V O L U M E 2 1 , NO. 8, A U G U S T 1 9 4 9
99 1
with a htundard deviation of 0.06. A calibration curve was drawn to pass through the intercept obtained by analysis of the best sample of purified benzyl alcohol. The data for these analyses are shown in Table 11.
Table 11. Absorbancy of Purified Beneyl Alcohol Absorbancy a t 283 mu 0.042 0.043 0.041 0.042 0.044 0,044
PROCEDURE
Pipet 5 ml. of the benzyl alcohol sample into a 100-ml. volumetric flask, dilute to the mark with methanol-water diluent, and invert several times to dissolve the sample. Transfer a portion to a 1-em. fused silica absorption cell and determine the absorbancy a t 283 mp. The blank cell should contain the methanol-water diluent.
hv. 0 . 0 4 3
Table 111. Benzaldehyde Found by Four Analysts Using Two Beckman Spectrophotometers
ACCURACY ANI) PRECISION
I t is possible that there was a slight residue of benzaldehyde in the benzyl alcohol having the lowest absorbancy a t 283 mp and so any statement regarding the accuracy is subject to criticism on this point. If pure benzyl alcohol has no absorption at 283 mp, it is evident that the best sample prepared contains material which, if calculated as benzaldehyde, would amount to 0.006% benzaldehyde. However, the actual error, if any, is probably less than 0.006% of benzaldehyde. To secure an indication of the precision of the determination, four analysts using two spectrophotometers analyzed a sample of benzyl alcohol (Table 111). One dilution was made by each analyst and in no case was a duplicate or check dilution prepared by any analyst. Variance analysis indicates that there is no significant systematic variability between the spectrophotometers or among the test operators. The best estimate of the variability of the method is the standard deviation of the eight values and this is 0.00022% henzaldehyde. The average error is about 1%.
Analyst
h
B C D .A\..
Spectrophotometer 1380, % 0.0126 0.0122 0.0125 0.0127 0.01250
Spectrophotometer 2323, % 0.0127 0,0126 0,0126 0.0130
Average, % 0.01265 0.01240 0.01255 0.01285
0.01272
0.01261
ACKNOWLEDGMENT
The benzaldehyde-free benzyl alcohol was prepared by D. B. Glass and Stephen Michel, Color Control Department, Eastman Kodak Company. LITERATURE CITED
(1) Donnally,L. H.,IXD.ESG.CHEM.,ANAL.ED.,5,91(1933). (2) Iddles, H.A., and Jackson, C. E., Ibid., 6,454(1934). (3) Natl. Bur. Standards, Letter Cir. LC 857 (May 19,1947). (4) Parkinson. A. E., and Wagner, E. C., IND. ENG. CHEM., ANAL.ED., 6,433 (1934). (5) Youden. W. J., ANAL. CHEY.,19, 946 (1947). RECEIVEDOctober 4, 1948.
Direct Colorimetric Method for Carbohydrates MALTOSE G. HARVEY BENHAM AND VIRGINIA E. PETZING Illinois Institute of Technology, Chicago, Ill. The molybdenum blue reaction as applied to glucose has been extended to maltose in such a way t h a t mixtures of maltose and glucose may be analyzed. Maltose reacts in conformity with Beer's law, b u t gives values which are about one tenth as great as values for glucose under identical conditions. Aliquots of mixtures containing not more than 5 mg. of the two sugars combined were analyzed to yield B, the absorption value a t 650 for the glucose-maltose mixture. Complete hydrolysis of the maltose without any destruction of glucose was effected in a n autoclave in 1hour by using small concentrations
T
HE molybdenum blue reaction a b used for small quaiititieb of glucose by Benham and Despaul ( 1 ) has been adapted for
the determination of maltose and mixtures of maltose and glucose. The method as previously outlined was used throughout this Rork, with strict attention to details of time and temperature. In order to analyze an unknown mixture of glucose and maltose in terms of each of these sugars, it was decided to evaluate the blue color hrfore hydrolvsis and after complete hvdrolvsis
of hydrochloric acid. i f t e r this hydrolysis the mixture contained only glucose, which was analyzed to yield A, the absorption value for all t h e glucose now present. By calculation from the most favorable points obtained from the standard curves, two 0.0245) equations were obtained: B = (0.084G (0.0097M - 0.0009) and A = 0.084 (G -M) 0.0245. where G = glucose and M = maltose. Solving for :M,A B = 0.0743 0.0009. Substitution of the values for M in the second equation yields the value for G. Mixtures so analyzed yield recoveries of 98 to 100% when 2 to 5 mg. are taken for analysis.
+
-
+
+
+
+
The value before hydrolysis ( B ) is due to glucose alone only if the maltose color is entirely eliminated. The value after hydrolysis ( A ) is due to the sum of the original glucose and the glucose obtained by total hydrolysis of the maltose present. This approach hinges upon three conditions: (1) the complete elimination of any color due to maltose in determination B , (2) the complete hydrolysis of maltose for determination A , and (3) absence of any destructive effect upon glucose with the conditions of hvdrolvsis chosen.
992
ANALYTICAL CHEMISTRY
Table I.
Effect of Acid Concentration on Hydrolysis of Maltose
(2.5 mg. of maltose in an autoclave for 1 hour at 120' C.) Acid Used Recovery
M1. 5 Concd. HC1 2 Concd. HC1 1 Coned. HC1 S 10% HCI 4 1 0 7 HC1 3 HC1
lOd
Mg.
%
2.0 2.2 2.3 2.4 2.45 2.45
80 88 92 96 98 98
0.50
a 0.40
a
z
Q
2 0.30 II 0
2I 0.20
Accordingly, the conditions for the determination of unknown mixtures of glucose and maltose were established as follows: A sample containing not more than 500 mg. of glucose and maltose is accurately weighed out and made up to 100 ml.; 50 ml. of this are taken and made up to 100 ml. with water, and 2 nil. of this solution, which contains not more than 5 mg. of the niised sugars, are used for direct analysis without hydrolysis. To it are added 5 ml., of 0.02 M potassium dihydrogen phosphate and 10 ml. of 7.5% ammonium molybdate. The solution is adjusted to the mark in 25-ml. volumetric flasks and mixed and the stoppers are removed. The flasks are introduced into a preheated autoclave and covered with a piece of sheet metal to prevent any condensation, then heated for exactly 30 minutes n-ith open steam at 100" C. to develop the blue color. They are removed after the heating period and plunged immetlistely into an ice bath to arrest the reaction: when cooled t o room temperature, the volumes are adjusted to the mark if necessary. The color is read in a Coleman )lode1 I1 spectrophotometer a t a wave length of 650 mg. This rending gives value B. To the other 50 nil. of original solution are added 3 ml. 10% hydrochloric acid, and hydrolysis is carried out in an autoclave at 68-kg. (15 pounds) pressure for 1 hour. The flask is cooled, and the solution is neutralized nith 0.1 S sodium hydroxide to the methyl red end point and made u p t o 100 nil. The presence of methyl red in the neutralized snllltion has no effect on the subsequent colorimetry. Two milliliters of this solution are taken and treated for development of the blue color in exactly the same \my as the unhydrolyzed sample. This gives value A .
In order to evaluate with some precision the maltose and glucose content of unknon-n mixtures from the two colorimeter readings B and A , the method of mean squares is employed to analyze the most probable curve for both sugars.
o(
0.10
Table 11. P
1
Figure 1.
3
4
5
Analjsis of Known 3lixtures of Maltose and Glucose Tranqmit tsncy after Hjdrolysis
Standard Curves for Glucose and Maltose A. B.
hZg. of glucose M g . of maltose
Benham and Despaul ( 1 ) found that under the standard conditions adopted for glucose, the presence of sucrose, except in high concentrations, could be discounted, inasmuch as the blue color developed with sucrose was substantially zero. Under the same conditions, maltose gives a slight blue color, as shown in Figure 1, which represents standard curves for glucose and for maltose plotted on the same graph. An attempt was made to eliminate the color due to maltose by shortening the heating time to 10 minutes. Under these conditions 1 mg. of maltose does not have time t o react, but 5 mg., the upper limit for the procedure, still yield a very slight blue color. Moreover, the shorter heating time decreases the blue color due to glucose to a point where the sensitivity of the method is considerably impaired. Accordingly, no attempt was made to eliminate the maltose color, but a procedure involving two steps was adopted for an unknown mixture of the two sugars. With dilute acid hydrolysis for 1 hour a t a temperature of 120' C. in an autoclave, the recovery of maltose as glucose after hydrolysis is 98 to 100%. The acid concentration in the final hydrolysis mixture was 3 ml. of 10% hydrochloric acid in a total of 50 ml. This is approximately 0.07 X with respect to hydrochloric acid and has a pH of 1.7. More concentrated acid causes some destruction, as shown in Table 1. Analysis of known mixtures of maltose and glucose, each containing the equivalent of 5 mg. of glucose when hydrolyzed, yields satisfactory agreement, as shown in Table 11. Standard curves were obtained for glucose alone after identical acid treatment followed by neutralization. In each case, these curves matched precisely the standard curves obtained for glucose directly. It is evident that glucose itself is unaffected when submitted to the acid treatment finally adopted for maltose hydrolysis.
1Ialtose
Gluco*e
Ifo.
.I1g 0
5 4 3
.
-
log G
Recoiery
,llg .
0.435 0.433 0,440 0.441 0,440
36.7 36.8 36.2 36.1 36.2
1 2 4 5
1 0
2
"0
4.95 4.95 5.0 5.0 5.0
Equations 1 and 2 are solved from the actual observed values for zand y listed in Table 111.
- m2z - nb = 0 - mZx2 - bZx = 0
2ny Zxy
(1) (2)
By substitution in Equations 1 and 2, Equations 3 and 4 are obtained for maltose and Equations 5 and 6 for glucose. 0.254 - 27m 0.908
- 96m
Table 111. x 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4 5 5.0 22 = 2 7 . 0
1.0 1.5 2.0 2.: 3.0 3.5 4.0
4.5 5.0 Zz = 2 7 . 0
-
(3) (4)
9b = 0
- 27b = 0
Standard Curves rY
!L 22 Maltose Standard Curve 1.0 0.010 2.25 0.014 4.0 0.018 6.25 0.023 9.0 0.027 12.25 0.032 16.0 0.038 20.25 0.044 25.0 0.048 Pu = 0 254 Pz* = 96.0
0.010 0.021 0.036 0.058 0.081 0.112 0.152 0.198 0.240 zzy = 0 , 9 0 8
Glucose Standard Curve 1.0 0.106 2.25 0,147 4.0 0.199 6.26 0,230 9.0 0.279 12.25 0.325 16.0 0.359 20.25 0.403 25.0 0.440 Zzz = 9 6 . 0 Zy = 2.488
0.106 0.221 0.398 0.575 0.837 1.138 1.436 1.814 2.20 zzy = 8 . 7 2 5
V O L U M E 21, NO. 8, A U G U S T 1949
- 27m - 9b = 0 8.725 - 96m - 2ib = 0
993 (5)
2.488
(6)
Solutions of these pairs of equations yield: For maltose
m = 0.0097 b = -0.00088
For glucose
m = 0.0840
b = 0.0245
Hence the values of 2 - log transmittance before hydrolysis ( R )and after hydrolysis ( A ) are represented by:
B A en c e
+ 0.0245) + (0.0097M - 0.00088) + M ) + 0.0246 A - B 0.0743 M + 0.00088
=
(0.0840 G
(7)
=
0.0840 ( G
(8)
= (9) The value of M so obtained is substituted in Equation 8 to ewluate G . A sample calculation is given in which not more than 5 mg. of a glucose-maltose mixture were taken. The 2 log G values were .4 = 0.435 and B = 0.146. Solving for M by Equation 9, M = 3.0 mg. and for G by Equation 8, G = 0.98 mg. Actually this mixture contained 4 mg. of maltose and 1 mg. of
glucose. The experimental results were satisfactory, as the ratio of maltose to glucose was exactly as taken. This type of determination and calculation may be applied to commercial products to analyze for maltose and glucose. One typical product (Dextrimaltose) was labeled as containing “55% maltose as total reducing sugars.” Analysis by the hlunson and Walker method gave a value of 57.5% as total reducing sugars. By using the method outlined in this paper, and resolving the equations, the results obtained were: 4i.7% maltose and 10% glucose. Such a procedure is rapid and extremely useful in cases where the ratio of maltose to glucose must be controlled, as in the preparation of partially hydrolyzed starch products. In these instances, it is necessary to remove oligosaccharides and dextrins of higher molecular weight in a quantitative manner prior to the determination.
-
LITERATURE CITED
(1) Betiham, G. H., and Despaul, J. E., ANAL.CHEM.,20, 933-5
(1948). RECEIVED October 10,1918.
Determination of lactose in Milk Products B. D. HITES
AND
C. W. ACKERSON
WITH THE TECHNICAL ASSISTANCE OF
G . H. VOLKJIER Agricultural Experiment Station, Lincoln, Neb. The ferricyanide method may be used for the determination of lactose and sucrose in dairy products. It is simple, convenient, and time-saving, and requires no special equipment. The method can be used for the analysis of dairy products containing lactose and lactose in the presence of sucrose, but cannot be used when the products contain other reducing sugars.
T
H E unsatisfactory nature of existing methods for the determination of maltose in flour led Blish and Sandstedt ( 9 ) to seek a method that possessed reliability, simplicity, convenience, and minimum requirement for special equipment. Accordingly they adapted the ferricyanide method of Hagedorn and Jensen (9) to the estimation of maltose in flour. This adaptation was modified by Sandstedt ( d ) , who also applied it to the determination of sucrose in flour (6). In view of the agreement in the maltose values secured by the ferricyanide method as compared to the official method of the association of official agricultural chemists (1), the procedure has been eytended to the determination of lactose in milk and milk products. Therefore, comparisons were made by both the ferricyanide and the official method on the lactose determination in milk and milk products. REAGENTS
Acid Buffer Solution. Dissolve 3 ml. of glacial acetic acid, 4.1 grams of anhydrous sodium acetate, and 4.5 ml. of sulfuric acid (specific gravity 1.84)in water and dilute to 1 liter with water. Sodium Tungstate, 12%. Dissolve 12.0 grams of sodium tungstate dihydrate in water and dilute to 100 ml. with water. Acetic Acid-Salt Solution. Dissolve 70 grams of potassium chloride and 400 grams of zinc sulfate heptahydrate in water, add slowly 200 ml. of glacial acetic acid, and dilute to 1 liter with water. Soluble Starch-Potassium Iodide Solution. Suspend 2 grams of soluble starch in a small quantity of cold water and pour slowly into boiling water with constant stirring. Cool thoroughly (or the
resulting mixture will be dark colored), add 50 grams of potassium iodide, dilute to 100 ml., and add one drop of saturated sodium hydroxide solution. Thiosulfate Solution, 0.1 N . Dissolve 24.82 grams of sodium thiosulfate pentahydrate and 3.8 grams of borax and make up to 1 liter. Standardize against pure copper. Dissolve 45 to 55 mg. of pure copper in 2 ml. of concentrated nitric acid, and heat carefully until brown fumes are driven off. Dilute to 10 ml. with water. Add concentrated ammonium hydroxide a drop a t a time until the last drop produces a deep blue solution. Add 5 ml. of concentrated acetic acid and 1 ml. of potassium iodide-starch solution and titrate with the thiosulfate solution until the starchiodide color fades out. From the milligrams of copper and the milliliters of thiosulfate used the normality can readily be calculated. Alkaline Ferricyanide Solution, 0.1 N . Dissolve 33 grams of pure dry potassium ferricyanide and 44 grams of anhydrous sodium carbonate and dilute to 1 liter. To standardize, add to 10 ml. of this solution 25 ml. of acetic acid-salt solution, and 1ml. of soluble starch-potassium iodide solution, and titrate with 0.1 N thiosulfate. Exactly 10 ml. should be required to discharge the blue color. STAKDARD LACTOSE CURVE
A a.ater solution of high purity a-lactose was prepared so that 1 nil. contained 50 mg. of the anhydrous sugar. Increasing amounts of this solution were added to 50-ml. volumetric flash which contained 43 ml. of acid buffer and 2 ml. of sodium tungstate solution. Sufficient water was added, when needed, to give a final volume of 50 ml. The mixture was vigorously agitated and 5-ml. aliquots were immediately added to 10-mi. quantities of the alkaline 0.1 &Vferricyanide contained in 50-ml. Pyrex test tubes (18 to 20-mm. diameter). The tubes were immersed in boiling
