lowing upper limits of A ; 350 for the NDR and 300 for the Jaffee and Sakaguchi reactions.
Table 1.
Urinary Excretion of Creatine and Glycocyamine
Glvco-
RESULTS
A recovery of 85 to 105% of the amount of creatine or glycocyamine added to urine was achieved by applying the isolation and colorimetric procedure described above. The recovery of creatine was checked by an independent method involving the addition of 1000 c.p.m. = 1 pg. of creatine-2 C14 to the volume of urine used routinely. The CI4activity recovered upon completion of the isolation procedure represented 88 to 102y0 of the initially added activity. By determining the specific activity of creatine isolated as described, it was ascertained that under the conditions employed and with the use of the equation for computing the concentration of creatine, only creatine was determined. The evidence for this assertion rests upon the finding that the specific activity remained unchanged upon rechromatographing in the solvent system butyl acetate-acetic acid-ethanol-water 3:2: 1:l (v:v:v:v) (7). The standard error of the mean for duplicate determination of creatine is 12% for concentrations lower than 35 pg. per ml. and 8% for higher concentrations. The standard error of the mean for duplicate determination of glycocyamine is 10%. The method described was applied to measurements of the excretion of creatine and glycocyamine in healthy as well as creatinuric subjects. Representative values for the 24-hour excre-
Creatine, cyainine, Mg./24 Mg./24 Hr. Hr. Normal (male)
57 35 42 31 28 52 46 47 ~~~
Normal (female) Accidental ex osure to ionizing raclation (male)
Radiation therapy for malignant disease (female)
37 52 48 ~. 35 21 64 35 37
340 875 580 490
45 38
235 385
65 69
..
..
under the alkaline conditions of the NDR, this secondary reaction constitutes the most important source of interference. The method described incorporated two feasible means of eliminating the contributions from creatinine-namely, a partial separation of creatine and glycocyamine, by which 80 to 90% of the creatinine as well as methylguanidine and arginine is removed, prior to colorimetry, and an independent determination of the amount of creatinine contaminating the creatine-glycocyamine fraction isolated as described. The independent determination of glycocyamine is designed to correct for the contribution of this compound to the A obtained with the NDR. REFERENCES
tion of creatine and glycocyamine in the urine of normal and creatinuric individuals are presented in Table I. The range of levels of creatine found in the urine collected over 24 hours from healthy and creatinuric subjects was 20 to 60 and 80 to 1000 mg., respectively. The daily excretion of glycocyamine in this group ranged between 30 and 70 mg. Since the procedure provides for the exclusion of factors interfering with the NDR, the values of creatine are lower than those usually given by other investigators. Since about 10% of the creatinine present may be converted to creatine
(1) Anderson, D. R., Williams, C. M., Krise, G. M., Dowben, It. M., Biochem. J . 67,258 (1957). (2) Chung, W., Arch. .Biochem. Biophys. 85,416 (1959). (3) Dubnoff, J. W., J . Biol. Chem. 141, 711 (1941). (4) Eden, E., Harrison, D. D., Linnane, A. W., Australian J. Expll. Biol. Med. Sci. 32, 332 (1954). (5) Ennor, A. H., Stocken, L. A., Biochent. J. 55,310 (1953). (6) ~, Folin. 0.. Wu. H.. J. Biol. Chem. 38, 81 (1919).' ' ' (7) Masamune, H., Yosizawa, F., TGhoku, J . Exptl. Med. 59, l ( 1 9 5 3 ) . (8) Raaflaub, J., Abelin, I., Biochem. Z . 321, 158 (1930). (9) Taussky, H., Clin. Chim. Acta 1, 210 (1956).
RECEIVEDfor review April 28, 1960. Accepted March 13, 1961. Work supported in part by a grant from the U. s. Atomic Energy Commission.
Irradiation Fluorometric Method for Estimation of Diethylstilb-estrol in Beef Liver Tissue J. M. GOODYEAR and N. R. JENKINSON Control Division, Eli Lilly 8 Co., Indianapolis, Ind. b The specific measurement of diethylstilbestrol in biological samples is difficult because of the absence of selective biological methods and sensitive chemical procedures. An irradiation fluorometric method, developed recently, allows estimation in the parts per billion range. This paper presents additional information on its application for the estimation of diethylstilbestrol. The method includes recovery of added diethylstilbestrol and compensates for extraneous fluorescence by use of a split sample at the final residue stage. Extraction steps in the chemical method were verified with C14-diethylstilbestrol recovery experi-
ments. Added diethylstilbestrol is recovered from tissue at approximately the 50% levei. The statistically designed experiment includes an interlaboratory study of added diethylstilbestrol standard to control tissue from different animals.
T
isolation of diethylstilbestrol from cattle liver tissue is based on methods for cattle feed (1, 3) with significant modifications in the preparation of the tissue prior to extraction and in the initial extraction. A double purification a t different p H values and final ether extraction are incorporated. HE
A recovery of 96.1% C14diethylstilbestrol at a concentration of 5 p.p.b. was obtained from a pure analytical system. This proved an efficient and practical method of purification; it was decided to use the procedure for liver samples. The total amount of diethylstilbestrol added to the pure system was 2.50 pg. A pure system is identical with the sample system, except that distilled water is substituted for the tissue. The labeled experiment with C14-diethylstilbestrol a t a concentration of 76.2 p.p.b. added to liver involved two initial extraction procedures and one purification method. An exhaustive extraction with chloroform in a Soxhlet VOL. 33,
NO. 7,
JUNE 1961
853
sample. The difference between these two fluorescent intensities is a measure of diethylstilbestrol. Table I demonstrates the consistent fluorescence background obtained with Animal irradiated radiated DifNo. Portion Portion ference different beef liver control tissues and the difference between the irradiated 200-Gram Sample Size and nonirradiated portions of the 326" 27.0 25.5 -1.5 sample. The majority of the experi25.0 +2.0 33ga 23.0 34.0 mental runs with control tissue show a 3400 31.0 - ~. ~ .+ 3 . 0 25.0 i3.0 860 22.0 ljackground fluorescence slightly greater 22.0 +3.0 870 19.0 than the reagent blank. Table I1 21.5 +1.5 870B 20.0 demonstrates the background fluores25.0 $4.0 914 21 .o cent intensities obtained from the re+4.5 19.5 24.0 933 946 19.0 21.5 +2.5 agent blank, while Table I11 indicates the consistent recovery of added di400-Gram Sample Size ethylstilbestrol to control liver ho291 27.0 31.0 +4.0 292 25.0 27.0 f2.0 - mogenates a t the 10-p.p.b. level. With control tissue, the difference between a All others .were steer a Heifer livers. preirradiation reading and an irradialivers. tion reading remains approximately the same. These observations indicate that Table 11. Background Fluorescent control tissue is not required. Reading of Reagent Blanks High preirradiation readings may be due t o faulty manipulations in the exNonIrirradiated radiated Diftraction of diethylstilbestrol from tissue. Run Portion Portion ference Degradation of extraneous fluorescence 11.o 0 1 11.0 may also occur upon irradiation when 2 15.0 18.0 3 high preirradiation readings are ob3 11.o 13.0 2 tained. An increase of ex%raneous 4 13 .O 16.0 3 fluorescence due to the addition of KOH 5 14.0 16.0 2 6" 12.0 72.0 60 is fully compensated by this method. 140.0 +126 7b 14.0 Three factors are involved in arriving a t Standard recovery of 1.0 pg. of didiethylstilbestrol values in liver tissue : ethylstilbestrol through pure system in 5 incorporation of the increment standml. of redistilled alcohol S.D. 3A. ard, the use of a split sample of the same b Standard from ether a t concentration residue, and the use of KOH to shift the of 2.0 pg. per 5 ml. of redistilled alcohol S.D. 3A. fluorescence spectrum. No significant differences in extraneous fluorescence background have occurred between Table 111. Recovery of Added sexes of the animals examined. Diethylstilbestrol at 10 P.P.B. Since most solvents display some from Liver Homogenates extraneous background and occasionally % show inhibitory effects on the standAnalysts Recovery Means ard, it is necessary t o standardize daily 54.8 AI 54.2,55.3 with reagents currently in use. The A2 50 .O,53.5 51.8 variation of the ultraviolet source is 57.8 AS 60.5.55.0 compensated by simultaneously ir50.1 A; 52.2; 48.0 radiating the sample, the sample plus standard, and direct standard. Preliminary data in regard to excitation and emission were obtained by the use of a n extractor and the reflux method deAminco Spectrophotofluorometer ; howscribed in this paper were examined. ever, fluorescent measurements on all After the extracts were processed liver samples were obtained with a filterthrough the complete purification procetype fluorophotometer. The characterdure, the count on the final residue inistics of the fluorophor from diethyldicated 60.9% recovery by the reflux stilbestrol have been described (2). method and 82.5% by Soxhlet extracExtraneous fluorescence excites a t the tion for 16 hours. same wave length as shown in Table I ; T o detect gross differences which may however, the background fluorescence occur in the isolation of diethylstila t a n excitation of 360 mp is three times besterol because of extraction difficulties the fluorescence a t 410-mp excitation. and tissue variation, it is advisable to The inhibitory effect from samples in obtain preirradiation and irradiation the irradiation step is not serious. It values before adding alkali. These is not dependent on background fluoresreadings are not used in calculating final cence intensity. C14-diethylstilbestrol diethylstilbestrol concentration but recovery experiments indicated meserve as an indicator of final residue puchanical loss rather than irradiation rity. All fluorescent measurements used product loss. for calculations are made on the alkaliChloroform has been a satisfactory treated solutions of both the irradiated solvent for many years in this labornand nonirradiated portions of the split Table 1. Background Fluorescent Readings of Liver Tissue NonIr-
++ + ++
854
ANALYTICAL CHEMISTRY
tory for the extraction of diethylstilbestrol from various samples. However, in tissue analysis, chloroform transfers a highly fluorescent substance from liver to the final residue. For this reason ether was selected for extraction at the final step from aqueous solution at p H 9.5. However, ether contributes a n inhibitory substance in the irradiation step. The substance is removed by three water washes of the final ether solution. This treatment produced consistent standard recoveries of the fluorophor from ether. The use of alcohol as an irradiation medium is satisfactory, provided one does not retain the alcohol solutions for more than 8 hours before irradiation. Apparently, diethylstilbestrol in alcohol a t these low concentrations decreases in activity in respect to the irradiation method. All standard stock solutions are first prepared in chloroform, because chloroform solutions appear to be more stable for this test. When the alcohol direct standard indicates low fluorophor yields, the output of the arc lamp is diminishing in the radiation required for photochemical conversion. EXPERIMENTAL
Apparatus. A Coleman Model C fluorophotonieter was modified to accommodate 2.5 ml. of sample for measurement of fluorescent intensity. The primary filter for excitation in the 410-mp range is a combination of Corning Nos. 3391 (mith a minimum transmittance of 37% betneen 400 and 416 mp) and 5850. The secondary filter, Corning N o . 3486 or 3385, transmits wave lengths above 510 mp. This filter system fulfills the requirements as dictated by previously published spectrophotofluorometric observations. A type such as the Hanovia analytical lamp, a high-pressure arc lamp without filter, is used for the photochemical conversion. The secondary standard is prepared from the U.S.P. XVI riboflavin reference standard. A concentration of 0.10 pg. of riboflavin per ml. in mater is satisfactory to set the instrument at 100 fluorescent intensity units. All solutions of riboflavin must be protected from direct light and the final working solution should be discarded after each test. Riboflavin was selected because i t excites in the 410-mp region. Calibration of Irradiation Time. (Caution. Protect the eyes from direct rays of ultraviolet light.) Prepare a standard solution which contains 0.40 pg. of diethylstilbestrol per ml. n i t h redistilled S.D. 3A ethyl alcohol, Transfer 5 ml. of this solution to four separate quartz tubes and irradiate at different time intervals a t a distance of 6 inches from the arc lamp. Examine a t 30-second i n t e r d s t o determine which time is required to develop masimum fluorescence from the standard solution. Select quartz tubes on the baqis of their ability to reproduce maximum fluorescent intensities.
Preparation of Tissue. Hoinogcnize a 200-gram s:~ni~ilcof liver tissue in a suit~alilc blender. Blend the homogenized liver with 150 ml. of a 1 t o 1 mixture of ethyl alcohol in chloroform for approximately 2 minutes a t high speed. Transfer this mixtu,rF: quantitatively to shallow aluminum or glass container. Place the containcr on a steam bath and evaporate the excess alcohol and chloroform mixture. Divide the liver honiogcnate while on the steam bath into small squares n i t h :t spatula. Cover the sample \\ itli a suitable mntcrial and dry in a vacuum ovcn a t 30" C. for 16 hours. Preparation of Internal Standard. Prcparc n n additional 200-gram sample of tlic same liver as tlcsc.rihed aiiovr. Atid a standard chloroform scilution of dicthylstilbcstrol crluiv:ilcsnt to 2 fig. to the honiogenizcd liver. I5lmd for 2 minutes before addition of tlw 1 to I mixture of ethyl alcoEiol and cliloroform. From this point, process the intcrn:il standard sample as in the preparation of tlic sample. Extraction Procedure. Transfer the di,icd powder to a suitable blender and niis until a fine powder is cbtained. ?'iansf(lr qilantitatively b y thoroughly Iinsing t h e container with small portions of chloroform. Transfer all washings to thc blender jar. Place a n additional amount of chloroform equivalent to a totnl of 250 mi. in the blender and further mix for 5 minutes a t high spccd. Transfer this mixture to a 600ml. b w k r r and actively boil on a steam bath for 1 hour. Cool the suspension of liver in chloroform and filter through paper with the aid of vacuum. Wash the filter cake with approximately four 100-ml. portions of chloroform t o ensure removal of extracted diethylstilbestrol from the liver tissue. Evaporate the clear filtrate to an approximate volume of 100 ml. on a steam bath with the aid of filtcwd air. Purification Procedure. Place t h e 100 ml. of chloroform obtained from the above evaporation in a 250-ml. separatorg funnel. Extract the chloroform with two 20-ml. portions of 1N sodium hydroxide, by very gently swirling the separatory funnel and inverting it while the liquid phases are in motion. Retain t,lie emulsion a t this stcp. Combine the sodium hydroxide extracts and the emulhion and wash with chloroform. Repeat the washings with the same swirling action as described above unt'il the chloroform is waterclear. Transfer the sodium hydroxide solution to a 250-ml. beaker and adjust the i)II i o 8.0 with 3 S phosphoric acid. Place the pH-adjusted solution in a clean 250-nil. separatory funnel and extract with thrw 25-ml. portions of cliloroform. This extraction may be shakcn vigoroiiply, since no emulsions will form at this step. Combine the chloroform fractions and extract with t\vo 15-ml. portions of IN sodium hydroxide. Combine the sodium hydroxide solutions and wash with chloroform as in the first stcp. If any emulsion is present in the alkali solution after b h i g washcd, transfer only the emulsion to a 150-rnl. brnkrr. 1q:vapo-
Table IV.
Results of lnterlaboratory Study
Sample
Levcl, P.P.B.
No. 933
Laboratory
Laboratory
1 2 2 3.2,2.5 2.6,2.9 946 2 3.2,2.5 1.9,2.6 933 8 6.6,6.4 9.3,7.3 946 8 8.1,5.9 6.3,7.0 Rcwlts expressed as parts per billion in wvct livcr tissue.
rate off chloroform in the emuliion on a steam bath with the aid of filtered air. Combine the alkaline solution n ith the small portion of sodium hydroxide in the beaker. Adjust the pH to 9.5 at this step with 3N phobphoric arid. Return this solution to the separatory funnel and extract with three 30-ml. portions of anhydrous ethyl ether. Place a 250-ml. separatory funnel in a horizontal position on a steam bath. Transfer a volume of standard chloroform solution equivalent t o 2 p g . to the separatory funnel for evaporation of the chloroform. Then add approximately 90 ml. of ether to the separatory funnel to dissolve the residue. Wash the ether n i t h three equal volumes of water. This renreseiits the standard from ether. Fill three coarse sintered-glass filter funnels with a 1-inch bed of anhydrous sodium sulfate. Wash each bed n i t h approximately 40 ml. of water-washed ethyl ether. Discard the ether aashings of the filter bed. Filter the waterwashed ether extracts of all samples through the previously prepared filter beds into beakers previously rinsed with anhydrous ether. Carefully evaporate the ether in each beaker to dryness on a steam bath with the aid of filtered air. Irradiation Procedure for Samples. Dissolve t h e residues obtained from the above purification procedure in exactly 5 ml. of redistilled S.D.3A ethyl alcohol. Transfer a 2.5-ml. aliquot of this solution to a quaitz tube. Transfer the remainder to a cuvette for fluorescent intensity measurement. This is the prcirradiation reading. Irradiate the sample in the quartz tube for a n optimum time predetermined by irradiation of the standard in alcohol. Transfer the irradiated solution to a cuvette and measure the fluorescent intensity of the sample before adding pota,,'w u m hydroxide. Make all sample5 alkaline n i t h 0.05 ml. of 111' potaqqiuni hldrox-
Table V.
idc. Mix the alcoholic solutions tliorouglily and allow to stand 10 minute5 bcfore recording the fluorescent intrnsity. The difference in reading obtained on a nonirradiated sample plus KOH and on the irradiated sample plus KOH is a measure of the diethylstilbestrol aetivity. Calculations. T h e nonirradiated aliquots of the final solution reprcsent nonspecific fluorescence which is subtracted from the fluorescence intensity obtained on t h e irradiated aliquots of the same solution. Diethylstilhestiol activity is calculated in parts per billion in the sample by t h e us^ of the increment standard. Per crnt recovery is calculwtid b y dividing the direct standard from etlier into the value obtained by tlit, incremcnt method. The standard addctl to the liver samples is equivalent to 0.40 p g . of dicthylstilbcstrol per ml. of final solution. STATISTICAL RESULTS
The data in Table IV were obtained from a collaborative interlaborstory study designed to evaluate the procedure statistically. An analysis of variance indicates no significant difference at the 5% level between laboratories, days, or liver samples to which the known quantitics of diethylstilbestrol were added. The relative standard deviation a t both the 2- and the 8-p.p.b. level was 1 1 6 % . A test of the hypothesis that the mean of each set of the data is equal to its theoretical value suggests that the hypothesis is correct for the 8-p.p.b. level but that a small positive bias is indicated a t the 2-p.p.b. level. Previous observations (Table I) have indicated such bias as a consistent small fluorescent increment observed in the comparison of nonirradiated amd irradiated extracts of control livvr samplcs. This bias also exists, of course, in the analysis of samples having 8 p.p.b. added diethylstilbestrol, but the magnitude of the standard deviation is such that it is obscured in comparison of mean observed value to thcoretical value. All of the above values werc obtained using a sample size of 200 gr:ims. The small residual positive contribution
Confidence Intervals about the Mean
s o . of
Replicate Runs 8 8 2 2
Theory, P.P.B. 2 8 2 8
Observed
95730 CIa 2.67510.356 7.112f0.930 2 . 6 7 5 0.712 7.112*1.860
*
Range 2,32-3.03 6.18-8.04 1.96-3.39 5.25-8.97
O C I = L S
di' VOL. 33, NO. 7, JUNE 1961
855
to the fluorrscwiw is rcslntivoly independent of sample size (compare column 4 of Table I to Table 11); conscquontly, if one has thc facilitiw for scaling u p the extraction process to accomrnodatc 400gram tissue samples, the certainty of results in the 2-p.p.b. range may be increased significantly. It is of some interest to set up 95% confidence limits on the reported mean diethylstilbestrol content of samples as determined by these experiments (Table
tivity. The combined use of the split sample technique, incremrnt standard, and a pH changc offers in the above described method a suitable procedure for the correction of extraneous fluorescence and me:mircmcnt of diethylstilbestrol in biological samples. Applications of this mcthod to t h r drtermination of trace quantities of diethylstilbestrol in other biological material are being considered for future publication.
17).
ACKNOWLEDGMENT SUMMARY
Photochemical reactions offer a high degree of specificity as well as sensi-
The authors thank R. Q. Thompson and F. A. Smith for confirmatory data and technical contributions, M. M.
Marsh for consult:ttion, and H. L. Breunig for the design and st:Ltistical analysis. The authors rtpprcciatc the combined efforts of the Agricultural Products Assay Dcpartmcnt and Analytical Development Department in obtaining experimental data. LITERATURE CITED
(1) Cheng, E. W., Burroughs, W., J . Assoc. Ogic. Agr. Chemisk 38, 147 (1955). (2) Goodyesr, J. M . , Jenkinson, N. R., ANAL.CHEM.32, 1203 (1900). (3) Munscy, V. E., J . Assoc. O h . Agr. Chemists 41, 316 (1958).
RECEIVED for review Septembcr 27, 1960. Accepted February 1, 1961.
Spectrofluorometric Determination of Total Bile Acids in Bile SAMUEL J. LEVIN, J. LOGAN IRVIN,' and CHARLES G. JOHNSTON Deportment of Surgery, Wayne State University College of Medicine, Detroit, Mich The method of Minibeck has been modified to permit the accurate spectrofluorometric determination of total bile acids in bile. This procedure is applicable to the mixtures of bile acids found in the bile of human beings and various experimental animals.
T
HE BILE ACIDS of the bile of human beings and various experimental animals consist principally of mixtures of the glycine and taurine conjugates of mono-, di-, and trihydroxycholanic acids (7). The determination of total bile acids in bile has been a difficult procedure which usually has required the summation of separate analyscs for several different bile acids. Although it is important to have analytical procedures which are specific for the individual bile acids, there is a need for a method which will permit the rapid determination of total bile acids in a single analysis. The method of Doubilet ( 4 ) is of uncertain specificity and lacks sensitivity for the determination of milligram and microgram quantities of bile acids. Previously dcscribed spectrophotometric (6) and fluorornctric (IO) methods for bile acids yield low recoveries with the dihydroxycholanic acids, and they arc not suitable for the determination of total bile acids in unresolved mixtures due to marked differences in absorbance and fluorescence of various bile acids Present address, Department of Biochemistry, University of North Carolina, School of Medicine, Chapel Hill, N. C.
856
ANALYTICAL CHEMISTRY
.
in concentrated sulfuric acid under the conditions previously described. In this paper thc method of Minibcck (IO) is modified to permit accurate fluorometric determination of total bile acids in mixtures of di- and trihydroxycholanic acids, and the method is applied in the determination of total bile acids in bile. EXPERIMENTAL
Sources and Purification of Bile Acids. Pure cholic a n d deoxycholic acids were obtained by repeated crystallization of commercial samples (Nutritional Biochemicals Corp.). Chenodeoxycholic acid was synthesized according to the method of Fieser and Rajagopalan (6). Glycocholic, glycodeoxycholic, glycochenodeoxycholic, and the sodium salts of taurocholic, taurodeoxycholic, and taurochenodcoxycholic acids were prepared according to the methods of Cortese et al. (1-3). Thc purity of all bile acid samples was confirmed by chromatography according to Sjovall (I.?) and by determination of characteristic physical constants and properties. Reagents. Peroxide-frce ethyl ether. Extract reagent grade ethyl ether with 2% ferrous sulfate solution, d r y over anhydrous calcium chloride, and distill. T h e ether will remain free of peroxides for several weeks if stored under nitrogen gas in a dark bottle. Some freshly opened samplrs of reagent grade ether are free of peroxides (starch-iodide test) and can be used without purification. Standard cholic acid. T o prepare a stock standard solution of cholic acid,
dissolve 25.00 mg. of cholic acid in reagent grade absolute ethyl alcohol. and dilute the solution with this solvent to 25 ml. in a volumetric flask. Prepare working standards by suitable dilution of the stock standard with reagent grade absolute ethyl alcohol. When stored in a refrigewtor and protected from evaporation, the standard solutions can be kept for sweral months without change. Procedure. H Y D R O L YAND S I ~ EuTRACTION OF BILE SAMPLE. Mix 1.0 ml. of bile with 5 ml of absolute ethyl alcohol (reagent grade) in a test tube. Cover t h e tube with a glass bulb, and heat the contents at the boiling point for 5 minutes to denature protein. Cool the tube, and filter the contents into a 50-ml. Rockefeller tube. Wash the original tube and the residue on the filter paper twice with 5 ml. of hot absolute ethyl alcohol, and collect the washings in the Rockefeller tube. Attach the latter to a Rinco vacuum evaporator, and evaporate the contents to dryness. T o the residue add 5 ml of 1.25N sodium hydroxide, close the mouth of the tube with a cotton plug or a loosely fitting aluminum cap, and heat the tube in a n autoclave for 3 hours a t 15 pounds pressure. Cool thc tube and acidify the contents to p H 4.5 with approximately 3 ml. of 2.1N sulfuric acid. During this acidification, test the pH by touching small drops of the solution to Nitrazine paper with a pointcd glass rod. Transfer the solution quantitatively to a separatory funnel with several washings with distilled water such that the final volume in the separatory funncl is 15 ml. Extract the solution with three 15-ml. portions of peroxide-free ethyl ether. Combine the ether ex-
