ; D &I = mixture of the two alcohols. Rhotographed by white light.
The techniques described, can be adapted to and, besides affording good separations, have the pensiveness. These methods have been applied to clesmosterol and can be applied to other difficultly We wish to acknowledge
=
acetate: DA = desby ultraviolet light.
desmosterol
(impure);
many shapes and sizes of vessels, attributes of simplicity and inexthe separation of cholesterol and separable compounds.
the assistance of Mr. I. CI-IMTJRA in preparing
CIwomatogva$hy Laboratory, Medical Research Laboratories, Clzas. Pfa’zev & Co., Inc., Groton, Coma. (U.S.A .)
Fig.
I.
T. M. LEES M. J. -LYNCH F. R. MOSWER
I J.M. BOBBITT, TJain-Layer CJtromatograpJt_l,, Reinhold, New York, 1963. z L. G~LDEL, W. ZIMMERMANN AND D. LOMMER,~. PJqsiol.Chem., 333 (1963) 35.,. 3 R. BENNETT AND E. HEFTMANN, J. CJzromatog., 12 (x963) 245. 4 M. ANWAR, J. Chsm. Educ., 40 (1963) 29. 5 ill.BRENNER AND A. NIEDERWIESER, Evperientia, 17 (IgGI) 237. 6 N. Z~LLNER AND G. WOLFRAM, Klilz. Wochscltr., 40 (1962) 1098. 7 HI. ~ISCI-IBACH AND J. LEVINE, .%iClZCe, 121 (1955) 602. 8 T. LEES AND DXMURIh, J. CitYOmatOg., 8 (1962) 108. g E.TRUTER, J. Cltromatog., 14 (1964) 57. IO S. TURINh, v. MARJANOVIC-MRhJOVhN hND &r. OBRADOVIC, A?zaZ.CJJenz., 36 (1964) 1905. I I I<. RANDER~TW, TJain-Layer Chromatography, Academic Press, New York, 1963. 12 L. WOLPMAN AND 33. ShcEIS, J. Lipid. Res.. 5 (IgG4) 127. 13 Thin-Layer CJtvomatogvapJty wills Adsovbents, Woelm Publication AL IO, M. Woclm, Rschwege, W. Germany, 1963. 14 J, AVIGAN, DEWITT S. GOODMAN AND D. STEINBERG, _I..i;ifik$ Res., 4 (1963) 100. First received June 3rd, 1964 Modified November myth, 1964 J, Cilzvomatog..
I 8 (1965) 595-598