Column Chromatography Isolation of Caffeine Downloaded via UNIV GENT on January 30, 2021 at 09:26:44 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. Douglass F. Taber and R. Scott Hoerrner University of Delaware, Newark, DE 19716 Column chromatography is the technique commonly used for routine purification of synthetic intermediates in organic chemistry research laboratories. We report an undergraduate laboratory procedure using this technique.1 In the first semester of the sophomore organic laboratory course, it is common to do an experiment with qualitative thin-layer chromatography. We have for many years used the separation of the common analgesics acetominophen, aspirin, phenacetin, and caffeine.2 We have now found that chromatographic purification of caffeine, extracted from tea,3 fits well with this experiment. Both procedures (qualitative TLC of analgesics, column chromatography) can be carried out within a single 3-h laboratory period, if the TLC determinations are done at slow periods of the extraction (e.g., while the hot aqueous extract is cooling). We currently take two laboratory periods and include quantitative determination of the same “unknown” analgesic mixture, using high performance liquid chromatography.4 Experimental Extraction Distilled water (40 mL) in tapping on the side of the column to free air bubbles. After the silica gel is settled, it is covered with another 0.5-cm layer of sand. The residue from the extraction (above) is added to the top of the column. The solvent is allowed to drop to the level of the upper layer of sand. The evaporating flask is rinsed with an additional 5 mL of CH2CI2, and this also is added to the top of the column. Eluant from the column is collected in test tubes, held in a rack. It is important to keep these tubes in order. The column is eluted with 5 mL each of 5%, 10%, 20%, and 40% ethyl acetate/CHLCL, followed by 5 mL of pure ethyl acetate. After each addition, the level of the solvent in the column is allowed to drop to the level of the upper layer of sand, and the column is switched to a new receiving tube. If elution is too slow, gentle air pressure can be applied to the fop of the column. Be sure to remove the air pressure before the solvent drops below the level of the sand. The contents of each test tube are checked by TLC, with comparison to an authentic sample of caffeine. Several tubes can be spotted on the same plate, if care is taken. It helps to mark the spotting points lightly with a pencil. If the column has worked properly, early yellow fractions (evaporate 4 sample, and smell it!) will be followed by colorless fractions that contain caffeine. Combine in a tared round-bottom flask the fractions that contain caffeine, evaporate the solvent on the rotary evaporator, and weigh the fluffy white residue. About 65 mg of caffeine should be obtained. concurrent 125-mL Erlenmeyer flask containing two tea bags is brought gently to a boil (foams!). After 2 min the heat is removed, and the solution is allowed to cool (may be packed in ice). The cooled aqueous extract is then extracted with CH2CI2 (4 X 10 mL). The CH2CI2 extracts are combined, dried over Na^SCL, and concentrated on the rotary evaporator (note: the distillate, and all other organic residues, should be retained for proper disposal) at reduced pressure until they just become cloudy (5-10 mL remains). a Chromatography eluting solvent Commercially available open glass tubes, 19 mm i.d. X 10 cm, drawn down at one end to a drip tip, were used (figure). A small wad of glass wool is tamped down in the bottom of the column, then covered with a 0.5 cm layer of sand. Silica gel (0.7 gm, 60-200 mesh, “silica gel for chromatography”) is added in a thin stream,5 with sand A very nice procedure for the purification of carvone by simple column chromatography was reported several years ago: Mitchell, R. H.; West, P. R. J. Chem. Educ. 1974, 51, 274. Despite this, isolation of a natural product by preparative column chromatography has apparently been considered to be too tedious to include in the first semester 1 of the beginning organic laboratory course. 2 Moore, J. A.; Dalrymple, D. L.; Rodig, O. R. Experimental Methods in Organic Chemistry, 3rd ed.; Saunders: Philadelphia, 1982; pp 7289. We have found that 2:9:9 n-butanol/methyl isobutyl ketone/ethyl acetate works well for TLC separation of the analgesics. We use TLC plates having fluorescent indicator, with UV detection of the analgesics. 3 Wilcox, C. F., Jr. Experimental Organic Chemistry: A Small-Scale Approach: Macmillian: New York, 1988; p 96. We have found that the CH2CI2 extract from coffee is too thick to apply to the chromatography column. We have not yet investigated other sources of caffeine (e.g., soft drinks). 4 Kagel, R. A.; Farwell, S. O. J. Chem. Educ. 1983, 60, 163. (b) Haddad, P.; Hutchins, S.; Tuffy, M. J. Chem. Educ. 1963, 60, 166. 5 The procedure outlined here, suitable for small-diameter columns, is a modification of the usual approach: Targett, N. M.; Kilcoyne, J. P.; Green, B. J. Org. Chem. 1979, 44, 4962 and references cited therein. silica gel sand glass wool plug 10 mLtest tube Silica gel chromatography. Note that with the 1.9-cm-i.d. column, it is not necessary to make provisions to stopper the column at the bottom. Column flow stops when the eluting solvent reaches the upper layer of sand. Volume 68 Number 1 January 1991 73
