January 15, 1931
17
INDUSTRIAL AND ENGINEERING CHEMISTRY
The method was applied to mixtures of soy bean oil with gilsonite and with Bermudea asphalt with less success. Difficulty was experienced in detecting the last maximum. It is recommended, therefore, that fltandard d~oholicbase be used in the analysis of mixtures which contain soy bean oil.
These results are also given in Table IV. Literature Cited (1) Abraham, “Asphalt and Allied Substances,” (2) Marcusson, Chem.-Ztg., 36, so3 (1912).
van
Nostrand, 1918.
Effect of Amino Acids upon Rotation of Glucose and Fructose and Its Significance to Determination of Sucrose by Double Polarization Methods’ D. T. Englis and F. A. Dykins CHEMICAL LABORATORY, UNIVERSITY OF ILLINOIS, URBANA, ILL.
The effect of increase in alkalinity upon the combination of glucose with glycine, as previously noted by Euler and associates, has been extended with studies of the effect of temperature and different molar relationships. The extent of combination has been followed polarimetrically and by an iodometric determination of uncombined glucose. The increase in combination with increase in pH, and the equilibrium constants for the reaction as determined by polarimetric and iodometric methods, were in agreement with each other and with results obtained by Euler from freezing point data. A t 25” C. the glycine-glucose complex was almost completely reversed by acidification while at a temperature
as low as 35” C. some of the stable type of melanoidal combination, not affected by acid, was evident. Near 0 ” C. the extent of the reaction was very limited. Similar experiments were carried out with asparagin and glucose and the same general effects noted although the two methods gave values of different magnitudes. Fructose showed no optical changes in the presence of glycine and asparagin with increasing pH which could not be attributed to alkalinity alone. The significance of the effect of pH, time of standing, and temperature upon the determination of sucrose in the presence of amino acids by double polarization method is discussed.
.............. HE importance of more exact knowledge of the effect of certain optically active nitrogenous impurities upon simple sugars and its significance to the determination of sucrose by the double polarization method is evident from the recent referee studies reported upon this subject by Zerban (15, 16). The information is especially necessary in studies of carbohydrate metabolism of plants in which the sugar concentration i s quite low and the concentration of the.nitrogenous materials relatively higher in proportion to the sugars than in the juice of plants used as sugar sources. Brown and Morris (%’),who are perhaps among the earliest workers to make a detailed study of the individual carbohydrates in plants, call attention to a disparity between the value for sucrose determined by polarization as compared to chemical methods. The magnitude of the difference varied with different plant materials and with different portions of the same plant. More recently Davis and co-workers, in estimating the carbohydrates of the mangold and potato, found polarization values as much as 80 per cent greater than the reduction values and sometimes as much as 90 per cent lower. The variation was generally greatest in the stem extracts. Davis (6) has made the assumption that the fluctuations between the optical and chemical values is due to asparagin and glutamin. In this laboratory, in the investigation of the effect of potassium salts upon the carbohydrate metabolism of the nasturtium, the results of the determination of sucrose by polarimetric methods have been consistently higher. This has led to a series of studies in an effort to determine the causes of the differences, and to find a means of eliminating the more serious sources of error.
T
1 Received
September 4, 1930.
Somers (14) investigated first the effect of reduction due to sucrose upon the chemical method; second, the effect of certain salts left after different defecation procedures; and finally, the influence of asparagin upon the optical values. These factors taken separately were found to have comparatively little effect, but some observations of the last mentioned are of special interest. An asparagin solution and invert solution were prepared and portions of each were mixed with an equal volume of water. Then equal portions of each original solution were mixed together. The mixture showed an increased levoI rotation over the sum of the optical values of the individual constituents. A few years later, Drake (6) studied the effect of the presence of a large number of amino acids upon glucose, fructose, and sucrose before and after its inversion. An effort was made to keep the solutions approximately neutral to avoid the effect of change in acidity upon the rotation of the amino acid itself. Both plus and minus differences in rotation of the mixtures were found but the value of the work suffers from lack of exact knowledge of the acidity and duration of the reaction period. I n 1925, Nueberg and Kobel (11) made observations of a somewhat similar character. These investigators were concerned with a probable reaction product between sugar and amino acid which accelerated the alcoholic fermentation of the sugar. Addition of d-l-alanine to a fructose solution increased the levorotation while with glucose very little effect was noted. Supplementary work by Neuberg and Kobel (1%’)indicated that fructose seems to react immediately with amino acids and amides at a pH of 7. Particularly striking increases in levorotation were given by aspartic
.
ANALYTICAL EDITJON
18
and glutamic acids. With the aldol sygars the effects were much less. It has been pointed out by Browne (3) and others whom he quotes that optical rotation in the ordinary sense of the term is not an additive property. It follows as a consequence that a discrepancy between the rotation of a mixture and the sum of the rotations of its constituents does not necessarily mean that a reaction has taken place between the constituents. Although the effect of the water concentration which Browne has emphasized would probably not be sufficient t o account for the total differences observed by Somers and other investigators cited, additional indications of a reaction should be furnished by other methods. This has been done by Euler and his associates. Euler and Josephson (IO) gave attention to the effect of decreasing hydrogen-ion concentration upon the reaction of glucose with glycine. At the isoelectric point of glycine no reaction took place, but with increasing pH, the apparent combination as indicated by change in polarization was 32 per cent a t a pH of 9.6. A more detailed study by Euler and Brunius (7, 8, 9) in which the reaction was followed by freezing point lowering, determination of uncombined amino acid by the Van Slyke method and glucose by the iodometric method, in addition to polarimetric change, confirmed the earlier observations. It was observed that the rate of combination of glucose with glycine is slow, but a t a pH of 9.5, based on observations of freezing point lowering, it reaches equilibrium a t 53 per cent combination in 48 hours. Glucose and alanine behaved in a similar fashion. The first explanation advanced for the increase in extent of reaction with increase in pH was that the alkalinity favored the change of the sugar from the lactone to the free aldehyde form which then condensed with the amino group. Preliminary treatment of the sugar with alkali had no effect on the final reaction, so any previous rearrangement to the acyclic form did not accelerate the combination. Additional evidence from refractometric observations seemed to indicate absence of a double-bonded compound, and it was concluded that the union between sugar and amino acid was of glucosidal character. While the experiments of Neuberg and Kobel, and Euler and his associates were carried out with a biological application in view, they are not less important from the standpoint of analysis. I n the present study some of the previously reported experiments with glycine have been extended and additional experiments carried out with asparagin, a representative of bne of the more common nitrogenous impurities in plant extracts. Experimental Methods
The amino acids and sugars had been carefully purified and were of high quality. Glycine was selected as one of the amino acids for study since it is optically inactive and hence the complications due to the change in rotating power of the acid itself are eliminated. Sodium hydroxide was added to the amino acid solution to bring it to a definite pH and then the solution made to volume with a carbonate-bicarbonate buffer. The sugar solution was similarly buffered. Amino acid and sugar solutions were then mixed in equal quantities, and for the purpose of control an equal volume of each was mixed with an equal volume of buffer solution. The hydrogen-ion concentration of each of the prepared solutions was measured. A Leeds and Northrup potentiometer was used with a Hildebrand hydrogen electrode and a 0.1 N calomel half-cell. The e. m. f. of the solution was measured and the pH calculated. An atmosphere of purified hydrogen was maintained above the solution to insure a steady potential. The reactions
Vol. 3, No. 1
were allowed to proceed a t various pH values and various temperatures until equilibrium was reached. I n most of the experiments the concentrations of the mixture were in a 1 to 1 molecular ratio. I n others the amino acid was reduced, keeping the molar concentration of glucose constant. I n all experiments the extent of the reaction was determined by decrease in optical value. The observations were made with a Schmidt and Haensch saccharimeter. I n some of the mixtures the uncombined sugar was determined by a slight modification of the Cajori (4) iodometric method for aldol sugars, and the uncombined amino acid estimated by the Van Slyke (13) method. Calculations of equilibrium constants, glucose-amino complex c glucose X amino acid, were made on the assumption that the combination is in an equal molecular ratio and that the compound formed is of negligible rotation so that the decrease in rotation is equivalent to the extent of combination. This assumption will be open to criticism. Euler has made a calculation of the probable optical activity of compound and found it to be comparatively high. However, if one accepts the theory of a glucosidal structure and if the amino acid reacts equally well with the a! and p forms of glucose, one would anticipate that little change in rotation would occur if the glycine glucosides differ in rotation from their respective forms of glucose as do the methyl glucosides. The detailed results of these experiments are given in Table I. of Glycine on Glucose a t Various pH Values Pa76 I-Optical Changes (2 dm. tube) Temperature, 25' C.; molar concentration, 0.0666
Table I-Effect
1 2tyEF;E GLUCOSE
TIME
PH
50 c c
1
Hrs. 8.11
EQUI-
DECREASE IN ROTATION
LIBRIUM
CONSTANT
V. 3.60 3.53 3.50
4s
0.43
9.52 4s
3.00
0.00 13.33 16.66
0.60
3.5
10.30
5.5
Part 11-Decuease pH
MIXTURE
10 lo
10
1
8 11
1;
!;
10.1116 N Ia SOLN.
1
1
in Appavent Glucose b y Iodometric Method
I
': a i 11 57
1
1
GLUCOSE GLUCOSE Lossoa PRESENT ACCOUNTEDGLUCOSE
116 56 102 92-97 20
FOR NOT
1
:;:: 1 3 44
2 87 22 14 83 53
i
Part III-Uncombined Amino Acid by V a n Slyke Method Temperature, 26" C.; barometer, 749 mm. Hg GLUCOSEGLYCINE MIXTURE
1 1 PH
1
NZFORMED G e i F F i N
1
TOTALGLYF A N 6 F ~ (1.000 GRAM P R EsEN T)
1
Part IV-Summary . . l r rll
1
APPARENT GLUCOSE
I Polarimetric I
Iodometric
of Results
APPARENT
I
COMBINED GLYCINE
Van Slyke method
INDUSTRIAL A N D ENGINEERING CHEMISTRY
January 15, 1931
Experiments with Asparagin
Effect of Variation in Glucose-Glycine Ratio upon Their Combination
A series of experiments analogous to those with glycine were carried out with asparagin and the results are recorded in Table 11. of Asparagin on Glucose a t Various p H Values Part I-Optical Changes (2 dm. tube) Temperature, 25' C.; molar concentration, 0.0666
Table 11-Effect
PH
I
TIME
I
I
Hrs.
I
O
0
10.64
0
24 48 72 Part II-Decrease
1 1
GLUCOSEASPARAGIN MIXTURE
10 10
O
I
v.
%
0.00
0.45 0.94 0.98
12:33 25.75 26.85
0.00
2.30 3.24 3.42
I
6i:Ol 88.76 93.72
i n Apparent Glucose b y Iodometric Method
1
0.1116 N
IZSOLN.
pH
DECREASE IN ROTATION
I
v.
3.65 3.20 2.71 2.67 3.65 1.35 0.41 0.23
24 48 72 11.55
I
GLYCINE 50 cc. G~~~~~~ 50 cc,
,
GLUCOSE GLUCOSE LOSS OF PRESENT ~ A c c ~ ~ ~ GLUCOSE T E D ~
107.40 72.89
I
1
$:$I
I
1
cc. 10 10 10
I
PH
1
I
1
I
8.75
I
cc. 4.16
SAMPLE
-
MIXTURE
IN
0.440GM.
I
1
Mg.
22.04
pH
8.75 10.64 11.55
I
I
Polarimetric
2 1 1 26.85 93.72
1
Iodometric
% 0.00
10.50 39.26
V.
0
12.24 9.66 8.39 8.12 8.32 8.04 8.12
6 18 24 30 42 48
Gm. 0
Van Slyke method
8.11 9.52 10.30
I
1
OV.
I
I
ov.
7.00 6.30 5.60
1
ov.
0.0
% 0.0
22.7 30.9 33.9 37.7 39.3 37.7
% 0.0
% 0.0
27.7 35.0 45.1 48.0 47.7 44.1
26.2 44.4 45.1 45.1 62.7 52.3
9.93 11.35 11.23 11.57
10.89 11.43 11.38 11.39
11.44 11.60 11.53 11.66
I
Hvs.
44.1 26.4 28.1 27.7
52.3 41.8 46.4 37.9
9.6 10.9 27.7 37.9 24.1 26.8 16.4 15.5
Initial glucose-glycine ratio
1:0.5 1:0.251:0.126
v.
v. 12.24 10.63 10.38 9.63 9.63 9.65 9.63
v. 12.24 11.35 10.98 10.70 10.56 10.60 10.51
0 6
12.24 9.50 8.10 7.89 7.86 7.77 7.82
0
7.82 9.63 10.51 11.01 11.21 11.07 11.01 11.03 11 10 10.93 11.21 11.00
18 24 30 42 48
37.7 14.5 16.5 10.9
APPARENT COMBINATION
Initial glucose-glycine ratio
a
33.7 10.3 8.7 9.6
p H , 10.3; Temperature, 3.5' C.; Molar Concentration of Glucose, 0.222
1:l
I
1:l
1:0.5 1:0.251:0.125
v.
%
12.24 11.80 11.28 11.16 11.00 11.07 11.03
0.0
0.0
22.4 33.8 35.5 35 8 36.5 36.1
26.3 30.4 42.6 42.6 42.3 42.6
O
%
%
%
0.0
0.0
29.0 41.2 50.3 54.9 53.6 56.6
28.9 62.7 70.6 81.0 76.4 79.1
36.1 42.6 56.6 10.0 16.8 38.2 10.0 19.8 37.2 10.7 16.8 40.5
79.1 79.1 90.2 88.2
AFTER ADDITION O F SOLID OXALIC ACID
66
11.03 11.03 10 86 10.89
(A) Per cent reduction of rotation due to destruction of glucose or formation of a non-dissociable compound. (B) Per cent combined at equilibrium after correction for (A),
10.7 16.8 40.6 88.2 25.4 25.8 16.1
,.
Method of Calculations
AFTER48 HOURS
DIL,
3.50 3.15 2.80
1:0.51:0.251:0.125
-
AEzc/ HC1
I
% 21.0 31.4 33.7 32.0 34.3 33.7
ROTATIONS REACTION PERIOD
of Acidification u p o n Glucose-Amino Acid Condensation Product Molar concentration, 0.1332
7.20 7.20 7.20
V.
12.24 11.84 11.56 11.55 11.55 11.28 11.44
non-dissociable compound. (B) Per cent combined at equilibrium after correction for (A).
Part 11-Original
%
AFTER48 HOURS
V. ' V . 12.24 12.24 11.39 10.35 11.17 10.16 10.86 9.93 10.77 9.83 10.78 9.93 10.89 10.85
(A) Per cent reduction of rotation due t o destruction of glucose or formation of a
0.0
Euler and Brunius (8) found that, when solutions of alanine and glucose with a pH of 10.4 had been allowed to stand until equilibrium was reached and then acidified with sulfuric acid, after 25 hours the iodine consumption was almost the same as the initial value, indicating a reversibility of the reaction. Such reversibility should characterize all glucoseamino compounds. To demonstrate this point solutions were prepared in double the concentration indicated in Table I, Part I, and polarized. They were allowed to stand 48 hours, polarized, and acidified with an equal volume of dilute hydrochloric acid. After 48 hours the solutions were again polarized. The results are given in Table 111.
GLUCOSE 50 cc. GLYCINE 50 cc.
8.12 10.97 11.18 11.06
2.04 1.36
Effect of Acidification on the Reversibility of the Reaction
Table 111-Effect
1:l
AFTER ADDITION OF SOLID OXALIC ACID
0.441 0.431 0.434
APPARENTCOMBINED GLYCINE
I
APPARENT COXBINATION Initial glucose-glycine ratio
1:0.5 1:0.25 1:0.125
Hvs.
66 APPARENT COMBINED GLUCOSE
I
ROTATIONS Initial glucose-glycine ratio
1:l
TOTAL ASPARAGIN
IN
Table IV-Effect of Changes i n Glucose-Glycine Ratio a n d pH u p o n R a t e of Change in Optical Value of Solution. Rate of Change o n Acidification Part I-Original p H s 9.52; Temfierature, 3.5' C.: Molar Concentration of Glucose, 0 222
10:50
39.26
I
Nz FORMED ASPARAGIN
The next experiments were designed to determine the effect of altering the ratio of the glucose and glycine upon the optical value a t different pH values. The majority of sugar juices or sirups contain sugar in large excess of the amino acids so that the mass action effect of the sugar is quite high, and one would expect almost all the amino acid to be in combination. I n this study the glucose concentration was kept constant, and that of the glycine reduced. The general method of procedure was the same as previously outlined. After equilibrium was reached, solid oxalic acid was added, and the rate of reversion to the original constituents followed. These experiments were carried out a t room temperature during an exceedingly warm summer and the thermometer registered close to 35" C. during the whole period. The results are given in Table IV.
REACTION PERIOD
Part 111-Uncombined Amino Acid by V a n Slyke Method Temperature, 26' C.; barometer, 749 mm. Hg GLUCOSEASPARAGIN MIXTURE
19
1
1
0
8 . .. 3.60 3.50 3.40
Calculation of the apparent percentage of glycine in combination may be indicated as follows: I n Table IV, Part 11, the ratio of glucose to glycine is 1 to 0.5. The decrease in rotation indicates an apparent loss of glucose equal to 21.3 per cent. Since one mol of glycine combines with one mol of glucose and its initial concentration is only half that
20
ANALYTICAL EDITION
of the glucose, then 42.6 per cent of i t may be considered as in combination. The equilibrium values at 48 hours would seem to indicate that as the glucose is increased in proportion to glycine, a greater percentage of glycine is brought into combination. However, when the solutions are acidified, they fall far short of returning to their original values. Apparently, a t this higher pH and temperature the type of melanoid reaction recently discussed by Ambler (1) begins to take place between sugar and amino compound. I n addition there may be some alteration of the glucose itself. Consequently calculations of even apparent equilibrium constants are out of the question. Effect of Temperatures of 0' C. and 50' C. upon Reaction of Glucose and Glycine at pH 10.3
Vol. 3, No, 1
to determine the effect of increasing alkalinity upon such sugar mixtures. Accordingly experiments were tried with glycine and asparagin. The results are given in Table VI.
ROTATION (4-DM. TUBE)
pH
T*ME
-HYS. S 15
9.52 10 30
0
79 0 79 0 79
Levulose and buffer
Levbloseglycine
v.
v.
-19.69 -19.55 -19.48 -19 44 -19.41 -19.42
-19.61 -19.52 -19 64 -19.50 -19.46 -19.44
Levuloseasparagin O
v.
-19.87 -19.78 -19.79 -19.82 -19 74 -19.72
The temperature of 35 O C. having given a non-dissociable compound of sugar and amino acid, a couple of experiments Discussion of Results were carried out to determine the effect of a greater range As can be noted in Table I, and in agreement with obserfrom ordinary room temperatures. The glycine and glucose were again used in equivalent amounts, and the solutions vations of previous workers, the effect of decreasing the buffered at a pH of 10.3. Blanks containing sugar and hydrogen-ion concentration is to increase the tendency of the buffer alone were examined at the same time. Experiments sugar and amino acid to react, and the rate of reaction is were run in duplicate, one series of flasks being placed in a fairly slow. The very satisfactory agreement between the refrigerator at 0' C., and the other in an oven at 50" C. The values for the extent of combination of glucose and glycine, as determined by polarimetric and iodometric methods, and the observations of rotation are given in TabIe V. almost complete recovery of amino acid when the mixture is Table V-Effect of Different Temperatures u p o n Reaction of analyzed by the Van Slyke method, are, however, somewhat Glucose and Glycine a t a p H of 10.3 at variance with the results of Euler and his associates for Part I-Tem#eralure, O n C.; Molar Concentvation, 0.222 the same pH values. It should be pointed out that their mixCONTROL: GLUCOSE-GLYCINE tures of glycine and glucose were in the ratio of 2 mols of GLUCOSE AND BUFFER AND BUFFER the former to 1 of the latter, and their iodometric method Rotation Change Rotation Change necessitated a much more strongly basic solution. The (a) (b) (a) (b) (a) (b) (a) (b) difference in the Van Slyke values may be attributed to a Hrs. OV. O B . % % O V . O V . % % longer period of time consumed in the analyses here reported, 0 12.24 12.24 0.0 0.0 12.24 12.24 0.0 0.0 with the result that under these conditions the amino-acid 24 12 12.16 1 2 . 2 0 1 22. 1. 81 6 ~ 0 . 3 0 . 46 l I 1I1. .88 0 1 1 . 7 8 1 3 . 5 3 . 7 sugar complexes had almost completely reverted to the 36" 12.02 12.02 1 . 6 11.76 11.76 3.9 3.9 72 12.00 12.00 1 . 8 11.76 11.76 3.9 3.9 original constituents. The equilibrium constants, as calculated, lie close to the curve which Euler and Brunius (7) obtained by the freezing GLUCOSE-GLYCINE CoN TR oL : GLUCOSE AND BUFFER AND BUFFER point method for the extent of combination at different pH values. This fact, in addition to the agreement between Rotation Change PERsoD Rotation Change (a) (b) (a) (b) (a) (b) (a) (b) the polarimetric and iodometric values of Table I, Part IV, lends support to the assumption that the glucose-glycine OV. OV' % % O V . O V . % % Hrs. compound is of negligible optical activity. 1 2 . 2 4 12.24 0 . 0 0 . 0 12.24 1 2 . 2 4 0.0 0.0 0 9.44 9 . 8 8 22.9 19.3 8.14 8 . 4 2 33.5 31.2 3 A complete dissociation of the glucose-glycine complex 8 . 3 8 8 . 5 0 31.5 30.5 7 . 7 6 7 . 7 8 36.6 36.4 6 7.22 7.22 4 1 . 0 41.0 6.98 7.02 42.9 42.6 12 is evident after acidification of the solutions of low alkalinity, 6.46 6.54 47.2 4 6 . 5 6.02 6.02 50.8 50.8 24 but in the solutions of higher alkalinity there appears to be 6.52 6 . 5 2 46.7 46.7 Dark Dark .. . . 36 either a slight destruction of glucose or the formation of a At end of 36-hour period the temperature had risen somewhat due to trouble with mechanical refrigerator. Experiments were continued in some non-dissociable product. an ice chest a few degrees above zero. The results with asparagin are similar except that an At the lower temperatures, the solutions remained per- enormous change in optical value takes place which is probfectly clear and uncolored throughout the experiment. Even ably due to the effect of the alkali upon the amino acid itself at relatively high pH values, the amino acid and sugar react in addition to the favoring of the condensation reaction. As can be noted in Table VI there is little change in into a very limited extent and the equilibrium value a t the low temperature is practically reached within the first 12 creasing alkalinity upon the keto sugar-amino acid complex. Any differences observed are probably due to the effect of hours. At the higher temperature, both the control and the mix- the alkaline solution upon the sugar itself. ture became dark colored, but the mixture was very much Significance of Effect of Amino Acids upon Determination darker than the control containing no amino acid. Addiof Sucrose by Double Polarization Methods tion of oxalic acid caused but little change in rotation, inThe effects of change in the optical value of the amino acids dicating that the products of the reaction were of a very stable and fructose with change in acidity of the solution, hydrolysis character. of reversion products, and chemical change in some of the Effect of Amino Acids on Fructose at Various pH amino acids themselves, upon the determination of sucrose Values by the double polarization method with different hydrolytic Neuberg and Kobe1 (12) have demonstrated that at a pH procedures, have been discussed by Zerban (15, 16). Howof 7, fructose tends to react immediately with amino acids to ever, he states that a preliminary study of the methods give products of a lower rotatory value. It seemed desirable following lead clarification gave such divergent results by
1
1
y;!
I
1
January 15, 1931
INDUSTRIAL AND ENGINEERING CHEMISTRY
two chemists working in the same laboratory that work along this line was postponed temporarily. Since raw sugar solutions are usually defecated with lead acetate and the excess lead removed with sodium carbonate or a similar reagent before the determination of sucrose, the pH of the solution may be very markedly increased by both the excess of deleading agent and the soluble acetate salt produced in the lead precipitation process. This may be expected to favor the amino acid-aldol sugar combination, the extent depending on the relative quantities of each present, the alkalinity of the solution, and the time. the solutions are allowed to stand before polarizing. Even if acid be added to bring to some definite pH near the neutral point, the rate of reversion of the complex to its original components in low acid concentration is slow and the time required to reach an equilibrium value may be several hours. I n addition, the temperature of the solution exerts a marked influence upon the extent and nature of the complex formed. Thus the apparent change due to the hydrolysis of the sucrose can vary widely and be either higher or lower than the theoretical value. The variations between polarimetric and reduction previously mentioned as observed by Brown and
21
Morris, Davis, and others probably result from all those factors. It is evident that satisfactory results for sucrose by the double polarization method must involve very careful consideration of this tendency of amino acids and glucose to combine as well as the many other factors which have received attention previously. Literature Cited (1) Ambler, IND. END. CHEM.,21, 47 (1929). (2) Brown and Morris, J . Chem. Soc., 63, 664 (1893). (3) Browne, Louisiana Planter, 67, 44 (1921). (4) Cajori, J . Bzol. Chem., 64, 617 (1922). (5) Davis, J . Agr. Sci., 7, 327 (1916-17). (6) Drake, B.S. Thesis, University of Illinois, 1924. (7) Euler and Brunius, Bey., 69B,1581 (1926). (8) Euler and Brunius, Z. physiol. Chem., 166,259 (1926). (9) Euler and Brunius, Ibid., 161, 265 (1926). (IO) Euler and Josephson, Ibid., 163, 1 (1926). (11) Neuberg and Kobel, Biochem. Z., 162, 496 (1925) (12) Neuberg and Kobel, 2. physiol. Chem., 174, 464 (1926). (13) Slyke, Van, J . B i d . Chem., 9, 185 (1911). (14) Somers, B.S. Thesis, University of Illinois, 1920. (16) Zerban, J . Assoc. Oficial A E Y .Chem., 11, 175 (1928). (16) Zerban, Ibid., 12, 158 (1929).
Determination of Glucose in Presence of Fructose and Glycine b y Iodometric Method' F. A. Dykins and D. T. Englis CHEMICAL LABORATORY, UXIVEKSITY OF ILLINOIS, URBANA,ILL.
HE selective oxidation
T
of aldol sugars by iodine in alkaline solution has received renewed attention on account of the ease and r a p i d i t y of the method (IO) and its special application to the analysis for glucose in plant m a t e r i a l s used as sources of fructose (9, $1.
A study has been made of the effect of the presence of fructose and glycine upon the determination of glucose by oxidation with alkaline iodine solution in the presence of sodium hydroxide, sodium carbonate, and phosphate buffers. The rate of oxidation is so rapid that the extent of condensation of the glucose and glycine is negligible under the conditions of the experiments, and there is no tendency for low results due to this cause if the solution is neutral when the analysis is begun. The results obtained are always higher than those due to glucose alone because of reduction of iodine by the fructose and glycine. Even if fructose may react immediately with the glycine, the compound has little Protective action against the oxidizing agent. Of the various basic reagents used with iodine, sodium Phosphate has a number of advantages and gave the most satisfactory results.
One of the objections assigned to the i o d o m e t r i c method is that it gives high results because ofu the f6t that substances other than sugars take up iodine under the conditions of the analysis. on the other hand, certain fattors may tend to cause low results. It has been demonstrated by Euler and Brunius (6,6), and Englis and Dykins (4) that when amino acids are present and the solution is alkaline, a stable condensation product of the monosaccharide sugars with the amino substances takes place and prevents its oxidation by iodine in alkaline solution. With glucose the rate of condensation is relatively slow near the neutral point but increases rapidly in rate and extent as the alkalinity is increased. With levulose the combination is immediate and at a lower alkalinity. If the amino substance can then combine with only an equal molecular quantity of sugar, it might be expected that since fructose is the predominating sugar in the hydrolyzed extracts of inulin-containing plants, it might combine with all the amino material and thus leave the aldol sugar to react as it normally would in the absence of the amino substance. If the amino substances still tend 1
Received September 12, 1930.
to react with the aldol sugar, the rate will be a function of the time, temperature, and alkalinity, and different values may be expected for the iodometric methods using different degrees of alkalinity and different reaction periods. To secure definite information upon these points was the object of these experiments. Procedure
Three methods involving different basic reagentsfor the iodometric procedures were used. I n the first, sodium hydroxide was the base and the conditions approximated those outlined by Kolthoff (7). I n the second, sodium carbonate was utilized under about the same conditions as specified by Cajori (1) except that the quantities of reacting materials were increased about 2.5 times. I n the third, a disodium phosphate-sodium hydroxide buffer mixture of about the same pH value as the sodium carbonate mixture was used. The iodine and sodium thiosulfate solutions used were approximately 0.1 normal. .The sodium thiosulfate solution was standardized against pure potassium dichromate and its value in terms of glucose was found to be 9.515 mg, per cc. I n each series of determinations, blank runs were made in which distilled water replaced the sugar solution. A concentrated stock solution of a high purity glucose containing 10.000 grams in 100 cc. was made the source of the glucose for all experiments. When 10 cc. of this were
