Rapid Spectrophotometric Determination of Caffeine N. H. ISHLER, T. P. FINUCANE,
AND EMAKUEL BORKER
Central Research Laboratories, General Foods Corporation, Hoboken, N . J . The characteristic absorption of caffeine at 272 mp is utilized to measure quantitatively its presence in coffees and crude caffeine. Interfering impurities found in these samples are removed by treatment with heavy magnesium oxide and zinc ferrocyanide, plus in some cases permanganate oxidation. Rapidity and specificity for caffeine are outstanding characteristics of the method. Results obtained by
the new method compare favorably with the BaileyAndrew procedure which is used as a reference for comparison. Detailed precision studies showing the new method to be equal to or better than the Bailey-Andrew method are reported, Decaffeinated coffees will require a modification of the methods herein described, which will probably involve a solvent extraction step.
T
H E operation of a coffee decaffeinating plant requires a great number of analytical caffeine determinations. For a number of years, the A.O.A.C. official Power-Chesnut (2) method was used in this laboratory. This method was satisfactory, but excessively time-consuming for control operations. In 1943, the Bailey-Andrew method (S), then official only for tea, was successfully adapted for use here by increasing the specified amount of magnesium oxide to 25 grams and by making suitable changes in sample weight to permit its application to decaffeinated products and solutions as well as to green or roasted coffee. This method has since been made official for caffeine in coffee (1). The procedure, although more rapid than the PowerChesnut, still required an elapsed time of a t least 7 hours per . determination. Even when the greatest number of these analyses was being performed, it was never possible in this laboratory t o perform the many manipulations in much less than 1 man-hour per sample. In 1946, an investigation into the possibility of measuring concentration of caffeine by utilizing its known absorption characteristics in the ultraviolet range Tyas begun. Absorption of light by caffeine was reported as early as 1905 by Hartley (8, 9). In 1919, Henri (10) reported that the absorption spectrum of caffeine had been studied. Castille and Ruppol (4)in 1928 reported quantitative measurements on caffeine indicating an absorption maximum a t 271 mp. Their work was substantially confirmed by others ( 7 , 11, 1 4 ) , .who reported the caffeine maximum between 271 and 275 mp a t differing pH. A4dherenceto Beer's laty was generally indicated. Molecular extinction coefficients ranging from 8000 to 11,530 were reported by these workers.
1. = length of light path through absorbing medium = concentration E = extinction coefficient c
For quantitative estimation of caffeine, an accurate determination of E?:!' or caffeine factor (optical density per mg. per 100 ml.) was mandatory. Optical densities [log (loo/% transmittance)] may be read directly from the scale of the Beckman spectrophotometer. Further, it was desired to establish the optimum concentrations of caffeine for reading in this instrument. Fifty observations were made to evaluate the caffeine factor. U.S.P. caffeine was sublimed and the sublimate dried in a vacuum oven a t 70" C. for 16 hours. From a solution of the sublimed caffeine, aliquots were taken to provide concentrations from 0.2 to 2.6 mg. of caffeine per 100 ml. Density values on the samples were then read in the spectrophotometer a t 272 mp a t a fixed slit width of 0.74 mm., which, according to the manufacturers, corresponds approximately to a 2 mp band width. At this narrow setting it is easy to detect any slight shifting of the absorption maximum of caffeine, an indication that impurities are present. hverages of the data are presented in Table I. The factor that best fits the data is 0.510 density unit per mg. of caffeine per 100 ml. of solution. All measurements were made in matched quartz absorption cells of 1-cm. light path. As an additional precaution the same cell was always used as a blank while another was reserved for the solution to be examined. (The same procedure was applied in the analytical examination of unknown solutions, as even in carefullp matched cells observable differences can be encoun-
EXPERIVENTAL
Table I.
A model DU Beckman spectrophotometer Xyith Ultraviolet accessories was used for all the absorption measurements presented in this report. An aqueous solution of sublimed and vacuum-dried caffeine was examined with this instrument. The findings of earlier investigators (4,7, 11, 14) were substantially confirmed; caffeine was found to exhibit a maximum absorption a t 272 and a minimum a t 245 millimicrons. Variation of pH between 5 and 9 has not been found in the authors' work to affect either the extinction or the absorption maximum of aqueous caffeine solutions. It' was necessary to ascertain the conformance of caffeine solutions to the Beer-Lambert law. log I/Io
=
Elc
(1)
where
I
IO
= intensity of light transmitted by solvent plus solute
a
= intensity of light transmitted by solvent
1162
Determination of Caffeine Factor
Caffeine, Average bIg./100 bll. Optical Density 0.257 0.131 0.498 0.262 0.265 0.506 0,514 0.263 0.384 0.747 0.390 0.760 0.770 0.396 0.996 0.512 0.518 1.013 0,523 1.027 1.245 0.633 1.266 0.643 0.650 1.284 0,760 1.494 0,768 1.519 0.776 1.541 0.903 1.798 1.04 2.054 1.16 2.311 1.29 2.568 Density/mg./lOO ml. solution.
Average Caffeine FactorG 0.511 0.525 0.524 0.512 0,514 0.514 0.515 0.514 0.511 0,509 0.508 0.508
0,505 0.509 0.506 0.505 0.503 0.508 0.501 0.501
V O L U M E 20, N O . 1 2 , D E C E M B E R 1 9 4 8 Table 11.
1163
Spectrophotometric Recovery of Caffeine from Zinc Ferrocyanide Clarification
(Temperature of ?ohition. 20.5O C.) Concentration. Caffeine Recoi-wed, c7 lI&/lOO 511. /c 1. o 101.0 2.5 Ri.8 5.0 94.1 10.0 9 2 , !1 2.5,0 92.2
Table 111.
Efrect of Reagent Blanlcs upon Recovery of
Caffeine Caffeine. lIg./lOO lI!: Spectrophotometer Theoretical ~
Density :it 272 nip Distilled water ( i n haiiiplc, cell) Filtrate f r o m zinc acetate ferrocyanide Control caffeine 4-distilled u-ater ziiic ferroCaffFine cyanide filtrate
+
+
Tahle IV.
.. ...
,..
0.034
0 518
1.02
1.03
0.521
1.03
1.03
0.016
..,
Spectrophotometric Recovery of Caffeine f r o m 3Iagnesium Oxide Clarification
Concentration, lIg./lOO MI. 1 5 10 50
Caffeine Recovered,
7%
100.5 99.9 98.3 100.5
tered.) The precision of the method depends in part upon thr reproducibility of optical density readings, and it is felt to be of vital importance to utilize all the precision which the spectrophotometer affords. Each independent investigator will need to establish his own factor for density in terms of caffeine concentration to allow for differences in instrument and particularly for differences in transmittance characteristics and light-path distances in sample cells. As Table I shows, caffeine solutions conform to the BecrLambert law, a t least within the limited range covered. Thus it is feasible to determine caffeine quantitatively in the spectrophotometer if there is a means of completely removing all substances in coffee extracts and crude caffeine samples which adsorb light a t 272 mp. Therefore, the first step in the solution of the problem \vas the discovery of highly selective clarifying agents to remove the interfering substances. Samples of crude caffeine and green coffee ext,racts were used in the first studies to find these clarifying agents. Heavy magnesium oxide, a n effective agent used in several current methods, was inadequate as the sole clarifier for spectrophotometric analysis. Other commonly used clarifying agents (15), such as zinc ferrocyanide, neutral lead acetate, ferric hydroxide, and alumina cream, after an initial magnesium oxide treatment of the samples, proved ineffective for complete removal of interfering substances. The order of clarification was reversed Kith encouraging results. ill1 four of the clarifiers displayed greatly improved ability to remove interfering substances, but only zinc ferrocyanidc successfully removed all interferences from the solutions when followed by magnesium oxide treatment. Using solutions of some compounds known to be present in coffee, the specificity of the clarifiers was determined. Magnesium oxide was found to be an effective reagent for the removal of chlorogenic acid, while zinc ferrocyanide removed trigonelline. I n unextracted green coffee the caffeine content is about lYO, trigonelline l.5%, and chlorogenic acid about 7Y0, The recovery from different concentrations of pure caffeine after zinc ferrocyanide clarification in 100-ml. volumetric flasks was determined. As described under hna!ytical llethods, 7.0
nil. of 1.0 M zinc acetate solution and 6.0 ml. of 0.25 M potassium ferrocyanide vere used to prepare zinc ferrocyanide for these clarifications. All clarifications vere made a t a constant temperature. Table 11 s h o w quantitative recovery below 2.5 mg. of caffeine per 100 ml. The observation had been made in earlier work that the reagent5 used in the clarification step exhibited some slight :tbsorption a t 272 nip, but a t that time the readings were considered negligible because of the low densities. I n order to ileterniine n hether a reagent blank was a compensating factor for caffeine absorbed, aliquots TTere taken from a standard solution of caffeine for samples and controls to be made up volumetrically nith the filtrate from the preparation of zinc ferrocyanide and with distilled 17-ater, respectively. The results in Table I11 indicate that vihile the zinc ferrocyanide by itself gave an optical density reading of 0.054 it did not contribute any error to the density of the caffeine solution prepared as above which was almost identical with the density of the caffeine-distilled Jvater control. This indicates that reagent blanks cannot be read directly and corrected for by simple subtraction because of the inaccuracy of very low density readings. I n this case, it is established that light absorption by zinc ferrocyanide does not introduce a n appreciable error to the density reading of a caflrine solution containing approximately 1 mg. per 100 ml. The recovery of pure caffeine a t different levels of concentration from magnesium oxide clarification was also checked and found to be quantitative spectrophotometrically in concentrations from 1 t o 50 mg. of caffeine per 100 ml. of solution (Table IT). Five grams of magnesium oxide \yere used for each 100 1111. of solution. Zinc ferrocyanide folloxed by a magnesium oxide clarification failed to remove all interfering substances in roasted coffee. The absorption maxima of the resulting solutions occurred between 27-1 and 276 mp instead of a t 272 mp. Additional clarification steps involving mercuric nitrate, ammonium sulfide, or neutral lead acetate were also found ineffective. Attempts to reduce the interfering material with sodium sulfite, stannous chloride, or potassium thiosulfate were unsuccessful. Oxidation by iodine, dichromate, ceric salts, perchlorate, or hydrogen peroxide also failed. The A.O.A.C. official Fendler-Stiiber method (1, 6) for caffeine in coffee uses potassium permanganate as one purification step in the procedure. This reagent has been used by others (12). Khen tried in conjunction with zinc ferrocyanide and magnesium oxide, encouraging results Fvere ob'tained. Table V shows that different sequences of reagents produced the characteristic caffeine maximum a t 272 m p in the final solution. I n each case the permanganate color vias discharged after 10 minutes, when sodium sulfite and glacial acetic acid were used. No reducing agent other than sodium sulfite was found satisfactory. 'I'ahle V. Clarification by Zinc Ferrocyanide, Magnesium Oxide, and Potassium Permanganate in Various Sequences (Roasted coffee extract, Bailey-.indrea, 5.59% caffeine) Maximum Apparent Caffeine, Absorption, Order of Sequence of Reagents
a
%
w
I i l I n O i treatment include! whsequent reduction with XazSOa.
Lepper (13) has phown that there i. a very slight loss of caffeine n ith permanganate oxidation a t room temperature, and that the loss is minimized when the treatment is made a t low tem;)ciatare. To determine the recovery of pure caffeine from permanganate oxidation at room temperature, a standard solution was prepared.
1164
ANALYTICAL CHEMISTRY
Table VI. Spectrophotometric Recovery of Caffeine from 1% Potassium Permanganate Oxidation and Reduction of KRln04 with 5 % Sodium Thiosulfate
Excess 5 % KatSOa Solution, M1.
(Temperature 25’ C.) Acetic Acid Added After Initial Prior t o Reduction Initial Reduction Caffeine recovered, neutral solution
5%
%
0.0 0.2 0.5 1.0 2.0 5.0 10.0
One-milligram aliquots were treated in 100-ml. volumetric flasks with 10 ml. of 1% potassium permanganate solution for 15 minutes. Treatment was in neutral solution and in solutions to which 5 or 10 ml. of 0.1 N sulfuric acid had been added. In the acid solutions, reduction to the manganous salt was made directly by approximately 5 ml. of 5% sodium sulfite solution. I n the neutral solutions one series was reduced directly with sodium sulfite after the addition of 0.5 ml. of glacial acetic acid; another series was reduced by the sulfite solution to manganese dioxide initially, and then after adding 0.5 ml. of acetic acid, reduction was continued to the manganous salt. I n the neutral solutions, varying excesses of 57c sodium sulfite solution were added. There \vas considerable loss of caffeine in the solutions containing acid permanganate. Table 1’1 indicates that satisfactory recovery of caffeine is obtained from the oxidation with 1%permanganate solution in neutral solution, followed by initial reduction in neutral solution and further reduction of manganese dioxide in acid solution. I t is also apparent that a considerable excess of sodium sulfite solution will not affect this quantitative recovery. This work confirms the observations of Leppcr (13) concerning the loss of traces of caffeine viith permanganate oxidation. A study was made of the effect of the length of permanganate oxidation time on two samples of a commercial brand of coffee. The caffeine value obtained spectrophotometrically with osidation times from 0.5 to 24 hours was compared to the values obtained from an oxidation time of 10 minutes. -4s shown in Table VII, there is no appreciable loss of caffeine when the permanganate oxidation is less than 1 hour. This oxidation occurred in alkaline solution as a result of the initial magnesium oxide clarification. Table VII. Effect of Potassium Permanganate Oxidation Time on Caffeine Content of Roasted Coffee Solutions Time of KMnOd Oxidation Hours 0.5 1.0 2.0 3.0 4.0 24.0
Caffeine Recoverv ComDared t o lO-JIinute Oxcdation-Time Sample A Sample B 0 10
%
99.2 97.4 95.5 95.5 89.3 56.7
,100.8 100.4 95.6 94.8 86.9 53.2
The recovery of caffeine from artificial and semiartificial mixtures of soluble coffee solutions )vas determined to discover any loss of caffeine resulting from the series of purification steps. Because it is most convenient, the sequence of magnesium oxide, potassium permanganate, and finally zinc ferrocyanide !vas used for the analysis. The artificial mixture TI-as prepared from a standard caffeine solution and a solution of decaffeinated soluble coffee, which provided the necessary coffee solids. The semiartificial mixture was prepared from the artificial mixture and a solution of soluble coffee. The theoretical caffeine values of these mixtures were calculated from the spectrophotometric determination * of the standard caffeine solution and the BaileyAndrew caffeine values of the two soluble coffees. As shown in
Table VIII, recovery of caffeine from the mixtures was quantitative. ANALYTICAL METHODS
Reagents. Magnesium oxide; heavy MgO, U.S.P. Potassium ferrocyanide solution, 0.25 molar. Buffered zinc acetate solution, 1.0 molar. Dissolve 438.0 grams of zinc acetate dihydrate in distilled water, add 60 ml. of glacial acetic acid, and make to 2000 ml. Sulfuric acid, 0.1 .V. Potassium permanganate solution, 1%. Glacial acetic acid. Sodium sulfite solution, 5%. Procedure for Crude Caffeine and Green Coffee. PREPARATION OF SAMPLE. Crude Caffeine. Weigh 0.1 gram of sample, transfer to a volumetric flask (500 ml.), add 400 ml. of hot water, and shake thoroughly. Cool the volumetric flask to room temperature and make to the mark with distilled water. Pipet a convenient aliquot containing approximately 2 mg. of caffeine to a 100-nil. volumetric flask. Add distilled water to a total volume of approximately 50 ml. Green Coffee. Weigh 2.0 grams of sample and transfer to a tared 1-liter Erlenmeyer flask. Add 50 ml. of 0.1 S sulfuric acid and 450 ml. of distilled water to the flask, heat to boiling, and boil 30 minutes. Cool flask to room temperature, make to weight (tare plus 502.0 grams), and filter. Pipet a 50-ml. aliquot into a 100-ml. volumetric flask. Table VIII.
Spectrophotometric Recovery of Caffeine from Artificial Mixtures
Caffeine Calculated M g . / l O O ml. Artificial soluble coffee 1 .oo Semiartificial soluble coffee 1.05 Sample
FERROCYANDE AKD lI.\GKESIUJl
Caffeine Recorered ml. % 0.997 99.5 1.06 100.4
.lfg./lOO
OXIDE CLARIFICbTIOX.
TO
the volumetric flask add 7.0 ml. of zinc acetate solution and swirl vigorously. Dropnise add 6.0 ml. of potassium ferrocyanide solution with constant swirling of the flask. (An excess of zinc acetate must be present to prevent interference by the ferrocyanide.) hlake to the mark with distilled water. Mix thoroughly, filter through a S o . 41 (15-cm.) Whatman filter paper, and discard first 10-ml. portion. Pipet a 50-ml. portion into a tared 250-nil. Erlenmeyer flask containing 5.0 grams of magnesium oxide, add 50 ml. of distilled water, and boil for 20 minutes. Cool to room temperature and make to weight (tare plus 105 grams). Filter and discard first portion (10 ml.). Read a portion of the filtrate in the spectrophotometer at 272 mp. Density reading a t 272 nip divided by the factor previously determined equals milligrams of caffeine per 100 ml. of filtrate. Time for crude caffeine, 1.5 hours. Time for green coffee, 2 hours. Procedure for Green, Roasted, and Soluble Coffee. PREPARATION OF SAMPLE AXD AIAGSESIUM OXIDE CLARIFICATION. Green and Roasted Coffee. Weigh 1.0 gram of roasted coffee or 1.2 grams of green coffee and transfer to a tared 1-liter Erlenmeyer flask containing 50 ml. of 0.1 N sulfuric acid. Add 250 ml. of distilled water and boil for 20 minutes. Then add 50 grams of magnesium oxide and continue boiling for 20 minutes. Cool and make to weight (tare plus 350 grams plus weight of sample]. Filter, pipet a 25-ml. aliquot into a 100-ml. volumetric flask, and add 25 ml. of distilled water. Soluble Coffee. Take a sample weight containing approximately 100 mg. of caffeine and transfer to a 500-ml. volumetric flask. Make to volume, mix thoroughly, and pipet a 25-ml. aliquot into a tared Erlenmeyer flask containing 25 ml. of 0.1 -Y sulfuric acid. Add 200 nil. of distilled water and boil for 20 minutes. Then add 25 grams of magnesium oxide and continue boiling for 2 0 minutes. Cool and make to weight (tare plus 275 grams). Filter and pipet a 50-ml. aliquot into a 100-ml. volumetric flask. I n the PERJIASGASATE ASD FERROCYANIDE CL.\RIFICATIOS. volumetric flask place 10 ml. of potassium permanganate solution. hfter 10 minutes, add 3.C ml. of sodium sulfite solution, then add 0.5 ml. of acetic acid, and titrate with sulfite solution to the disappearance of the manganese dioxide precipitate. Add 7.0 ml. of zinc acetate solution and swirl vigorously. Dropwise add 6.0 ml. of potassium ferrocyanide soluticn with constant swirling of the flask. Make to volume with distilled water. Mix thoroughly and filter through No. 42 Whatman filter paper, discarding the first portion. Read a portion of the filtrate in the spectrophotometer. From the density reading, calculate the per cent caffeine in the sample. Time for green, roasted, or soluble coffee, 2 hours.
V O L U M E 20, NO. 1 2 , D E C E M B E R 1 9 4 8
1165
able for any given sample, as a similar extraction procedure is used in both methods. [Sequence of reagents, spectrophotometric method, Zn?Fe(CS)a, M g O ] From Tables IX and XI, i t is apparent that the agreement of Per Cent Caffeine No. of results between the Bailey--4ndrew and the spectrophotometric Detns. per BaileySpectroSample Method .kndre\~ photometric method is very good. However, in about 70% of the samples analyzed, the spectrophotonietric caffeine value is lower. 'Crudes I 10 94.8 94.2 The precision data reported are intended to be only relative I1 10 67 7 63.8 I11 10 28 9 26.9 and are not necessarily limiting for either method, because comIV 10 13.3 12.3 .I 2 82.9 84.0 parative runs were made simultaneously by the same analyst. The B 2 92.5 91 . o dat,a do give an excellent comparison of the two methods under > C 41.3 41.3 D 2 73.8 73.0 identical conditions. Table X demonstrates a much greater E 2 71.1 70.0 F 2 54.3 54.0 precision for the spectrophotometric procedure involving a G 2 83 5 ., 33 83 5 .. 0 0 zinc ferrocyanide and magnesium oxide clarification. Table H 2 7 7 J 2 68.6 69.0 XI1 demonstrates that when potassium permanganate is used in x 2 69.6 71 . O the spectrophotometric method, the precision of the BaileyG r e e n coffee Columbian, coarse grind 2 1.11 1.07 a 4 n d r e method ~ is the same as or slightly better than the spectroColumbian, fine grind 2 1.19 1.14 photometric procedure. Central American 2 1.19 1.15 Aged Venezuelan 2 1.17 1.13 The greater precision obtained spectrophotometrically with Special Santos, fine grind 2 0.95 0.93 2 0.97 0.96 only a ferrocyanide and a magnesium oxide clarification implies Special Santos, flaked Medellin 2 1.12 a correspondingly greater acSantos 10 1.03 01 .. 90 99 ~~. __ -~ curacy. Even when potassium permanganate is used in the Table X. Precision ( 1 6 ) of Ten Determinations spectrophotometric procedure, [Sequence of reagents, spectrophotometric method, ZnnFe(CN)e, MgO] % Caffeine b y Bailey-Andrew Method % Caffeine b y Spectrophotometric AIethod the average value obtained Sample Av. Range s" L.C.nr% b Av. Range sa L.C.sa%b spectrophotometrically is prob'Crude caffeine ably more accurate than the I 94.8 91.9 -97.3 2.4 6.3 94.2 93.6 -95.5 0.59 1.5 Bailey-Andrew value. This I1 67.7 65.7 -68.7 1.1 2.9 63.8 6 3 . 1 -64.6 0.55 1.4 I11 28.9 2 7 . 8 -30.6 0.87 2.3 26.9 2 5 . 9 -27.7 0.47 1.2 assumption is based on the IV 13.3 12.2 -14.6 0.83 2.2 12.3 1 2 . 1 -12.4 0.10 0.26 -Green coffee known presence of noncaffeine Santos 1.03 1.00- 1.04 0.012 0.031 0.99C 0.98- 1 . 0 0 0,007 0.018 nitrogen in the Bailey-Andrew Santos 1.03 1.00- 1 . 0 4 0.012 0.031 1.04d ... ... .. chloroform extract ( 5 ) that is Standard deviation = where d = del-iation of each observation from sample mean. calculated as caffeine in the * Water L.C.959i = h . 0 5 s = f limits f r o m true value within which 95 out of 100 single determinations will fall. final result, whereas quantitaextracted, neutral solution. d Bcid extractc'd, 5 determinations. tive recovery of caffeine is ~ ~ ~ ~ - _ _ _ ~__ obtained spectrophotometrically. These procedures involving the direct clarifications of aqueous The greatest difference in caffeine analyses made spectroextracts of coffee are not directly applicable to decaffeinated photometrically and by the Bailey-AndreLY method is in the time coffee. A modification of the procedure involving an initial required per analysis. Whereas 0.5 hour per analysis and 2 solvent extraction applicable to these samples is being developed. However, there are insufficient data at, this time to present a Table IX.
Agreement of Results
~
~
VX,
~~
~~
~
~
Table XI.
.finished procedure for decaffrinated coffre. COMPARISON OF METHODS
Agreement of Results
[Sequence of reagents, spectrophotometric method, N g O , KAInOa, ZnnFe(CN)a] No. of - Per Cent Caffeine Detns. per BaileySpectrolndrew photometric Method Roasted coffee Sample Blend I Blend I 1 West Central American
A number of samples of each type were analyzed by the appropriate spectrophotometric procedure and by the BaileyAndrew method. The results obtained are given in Tables IS, X, XI, and X I . There is some doubt as to the completeness of the extract,ion ~~$~,"n,",o,',~~ian L 1.35 1.33 1.20 1.21 of caffeine from coffee by any of these methods, particularly Santos 2 Brand I 10 1.34 1.27 green coffee. I t is known, for example, that the finer the grind Brand 11 10 1.28 1,25 Brand I11 10 1 . 2 8 1.22 the greater the extraction of caffeine and that flaking further Brand 10 1.16 1.17 Green coffee increase the yield of extractable caffeine. The increasing yield with finer grinding and flaking is clearly demonstrated in thr Soluble Santoscoffee 10 1.03 1.03 Brand .I 10 5.59 5.36 analyses of Columbian and Brand B 10 3.98 3.93 special Santos samples (Table I X ) . It is also known that extraction is more effiTable XII. Precision ( 1 6 ) of Ten Determinations cient in acid or alkaline rather [Sequence of reagents, spectrophotometric method, MgO, KAtnO4, ZnxFe(CN)a] Caffeine by Spectrophotometric Method 7c Caffeine by Bailey-Andrew %lethod than neutral solution. This difference in extraction is demSample .Ir. Range s L.C.sa% Av. Range S L.C.96% Green coffee onstrated in the analyses of Santos 1.03 1.00-1.04 0.012 0.031 1.03 1.00-1.05 0.019 0.050 Santos samples (Table 9). Roasted coffee 1.27 1.22-1.32 0.032 0,083 0.057 Brand I 1.34 1.32-1.38 0.022 Acid extraction resulted in a Brand I1 0,022 0.058 1.28 1.25-1.33 0,022 0.057 1.25 1.22-1.29 0 . 0 3 1 1 . 2 2 1 . 2 0 1 . 2 4 0 , 0 1 6 0.042 Brand I11 1 . 2 8 1.26-1.30 0 . 0 1 2 caffeine value slightly greater 0.020 0.032 0.045 1.17 1.13-1.19 Brand I\' 1.16 1.14-1.18 0,017 than that obtained by alkaSoluble coffee Brand A 5.59 5,50-5,68 0.047 0,123 5.36 5.21-5.47 0.069 0.178 line extraction. However, the Brand B 3.98 3.93-4.05 0.051 0.133 3.93 3.86-4.03 0.050 0.131 methods are directly compar~~
ANALYTICAL CHEMISTRY
1166 hours’ elapsed time are the maximum required for a spectrophotometric analysis, the Bailey-rlndrew procedure requires 1 manhour per analysis and 7 hours’ elapsed time. Thus the spectrophotometric method reduces the time required to one third that previously necessary without sacrificing precision or accuracy. ACKNOWLEDGMENT
The authors wish to acknowledge the guidance and assistance received from L. W. Elder. Thanks are due to R. G. Moores for having provided samples of trigonelline and chlorogenic acid and for having given the authors the benefit of his experience with similar problems. I t is also desired to express appreciation for the great number of analytical determinations made for comparative purposes by J. J. Kelly, G. F. Lata, E. J. Sarna, and R. C. Sylvester. LITERATURE CITED (1) Assoc. O5cial Agr. Chem., J . Assoc. Official A g r . Chem., 30, 70-1 (1947).
( 2 ) hssoc. Official Agr. Chem., “Official and Tentative Methods of Analysis,” 6th ed., 18.14, p. 217, 1945. (3) Ibid., p. 220. (4) Castille, A., and Ruppol, E., Bull. soc. chim. biol., 10, 623 (1928), ( 5 ) Clifford, P. A,, J . Assoc. OflciaZ A g r . Chem., 14, 533 (1931). (6) Fendler, G., and Sttiber, W., 2. -Vahr. Genussm., 28, 9 (1941). (7) Gulland, J. M., Holiday, E. R., and Macrae, T. F., J . Chem. SOC.,1934, 1639-44. (8) Hartley, W.N., J . Chem. Soc., Trans., 87, 1796 (1905). (9) Hartley, W. N., Trans. R o y . Soe. (London), A, 176, 471 (1885). (10) Henri, V., ”Etudes de Photochimie,” Paris, Gauthier-Villars, 1919. (11) Holiday, E. R., Biochem. J., 24, 619 (1930). (12) Lendrich, K., and Nottbohm, F. E., Z . Nahr. Genussm., 17, 241 (1914). (13) Lepper, H. A,, J . Assoc. Official Agr. Chem., 4, 526 (1921). (14) Loofbourow, J. R., Stimson, M . M.,and Hart, M . J., J . A m . Chem. SOC.,65, 148 (1943). (15) .Moir, D. D., and Hinks, E., Analyst, 60, 439 (1935). (16) Snedecor, G. W., “Statistical Methods,” 4th ed., Ames. I o w a Collegiate Press, 1946. RECEIVED June 29, 1948. Presented before the Division of Analytical and Micro Chemistry a t the 113th Meeting of t h e AMERICAN CHEMICAL SOCIETY, Chicago, Ill.
Photometric Determination of Epinephrine in Pharmaceutical Products . I . HOY UOTY, Bureau of Chemistry, American Dental Association, Chicago. I l l . ired-blue color results from the addition of a solution of ferrous sulfate and a suitable buffer to a dilute solution of epinephrine. The transmittancy of this solution as determined at a wave length of 530 millimicrons is used to measure the amount of epinephrine. This method may be applied directly to many pharmaceutical products. The procedure is extremely simple and rapid and is capable of an accuracy of about 1% of the amount of epinephrine present. The optimum conditions for development of color are reported in some detail.
T
HE need for a reliable clieniical method for the t9stimatiori of epinephrine in solutions of local anesthetic agents led to the initiation of this investigation. -1 colorimetric procedure has been developed and tested over a period of approximately 3 years. The method is a modification of one described in 1923 by Mitchell (3) for the estimation of tannins and extended by Price (4)and Glasstone ( 2 ) to the analysis of related polyphenolic substances. The color varies with the pH of the solution. When an alkaline buffer is added to a slightly acid solution containing epinephrine and a ferrous salt, a blue color begins to develop at about pH 6.5 and gradually changes to the characteristic red-blue color which attains a maximum intensity at about pH 8 to pH 8.5. A similar procedure Fas mentioned unfavorably by Barker, Eastland, and Evers ( I ) , who did not find the reaction sufficiently sensitive for the estimation of epinephrine in adrenal glands. At least part of their difficulty may have been due to an attempt to use the blue color of lower intensity rather than the red-blue color that develops a t a higher pH. The present procedure may be employed to best advantage when the epinephrine content is a t least 10 p.p.m. Vogeler ( 5 ) reports briefly concerning a photometric study of the “ferrous iron-adrenaline complex.” Unfortunately his data are too meager to be of much value in formulating an analytical procedure. Yoe and Jones ( 6 ) , on the other hand, have suggested the use of disodium-1,2-dihydroxybenzene-3,5-disulfonate for the estimation of ferric iron. In the early part of this investigation a solution of a ferric salt was employed to develop the colored ironepinephrine complex. On the basis of further experience, however, it vias decided that the ferrous salt possessed some advantages and it was used throughout the remainder of this work.
EXt’EKI\IEYTAL
The data for the absorption curve in Figure 1 were obtained with a Beckman Model DU spectrophotometer. Maximum absorption occur> at a wave length of 530 millimicrons. There is very little absorption due to the reagents alone a t this same wave length. A Coleman Model 10s spectrophotometer with a light slit designed to give an effective spectral band width of 30 milli100
I
Reapents no epinephrine 0.4 mg. epinejlhrine Beckman Spectrophotometer Bandwidth 2 millimicrons Cell Length = 1.000 cm. A.
8 . xeaeents
J
-
90 ~
80
70
60
Figure 1.
Spectral Absorption Curve for IronEpinephrine Complex
I
