II
Addison Ault and Raymond Kraig Cornell College Mount Vernon, Iowa 52314
Isotopit Dilution Analysis Cholesferol in oallsfones
I
. 2
and caffein in tea, cola, and NoDoz
The procedures described here represent the results of one of our attempts to develop more interesting experiments in quantitative analysis. They illustrate the use of a diierent, obviously modern, and widely useful technique, and also involve the analysis of substances of considerable interest to students. Isotopic dilution analysis is a powerful modern method of quantitative analysis. The method normally involves addition to the sample being analyzed of a known amount of the compound for which the analysis is being performed,' the added sample containing a known concentration of isotopically substituted molecules. When the compound is then reisolated, the reduction of the concentration of the isotopically substituted molecules (dilution) is a measure of the amount of that compound originally present in the sample being analyzed; the lowcr the concentration (the greater the dilution), t6e more of the compound there must have been present originally in the sample. The exact mathematical relationship is easily derived using the fact that the number of isotopically substituted molecules is conserved (isotopic cone. of cpd added)(wt. epd. added) = (isotopic eonc. of cpd. recovered)(wt. cpd. added wt. cpd. orig. present)
+
Solving for the weight of the compound originally present in the sample wt. cpd. orig. present = (isotop~ecane. of cpd. added) (isotopiccone. of cpd. recovered)
{
1
(wt cpd added) '
'
The success of the method is seen to be independent of the amount of the substance isolated. It depends only upon knowing the weight of the isotopically substituted compound added and the ratio of the isotopic concentrations of the materials added and recovered. I n the experiments to be described, 14C-labeled compounds are used, and the ratio of isotopic concentrations is determined by means of a liquid scintillation counter. First a sample of the recovered compound is counted, and then a sample of the original addend is added and the sample is recounted. From these data, the desired ratio of isotopic concentrations may be calculated
wt. of recovered cpd. counted
With this procedure, the overall counting efficiency (machine efficiencyand quenching) is the same for each
sample, and the ratio of observed count rates per unit weight is the same as the ratio of isotopic concentrations. Procedure Cholesterol i n Gallstones.' Place a 2-g sample of crushed gallstones in a 50-ml Erlenmeyer flask. To this, add 1 g of "Clabeled cholesterol (see Note 1). Now add 17 ml dioxane and heat the mixture on the steam bath with occasional swirling for about 5 min. During this time, most of the solid in the flask should dissolve. While the mixture is still hot, filter off the brown residue of bile pigments, collecting the filtrate in a 50-ml Erlenmeyer flask. Dilute the filtrate with 17 ml of methanol, add some decolorizing carbon, and warm the mixture on the steam bath for a minute or two. While the mixture is still hot, filter off the carbon and collect the filtrate in a 50-ml Erlenmeyer flask. Reheat the filtrate on the steam bath, add 1 ml of hot water, and swirl to dissolve any precipitated solid. (If i t does not all dissolve, add a little methanol and heat with swirling until i t does.) Allow the solution to cool to room temperature, during which time cholesterol will separate as colorless plates. Collect the crystals of cholesterol by suction filtration, and wash them with a few milliliters of cold methanol. Leave thevacuum on for afew minutes to suck the crystals aq dry as possible. Recrystallize the cholesterol from methanol, using 35 ml/g. Count the recovered cholesterol both alone and in the Dresence of the orieinal addend (Notes 2 and 3). Caffein i n Tea.* Dissolve 100 mg of L'Glabeled caffein (Note 4) in 125 ml of water in a 500-ml Erlenmeyer flask with warming on the steam bath. When solution is complete, add 12.5 g of tea and 12.5 g of powdered calcium carbonate. Boil the contents of the flask wit,h constant stirring for about 20 min. At this time, filter the hot mixture with suction, and press out the liquid from the tea. leaves with a large cork. Transfer the filtrate to a 250-ml Erlenmeyer flask and add 100 ml chloroform. Swirl or gently stir t,he two layers together for about 10 min (Note 5), and then separate t,hem by means of a separatary funnel. Distill the chloroform layer until only about 10 ml remains, transfer the remaining solution to a tared 25-ml Erlenmeyer flask ( f i k e ~ ifg necessary) and evaporate to dryness on the steam bath. Recrystallize the solid residue of caffein from 9.574 ethanol, using 5 ml/g (Note 6). Count the recovered caffein both alone and in the presence of the original addend (Notes 2 and 7). Caffein i n Cola.8 Dissolve 100 mg 'Glabeled caffein (Note 4) in 50 ml of water in a 250-ml Erlenmeyer flask with warming on the steam hat,h. When solution is complete, add 50 ml cola syrup (Note 8) and 10 ml concentrated ammonium hydroxide solution. Mix the solution thoroughly, add 100 ml chloroform, and swirl or gently stir the two layers together for about 10 min (Note 5). Continue as in the isolation of caffein from tea. Caffein i n NoDoz. Dissolve 100 milligrams "C-labeled caffein (Note .~ 4). in 100 ml of water in a. 2.50-ml. Erlenmever flask with I P I, W O n .I I : I . \Vltcu - d $ 1 t i l ) 1 ~i~ I ~ O ~ ~ F :xdd e ' h Do, t,lIjIe!. n.l.ivlt huvn i,t.r.,n e r d w d a d wrirhwl. Hwr the 7 0 hiliug r o t > w >re. t , l ~ t r hc ~ ~ fthpcn-it,t~
'FIESER, L. F., "Organic Experiments," D. C. Heath and Co., Boston, 1964, p. 70. HELMKAMP, G. K., A N D JOHNSON, H. W., JR., "Selected EXperiments in Organic Chemistry," (2nd ed.), W. H. Freeman and Co., San Francisco, 1968, p. 157. RCLMKAMP A N DJOHNSON, op. cit., p. 158.
Volume 46, Number I I, November 1969
/ 767
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caffeinfrom tea.
Results
Notes 1) Fifty microcuries of Cholesterol-"C (Cholesterol-4-"C, Schwarts BioResearch, Inc., Cst. No. 6623-ORE) was added to a hot solution of 150 g cholesterol in 900 ml ethanol and 400 ml methanol. After addition of another 200 ml methanol, the solution was allowed to cool for crystallization, The resulting cholesterol, which cfystallized as long narrow blades, hes approximately the expected level of activity of 73,000 dpmper 100 mg. 2) The counting solution which was used consisted of 2.5 g 2,s-diphenyloxasale (PPO) dissolved in 500 ml of toluene. A Beckman Model LS-100 liquid scirit,illation counter was used. 3) Cholesterol is soluble in the counting solution to the extent of inore than 1 g/15 ml. Two hundred fifty-milligram samples of recovered cholesterol and 100-mg samples of original addend were used for eounting. 4) Fifty microcuries of eaffein-14C (CaKein Methyl-1-"C, Tracerlab, Cat. No. G172) was added to 2.5 g caKein during reervstallisi~tionfrom 375 ml ethanol. The resoltine caffein had ap"proximate1y the expected level of activity of 110$00 dpm per 25 mg. 5) )More vigorous extraction procedures often result in very troublesome emulsions. Gentle stirring of the mixture with a magnetic stirrer was quite satisfactory. The NoDoa extraction is least likely to farm an emulsion. 6) One recrystallisation from ethanol is sufficient and yield^ caffein in the form of small needles. A reasonable expected recovery of recrystallized caffein is about 2 5 5 0 % of the total present (original plus addend), depending upon the thoroughness of the extraction. The greenish color which is present in the case of the tea extraction can be completely removed by careful washing of the crysta4s with ethanol. 7) Caffein is soluble in the counting solution to the extent of
768
/
60 mg/15 ml. Twenty-fivemilligram samples of both recovered caffein and original addend were used far counting. 8). Peusi other caffein-containinp: . svruu was used:. ~resumablv . syrups would besnitable.
Journol of Chemical Education
Cholesterol in gallstones: Thirty-eight different runs were made. Since twenty-nine students made two trials in counting their samples, a total of 67 results was obtained. The average of the 67 results indicated a cholesterol content of 76%, with a standard deviation of 7.4%. The gallstones were obtained from The University Hospitals, Iowa City, Iowa. Caffein i n tea: Our sample of tea (William Whiteley, Ltd., London) contained approximately 400 mg caffein per 12.5 g, or about 3% by weight. Caffein in Cola: Our bottle of Pepsi syrup contamed about 33 mg caffein per 50 ml syrup. At the recommended rate of 1 fl. oz./glass, a glass of Pepsi should contain about 20 mg caffein. Caffein in NoDoz: We found a caffein content of about 95 mg per tablet; the label stated a caffein content of 0.1 g/tablet. Presumably, this method of analysis for caffein could be extended to other substances (instant coffee and tea. for example) with appropriate Godification; it should be very easy to interest students in doing this. We wish to acknowledge the assistance of Rachel Kopet and Arthur Serianz in working out some of the experimental details. We thank the Pfeiffer Foundation for funds for the purchase of the liquid scintillation counter.
