Method for the Separation and Determination of Theophyllin, Theobromine, and Caffeine ANGUS J. SHINGLER The Coca-Cola Co., Atlanta, Go.
JACK
K. CARLTON
Division of Sciences, Louisiana State University in New Orleans, New Orleans, la.
b
The purpose of this investigation was to develop a rapid procedure for the accurate determination of caffeine, theophyllin, and theobromine in mixtures of the three. By using a gradient e1ut.m partition chromatographic technique and by determining the purines spectrophotometrically, accurate resuits were obtained in the 10- to 2500-p.p.m. range. Caffeine, theophyllin, and theobromine occur together in many foods and beverages. This method does not alter the chemical structure of the purines; therefore they may be identified qualitatively after separation.
S
paper chromatographic methods have been described for the separation of caffeine, theobromine, and theophyllin (S? 3, 7), and spertrophotometric methods for the deterniination of catreine and/or theobromine. The purpose of this investigation vas to develop a rapid procedure for the accurate determination of caffeine, theobromine, and theophyllin in mixtures of the three. The method employs a gradient elution partition chromatographic technique similar to that of Marvel and Rands (5)in which butanolEVERAL
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enriched chloroform solutions are used to develop a water-saturated silicic acid column. Subsequent to the separation, the isolated purine was determined spectrophotometrically.
This agrees well with recently reportfci kalues of this constant ( 4 ) . Absorbance measurements of theobromine and theophyllin were made on solutions in which the solvent was 5% butanol in materwashed chloroform. This solvent wah used because in the chromatogrq n.i separation it was found to elutr theobromine and theophyilin, and iz nas convenient to measure absorbanc*+tctirectly on the riuate. Theophyiiin e:.hibits absorptivity of 575 a t 276.5 mp and conforms to Beer's la\\ in the range from 0.60 to 1.50 nig. prr 100 mi. Theobromine exhibits absorptivity of 537 a t 276.5 nip and conforms to Bwr's law- in the range from 0.60 to 1.50 nigprr 100ml.
APPARATUS AND MATERIALS
Cary Model XIV Spectrophotometer (Applied Physics Corp.) Nitrogen (National Cylinder Gas Co.) Chromatographic tube 68 cm. long and 1.5 cm. in diameter (a 100-ml. buret was used for this work) Mallinckrodt silicic acid (Ramsey and Patterson) Chloroform, ACS, analytical reagent (Mallinckrodt Chemical Works) Butanol, reagent (J. T. Baker Chemical Co.) Caffeine, USP Theobromine (Matheson Coleman RBell Division) (6971) Theophylliii (Matheson Colcnian RBell Division) (7094)
Preparation of Partition Column. With a mortar and pestle, mix thoroughly 20 grams of dry silicic acid and 12.5 ml. of distilled watei. The amount of water may vary aceorciing to the initial n-ater content of silicic acid. Add enough waterwashed chloroform to form a slurry; if a 100-mi. buret is to be used for the tube, wret a small wad of rotton with chloroform and drop it into thr buret to
EXPERIMENTAL
Caffeinr, theobromine, and t h e e phyllin were purified by sublimation, the latter two at 7-mm. pressure. Purified caffeine dissolved in materwashed chloroform (100 ml. of chloroform washed with 10 mi. of water) exhibited maximum absorbance at 276.5
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FIACTION IUYBLR (5d.h.GUl.)
Figure 1. Typical separation of caffeine, theophyllin, and theobromine
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the range from 0.25 to 4 . ~
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Figure 2. Separation showing how peak effluent volumes change with concentration VOL 31, NO. IO, OClOBER 1959
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support thr silicic acid column. Wash the slurry into the buret and remove air bubbles by working the slurry carefully with a long stirring rod (6). Drive excess chloroform from the bottom of the column with nitrogen, exercising care not to let the level of the chloroform go below the top of the column. If the column is allowed to become dry, reslurry the silicic acid and repeat the packing procedure. Drop a circle of stiff filter paper, the diameter of the tube, onto the top of the coiumn. Now wash the column with rhloroform until no absorbance is reg:strcd by the eluate a t 276.5 mp. About 35 ml. of chloroform are required for this step, and it should be discarded afterward. Add about 3 ml. of chloroform to the top of the column and close the stopcock. Mix thoroughly 2 ml. of aqueous sample solution with 3.2 grams 3f silicic acid (1) (or in the same proportion of water to silicic acid as used in column preparation) and introduce this dry sample onto the top of the column carefully, adding niinimal quantities of chloroform to ensure a wet column a t all times. When sufficient chloroform has been added to form a slurry, stir gently to el iminnte bubbles. Work the sample into the column by adding small volume increments of chloroform and driving the liquid headinto thecolumn after each addition. Take care that the column does not become dry while driving these additions into the column. Use the first 10 mi. of eluate as the spectrophotometer blank. Collect the eluate in &nil. fractions until ail caffeine has passed from the column. This is indicated by finding no difference in absorbance when the sample is meamred at 310 mp and 276.5 mp. The number of milliliters of chloroform needed for complete elution depends in part upon the silicic acid, the water it contains, and the amount of chloroform used to introduce the sample onto the column, as well as
Table
1.
Added, Mg.
Quantitative Results with Known Solutions
Found, Mg.
?&
Caffeine 65.0 10.0 65.7 253.0
10.0
64.6 10.4 65.9 257.4 9.96
99.4 104.0 100.3 101.7 99.6
Theophyllin 22.1 20.0 9.85 20.8 1.64
20.9 19.2 9.9 20.7 1.64
94.6 96.0 100.5 99.5 100.0
Theobromine 75.8 10.0 29.0 22.6 1.13
73.1 9.5 28.5 21.9 1.08
96.4 95.0 98.2 96.9 95.6
the amount of caffeine in the sample. When 'absorbance measurements show that caffeine has been completely eluted (Figures 1 and 2), pour off the excess water-washed chloroform and change the solvent to 5y0 butanol in chloroform. Wash the latter solution with water (10 to 1 solvenbwater) immediately prior to we. W i n g the absorbance at the same wave lengths as before, but with 5y0 butanol in chloroform in the reference cell, elution is continued until first theophyllin and then t h s bromine have been stripped from the column (Figores 1 and 2).
using volrinies called for in the method. By gentle evaporation, so as not to sublime the ingredients present, satisfactory results may be obtained in the microgram range. The differenae in absorptivity of the purines in the water-washed chloroform and the 5% water-washed butanolchloroform mixtures was large enough to suggest the use of the latter as a spectrophotometric blank when measuring the absorbance of the purines in that mixture. For example, theobromine exhibits an absorptivity of 542 in chloroform as compared to 537 in the 5% butanol-chloroform solutions. Both silicic acid and butanol were found to contairi occasional absorbing impurities which rendered them unsuitable when dealing with very small quantities of purines. Distillation of the butanol over potassium carbonate reduced the absorption of the solvent below an objectionable level, but washing the contaminated silicic acid with chloroform proved to be of small value. LITERATURE CITED
(1) Bulen, W. A., Kames, J. E., Burrell, R. D., ANAL.CHEM.24, 187 (1952). (2) Eisenbrand, S., Pfeil, D., 2. anal. C h . 151. 241-58 (1956). (3) Fleische;, G., Pkunnazie 11, 248-54 (1R.Ml. \ - - - - I -
RESULTS AND DISCUSSION
(4) Jones, M. S,, Thatcher, R. L., ANAL. CHEM. 23,957 (1951). (5) Marvel, C. S., Rsnds, R. ID., J . Am. Chem. Soc. 72, 2642-6 (1950). f6) Rankin. W. L.. ANAL.CHEM.28. 288 (1956). ' (7) Teshima, I., Matauurx, I., Trukai, Y . , Iclukawa, Y . , Research Repk. Nagoya I d . Sci. Research Inst. No. 8 , 62-3 (1955).
Results in the milligram range, summarized in Table I, are satisfactory
RECEIVED for review February 16, 1959. Accepted June 1, 1959.
.
Photometric Determination of Antimony and Thallium in Lead C. L.
LUKE
Bell Telephone Laboratories, inc., Murray Hill, N. J .
b Traces of antimony and thallium in iead can be determined by separating the metals from the bulk of the lead by coprecipitation with manganese dioxide and then determining them by photometric rhodamine B methods.
of antimony or thallium in Iead can be determined by photometric rhodamine B methods after removal of the lead as sulfate (3,5). To avoid adsorption losses, however, RACES
it is preferable to separate the trace metals from the lead by coprecipitating them in dilute nitric acid solution with manganese dioxide (2,6). Because the optimum conditions for this coprecipitation and for the photometric determinations are not adequately covered in the literature, recently developed methods employing these techniques are described. REAGENTS
Standard Antimony Solution (10
y
of antimony per ml.). Dissolve 0.2743 gram of potassium antimonyl tartrate in 1 liter of water. Dilute 100.0 ml. of this solution to 1 liter. Standard Thallium Solution (10 7 of thallium per ml.). Dissolve 0.2346 gram of thallous chloride or 0.2608 gram of thallous nitrate in about 300 ml.of warm water. Cool and dilute to 1 liter. Dilute 50.0 mi. of the solution to 1 liter. Manganese Nitrate Solution. Dilute 2 ml. of manganese nitrate (50% solution) to 100 ml.
