Enzymatic Spectrophotometric Determination of Sucrose in U.S.P.

Chem. , 1959, 31 (1), pp 109–110. DOI: 10.1021/ac60145a022. Publication Date: January 1959. ACS Legacy Archive. Cite this:Anal. Chem. 31, 1, 109-110...
0 downloads 0 Views 268KB Size
(8) hIcClure, F. J., IXD.ENG. CHESI., ANAL.ED. 11, 171 (1939). (9) Megregian, Stephen, ANAL.CHEM.26, 1161 (1954). (10) Nielsen, H. X., Ibid., 30,1009 (1958). (11) Richter, F., Z. anal. Chem. 124, 19% (1942). (12) Samachson, Joseph, Slovik, Norman, Sobel, -4.E., AXAL.CHEM.29,1888 (1957).

(13) Smith, F. A., Gardner, D. E., G. S. Atomic Energy Commission unclassified document, U. R.46 (1948). (14) Smith, F. A., Gardner, D. E., Hodge, H. C., J . Dental Research 29,596 (1950). (15) Venkateswarlu, P., Karayana Rao, D., h A L . CHE~LI. 26, 766 (1954). (16) Venkateswarlu, p., Karayana Rao, D., I n d i a n J . M e d . Research 42, 135 (1953).

(17) Willard, H. H., Winter, 0. B., IXD. ENG.CHEM.,ANAL.ED. 5 , 7 (1933). (18) Wulle, Hertha, Z . physiol. Chena. Hoppe-Seyler's 260, 169 (1939). RECEIVED for review May 24, 1958. Accepted August 2, 1958. Work supported by the Research and Develo ment Division, Office of the Surgeon &eneral, Department of the Army, Contract KO. D A-49-007-MD-390.

Enzymatic Spec t ro photo metric Determinat io n of Sucrose in U.S.P. Sirups J.

P. COMER

and

H. F. BRICKLEY

Analytical Department, Eli M y and Co., Indianapolis, Ind.

b Dilutions of U.S.P. XV sirups were hydrolyzed in hydrochloric acid solution to yield glucose. The glucose was determined colorimetrically by the specific glucose oxidase-horseradish peroxidase-dianisidine system. The average difference of found and actual concentration from 10 U.S.P. XV sirups was -0.270 and the average coefficient of variation of five replicates for each sirup was +0.9370.

T U.

are no methods, with the exception of specific gravity, in S. Pharmacopeia XV for the assay of sucrose in the various sirups (8). Procedures for sucrose generally require modifications for each sirup. Keston (5) described a method for testing for glucose utilizing the two enzymes, glucose oxidase (6, 4 ) and horseradish peroxidase (7'). The oxidation of glucose b y oxygen is catalyzed b y glucose oxidase, n i t h the production of gluconic acid and hydrogen peroxide. The reaction of hydrogen peroxide with a n organic substrate to form a colored product occurs in the presence of the horseradish peroxidase. The result of this sequence is a very specific reaction for glucose. This reaction has been used in commercial semiquantitative ( 1 ) and qualitative (3) test papers for glucose. Commercially available reagents have been used for the photometric microdrtermination of blood glucose (6). The method reported here is based on the enzymatic determination of glucose following the acid hydrolysis of sucrose. Other sugars may yield glucose on hydrolysis, so the method is not specific for sucrose, b u t i t is more specific than the usual reducing sugar methods following hydrolysis. A combination of glucose and sucrose may HERE

also be assayed b y color formation, before and after hydrolysis. EXPERIMENTAL

Reagents.

Enzyme. Dissolve approximately 2900 units of glucose oxidase (Takamine Laboratory, Clifton, N.J.), 1815 Pz units of horseradish peroxidase (Worthington Biochemical Corp., Freehold. X.J.), 0.125 gram of dianisidine citrate (prepared b y addition of citric acid t o a n ether solution of dianisidine), 0.200 gram of citric acid monohydrate, and 1.20 grams sodium citrate dihydrate in 500 ml. of water, and filter before using. The enzyme reagent may be stored for a week if refrigerated. The gradual loss in potency is corrected by standard assays. Glucose Standard Solution. This contains 0.2 mg. of anhydrous dextrose per ml. Allow this solution to stand a t least 1 hour, so that the equilibrium amounts of a- and @-glucose can be established. Glucose oxidase is specific for 8-D-glucose and insufficient standing time for the standard solutions will give low standard readings and erroneously high sucrose results. Sirup Dilution. Dilute the sirups to contain approximately 4 mg. of glucose per ml. If the actual glucose content of a mixture of glucose and sucrose is t o be determined, the initial concentration of glucose should be about 0.2 mg. per ml. for the assay before hydrolysis.

Hydrolysis. H e a t 5 ml. of t h e sirup dilution, 25 ml. of distilled water, a n d 5 ml. of concentrated hydrochloric acid for 30 minutes on a steam b a t h . Cool, adjust t o p H 5.6, and adjust t h e volume t o 100 ml. with water. Enzymatic Color Formation. Test tubes of t h e same dimensions should be used. T h e oxygen content of t h e enzyme reagent will regulate t h e rate of color formation, a n d differing surface areas of t h e enzyme reagent i n t h e

Table 1.

Absorbance of Glucose Solutions

Glucose Found Using Actual Mg. Absorb- 0.2 Xfg./Ml. Glucose/Ml. anceStd.7 % 0.1 0.201 100 0.15 0.302 100 0 20 0.401 100 0.25 0.507 101 0.607 101 0.30 0 699 99.7 0.35 0.799 99 6 0.40 400 mp 1-em. cells us. reagent blank. Table

II.

Recovery Data of GlucoseSucrose Mixtures

Total Glucose Recovered after Actual Glucose, yG Hydrolysis, 7 0 From From Analyst Analyst sucrose glucose A B 100 0 100 100 96 2 20 80 97.8 40 60 100 100 100 60 40 99.4 20 100 96.2 80 0 100 99.3 100 98.6 Av. 99.2 f1.5 Std. dev. =tO.88 tubes will a1loFv unequal atmospheric oxygen diffusion. Pipet 1 ml. of water into the reagent blank tube, 1 ml. of standard glucose into three tubes, and 1 ml. of the sirup dilutions into an appropriate number of tubes. If a sirup is highly colored, a sirup blank should be made b y adding 1 ml. of hydrochloric acid (1 to 1) to another tube labeled sirup blank. At definite timed intervals, pipet 10 ml. of the enzyme reagent into each tube. After exactly 10 minutes, add 1 ml. of hydrochloric acid (1 t o 1) t o each tube in the proper sequence, except for the ones containing the sirup blanks. T o VOL. 31, NO. 1, JANUARY 1959

109

100% glucose after hydrolysis. Table Table 111.

Sirup Acacia

Assay of U.S.P. XV Sirups

Av. found 82 6

Cocoa

Citric acid Ferrous sulfate Glycprrhiza Ipecac Orange Simple Tolu balsam Kild cherry

63 1 80 8 82 2 61 5 70 6 81 7 83 6 81 0 66 8

Sucrose, yo Actual 80 0 60.0 82.4 82 5 63 8 70 5 82 0 85 0 82 0 67 5

the Firup blank add 1 ml. of the sirup dilution. N i x all solutions by mild agitation. Measure the absorbance of these solutions in 1-em. cells a t 400 mg in a suitable spectrophotometer, using the reagent blank as the reference solution. Calculations. Mg, of sucrose per nil. of sirup (after

hydrolysis) =

ahs. sirup diln. - abs. sirup diln. bla& X abs. std. glucose (av.) 1.9

100

0.2 X - X - X sirup diln. factor 1 5 lfg. of glucose per ml. of sirup (before

hydrolysis) =

abs. sirup diln. - abs. sirup diln. blank X abs. std. glucose (av.) 0.2 X sirup diln. factor DISCUSSION

The enzymatic color reaction is stopped b y the addition of hydrochloric acid, so the timing intervals of

Dif. +2 6 1-3.1 -1 6 -0 3 -2 3 1 -0 3

+o

-1 4 -1 0 -0 7

I1 shon s their results and that variaStd. Dev., 5 Replicates 0.95 0 47 0 88 0 24 0 72 0 81 1 63 0 64 0 30 0 43

the addition of samples and acid should be accurately controlled. The daily standard glucose absorbance value (400 mp, 1-cm. cells, Beckman Model DU spectrophotometer) from the average of three determinations per day, for 20 days, was 0.423 and the standard deviation was 10.016. The enzyme reagents for the above study were prepared from the same raw materials, but were made every 2 days. If a greater accuracy than =t4% is desired, standard solutions should be assayed with the samples. A sequence of glucose concentrations was assayrd simultaneously and linearity was observed in concentrations of 0.1 to 0.4 mg. per mi. of glucose, as shown in Table I. The precision and accuracy of the hydrolysis procedure were established b y six replicates on a sucrose sample, yielding a n average of 99.1% and a standard deviation of 10.56%. TWO analysts assayed mixtures of glucose and sucrose containing a theoretical

tions in glucose or sucrose ratios do not alter the accuracy of the assay of glucose after hydrolysis. The U.S.P. sirups mere prepared as directed from reagent sucrose. Fresh cherrics and raspberries were not available, so cherry and raspberry sirups n-ere omitted. Liquid glucose, hai ing an indefinite amount of glucose, x i s omitted from the cocoa sirup. Five complete rrplicate assays were made on the initial dilution of each sirup and the results condensed into Table 111. The average differences of actual and found sucrose for the ten sirups are about -0.2% and the average coefficient of variation (S/Z x 100) for the five replicates is 10.93%. LITERATURE CITED

(1) Comer, J. P., AKAL. CHmr. 28, 1748 ( 1956).

(2) Coulthard, C. E., et al., Biochem. J . 39, 24 (1945). 13) Free. A. H.. et al.. Clin. Chem. 3. 163 \

,

\

,

(1957). (4) Keilin, D., Hartree, E. F., Biochem. J . 50, 331 (1952). ( 5 ) Keston, A. S., Abstracts of Papers, p. 31c, 129th Meeting, ACS, Dallas, Tex., April 1956. (6) Saifer. A . Gerstenfeld. S.. J . Lab. Clin. M e d . 51, 448 (1958j. ( 7 ) Theorell, Hugo, Arkiv. Kemi.Nzneral. Geol. 2, 1 (1942). (8) “United States Pharmacopeia,” 15th Revision, p. 711, Mack Publishing Co., Easton, Pa., 1955. RECEIVED for review May 8, 1958. Accepted September 2, 1958.

Determination of Cadmium by Flame Photometry PAUL T. GILBERT, Jr.‘ Nuclear Engineering and Manufacturing Deparfmenf, Norfh American Aviation, Inc., Downey, Calif.

A , survey of the excitation of cadmium in various flames has shown that the air-hydrogen flame i s particularly advantageous for the determinaiion of cadmium a t 326.1 mp. A sensitivity of 0.1 p.p.m. and a precision to 0.1% have been demonstrated with a Beckman flame photometer with multiplier phototube. Interferences of aluminum, iron, copper, and calcium with cadmium in the air-hydrogen flame, and of aluminum and iron in the oxyhydrogen flame have been measured. The working curve for cadmium i s nearly linear below 500 p.p.m. To circumvent interferences the method of successive dilutions i s used. The possibility of avoiding aluminum inter-

110

ANALYTICAL CHEMISTRY

ference b y use of oxyhydrogen or b y flame determination of aluminum i s also considered.

c

inrestigated by Lundegirdh ( I O ) in the air-acetylene flame; he mentioned a detection limit of 200 p.p.ni. a t 326.1 mp. The inner cone of air-acetylene emits also the resonance line 228.8 mp (8,9). Rusanov and Alekseeva (12) determinrd cadmium in ores semiquantitatively with a visual air-acetylene flame photometer. Rusanov (11) subsequently determined cadmium in solution by photographic photometry with the air-acetylene flame, and in mineral powders with the oxy- m x u c h f TWS



acetylcnc flame. The oxygen-coal gas flame is said to excite cadmium only n-eakly (15). The oxygen-methane flame is also poor for this metal (7); with a Beckman DU spectrophotometer and rnultiplicr phototube a detection limit of about 50 p.p.m. might be expected a t 326.1 nip. Egerton and Rudrakanchana ( 2 ) studied the flame spectrum of dimethyl cadmium, and Alekseeva and Mandel’shtam ( I ) examined the emission of cadmium in the inner and outer cones of the air-acetylene flame, but 1 Present address, Beckman Instruments, Inc., Fullerton, Calif. * Xow Atomics International, Canoga Park, Calif.