Nonashing Technique for Determination of Traces of Copper in Malt Beverages IRWIN STONE, ROMOLA ETTINGER, AND CARLYLE GANTZ Wallerstein Laboratories, New York 16, N. Y .
Traces of copper in beers and ales act to accelerate oxidation changes which adversely affect flavor and shelflife of the packaged beverage. The brewing industry requires a simple, rapid, and precise method for the determination of these traces of copper. A colorimetric procedure has been developed which requires no ashing of the sample and yields results in about 1 hour. The reagent employed is zinc dibenzyldithiocarbamate, which gives a brownish, solvent-soluble, copper complex in strongly acid solutions of the sample. The colored solvent layer is separated and measured in a photometer. No interference is encountered from the metals normally present in beer. Good recovery of added copper is obtained. Routine application of this method will provide another quality control tool, enabling the brewing industry to produce more uniform and stable products.
tometer used in this work was a Klett-Summerson photoelectric colorimeter, but the method is adaptable to all of the commercially available instruments. REAGEYTS
Dilute sulfuric acid (1 plus 3) Hexyl alcohol Solution (0.5%) of zinc dibenzyldithiocarbamate in carbon tetrachloride. Filter and store in a brown bottle in a cool dark place. Standard copper solution: Weigh out 3.93 grams of clean, C.P. copper sulfate crystals (CuS04.5H,0) free of any whitish deposit of anhydrous copper sulfate, dissolve in water, and dilute to 1 liter (1 ml. = 1 mg. of copper). For the calibration curve, prepare a more dilute standard immediately before use by pipetting 5.0 ml. into a 500-ml. volumetric flask and diluting to the mark with water (1 ml. = 0.01 mg. of copper). CALIBRATION OF PHOTOMETER
Into a series of corked centrifuge tubes of the same type, and cleaned in the manner noted below, pipet the following amounts of dilute copper solution: 0.0, 1.0, 2.0, 3.0, 4.0, and 5.0 ml. equivalent to 0.0, 0.4, 0.8, 1.2, 1.6, and 2.0 p.p.m., respectively. Add 25 ml. of beer and 3 ml. of the dilute sulfuric acid solution and follow through the method below. From the photometer readings obtained, calculate a factor converting these readings into parts per million of copper. The color over this range appears to follow Beer's law, but if the response of the instrument is not linear, draw a calibration curve on graph paper.
T
H E presence of traces of copper and iron of the order of a fraction of a part per million to several parts per niillion has a strong catalytic effect on the oxidation reactions occurring in beer. These reactions adversely affect the shelf-life and taste of the packaged product. The industry is cognizant of this and requires simple, sensitive, and rapid test methods for these elements. The problem with regard to the determination of iron has recently been resolved by the adoption of a direct colorimetric method by the Anierican Societyof Brewing Chemists andpublication in its book of methods ( 1 ) . I n the case of copper, the previous available methods ( 5 ) have involved procedures for destruction of the organic matter, either by wet or dry ashing, before the copper could be determined. This destruction of the organic matter made the method cumbersome and time-consuming and was unsuited for routine work in the laboratories of the industry ( 3 ) . This papei present. a simple, direct, nonashing method for the determination of the traces of copper which may be found in malt beverages. -4recent paper by Martens and Githens ( 4 ) discuwed the use of zinc dibenzyldithiocarbaniate for the colorimetric determination of small amounts of copper in dyes and rubber chemicals. This reagent complexes with the copper to give a yellowish or brownish, organic solvent-soluble color in strongly acid solution. The commercial availability of the reagent adds to the convenience of the method. (Zinc dibenzyldithiocarbamate may be obtained from Naugatuck Chemical, Naugatuck, Conn., under the trade name Arazate.) The use of this reagent ivas adapted to beer as a direct nonashing technique and the method was suitably modified for this purpose. I n the strongly acid solution employed, none of the common metallic constituents which may be present in beer interfere with the copper color tlevelopment. The test is conducted entirely in a corked, 50-ml. centrifuge tube and results may be obtained within 1 hour. The sample, acidified to 1 sulfuric acid, is heated in a boiling water bath for 0.5 hour, cooled, and then shaken out with a measured portion of a carbon tetrachloride solution of the reagent. The tube is centrifuged and the colored carbon tetrachloride layer is transferred to a cell and read in a photometei The copper content i q calculated from the photometer rending. The pho-
METHOD
The entire test is conducted in a corked, 50-ml. Pyrex centrifuge tube (Pyrex S o . 8420). Before running the test, render the centrifuge tube copper-free by taking the cleaned and rinsed tube. stoppered with the cork to be used during the analysis, and adding about 15 ml. of distilled water, 3 ml. of dilute sulfuric acid, and 5 ml. of the carbon tetrachloride solution of zinc dibenayldithiocarbamate. Shake thoroughly, discard, and allow the tube to drain. Toavoid copper pickup duringdegassing, the beer sample is taken directly from the bottle. The bottle should be cold and shaken immediately before opening. Allow the gas bubbles to leave the liquid before removing the cap. Carefully pour the cold beer into an accurate 25-ml. graduate. If foam interferes with the measurement of the 25-ml. sample, one drop of hexyl alcohol will remove this interference. To the cleaned tube, add 25 ml. of the cold beer sample. Add 1 drop of hexyl alcohol and 3 ml. of dilute sulfuric acid solution. Mix and place the tube in a boiling water bath for 0.5 hour This heating is conducted to hydrolyze, partially, foam-forming materials in the sample to prevent emulsification difficulties 1% hen separating the carbon tetrachloride and also to break down an) possible copper-organic complexes. During the heating, some samples may require addition of an extia drop of hexyl alcohol to control excessive foaming. Remove the tube and cool to 25" C. ildd 5 or 10 ml. (accurately pipetted) of the zinc dibenzyld~thiocarbonate solution, depending on the size of the photometer cell used. Cork the tube and extract a t 25" C. by vigorously shaking 60 times. Thorough and complete emulsification should be obtained during each series of shakings. Low results may be obtained if the samples are not vigorously and thoroughly extracted. Reshake four times again, giving 60 snappy shakings each time to obtain a fine emulsion; allow the carbon tetrachloride to separate partially betxeen shakeouts. Centrifuge the tube and draw off the clear-colored carbon tetrachloride layer. Centrifuging a t 2500 r.p.m. for a couple of minutes is usually sufficient to obtain a good, clean-cut separation and the carbon tetrachloride layer may be easily drawn off with a pipet. If small droplets of the aqueous layer are carried into the pipet, these may be removed by allowing the stream of carbon tetrachloride from the pipet to flow down
893
ANALYTICAL CHEMISTRY
a94
the wall of a cleaii dry test tube preliminary to transfer to the photometer cell. The water droplets will adhere to the test tube and the clear carbon tetrachloride can be poured off. Transfer the clear solution t o a suitable cell (the same size as used in the calibration) and read in t.he photometer, using a green light filter. Set the zero reading of the photometer by means of the cell filled with carbon tetrachloride. A blank determination on the reagents is conducted by carrying through a shakeout on the reagents involved in the method. I t is conducted in the usual centrifuge tube by shaking out. a t 25" C., 3 nil. of t>hesulfuric acid plus 25 ml. of copper-free water with 5 or 10 ml. of the zinc dibenzyldithiocarbamate reagent'. If the method is used frequently, the centrifuge tubes can be provided with glass stoppers, giving a more easily cleaned all-glass surface. (Tubes with interchangeable glass stoppers are available from E. Machlett B: Sons, Kew York 10, 9. T.) Also determine the color extracted by carbon tetrachloride and correct accordingly. Carbon tetrachloride extracts a slight amount of color from the acid-digested sample. The amount of solvent-soluble color is not directly related to beer color but is probably due to the presence of traces of colored resinous materials from the hops. The amount of color extracted is nearly constant for any particular type of sample so that it, is not necessary to determine this correction for every sample examined. The use of a n average correct,ion representative of the color extracted from the type of material under examination will not introduce a significant error.
Table I. Sample Beer
Recover? of Added Copper
Copper S d d e d , P.P.hZ. None
Copper Found, P.P.M. 0.19 0.70 0.69 1 00 0.99
0.50 0.50
0.75
0.75
Ale
None 0.50 0.50 1 00 1.00 1.30 1.50 None 0.50
Korr
0.32 0 81 0 82 1 28 1.27
1.75 1 78 0 24 0.74 1 28
1.00
Copper Recox'ered P.P..\I. % 0:il 0.50 0.81 0 80 0: 49 0.50 0.96 0.95 1 43 1 46
..
...
102 100 10;
105
...
98 100 96 95 9i 9; , .
0 50 1 04
100 104
recoveries are obtained for these traces of added copper. Some n-orts containing added copper were also examined with similar I~t!.Ult Q.
INTERFERENCE O F OTHER METALS
The most common metallic contaminant in beer Lvhicli could affect the copper determination is iron. The normal iron content of beer is usually of the order of 0.1 p.p.m. or less, but d i e n preeent as a contaminant, it may go as high as several p u t s per million. Beer containing an added 0.5 p.p.ni. of copper wad treated to contain 2 and 10 p.p.m. of iron. I n addition, other bottle$ were prepared to contain these levels of tin, nickel, and chromium although contamination by these metals would be rare. Copper \vas determined on the beers with the added metala and the results are presented in Table 11. Iron and the other metal3 in amounts up to 10 p.p.ni. did not produce any interference with the copper determination. EXTRACTION OF BEER COLOR BY CARBON TETRACHLORIDE
COppFA
#AflTJ
CCI N/Li/OA'
Figure 1. Calibration Curve
Samples of beer were heated with sulfuric acid by the niethoi given and then were extracted with carbon tetrachloride CORtaining no zinc dibenzyldithiocarbamate in order to determine the extent of extraction of any color from theacid-digested wiiple. The results are recorded in Table I11 and show a slight extractiop 171 solile color component. The extent of color extraction ap-
Using Klett-Summerson photoelectric colorimeter. No. 54 green filter, 5 ml. of reagent, and a 13-mm. micro test tube
Calculate the copper in parts per million by deducting the blaiik and extractable color readings from the sample readings and multiplying the difference by the factor (converting scale readings to parts per million). If the response of the photometer is not linear, use a calibration graph instead of a factor to obtain the copper content. CALIBRATIOX D4T.4
-4plot oi the calibration data obtained on a Iilett-Summerson photoelectric colorimeter is given in Figuie 1. A straight line is obtained indicating that the color-copper concentration function obeys Beer's law in this range. The factor fol converting photometer readings to parts per million of copper a- calculated for tliiq calibration is 0.0084. RECOVERY O F ADDED COPPER
Bottled beer from a single uniform hatch was obtained from the filling line of a brewery. The bottles were cooled and opened, and varying known amounts of copper were added thereto. The bottles were recapped, thoroughly mixed, and allowed to stand for several weeks a t room temperature. The beers were then tested and the recoveries obtained are given in Table I. Good
Table 11. Recovery of Copper in Beer Containing 0.3 P.P.31. of .-idded Copper in the Presence of Other 3IetalJ Metal Added Sone Iron Iron Sickel Sickel Chromium Chromium Tin Tin
Table 111. Sample
Amount .4dded, P.P.hl.
Copper Recovered, P.P.11.
..
0.50 0.49 0.50 0.49 0.50 0.50 0.50 0,50 0.49
2 10 2 10
2 10 2 10
Extraction of Color by Carbon Tetrachloride Beer Color'
Extractable Color Photometer scale Equivalent readings p.p.m. coppei o 03 0.03 0.03 0.04 0.03 0.03 0.08
a Beer color determined by Am. Soc. Brewing Chemists photometric method ( 1 ) . Samples I t o VI were of normal color of present beers and ale. Stout sample was very dark brown.
. 895
V O L U M E 25, N O . 6, J U N E 1 9 5 3 COMPARISON W-ITH ASHIhG METHOI)
Table IV. Copper Determinations on Beers by Two Methods Sshing Techniquea, P P.M. 0.05
Sample S o .
I I1 SI1 SV V VI VI1 a
Nonashing Technique, P P.11. 0.05 0.28
0.26 0.10 0.19 0.29 1.37 1 16
0.11 0.28 0.32 1.43 1.23
hIethod given in (6).
pears to have no direct relationship to the visible beer color of the sample. The colored material being extracted is probably some modified solvent-soluble resinous material from the hops. Foi precise results on samples, especially those with very low copper content, it will be necessary to apply a correction for this small amount of extractable color before calculation of the copper content. Inspection of the results of the samples in Table I11 shows that the amount of extractable color coveronly a narrow range so that it would appear unnece-ary to detrrmine the extractable color on each sample eyamined. The u ~ of e an average correction representative of the type of ~ a n i p l e unclt~it,\am~nation would tw an allon-able time-saving c\pctlient
Tests n-ere conducted comparing the results obtained by this direct method with an ashing method ( 6 ) now in u.se in the industry. The technique of this latter method involves ashing of the sample, solution of the ash in acid, addition of 2,2’-bil)~-ridine and neutralization to complex the iron and to prevent its interference, then addition of sodium diethyldithiocarbamate, shaking out with amyl acetate, and reading the colored solvent in a photometer. This method is somewhat lengthy and requires skill and care to avoid contamination and losses. The results of this comparison are contained in Table I V and show that slightly higher r e d t s were obtained by the new direct method on nearly all of the samples. These higher resulte might be expected because previous collaborative tests of the ashing technique indicated it to give somewhat low recoveries ( 2 ) . LITERATURE CITED
(1) .\nierican Society of Brewing Chemists, ”Methods of Anaiysis,”
Beer-20, 1951. ( 2 ) 9 m . Soc. Brewing Chemists, Proc., 1951, 147. (3) A m . Soc. Brewing Chemists, Pioc., Report, S u b c o m m i t t e e 011 Copper in B e e r , 1952 (in press). (4) M a r t e n s , R. I., and Githens, R. E., Sr., -4x.4~.CHEM.,24, 991
(1952). (5) Stone, I., IND.ENG.CHEM.,ANAL.ED.,14,479 (1942). RECEIVED for review November 3, 1952. Accepted M a r c h 6 , 1953.
Determination of the Pyrazinamide Content of Blood and Urine WILLIAM S. AL EN, S. M. ARONOVIC’,L. 31. BRANCONE, AND J . H. WI LIAM S Lederle Laboratories Division, American Cyanamid Co., Pearl River, ,V.Y . EXPERIMENTAL
HE therapeutic value of pyrazinamide (Aldinamide.American
T C , - , namid Co.) in the treatment of tuberculosis. as
S~OTYII
bj- clinical trials ( 1 , 2), has indicated a need for a method of determining the blood and urine levels of the drug. .Is pyrazinamide is easily hydrolyzed to pyrazinoic acid x-ith dilute alkali, S
j\
--
)COSH? Dilute
~1
S
/
KOH
Y
d- )COOK
+
,
s
.
a method for estimating pj-razinoic acid was developed. Pyrazinoic acid reacts with ferrous ammonium sulfate (LIohr’s salt) to produce an orange-red complex n-hich absorbs light in accordance with Beer’s law a t a wave length of 460 nip (Figures 1 and 2). This complex is stable a t room temperature for -2 hours. Pyrazinamide is hydrolyzed to a large extent in the bod!yielding products which give an orange-red complex Lvith LIohr’s salt analogous to the complex obt,ained with pyrazinoic acid. Thus calibration curves of pyrazinoic acid-Mohr’s salt can be used to estimate the amount of hydrolyzed pyrazinamide and by treating the appropriate extract with dilute alkali, the pyrazinamide not hydrolyzed naturally in the body can be estinxited in the blood and urine. It is thought that the principal hydrolysis product in the body is pyrazinoic acid and future references in this paper to the hj-drolysis product will he as pyrazinoic acid. Isolation Jvork is nonheing carried out to establish the identity of the hydrolypis products. 1
Present address, Department of Chemistry, University of Wiiconsin,
3Iadison, Wis.
Method for Blood. Rmc;Es,ra REQUIRED. l o yosodium tungstate solution. 1OY0 sulfuric acid solution. 10% barium chloride solution. 50% potassium hydroxide solution. 1% sodium chloride solution. Ferrous ammonium sulfate crystals. De-;Zcidite anion exchange resin (The Permutit Co., S e w Tork, S . Y.). 0.800 0.700
2
u 0.900
-
/
340 360 380 400 420 440 460 480 500 520 540 563
WAVE LENGTH, M r
Figure 1.
-4bsorption Curve of Py-razinoic Acidllohr’s Salt Complex in Water
Concentration, 180 micrograms per milliliter
The blood cells, color, and proteins are removed by a tungstic a d precipitation. One milliliter of blood is pipetted into each of two caentrifuge tubes containing 3 ml. of distilled water and both are t w a t e d as follows: 1 ml. of 10% sodium tungstate is added and. after mixing, 2 ml. of 10% sulfuric acid are added. The
