A Cheap and Practical Procedure
for Medium
Performance Liquid Chromatography
J. Chem. Educ. 1988.65:903. Downloaded from pubs.acs.org by UNIV OF GOTHENBURG on 01/23/19. For personal use only.
Christophe Morin Laboratoire d’Etudes Dynamiques et Structurales de la Selectivity, University Scientifique, Technologique et Medicale de Grenoble, BoTte Postale 68, 38402-St. Martin d'Heres, France
Preparative chromatography is frequently used by the synthetic organic chemist and often remains a decisive step in product purification. High-performance liquid chromatography, HPLC, (1-3) is now a major tool for this purpose, but its use may be somewhat restricted due to high investments of time and operation costs for nonroutine purifications. Several compromises between these investments and efficiency have been proposed, thus leading to medium-performance liquid chromatography, MPLC (4-10). A version is now presented which combines readily available equipment, short running time, and reasonable efficiency. This is obtained by use of a short column (its diameter being greater than its height) of silica gel H (calcium sulfate-free), using uacuum for both packing and elution. Although similar systems have been alluded to (11,12) or described (13,14), credit for the use of vacuum must certainly be attributed to the recognized discoverer of chromatography, Tswett, who as early as 1906 connected chromatographic columns to vacuum suction flasks (15). On the other hand, preparative techniques using silica gel H for large-bore columns were proposed some 15 years ago (16). A combination of several of the above observations has led to a fast and easy MPLC procedure that has now found daily bench use and that has been successfully applied to various fields: plant extracts or crude reaction mixtures purifications (carbohydrates, nucleotides, antibiotics, heterocycles, etc.). Selection of the Column Size As a general rule, the ratio of absorbent to the amount of material to be purified should be less than usual practice.
Selection of Chromatographic Conditions* Amount of silica ge! 60 H weight (g)
volume (mL)b
3 5
12
20 30 40 50 60 80
7.5 10
12.5 15
20 25 50 75 100 125 175 250
Column diameter (cm)
100
200 300 400 500 700 1000
3 3 4 4
4 5 5 5
Crude malerial to be purified
weight (g)
0.1-0.25^ 0.2-0.5* 0.5-0.75 0.7-1 0.9-1.2 1.2-1.5 1.5-2
6 8 8 10 10 13
2-3 3-5 5-7 7-10 9-12 10-15 20-50c
3
These figures are meant to be indicative. bThis is for unpacked dry silica gel; about 25-30% contraction occurs upon packing. c Due to the quantities involved, a deviation from the usual crude material/adsorbent ratio (1/10-15) can be observed. dTo keep proper elution rate, minimal vacuum is applied in this case.
The quantity of silica gel used should be 10 to 15 times the weight of sample applied. The diameter of the packed silica column should be 1-2 times its height. It is essential that the column be cylindrical (see figure). Dry silica gel 60 H (15-nm particle size) has an apparent density of ~0.25, and it is convenient to measure by volume (see table).
Volume 65
Number 10
October 1988
903
mixture to be separated so as to get Rf in the 0.3-0.7 range helps in the determination of adequate eluting conditions. s
cylindrical glass column packing aid {flat bottomed) _excess solvent
filter paper si/ica gel sintered glass to vacuum rubberadapter glass adapter ground-glass neck
receptor
Diagram of the column.
Preparation of the Column The silica gel is slurried and homogenized in a beaker with an excess of the starting solvent system (usually pure cyclohexane or dichloromethane). It is then poured into the column (porosity of the sintered glass: 2 or 3) and gently stirred to liberate any trapped air bubbles. Afterwards it is set aside for 10 to 15 min making sure that the level of the liquid is horizontal. Two layers can then be observed: moist settled silica gel with excess solvent above. From this stage up to the completion of the chromatography, one should be careful never to let the column run dry. The column is then connected to the vacuum line (see figure) and the water aspirator is opened, slightly at first, then more and more fully. Care must be taken, especially at the beginning, not to disturb the top of the silica gel when adding fresh solvent. As the solvent runs through, and as full vacuum is applied the silica becomes more and more packed. A piece of filter paper is then placed on top and the silica gel is pressed down (with a packing aid: for example, a flat-bottomed piece of glassware—see figure) more and more firmly until it becomes very hard. The column is ready for use. Choice of Solvent Systems tor Elution Although many solvents can be used, the following have been found satisfactory: cyclohexane, dichloromethane, methanol, and their combinations; they have less toxicity and display simple residual NMR absorptions. Other combinations include cyclohexane/ethyl acetate and, for more polar compounds, ethyl acetate/acetone/water (keeping a 4/1 ratio between acetone and water). Running a TLC of the 904
Journal of Chemical Education
Sample Application As for classical columns, the sample can be deposited in two ways: adsorbed onto dry silica gel 60 or dissolved in small volumes of the starting solvent. In both cases it should be spread as evenly as possible on the surface of the filter suction is paper. Either natural gravity or gentle vacuum used for the sample to penetrate the upper part of the silica in case of liquid application. This is the only delicate part of the whole process, as no air should be introduced into the column. Running the Column Full vacuum is then applied, which enables proper flow rates (when quantities of material to be purified do not exceed 100 mg—a lower practical limit—the flow rate is lowered to 10-20 mL/min, due to the column size). Starting with a pure solvent the eluting power will be increased to the following A/B ratio: 100/0, 99/1, 98/2, 95/5, and 90/10. Fractions of a volume roughly equal to the volume of packed silica gel are collected (no special precautions are to be taken when breaking vacuum; the lower part of the column is sucked dry when low-boiling/high-volatility solvents are used, but this is without major interference with the separation). Usually one fraction (or at most two) per eluting system is collected, which amounts to a total of five to seven for the whole chromatography, a key feature of this process. Sometimes bands are clearly visible. These have horizontal baselines if the chromatography is properly run; it is therefore advantageous to use this visual information to make appropriate fraction cuts. These fractions are collected in vessels that can be directly connected to rotatory evaporators (see figure). Quantities that have thus been separated range from 100 mg to above 50 g. This has taken less than an hour (overall time), the limiting step being evaporation of
the fraction volatiles. Quite often no further purification is necessary. The main advantages of this system lies in the fact that no special equipment is necessary. Furthermore, surprisingly good results are obtained when one considers the “roughness” of the procedure. The above system is a compromise; it is hoped that its rapidity, low cost, and reasonable efficiency will speak for itself.
Acknowledgment IOCD (International Organisation for the Chemical Sciences in Development) (17-19) is thanked for interest and financial support. C. Pougault is thanked for her assistance in drawing and C. Williamson for streamlining the English version of the manuscript. This paper is dedicated to the memory of Pierre Crabbe, founder of IOCD. Literature Cited 1.
Bidlingmeyer, B. A.; Warren, F. V., Jr. J. Chem. Educ. 1984, 61,716.
2.
Sitrin, R.; De Phillips. P.; Dingerdissen, J.; Erhard, K.; Filan, J. LC.GC 1986, 4, 530. Verzele, M.; Dewaele, C. Preparative High Performance Liquid Chromatography, A Practical Guideline; TEC: Gent, 1986. Loev, B.; Snader, K. M. Chem. Ind. (London) 1965, 15. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 13, 2923. Meyers, A. I.; Slade, J.; Smith, R. K.; Mihelich, E. D.; Hershenson, F. M.; Liang, C. D. J.
3. 4. 5. 6.
Org. Chem. 1979,44, 2247.
Loibner, H.; Seidl, G. Chromatographia 1979, 12,600. 8. Targett, N. M.; Kilcoyne, J. P.; Green, B. J. Org. Chem. 1979,44, 4962. 9. Taber, D. F. J. Org. Chem. 1982, 47,1351. 10. Thomson, W. J.; Hanson, B. A. J. Chem. Educ. 1984,61, 645. 11. Ravi, B. N.; Wells, R. J. Aust.J. Chem. 1982,35, 129. 12. Babin, D.; Fourneron, J.-D.; Julia, M. Bull. Soc. Chim. France 1980, (2), 588. 13. Pelletier, S. W.; Chokshi, H. P.; Desai, H. K. J. Nat. Prod. 1986, 49,892. 14. Coll, J. C.; Bowden, B. F. J. Nat. Prod. 1986, 49, 934. 15. Tswett, M. Ber. Deut. Bot. Ges. 1906, 24, 384 + Table XVIII. 16. Godbille, E.; Devaux, P. J. Chromatog. Sci. 1974,12,564. 17. Seaborg, G. T. Science 1984, 223 (Jan. 6), 9. 18. Chem. Eng. News 1984, Nov. 19,8. 19. L'Actual, Chim. 1986, Mai, 38. 7.
