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Liquid column chromatography using thin-layer chromatographic silica gel
of Chromatography,450 (1988) 443-447 Elsevier Science Publishers B.V., Amsterdam ~ Printed in The Netherlands
Journal
CHROM. 20 758
Note
Liquid column silica gel
chromatography
DAVID J. BEVERIDGE,
using
thin-layer
chromatographic
DUNCAN M. HARRISON and ROBERT M. McGRATH*
Department qf Biochembtry, Africa)
Universit_y of the Witwatersrand, P.O.
WITS 2050, Johannesburg
(South
(First received May 4th, 198X; revised manuscript received June 25th. 1988)
Thin-layer chromatographic (TLC) grade silica gel has already been used in column form to separate intermediates in aflatoxin biosynthesis but was packed dry’,‘. The method depends on capillary attraction to pull the solvent through the column similar to solvent movement on a TLC plate. No large externally applied forces are required to either push or pull the solvent. However, in some cases, particularly when the solvent is alkaline, and in spite of precautions such as dampening the silica gel with solvent before packing, the gel cracks and channels during development. To obviate this problem we packed the column wet, as in traditional column chromatography, reasoning that the continual evaporation from the bottom of the column would create the capillary forces needed to move the solvent through the gel. This is necessary because the TLC-grade silica gel is so fine that the solvent cannot move through it under gravity or indeed under applied moderate pressure. EXPERTMENTAL
The column design is shown in Fig. 1 and is self explanatory. It was packed with a slurry of TLC silica gel 60 GFz54 (Merck, Darmstadt, F.R.G.) prepared by weighing an amount of dry gel equal to the volume of the column multiplied by the factor 0.37 and vigorously shaking it with the column volume of solvent. The slurry was briefly and carefully degassed at a water vacuum pump before being poured all at once into the column. The silica was allowed to settle and the solvent to run until a little remained to cover the gel, when the end of the column was sealed with a household cling wrap. The sample was loaded by dissolving it in volatile solvent, adding 1 to 3 g of silica gel and taking to dryness in a rotary evaporator. The free running dry gel with sample was then poured onto the column. A little eluent, bearing Sudan blue dye (fat soluble) to act as indicator for the front, was carefully added to the dry load sufficient just to mobilise it. Next, glass wool was pushed on top of the loaded column and eluent added carefully before inserting the reservoir into the top of the column which was left to develop. Separation was deemed complete when the Sudan blue just exited from the column. At this point the silica gel was extracted from the column using a suitable plunger onto a sheet of Whatman 3MM chromatography paper. OO21-9673/88/$03.50
6
1988 Elsevier Science Publishers B.V
NOTES
The various components could be detected under short-wave length UV light and simply sliced out to be eluted with a suitable solvent or the silica column could be sliced into equal portions and each extracted with methanol for TLC analysis. The extruded gel must not be allowed to dry otherwise it collapses into a fine powder. Flow-rate depended on the column diameter, volatility and mobility of the solvent system. The mobility of a solvent system can readily be gauged on a TLC plate and slow moving systems, such as propanol and butanol, could not be used as described here. Very suitable solvents include methanol, ethanol, ethyl acetate and light petrolum (b.p. SO-100X$ RESULTS AND DISCUSSION
Fig. 1 shows the separation of 125 mg of a chloroform extract of acidified mycelium and medium of a culture of Peniciliium cyciopium known to produce cyclopiazonic acid mycotoxins. The silica gel column measured 30 x 2.5 cm and required 8 h for development. Fig. 2 shows a TLC analysis of the separation obtained on this column of silica gel. The RF differences between the original material {lane 8) and separated material (lanes O-7) are presumably due to the amount loaded and also to the difference in environment each component causes for the others.
Truncated 250 mQ
0I23-
Glass wool Load Fine Silica Gel (TLC) Extruded Gel Cut
4 5-
l+Cyclopiazonic Acid
67-
a-Cyclopiazonic Acid
Silicone Rubber Tubing
Fig. 1.A line drawing of a wet-poured TLC-grade silica gel 64 GF 29430 x 2.5 cm, with an extract (125 mg) from P. cyclopium partially separated using ethyl acetate-methanol-cone. ammonium hydroxide (7k25.5 v/v/v) as solvent. The numerals indicate the cut sections of the extruded gel that were extracted witd methanol and analysed as described in Fig. 2.
445
NOTES FRONT
Original
0
I
2
3
4
5
6
7
8
Fig. 2. A line drawing copy of the analysis by Merck silica gel 60 GF,,, TLC plates of the column chromatography described in Fig. 1. The mobile phase was ethyl acetatePmethanol-conc. ammonium hydroxide (60:35:5, v/v/v) and the components detected under UV at 2.54 nm.
Fig. 3 shows a similar investigation by TLC of the results achieved in the separation of the components of nutmeg oil on a gel column 25 x 1.2 cm using light petroleum (b.p. 8&100”C)-diethyl ether (80:20, v/v)_ Here the time required for separation was only 3 h, and even so a fast moving component was lost. It was this loss that prompted the use of a fat-soluble dye as a marker for the front and for column performance. The components were located under UV at 254 nm and cut from the extruded gel to be eluted with methanol. The relationship between the RF values on TLC plates and columns was investigated (the column RF values were calculated using Sudan blue as the reference FRONT 1
,_-. ,---.\ \,’
I :*__,,
I
s.__,’
0 1
1
2
3
4
5
7
6 i
8
L
ORIGINAL
Fig. 3. A line drawing copy of the analysis by Merck silica gel F,,, high-performance TLC plates of the column chromatography of the constituents of the oil of nutmeg. The mobile phase was light petroleum (b.p. SO-100”Cj-diethyl ether (80:20, v/v). The shaded area represents iodine uptake and the clear area is absorption at 254 nm-
446
NOTES
Fig. 4. The separation of the dyes of 175 mg black ink on a column of TLC-grade silica gel 60 GF,,, (38 x layer chromatographic plate (20 x 20 x 0.2 cm: I of the 2.5 cm) and of 17.5 mg black ink on a preparative ammonium hydroxide (60:35:5, v/v/v). same silica gel. The mobile phase was ethyl acetate -methanol-cone.
TABLE I VALUES OBTAINED BY TLC OF THE SECONDARY METABOLITES OF P. CYCLOPIUM HIGH-PERFORMANCE SILICA GEL PLATES (10 x 10 cm) AND ON 400 x 25 mm COLUMNS OF TLC-GRADE SILICA GEL PACKED WET
R, ON
Abbreviations: A = a-cyclopiazonate; B = fl-cyclopiazonate; C = cycloacetoacetyltryptophanyl; D and E = unknown. R, values
Columns Plates
Ethyl acetate-methanol-cone.
ammonium
70:25:5, v/v/v
60.35.5, v/v/v
A(0.57), B(0.46), C(O.28),D(O.10). E(0.05) A(0.39), B(0.38). C(O.18),D(0.06), E(O.03)
A(0.80), B(0.67), C(O.39),D(O.l5), E(O.10) A(0.44), B(0.32), C(O.22),D(0.09), E(0.05)
Average ratio of R, (column) to R, (plate) 1.60 f 0.18
hydroxide
1.87 f 0.15
front) and the results are shown in Table I. While the RF values are not the same between plate and column, a reasonably constant ratio is maintained. Thus, unlike traditional column chromatography on coarser gels, one can fairly accurately predict where the metabolite should be located. One can use TLC plates to investigate a mixture for the best eluent and translate this directly into a column technique. Though we have described an isocrfatic use, the column technique should lend itself to stepwise elutions. The effectiveness of the method is portrayed in Fig. 4 where the dyes of black ink are shown separated on a TLC silica gel column and, for comparison, on a preparative layer chromatographic plate (20 x 20 x 0.2 cm) of the same silica gel, i.e. 60 GFz54; the column was loaded with 175 mg ink compared to 17.5 mg on the plate. Though the ink load on the column was ten times that on the preparative plate the volume of silica gel used in the column was 190 ml compared to 80 ml on the plate. Thus the ratio of load to silica gel was 0.92 for the column and only 0.22 for the plate, and yet the plate was being used almost to its maximum capacity. The reasons for the difference probably lie in the ease of loading of the column, which gave a more uniform band, and the longer development time for the column (16 h) compared to the plate (2 h). The main advantages of this wet packing compared to dry packing of columns are that it is easier to do, it gives more reproducible results, and can be used for alkaline solvent systems. On the other hand it cannot be used for low-mobility solvent systems when dry packing can. And copmpared to preparative layer chromatography it is cheaper, easier to load, simpler to reclaim the separated components, and calcium sulphate can be omitted since no binding is needed, and it takes a heavier load of sample. REFERENCES 1 J. J. Dunn, L. S. Lee and J. W. Bennett, Biotech. Lett., 2 (1980) 17. J. W. Bennett, F. G. Kromberg, L. A. Goodman and M. A. Seltman, Mycologica,
2
75 (1983) 202.
Journal
CHROM. 20 758
Note
Liquid column silica gel
chromatography
DAVID J. BEVERIDGE,
using
thin-layer
chromatographic
DUNCAN M. HARRISON and ROBERT M. McGRATH*
Department qf Biochembtry, Africa)
Universit_y of the Witwatersrand, P.O.
WITS 2050, Johannesburg
(South
(First received May 4th, 198X; revised manuscript received June 25th. 1988)
Thin-layer chromatographic (TLC) grade silica gel has already been used in column form to separate intermediates in aflatoxin biosynthesis but was packed dry’,‘. The method depends on capillary attraction to pull the solvent through the column similar to solvent movement on a TLC plate. No large externally applied forces are required to either push or pull the solvent. However, in some cases, particularly when the solvent is alkaline, and in spite of precautions such as dampening the silica gel with solvent before packing, the gel cracks and channels during development. To obviate this problem we packed the column wet, as in traditional column chromatography, reasoning that the continual evaporation from the bottom of the column would create the capillary forces needed to move the solvent through the gel. This is necessary because the TLC-grade silica gel is so fine that the solvent cannot move through it under gravity or indeed under applied moderate pressure. EXPERTMENTAL
The column design is shown in Fig. 1 and is self explanatory. It was packed with a slurry of TLC silica gel 60 GFz54 (Merck, Darmstadt, F.R.G.) prepared by weighing an amount of dry gel equal to the volume of the column multiplied by the factor 0.37 and vigorously shaking it with the column volume of solvent. The slurry was briefly and carefully degassed at a water vacuum pump before being poured all at once into the column. The silica was allowed to settle and the solvent to run until a little remained to cover the gel, when the end of the column was sealed with a household cling wrap. The sample was loaded by dissolving it in volatile solvent, adding 1 to 3 g of silica gel and taking to dryness in a rotary evaporator. The free running dry gel with sample was then poured onto the column. A little eluent, bearing Sudan blue dye (fat soluble) to act as indicator for the front, was carefully added to the dry load sufficient just to mobilise it. Next, glass wool was pushed on top of the loaded column and eluent added carefully before inserting the reservoir into the top of the column which was left to develop. Separation was deemed complete when the Sudan blue just exited from the column. At this point the silica gel was extracted from the column using a suitable plunger onto a sheet of Whatman 3MM chromatography paper. OO21-9673/88/$03.50
6
1988 Elsevier Science Publishers B.V
NOTES
The various components could be detected under short-wave length UV light and simply sliced out to be eluted with a suitable solvent or the silica column could be sliced into equal portions and each extracted with methanol for TLC analysis. The extruded gel must not be allowed to dry otherwise it collapses into a fine powder. Flow-rate depended on the column diameter, volatility and mobility of the solvent system. The mobility of a solvent system can readily be gauged on a TLC plate and slow moving systems, such as propanol and butanol, could not be used as described here. Very suitable solvents include methanol, ethanol, ethyl acetate and light petrolum (b.p. SO-100X$ RESULTS AND DISCUSSION
Fig. 1 shows the separation of 125 mg of a chloroform extract of acidified mycelium and medium of a culture of Peniciliium cyciopium known to produce cyclopiazonic acid mycotoxins. The silica gel column measured 30 x 2.5 cm and required 8 h for development. Fig. 2 shows a TLC analysis of the separation obtained on this column of silica gel. The RF differences between the original material {lane 8) and separated material (lanes O-7) are presumably due to the amount loaded and also to the difference in environment each component causes for the others.
Truncated 250 mQ
0I23-
Glass wool Load Fine Silica Gel (TLC) Extruded Gel Cut
4 5-
l+Cyclopiazonic Acid
67-
a-Cyclopiazonic Acid
Silicone Rubber Tubing
Fig. 1.A line drawing of a wet-poured TLC-grade silica gel 64 GF 29430 x 2.5 cm, with an extract (125 mg) from P. cyclopium partially separated using ethyl acetate-methanol-cone. ammonium hydroxide (7k25.5 v/v/v) as solvent. The numerals indicate the cut sections of the extruded gel that were extracted witd methanol and analysed as described in Fig. 2.
445
NOTES FRONT
Original
0
I
2
3
4
5
6
7
8
Fig. 2. A line drawing copy of the analysis by Merck silica gel 60 GF,,, TLC plates of the column chromatography described in Fig. 1. The mobile phase was ethyl acetatePmethanol-conc. ammonium hydroxide (60:35:5, v/v/v) and the components detected under UV at 2.54 nm.
Fig. 3 shows a similar investigation by TLC of the results achieved in the separation of the components of nutmeg oil on a gel column 25 x 1.2 cm using light petroleum (b.p. 8&100”C)-diethyl ether (80:20, v/v)_ Here the time required for separation was only 3 h, and even so a fast moving component was lost. It was this loss that prompted the use of a fat-soluble dye as a marker for the front and for column performance. The components were located under UV at 254 nm and cut from the extruded gel to be eluted with methanol. The relationship between the RF values on TLC plates and columns was investigated (the column RF values were calculated using Sudan blue as the reference FRONT 1
,_-. ,---.\ \,’
I :*__,,
I
s.__,’
0 1
1
2
3
4
5
7
6 i
8
L
ORIGINAL
Fig. 3. A line drawing copy of the analysis by Merck silica gel F,,, high-performance TLC plates of the column chromatography of the constituents of the oil of nutmeg. The mobile phase was light petroleum (b.p. SO-100”Cj-diethyl ether (80:20, v/v). The shaded area represents iodine uptake and the clear area is absorption at 254 nm-
446
NOTES
Fig. 4. The separation of the dyes of 175 mg black ink on a column of TLC-grade silica gel 60 GF,,, (38 x layer chromatographic plate (20 x 20 x 0.2 cm: I of the 2.5 cm) and of 17.5 mg black ink on a preparative ammonium hydroxide (60:35:5, v/v/v). same silica gel. The mobile phase was ethyl acetate -methanol-cone.
TABLE I VALUES OBTAINED BY TLC OF THE SECONDARY METABOLITES OF P. CYCLOPIUM HIGH-PERFORMANCE SILICA GEL PLATES (10 x 10 cm) AND ON 400 x 25 mm COLUMNS OF TLC-GRADE SILICA GEL PACKED WET
R, ON
Abbreviations: A = a-cyclopiazonate; B = fl-cyclopiazonate; C = cycloacetoacetyltryptophanyl; D and E = unknown. R, values
Columns Plates
Ethyl acetate-methanol-cone.
ammonium
70:25:5, v/v/v
60.35.5, v/v/v
A(0.57), B(0.46), C(O.28),D(O.10). E(0.05) A(0.39), B(0.38). C(O.18),D(0.06), E(O.03)
A(0.80), B(0.67), C(O.39),D(O.l5), E(O.10) A(0.44), B(0.32), C(O.22),D(0.09), E(0.05)
Average ratio of R, (column) to R, (plate) 1.60 f 0.18
hydroxide
1.87 f 0.15
front) and the results are shown in Table I. While the RF values are not the same between plate and column, a reasonably constant ratio is maintained. Thus, unlike traditional column chromatography on coarser gels, one can fairly accurately predict where the metabolite should be located. One can use TLC plates to investigate a mixture for the best eluent and translate this directly into a column technique. Though we have described an isocrfatic use, the column technique should lend itself to stepwise elutions. The effectiveness of the method is portrayed in Fig. 4 where the dyes of black ink are shown separated on a TLC silica gel column and, for comparison, on a preparative layer chromatographic plate (20 x 20 x 0.2 cm) of the same silica gel, i.e. 60 GFz54; the column was loaded with 175 mg ink compared to 17.5 mg on the plate. Though the ink load on the column was ten times that on the preparative plate the volume of silica gel used in the column was 190 ml compared to 80 ml on the plate. Thus the ratio of load to silica gel was 0.92 for the column and only 0.22 for the plate, and yet the plate was being used almost to its maximum capacity. The reasons for the difference probably lie in the ease of loading of the column, which gave a more uniform band, and the longer development time for the column (16 h) compared to the plate (2 h). The main advantages of this wet packing compared to dry packing of columns are that it is easier to do, it gives more reproducible results, and can be used for alkaline solvent systems. On the other hand it cannot be used for low-mobility solvent systems when dry packing can. And copmpared to preparative layer chromatography it is cheaper, easier to load, simpler to reclaim the separated components, and calcium sulphate can be omitted since no binding is needed, and it takes a heavier load of sample. REFERENCES 1 J. J. Dunn, L. S. Lee and J. W. Bennett, Biotech. Lett., 2 (1980) 17. J. W. Bennett, F. G. Kromberg, L. A. Goodman and M. A. Seltman, Mycologica,
2
75 (1983) 202.