1214
ANALYTICAL CHEMISTRY of 15 to 30 p.p.m. Since the maximal concentration of calcium fluoride in water a t 25" C. (16) wouId yield only 8.7 p.p.m. of fluorine, the high concentrations of fluorine in the lysimeter leachings from quenched phosphate furnace slag have been explained by assuming that the outgo is calcium silicofluoride, rather than the simple fluorides (8, 9, 10).
Table 111. Determination of Fluorine in Lysimeter Leachings"
Water Sample
Dilution before Determination
Fluorine. P . P . N . Colorimetrically after Contact with Perchloric acid -4gents distillation of 1 hour 24 hours clarified natersb
LITERATURE CITED C: 13 red
1952 leachings 1 1 9
^^
30.0 30.0
30.0 30.0
... ... ...
a From soils into which various fluorides had been incorporated. b The findings by the perchloric acid distillation and thorium nitrate titration procedure (19) were obt,ained by Mary Hardison.
( 5 - t r ) 20-io1 I ) were made against standards with 0.1 p.p.m. intervals. Consideration was given to the precipitative action that the additive calcium sulfate might exert upon the fluorine of high concentrations in the lysimeter leachings. This possibility was tested by allowing a 24-hour contact between calcium sulfate and the leachings that carried fluorine concentrations of 22 and 30 p.p.m. and by comparing the results with those obtained on dilution of the 30 p.p.m. sample, prior to the addition of the sulfate. I t was assumed that both factors-extension in duration of contact and increase in fluorine concentration-11-ould enhance the fluoride precipitation. The results of Table I11 show that after the 24hours' contact, calcium sulfate had caused no decrease in the fluorine recovery for any concentration in the range
(1) Am. Pub. Health Assoc., New York, "Standard Methods for the Examination of Water and Sewage," 9th ed., p. 76, 1946. (2) Am. Pub. Health dssoc., Committee Report, J . A m . Water W o r k s Assoe., 33, 1936 (1941). (3) Association of Official .kgricultural Chemists, Washington, D. C., "Official Llethods of Analysis," i t h ed., 1950. ( 4 ) Emerson, P., SoilSci., 12,413 (1921). ( 5 ) Harper, H. J., I n d . Ena. Chem., 16, 180 (1924). (6) Hester, J. B., Va. Truck Expt. Sta., Bull. 82 (1934). (7) Lamar, Wm. L., IRD.ENG.CHEM.,AXIL. ED.,17, 148 (1945). (8) MacIntire, W. H., and Sterges, A. J., J . Agr. Food Chem., 1, 370-8 (1953). (9) IIacTntire, W. H., and Sterges, A. J., Soil Sci., 74, 233-47 (1952). (10) .,MacIntire, W. H., Winterberg, S.H., Clements, L. B., Jones, L. S.,and Robinson, Brooks, I n d . Eng. Chem., 43, 1797-9 (1951). (11) hlorgan, 11.F., Conn. Agr. Expt. Sta., Bull. 450 (1941). (12) Reitemeier, R. F., IND.ERG.CHEX.,ASAL. ED.,15, 393 (1943). (13) Sanchis, J. M., Zbid.. 6 , 134-5 (1934). (14) Schreiner, O., and Failyer, G. H., U. S. Dept. dgr., Bur. Soils, Bull. 31 (1906). (15) Scott, R. D., J . Am. Water W o r k s Assoc., 33, 2018 (1941). (16) Seidell, A, "Solubilities of Inorganic and Metal Organic Compounds," 3rd ed., Yew York, D. Van Nostrand Co., 1940. (17) Smith, H. V.,IND.ERG.CHEY.,ANAL.ED.,7, 23-5 (1935). (18) Walker, 0. J., and Finlay, G. R., Can. J . Research, 18, 151-9 (1940). (19) Willard, H. H. and Winter, 0. B., IND. EXG.CHEM.,ANAL.ED., 5 , 7-8 (1933). (20) Wolf, B., Ibid., 15, 248 (1943). RBCEIVED for review April 24, 1952. Accepted February 24, 1951.
Spectrophotometric Determination of Theobromine And Caffeine in Cocoa Powders DUANE T. ENGLIS and JAMES W. MILES Department o f Chemistry, University o f Illinois, U r h a ,
T
HE existing methods for the determination of the allraloids in commercial cocoa powders are used upon their extraction from the plant materials by the use of aqueous (2-4, 6, 9, 12, 15, 17, 18, 20) or organic solvents (7, 11, IS, 14, 16, 19, 21). Critical studies of some of the latter methods of Kay and Haywood ( 1 1 ), Moores and Campbell (18), Holmes ( 6 ) , and others have indicated that complete extraction is usually not obtained with organic solvents. RFoores and Campbell ( 1 8 ) and Holmes ( 6 ) have demonstrated that the cocoa alkaloids can be evtracted quantitatively by the use of hot water in the presence of magnesium oxide. After the extraction of the caffeine and theobromine from the plant material, the resulting solution must be purified before the alkaloids can be determined. When the extraction is made with aqueous solvents, the initial purification is usually accomplished by the addition of a clarifying agent such as mercuric acetate, lead subacetate, or zinc ferrocyanide to remove soluble protein and other interfering substances. The clarification iq usually followed by treatment of the clarified extract with an organic solvent to isolate the alkaloids. 1 Present address, College of Pharmacy, University of Kentucky, Louiswlle. Ky.
111. Various methods have been proposed for the final determination of the alkaloids following the purification steps. These include gravimetric ( 3 , 4, 7, 12, 16, Z l ) , iodometric ( 5 ) ,Kjeldahl (17, 18), acidimetric ( l ) ,and electrometric ( 1 8 ) methods. T o date no spectrophotometric methods for the determination of caffeine and theobromine in cocoa samples have been presented, although they have been determined spectrophotometrically in other products (8, 10). The absorption peaks of high intensity exhibited by theobromine and caffeine in the ultraviolet region (Figure 1) suggested their use as a basis for a very sensitive method for their determination in cocoa samples. The purpose of this investigation was to find a procedure for the isolation of the alkaloids from cocoa samples suitable for the quantitative determination of these substances by a spectrophotometric method. PROCEDURE FOR DETERMINATION OF THEOBROMINE
Apparatus and Materials. Thiamine adsorption tubes, standard type. The column is 9 mm. in diameter and 15 cm. long. The reservoir should have a capacity of about 30 ml. Spectrophotometer, Beckman Model DU, with ultraviolet accessories. Cells are 1-em. quartz. Sodium hydroxide, approximately 0.1N.
V O L U M E 26, NO. 7, J U L Y 1 9 5 4
1215
Sulfuric acid, approximately 0.1N. Hydrochloric acid, approximately 1.On.. Zinc acetate, 1-M. Dissolve 220 grams of zinc acetate, C.P. [Zn(C2H302)2,2H20]in water and make to 1 liter with water. Potassium ferrocyanide, 0.25M. Dissolve 106 grams of potassium ferrocyanide, C.P. [K4Fe(CN)6.3H20lt in water and make to 1 liter with watei. Rlaenesium oxide. U.S.P. heavv. CeGe 545. Fuller’s earth. English Superfine XL, obtained from L. A. Salomon Bros., 216 Pearl St., New York, N. Y. Method. PREPARATION OF ADSORPTION TUBES.Place a small plug of glass wool in the bottom of the tube and cover with about 5 mm. of Celite. Pour in a dry mixture containing 0.55 gram of fuller’s earth and 1.45 grams of Celite. Fit the tube over a suction flask and apply vacuum to pack the column. Release the vacuum and pour about 25 ml. of water into the reservoir of the adsorption tube. After the water has penetrated the length of the column by gravity, apply vacuum again and draw the water through the column until the level is only 1 or 2 mm. above the top of the column. Never allow the water level to fall below the top of the column. The column is now ready for the adsorption step. I
I\
250
300
WAVE LENGTH I N hi4
Figure 1. Ultraviolet .4bsorption Spectra 1. Theobromine i n 0.1N hydrochloric acid 2. Caffeine i n chloroform Concentrations 25 mg. per liter Cell length 1 cm.
EXTR.4CTION O F THE A L X 4LOIDS. Weigh accurately 0.2 to 0.3 gram of cocoa powder into a 150-ml. beaker. Add 0.1 gram of magnesium oxide and 0.5 gram of Celite. Mix with these materials a small amount of water to make a smooth paste, then add 50 ml. of water and boil for 20 minutes. Filter the hot solution on a small Buchner funnel and return the residue and filter paper t o the beaker. Extract twice again with 25-ml. portions of water, boiling for 10 minutes each time and filter as before. R a s h the last residue with hot water and combine the filtrates. Keutralize the combined extracts with 0.1N sulfuric acid to a pH of 6.5. Evaporate to a volume of about 40 ml. and trnnsfer to a 50-ml. volumetric flask. ildd 1 ml. of zinc acetate and 1 ml. of potassium ferrocyanide solutions. Mix well by shaking and make to volume with water. After several minutes, filter on a fluted filter. Discard the first few milliliters of filtrate. Pipet 25 ml. of ADSORPTIOX A N D ELUTION OF THEOBROMINE. the filtrate into a thiamine adsorption tube prepared as described ahove. Apply vacuum and adjust the rate of flow so that the 2.5-ml. portion passes through the column in 10 to 15 minutes. Wash the column with 10 ml. of distilled water, replace the suction flask with a clean flask, and elute the column by drawing 7 . 5 ml. of 0.1.V sodium hydroxide through. The elution step should require about 5 minutes. Transfer the eluate to a 100-ml. volumetric flask, add 10 ml. of 1.O.V hydrochloric acid, and make t o volume with water. SPECTROPHOTOMETRIC DETERMIN4TIOS O F T H E O B R 0 3 I I h E . Place some of the solution from the above step in a 1-cm. quartz cell and observe the absorbancy a t 272.5 and 310 mp. Subtract the value a t 310 mp from that a t 272.5 mp to obtain a corrected absorbancy value. Refer t o a calibration curve for theobromine in arid Eolution to obtain the concentration in the final
solution. Calculate the weight of theobromine in the final solution and multiply by 2 to obtain the weight in the original sample. DISCUSSION OF METHOD FOR THEOBROMINE
The method for extraction and purification described above was adopted after an exhaustive study of existing methods which were considered applicable for the isolation of theobromine from small samples. Except for the spectrophotometric determination, the method represents a modification of existing procedures. Two factors led to the decision to use the aqueous extraction technique. Recent workers (6, 11, 18,) have demonstrated that these methods are superior to methods employing organic solvents, and water is one of the most satisfactory solvents for ultraviolet absorption studies. The percolation method of lloores and Campbell (18) was used for the extraction of some samples and even though good results were obtained, i t was found that the batch extraction method of Dekker ( 4 ) was more expedient. I n both processes, magnesium oxide was added to ensure complete liberation of the theobromine. Holmes (6) was able to extract the theobromine completely from a 10-gram sample by refluxing three times with 250-ml. portions of water. I n the proposed method, the volume of water used, based on equal weights of sample, is about five times the amount used by Holmes. In order to prove that extraction was complete, one of the samples was subjected to a fourth extraction by boiling with 25 ml. of water. The filtrate was made to 100 ml. with l.OdV hydrochloric acid and examined in a Cary recording spectrophotometer, hlodel 11. S o absorption peak was observed in the region of 272.5 mp, although some general absorption was observed throughout the whole ultraviolet region (220 to 310 mp). Even though this was good evidence of the absence of theobromine, in order to prove the point, another sample was treated in the same manner and the fourth extract was collected. This solution was made slightly acidic (pH 6 ) with 0.LV hydrochloric acid and shaken out with five 20-ml. portions of chloroform. The combined chloroform extracts were made to 100 ml. and examined in a Beckman spectrophotometer. The solution showed no absorption a t 272.5 mp, thus proving the complete absence of theobromine in the fourth aqueous extract. The aqueous extract from cocoa powders contains some soluble protein and other objectionable plant materials. Several reagents have been proposed for use as clarifying agents to remove these substances. Ceriotti ( 3 ) used mercuric acetate, Holmes (6) used lead subacetate, and Moir and Hinks ( 1 7 ) used zinc ferrocyanide. Later, Moores and Campbell ( 1 8 ) found the ferrocyanide to be most satisfactory. They noted, however, that some loss of theobromine occurred in the clarification step owing to the adsorption of the alkaloid on the zinc ferrocyanide. In solutions containing about 250 mg. of theobromine per liter. they observed a loss of approximately 5% for solutions of pure theobromine of 3% for solutions obtained by extraction of cocoa powders. In order to determine if a similar loss of theobromine occurred in solutions containing smaller amounts of theobromine, the following experiment was conducted. Ten milliliter portions of a stock solution of theobromine containing 0.8 mg per liter were pipetted into five 100-ml. volumetric flasks. After the addition of about 50 ml. of water, 2 ml. of zinc acetate and 2 ml. of potassium ferrocyanide solutions were added with shaking. The solutions were made to volume with water and filtered after 3 or 4 minutes. The first few milliliters of the filtrates were discarded and 25-ml. portions of each 61trate were pipetted into five 100-ml. volumetric flasks. Ten milliliters of 1.ON hydrochloric acid were added to each flask and the solutions were made to volume. The theobromine content of the resulting solutions was determined from the absorbancy of each measured a t 272.5 mp. Tests were conducted which proved that the reagents used in the experiment did not contribute to the absorbancy a t 272.5 mp.
1216
ANALYTICAL CHEMISTRY
Table I.
Recovery of Theobromine from Solutions Clarified with Zinc Ferrocyanide
Theobroiiiine Taken, M g .
o.2t
Absorbancy a t 272.5 Mfi
Theobromine Recovered, Ng.
Theobromine Recovered,%
SAMPLE 3
I
= I
the ultraviolet region. The absorbancy of these solutions was on the order of 0.01 to 0.03. I t is thought that the general absorption observed in the eluates from the adsorption tubes is due to a suspension of finely divided particles of silica from the Celite. After this work, it was assumed that any absorbance observed at 310 mp was due to light scattering by fine particles from the column and the absoibancy observed a t 310 mp was subtracted from that at 272.5 mp to obtain a corrected value a t the latter wave length. Results obtained by this method Lvere very consistent. A4fterexperimenting with various columns, it was found that a column containing 0.45 gram of fuller's earth and 1.55 grams of Celite was adequate for the adsorption and elution of -1 to 6 nig. of pure theobromine. This represented the total theobromine content of 0.2- to 0.3-gram samples of average cocoa powders. Standard thiamine adsorption tubes were found to make ideal containers for the columns. -4 comparison of results obtained using large and small samples of the same cocoa powder is given in Table 11.
z
Table 11. Comparison of Results of knalyses \Iade Using 2.5- and 0.25-Gram Samples Size of Sample, Grams 2 500 0 2500
Theobromine Found % 2 22 2 24 2 24 2 23 2 19 2 25
Theobromine Found, A x erage
I 23 2 22
Table 111. Comparison of Analyses by (A) Moores and Campbell Method and (B) Proposed Method 250
370 WAVE LENGTH IN M P
Figure 2. Ultraviolet Absorption Spectra of Blank Solutions Passed through Fuller's Earth-Celite Columns
Results are given in Table I. From this experiment it \vas concluded that there was no loss of theobromine in the clarification step for solutions having this low concentration of the alkaloid. I n this case, the concentration of theobromine in the clarified soIution was 80 mg. per liter. Although other methods of separation were considered, the adsorption method of Moores and Campbell (18) appeared to be the most suitable for the spectrophotometric method. They established optimum conditions for isolation of the theobromine from 2.0- to 3.0-gram samples by trial and error methods. A number of analyses were carried out on 2.5-gram samples in order to duplicate the work of Moores and Campbell, even though the quantity of theobromine extracted was far greater than necessary for determination by the spectrophotometric method. The' eluate from one of the columns was made up to 200 ml. with water and 50 ml. of this solution were made to 250 ml. with O.IN hydrochloric acid. The resulting solution was examined Tvith a Cary recording spectrophotometer. The absorption curve was a perfect reproduction of that for pure theobromine and indicated the high degree of purity of the product obtained. A slight absorption was observed a t 310 mp for the eluted theobromine whereas pure theobromine shows no absorption a t this wave length. It was later found that this is characteristic of all solutions which pass through the fuller's earth-Celite columns. Experiments were conducted in which blank solutions were passed through the adsorption columns and their ultraviolet spectra examined. I n all cases, as illustrated in Figure 2, the absorption was found to be practically constant throughout
Theobromine, % ( D r y Basis) Theobromine .Iv. % Moisture, Method Xfetpd No. Found, % Theobromine % B A 170.4 2.46 2.50 2.48 2.48 7.48 2.68 2.68 170.5 2.18 2.14 2 35 2.46 2.14 2.15 8.46 170.6 2.52 2.50 2.80 2.48 2.50 7.80 2 71 4 .\nalyses from laboratory of General Foods Corp., Hoboken. 9. J.
Sample
PREPAR.4TION O F CAIJBRATION CTRVES FOR THEOBROMINE. A curve of the ultraviolet absorption spectrum of theobromine in 0.lN hydrochloric acid was prepared using a Cary recording spectrophotometer. The resulting curve, shown in Figure 1, exhibited an absorption peak a t 272.5 mp. For the preparation of a calibration curve, a sample of theobromine, N.F., was purified by sublimation. This was accomplished by placing the sample in a 500-ml. flask with a cold finger projecting into it. The system was sealed and vacuum was applied. The flask was heated on an electric hot plate and the sublimed crystals were collected on the cold finger. These crystals were recovered and dried a t 110" C. Solutions containing from 5 to 25 mg. per liter were prepared from the dry sublimed crystals and examined with a Beckman spectrophotometer a t 272.5 mp. The data, when plotted, showed excellent conformity to Beer's law. RESULTS. Some samples of cocoa powders, which had been analyzed by the Moores and Campbell method, were obtained from the General Foods Corp., Hoboken, S . J., and analyzed by the spectrophotometric method using 0.25-gram samples. Moisture determinations were made in order to compare results with the reported values which were based on dry samples. The results are compiled in Table 111. PROCEDURE FOR DETERMINATION OF CAFFEIhE
Apparatus and Materials. The equipment and many reagents used are the same as for the theobromine analysis. Additional reagents are listed below.
V O L U M E 26, NO. 7, J U L Y 1 9 5 4 Ammonium hydroxide, c.P.,28%. Chloroform, U.S.P., redistilled. Method. EXTRACTIOS OF ALKALOIDS.Weigh accurately about 1 gram of cocoa powder into a 150-ml. beaker and add 0.5 gram of magnesium oxide and 2.0 grams of Celite. Add enough water to make a smoot,h paste, then add 50 ml. of water and boil for 20 minutes. Filter the hot solution on a small Buchner funnel and return the residue and the filter paper to the beaker. Extract tnice again with 25-ml. portions of water, boiling for 10 minutes each time, and filter as before. Wash the last residue with hot water and combine the filtrates. Neutralize the combined extracts with 0.1.V sulfuric acid to a pH of 6 . 5 . Evaporate to a volume of about 75 ml. and transfer to a 100-ml. volumetric flask. -4dd 2 ml. of zinc acetate and 2 ml. of potassium ferrocyanide solutions. Mix well by shaking and make t,o volume with watei. After several minutes, filter on a flut,ed filter. Discard the first few milliliters of filtrate. SEPAR.4TION O F CAFFEIXE. Transfer 50 ml. Of the filtrate to a separatoiy funnel and add 6 nil. of concentrated ammonium hydroxide. In some cases a slight precipitate will form upon the addition of ammonia at this point. This will not interfere with the determination, but may contribute to emulsion foimation when the solution is shaken with chloroform. If this becomes ohjectionahle, i t is advisable to filter again and take a second aliquot hefore extracting with chloroform. rlfter making the solution hasic, extract ivith 25-, 20-, 1 5 , lo-, and 10-ml. portions of chloroform, shaking for 1 minute each time. Combine the chloroform extracts and viash with 10 ml. of 1N sulfuric acid. Filter through a small plug of cotton into a 100-ml. volumetric flask and make to volume n-ith chloroform. SPECTROPHOTOMETRIC DETERMISATION.Transfer some of the chloroform extract into a 1-cm. quartz cell and observe the absorbancy at 276 mp and a t 310 mp. Subtract the value a t 310 m p from that' a t 276 mp to obtain a corrected absorbance value. Refer to a calibrat'ion curve for caffeine in chloroform to obtain the concentration in the final solution. Calculate t.he weight of caffeine in the final solution and make the necessary correction for the aliquots taken to obtain the weight of caffeine in the original sample. DISCUSSION OF METHOD FOR CAFFEINE
As in the method for theobromine, the most applicable existing methods for extraction and isolation of the alkaloid were modified for use with the spectrophotometric determination. The batch extraction method of Dekker (4)n-as used along with the chloroform extraction method of Noores and Campbell (18) for removing the caffeine from t'he sample. E x T R a c ~ I o sA S D CL.~RIFICATIOXSTEPS. The method described above for the aqueous extraction of the sample was found t o afford complete extraction of the caffeine. This was proved by subjecting several samples to a fourth extraction and running a caffeine determination on the resulting solutions. S o traces of caffeine were found in the extracts, K O additional work was done to prove that the zinc ferrocyanide clarification did not interfere, as other workers had used it n-ith satisfactory results. Furthermore, samples of pure caffeine were recovered without loss after subjecting them to both clarification and liquid-liquid extraction steps. This indicated that no loss resulted from use of zinc ferrocyanide. CHLOROFORM SEPAR.~TIOS. Solutions containing known quantities of pure caffeine and theobromine were made basic with trisodium phosphate and extracted with chloroform according to the specifications of Noores and Campbell (18). Consistently high results were obtained n-hen the caffeine was determined spectrophotometrically in the chloroform extracts. Khen sodium hydroxide or ammonium hydroxide was substituted for trisodium phosphate, theoretical results were obtained. Furthermore, the use of ammonium hi-droxide had an added advantage in that filtrates from the zinc ferrocl-anide clarification formed no precipitate when this base !vas added. The same solutions yielded a precipitat,e of zinc hydroxide upon the addition of trisodium phosphate or sodium hydroxide. The formation of a zinc-ammonia complex kept the zinc in solution and made possible t,he elimination of one filtration step. Other than the substitution of ammonium hydroxide for trisodium phosphate, the hloores and Campbell procedure \vas found to be admirably suited to the isolation of t>hecaffeine for spectrophotometric determination.
1217
PREPARATION OF CALIBRATION CURVESFOR CAFFEISE IN CHLOROFORM. The absorption peak for caffeine in chloroform was found by observing the ultraviolet spectrum recorded with a Cary spectrophotometer. The curve for a solution containing 25 mg. per liter is shown in Figure 1. Pure caffeine was obtained by placing several grams of caffeine, U.S.P., in an evaporating dish covered by a watch glass and heating over a hot plate. The sublimed caffeine was collected from the watch glass and dried a t 110" C. for 1 hour. Chloroform solutions containin from 5 to 25 mg. per liter of the pure caffeine were examine8 with a Beckman spectrophotometer, Model DU. Readings were made a t 276 mp. A plot of absorbancy 2.s. concentration gave a straight line showing excellent conformity to Beer's law. RESLLTS. A series of commercial samples was taken for analysis. One-gram samples were used and aliquots of either one fourth or one half of the zinc ferrocyanide filtrates were extiacted with chloroform. A slight general absorption was observed a t 310 mp for both blanks and samples carried through the process. This was probably due to a slight turbidity of the chloroform extracts. These absorbancy values a t 310 mp, usually amounting to approximately 0.01, were subtracted from the readings at 276 mp to obtain an absorbancy value corrected for this general absorption. Results obtained for four samples are given in Table IV. These values m-ere lower than thoje obtained by the Moores and Campbell method on the same eamples. It !vas concluded that higher values by the Kjeldahl method may have been due to other nitrogenous plant products e\tracted with the chloroform. If any such extractives were present, they did not absorb light in the ultraviolet region. This vias demonstrated by recording the spectrum of the chloroform extracts from cocoa samples. The curves were identical to those of pure caffeine in chloroform.
Table IV.
Caffeine Analyses by the Spectrophotometric Method Averaee Or,
Ar. Sample No.
1912 1 : l
170 1
170 i 170
C,
Caffeine, "c 0 17 0 17 0 1R 0 18 0 46 0 0 0 0 0 0
4n 45 46
5%
Caffeine Bloisture,
70
Caffeine. (Dry'Basis) Spectrophotometric Kjeldahl
0.175
7 53
0 19
0 455
7 48
0 49
1 07
0 207
8 4fi
0 23
0 63
0 275
7 80
0 30
0 65
19
22 21 0 27 0 28
Several advantages may he cited for the spectrophotometric procedures as applied to cocoa pouder. As the intensity of the absorption of the caffeine and theobromine in the ultraviolet region permits the use of smaller samples, this makes possible the complete extraction of the slightly soluble constituents without use of large volumes of solvent. Much time is hereby saved in the extraction operations and subsequent evaporations. Cnder the conditions of operation, and with the more dilute solutions of the dissolved materials, no loss of theobromine is experienred in the clarification step involving zinc ferrocyanide. The spectrophotometric method seems specific for the determination of caffeine,as no other substances absorbing in the specified region appear to be present under the conditions of its separation. In contrast, the estimation of caffeine from the determination of total nitrogen by the Kjeldahl method would be subject t o error from the presence of any other nitrogen-containing materials. ACKYOWLEDGWlEVT
The authors wish to express their appreciation to R. G. Moores and H. A. Campbell of the General Foods Corp., Hoboken,
1218
ANALYTICAL CHEMISTRY
N. J., for providing the analyzed samples of cocoa. powders used in this investigation. LITERATURE CITED
(1) Boie. Heirrich. Pham. Z ~ Q75,968 ., (1930).
(2) Cappelli,G.,Ann.chim. appt., 14,254 (1924). (3) Cenotti, A., Anales a8m. pub. argentinn. 8 , 400 (1920). (4) Dekker. M. J., Rec. trau. chim.. 22,143 (1903). ( 5 ) Emery. W. O.,and Spencer, G. C., J . 7 d .Eng. Chem., 10, 605 (1918). (6) Holmes. K. E., Analyst. 7 5 , 457 (1950). (7) Humphries. E. C., 8th Ann. Rept. Cocoa Research, 7mp. Coll. Trop. Agi.. Trinzdad, 1938.36. (8) Ishler, N. H., Finucane, T. P., and Borker, Emanuel, ANAL.
CHEM.,20,1162 (1948). (9) Jalade, M., Ann.fals. etfmudes, 22,396 (1929).
(IO) Jones, Marie, and Thatcher. R. L., ANAL. CHEM.,23, 957 (1951). (11) Kay. J., and Haywood, P. J. C., Analyst, 71, 162 (1946). (12) Kunae, W. E., Z . anal. Chem., 33, 1 (1894). (13) Lowe, E. H.. Analysl, 73, 679 (1948). (14) Macdonald, J. A,, Ann. Rept. Cocoa Reseaxh, Imp. Coll. T ~ o P . Agr., Trinidad, 1936,43. (15) Martin, F., and Clerpue, H., Ann. chim. anal.,24, 202 (1942). (16) Maupy, L., Analyst. 22, 191 (1897). (17) Moir, D. D., and Hinks. E., Ibid.,60, 439 (1935). (1s) Moores. R. G.. and Campbell, H. A,. ANAL. CHEM.,20, 40 (1948). (19) Parks,A. E., andParks, H.A., Analyst. 62,791 (1937). (20) Pritzker, J.. and Jungkuns, R.. Mill. Lebensm. H y g . . 34, 185 (1943). (21) Wadsorth, R. V.,Analyst, 46,32 (1921). R ~ c n v for ~ oreview Ootober 27, 1953.
Accepted Maroh 20. 1954.
Partial Resolution and Detection of Some Cations by Differential Diffusion MARVIN ANTELMAN Maranter Laboratories, Ventnor City,
N. J.
ANALYTICAL
sep,arat? have been accomplished by the apphcatlon of diffusion techniques. In these procedures chemical potential gradients produce differential migration by diffusion (4). Uranium-235 is separated from uranium-238 for atomic purposes by allowing volatilieed uranium hexafluoride t o diffuse in multistage apparatus. Gaseous mixtures may be determined by microeffusiometry ( 1 ) . Porphyrins (a), azo dyes (S), radioactive ions (7), and ordinary eations (5, 6) have bsen resolved in solution by diffusionprocedures. The migration processes usually are performed in porous or gelatinous media in order t o prevent mixing. This paper describes the resolution of various cations by radial differential diffusion taking place in a concentrated gel of gelatin under the influence of a chemical potential gradient. No electric current is applied in the procedure. EXPERIMENTAL PROCEDURE
A thick concentrated gel was prepared by softening 16 grams of C.P. gelatin in 50 ml. of cold water and then adding 150 ml. of boiling water. Distilled water always was used. The gelatin was Domed into watch glasses (1.5 em. deep a t the center) and
his. pipet to the approxirhate center of the plates.
~~~
~~
paper. If the gels are alloivcd to stay for scveral days, they will harden. In most cases the concentric rings are still present in the hardened gel. The procedure of selective diffusion originally was attempted on gels formed in test tubes but failed to give good results. DISCUSSION
This diffusion procedure serves to resolve cations partially. When a cation mixture is $laced centrally on a gelatin surface, each component migrates under the influence of the chemical concentrations or potential gradient. The zone of each component extends from the starting position to the leading boundary. Each cation contaminates the zone of every more slowly diffusing cation; therefore, only a small fraction of the fastest migrating ion can be separated from others. Cross contamination of two and three cations, respectively, is illustrated schematically in Figure 3.
~~~~~
After B few minutes, the surfaces became dry, for the solutions had diffused through the gelatin. Developing reagents were added in a sufficient amount to cover the surface and to develop distinct cancentric rings. The developing may take from less than a minute to a n hour depending on the type of cations and the developing agents. Same cornbinations of cations and developing reagents failed to give good results. RESULTS
Many cation combinations were tested and are listed in Tables
I and IS. The difference in color of the contaminated aones and their position 8ewe t o identify the cations present in the mixture. The photographs show two partial seprtrations. Fignre 1 illustrates the psrtisl resolution of mercury(1) and lesd(1Ij from the middle and out developed with potassium iodide, whereas Figure 2 is the separation of iron(ISI), copper(SS), and cobalt from the middle and out developed with potassium ferrocyanide. Potassium iodide is superior to the chromate as a developer for group I metals, Potassium ferrocyanide is superior t o sodium phosphate as a developer of iron(SSI), copper(II), and cobalt ione. Results of Elective diffusion procedures may he recorded conveniently in colored pencil or crayon on polar coordinate
Figure 1. Partial Resolution of Me, Developed with Potassium I
