THE ACIDIC PROPERTY OF SUGARS

BY FRANK URBAN AND PHILIP A. SHAFFER

(From the Laboratory of Biological Chemistry, Washington University School of Medicine, St. Louis)

(Received for publication, October 26, 1931)

Work reported in an earlier paper (Shaffer and Friedemann, 1930) on the formation of lactic and other saccharinic acids from reducing sugars led one of us some years ago to the hypothesis that the “activation” of reducing sugars which takes place in alkaline solution is the consequence of salt formation of the sugars, not only of one but of two or more acidic groups. (Similar views had been advanced much earlier by Nef and by Mathews.) Although it is well known that sugars behave as weak acids, there was until recently no valid evidence to justify the assumption of more t.han one acidic group. In order to test the hypothesis that sugars possess not only one, but several acidic groups and act as di- or polybasic acids, we undertook, about 3 years ago, the task of determining, by means of hydrogen electrode measurements, the amounts of base bound by glucose, fructose, and sucrose in rather concentrated NaOH solutions. High alkalinity was necessary to call forth to a detectable extent the postulated second or third acidic groups. Determinations were made also in moderately dilute hydroxide within the range used by previous workers on this subject, at which less than 1 equivalent of base is bound. From the data in the lower range of alkalinity the first dissociation con- stants have been again calculated.

In concentrated hydroxide solutions the difficulties, both experi- mental and theoretical, are considerable. The hydrogen electrode behaves rather poorly under such extreme conditions, while the strong reducing property of alkaline sugar solutions and the grad- ual jomation of the relatively much stronger saccharinic acids from sugar decomposition were regarded as possible sources of

697

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698 Acidic Property of Sugars

error. Besides these, the liquid junction potential between NaOH (or NaOH + sugar) and KC1 of bridge or calomel half-cell is of considerable and somewhat uncert,ain magnitude. After a good deal of work these difficulties were, to a large extent, overcome, and results obtained which appear to leave no doubt that glucose, fructose, and sucrose possess at least two and, at high alkalinity possibly three acidic groups. At least two, and possibly three different sugar ions, quite apart from decomposition or “dissocia- tion” (Nef), may, therefore, be present in alkaline sugar solutions, the relative concentration of each ion depending of course on pH.

In a paper published in May, 1929, but which first came to our attention after a preliminary report’ of our work had been pre- sented, Hirsch and Schlags (1929) showed the dibasic character of a number of sugars. The data here reported may, therefore, be regarded as confirmation and extension of the results of these authors. (In their paper the earlier work on the acidic property of sugars is reviewed.) Hirsch and Schlags based their calculations of the amount of base bound on measurements of electrical con- ductivity of NaOH solutions in t,he presence and absence of added sugar, the pH of the solutions containing sugar being determined, as in our work, by the hydrogen electrode. The highest concen- tration of NaOH and of the several sugars used by them was 0.25 M. They calculated both first and second (apparent) dissociation constants, the latter on the assumption that the sugars react (in the solutions used) only as dibasic acids. Their values for glucose, fructose, and sucrose are shown for comparison with our own in Table VI. The fact that we obtain, in general, similar values by a different method gives weight to the results of both.

Theory of the Calculation of Base Bound

The addition of sugar to an aqueous alkali solution causes a shift (lowering) of the pH, due to salt formation between sugar acid and base. This shift is reflected by a change in E.M.F. of the cell,

Pt / H,, NaOH, 1 KC1 (saturated), HgCl 1 Hg

(a) CT) (b)

1 R,eported at the meeting of the American Chemical Society at Minne- apolis, September, 1929.

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F. Urban and P. A. Shaffer 699

and conversely, from the change in E.M.F. the amount of sugar salt formed may be calculated. Given the two cells

Pt 1 Hz, NaOH, 1 KC1 (saturated), HgCl 1 Hg (d (4 lb)

Pt 1 H,, NaOH, + sugar / KC1 (saturated), HgCl 1 Hg (4 (h) (b)

in which the initial NaOH concentration is the same in both, it follows that

(1) *El* l 1 F 1

(H+) + E(b) + rl

where log 1

- and log 1

&I+), - represent the pH of the NaOH and W.+)2

NaOH + sugar solutions’ respectively, E(b), the potential of the calomel half-cell, and rr, the liquid junction potentials.

By subtracting Equation 2 from Equation 1, we obtain the shift of E.M.F. (except for the difference in liquid junction potential a in the two cells considered below).

(3) E = ST In (H+)z El- 2 F -

@+)I

Substituting for (H+)l and (H+)z the correspondin,g values of Ki . (H2O) gives.

(OH-) *

(4) E _ E = g ln (OH-h . KaOWh

1 2 F (OH-h . K&50)1

The value of the last member of Equation 5 may be disregarded in our experiments by the assumption that the activity of water in

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700 Acidic Property of Sugars

an NaOH solution is only slightly changed by the formation of sugar salt. Equation 5 may then be written,

(6)

where y1 and yz are the activity coefficients, and C1 and Cz the concentrations of NaOH, without and with the sugar, respectively.

Since the ionic strength of the solution remains about the same after the addition of sugar, we may further assume the two activity coefficients for each pair of solutions to have the same value, and t,hus obtain the simplified equation (applicable only to each pair of solutions, of the same initial NaOH concentration, one with and one without sugar)

(7) E, - E, = F In ; = 2.303 $ log 2 2 2

which for 25” becomes

(8) EI - Ez 0.05912

= log 2 2

By means of Equation 8, the concentration of free NaOH in the presence of the sugar Cz, can be calculated from the “shift” in E.M.F. (C,, the concentration in absence of sugar being known by titration), provided that the shift is first corrected for the di$er- ence of liquid junction potential at (r) between Equations 1 and 2.

For calculation of the liquid junction potentials, we have used the Henderson equation, in spite of the fact that it is theoretically applicable only to dilute solutions. The modified equation of Cumming (1912), developed for higher concentrations, was less readily applicable because the necessary transport numbers were not available for 25”.

This equation was tested experimentally by one of us (U.) by measurements of the three cells

Pt 1 H,, NaOH, 1 saturated KCI, HgCl 1 Hg

Pt 1 Hz, NaOH, ) 0.1 M KCl, HgCl 1 Hg

fig / HgCl, saturated KC1 1 0.1 M KCl, HgCl 1 Hg

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F. Urban and P. A. Shaffer 701

and by measuring also another set differing only by the addition of glucose to the NaOH solutions. The results were regarded as justifying application of the equation to the range of concentration of our solutions. Use of Cumming’s equation would have given slightly greater values, and a small increase in values for the “shift.” The correct fractions of base bound may, therefore, be slightly larger than the values calculated; but the differences be- tween results by the two equations are probably within the experi- mental error.

In the calculation of liquid junction potential, which is simple with pure NaOH solutions, difficulty is encountered when the equation is applied to the alkali-sugar solutions in which the concentrations of free NaOH and of sugar-salt are not exactly known. The pro- cedure followed was first to use in Henderson’s equation the approximate concentration of free NaOH obtained from Equation 8. The resulting junction potential was then used to correct the value of the shift (El - Ez), and free NaOH again computed. This new value when substituted in the Henderson equation yielded a new junction potential value which, in most cases, was close to the first. When necessary the cycle was repeated. As a rule the third repetition yielded results within 0.1 mv. of the second. The sign of the liquid junction potential is such that for NaOH alone it is to be added to the observed E.M.F. (except at the two lowest NaOH concentrations). Being due chiefly to the hydroxyl ion, it is lowered by the presence of the sugar; hence, the true shift is greater than the observed difference of potential, E, - Ez, by the di$erence between the liquid junction potential in the presence and absence of sugar (by from 0.4 to 7.0 mv.). When correction for liquid junction potential is omitted, although the fraction of base bound per mol of sugar is somewhat decreased, the dibasic character of the sugars is still clearly shown.

The shift, El - E2, corrected for diference in liquid junction potential, divided by 0.05912, gives by Equation 8 the logarithm

Cl of C, the ratio (R) of free NaOH concent,rations without and

2 with sugar, the concentration in absence of sugar being known by titration. Then

(9)

(10)

(NaOH),, + R = (NaOH),,, and

(NaOH),, - (NaOH),, = (Na) salt

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702 Acidic Property of Sugars

the amount of base bound by sugar as salt. Dividing this value by the molar concentration of sugar used gives the molar equivalents of base neutralized per mol of sugar at the observed pH. These quantities are recorded in Tables II to V under “base fraction.”

The pH of the NaOH + sugar solutions was determined as usual from Eh (corrected for liquid junction potential). The value of the calomel cell (saturated KCl) used throughout as work- ing reference electrode was based upon its potential when com- bined with the hydrogen electrode in 0.05 M acid potassium phthalate, assuming its pH to be 3.97 (Clark, 1928) and ignoring the liquid junction potential between phthalate solution and saturated KCI-agar bridge. Corrections were made in all cases for Hz pressure.

The apparent dissociation constants were calculated as follows: The constants are”apparent”in that they include the activity coefh- cient of the salt. Change of the “apparent” constants with ionic strength of the solution is ignored. The values of pK1’ (the first constant) were obtained by applying the Henderson-Hasselbalch equation

(11) pK’ [salt]

= PH - log [HA]

to the data of experiments within the pH range at which less than 0.5 base equivalent is bound, and the average values taken. This equation is valid only at pH regions where it may be assumed that the amount of base bound by the second acidic group is negligible. Its use has the advantage of giving values for pK1 independent of the increasing errors at higher pH range.

The second constants (OK were calculated from the expression

(12) 2a - pK1’ = p&’

in which a is the pH at which the acid (sugar) binds 1 mol-equiva- lent of base. This equation is the same as Hirsch and Schlags’ (1929), 3 (Ml + M.J = a, and is derived as follows: Multiplying together the equations for the first and second dissociations of a dibasic acid,

(13) K 1

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F. Urban and P. A. Shaffer 703

substituting B,A (dibasic salt concentration) for A=, K’ for K, and taking the logarithm of both sides, gives

04) @LA)

2 pH - pK,’ - pK2’ = log - O&A)

Assuming the total sugar to be present as H2A, BHA, and BZA, the total base of (BHA) + (BZA) equals the total sugar of HZA + HBA + BSA; i.e., the sugar binds 1 equivalent of base, when (B&A) = (H2A). Let the pH at which this is true be a, then 2a - SKI’ = pKzz’. The values of a were determined graphically from curves constructed from the data (see Figs. 1 to 5).

Experimental Details-The NaOH solutions were prepared free from COZ by the method of Sorensen (1909), and standardized by titration against standard HCl and acid potassium phthalate solutions. The sugars used were of high purity. The glucose was from Eastman; fructose was obtained through the courtesy of Dr. Hudson, from the United States Bureau of Standards. Sucrose was purified from commercial cane sugar by decolorization with norit, crystallization by addition of 4 volumes of 95 per cent alcohol, and drying in vucuo.

The final NaOH concentrations ranged from 0.012 to 1.197 M,

and the sugar concentrations from 0.1 to 0.4 and 0.6 M. The cell measurements were made in a constant temperature bath, usually at 25” f 0.05”. The hydrogen was commercial tank electrolytic gas, purified by passage over platinized asbestos, in a quartz tube, heated by an electric furnace to a dull red, then bubbled first through water, then through a sample of the alkali solution being analyzed (in the bath), and finally into the hydrogen electrode vessel.

Saturated KCl-calomel electrodes and bridges of saturated KC1 in 1.5 per cent agar were used. The potentiometer was type K of Leeds and Northrup, with a calibrated Weston cell. Two different samples of each NaOH solution, alone and with each sugar concentration, were prepared and read separately. Potentiometer readings were made approximately 10 minutes after the bridge was in position, or 15 to 20 minutes after mixing the solutions. Dupli- cate determinations often agreed within 0.1 mv. When less satis- factory, often the case with the high NaOH concentrations, an

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704 Acidic Property of Sugars

average was taken, or the experiment was repeated until st,eady and apparently reliable values were obtained.

Error due to saccharinic acid formation was minimized by mak- ing the measurements as rapidly as possible. Readings over a 2 hour period of 0.4 M glucose in 0.666 M NaOH at 25” showed a drift, detectable after 10 to 20 minutes, of about 0.1 mv. per 10 minutes. This indicates that at high alkalinity (or temperature) a consider- able error may be introduced in the case of reducing sugars, if the potential readings are much delayed. Error due to this behavior possibly may account for some of the scattered results observed especially with glucose at high alkali concentration. It was necessary to replatinize the electrodes frequently when used in the alkali-sugar solutions; otherwise erratic results were often obt,ained.

Results

NaOH Solutions without Sugar-Table I gives the concentra- tions (titration values) of the NaOH solutions, the calculated liquid junction potentials, the corrected Eh values, and the corre- sponding pH numbers, in absence of sugar. These data not only represent the base-line for calculation of results obtained in the presence of the sugars, but indicate also the probable limits of error in the measurements and in the estimates of liquid junction potentials.

Multiplying the NaOH concentrations [NaOH] by the corre- sponding activity coefficients (determined by Harned (1925) at 25” in cells without liquid junction) gives hydroxyl ion activities, (OH-). The product (OH-) X (H+) should then equal the ionic product of water, Kn,o, or be slightly less than this value, to the extent that the activity of water in NaOH solutions is less than 1. Taking the logarithmic form of this relation

POH + PH = pKHpo + log &

Column 8 of Table I gives the sum pOH + pH and Column 9 the deviation of this sum from 13.99, the “best” value for pKn*o at 25” (InternaGonal Critical Tables, 6, p. 15‘2). Instead of the deviation approaching zero at the lowest NaOH concentration it does so around 0.4 to 0.5 M. drifting with a semblance of regularity on both sides of this region. The apparent, approach to systematic

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F. Urban and P. A. Shaffer 705

deviation suggests some theoretical basis, such as error in estimates of liquid junction potentials or in some of the constants. But we have not succeeded in reaching an acceptable explanation and shall,

TABLE I

NaOH Solutions. (No Sugar Present)

NaOH No.

(1)

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17

M ?,Ll~.

0.011 +0.7 0.023 $0.3 0.046 -0.4 0.069 -0.7 0.083 -0.9 0.093 -1.1 0.111 -1.3 0.166 -2.1 0.222 -2.7 0.333 -3.8 0.444 -4.6 0.500 -5.1 0.555 -5.5 0.666 -6.3 0.777 -6.9 0.888 -7.6 1.197 -9.1

-0.7037 11.903 0.91 -0.7232 12.233 0.87 -0.7408 12.530 0.825 -0.7505 12.695 0.80 -0.7537 12.750 0.79 -0.7579 12.82 0.785 -0.7630 12.906 0.77 -0.7723 13.064 0.755 -0.7795 13.185 0.74 -0.7889 13.343 0.72 -0.7975 13.490 0.705 -0.8002 13.535 0.70 -0.8031 13.585 0.695 -0.8080 13.667 0.685 -0.8118 13.732 0.677 -0.8145 13.777 0.675 -0.8246 13.948 0.695

17O

For 25"

20 0.095 -(1.1) -0.7556 13.133 21 0.190 - (2.3) -0.7723 13.424 22 0.285 -(3.3) -0.7833 13.615 23 0.380 - (4.1) -0.7906 13.74 24 0.570 - (5.6) -0.7992 13.89 25 0.720 - (6.6) -0.8068 14.023

-

-

NaOH

(2) i

Liquid

I !

Activity

junction Eh PH coefficient

y NaOH POH potential (Harned)

(3) (4) (5) m (7)

25" -

-

1.977 13.88 1.693 13.93 1.416 13.95 1.253 13.95 1.182 13.93 1.135 13.96 1.068 13.98 0.903 13.97 0.785 13.97 0.621 13.97 0.505 13.99 0.457 13.99 0.414 14.00 0.340 14.00 0.281 14.01 0.223 14.00 0.080 14.03

(8)

-

-

-

Devia- tion from

13.99

(9)

-0.11 -0.06 -0.04 -0.04 -0.06 -0.03 -0.01 -0.02 -0.02 -0.02

0 0

+0.01 i-o.01 +0.02 +0.01 f0.04

-

therefore, merely accept the variations as indications of the prob- able errors. Eleven of the seventeen values are within 0.03 unit and fourteen within 0.04 unit of the theoretical, the average devia-

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706 Acidic Property of Sugars

tion being +0.025 and - 0.013. It seems likely that the probable error in pH determinations does not exceed f0.028. This corre- sponds to f1.66 mv. as the probable error in Eh, from which the amounts of bound base are calculated. In the lower range of alkalinity where the “shift” is large an error of this magnitude would be of small consequence, but at the highest range of alka-

TABLE II Sucrose at 25’

NaOH NO.

(1)

1

2 3 4 5 6 8

10 12 14

M

0.0116 0.0233 0.0466 0.0698 0.0832 0.0931 0.166 0.3326 0.500 0.666

1 0.0116 3 0.0466 6 0.0931 8 0.166

10 0.3326 12 0.500 14 0.666

5 0.0832 8 0.166

10 0.333

NaOH

(2)

SllCIOBl3

(3)

M

0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

0.2 0.2 0.2 0.2 0.2 0.2 0.2

0.4 0.4 0.4

A liquid junction potential

(4) I’

mu.

+0.3 +0.2 $0.9 +1.0 +1.0 +1.1 $1.4 +1.5 $1.7 +1.7

(5) --

volt

0.0259

0.0243 0.0233 0.0213 0.0186 0.0189 0.0158 0.0100 0.0089 0.0069

0.07 11.46 0.14 11.82 0.28 12.15 0.39 12.341 0.43 12.44 0.49 12.51 0.76 12.80 1.08 13.17 1.47 13.39 1.58 13.56

$0.4 0.0382 0.05 11.26 +1.4 0.0367 0.18 11.90 +1.7 0.0354 0.35 12.22 +2.1 0.0292 0.56 12.57 $2.7 0.0209 0.92 13.00 +3.1 0.0180 1.26 13.24 f3.2 0.0153 1.49 13.41

$1.8 0.0522 0.18 11.87 +2.9 0.0496 0.36 12.24 +4.3 0.0430 0.67 12.62

Shift corrected

-

PH

(7)

PIG

(8)

12.584 12.608 12.56 12.54 12.56 12.53

(12.30)

12.54 12.56 12.49 12.46

(11.94)

12.53 12.49

(12.30)

linity with low values for the “shift” it amounts to as much as 20 per cent or more. In the latter region the liquid junction potential correction also becomes large, which further limits the accuracy attainable by this method. These fact,ors are probably responsible for the scattering of the data at highest alkalinity, to be observed by inspection of Figs. 1 to 5.

The data for sucrose, glucose, and fructose are shown in Tables

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TABL

E 11

1

Gluc

ose

at

16’

“CH

1 2 3 4 5 B 7 8 9 10

11

12

13

14

15

16

17

NIX0

11

M

0.01

:

0.02

: 0.

04;

0.07

( 0.

08:

0.09

: 0.

111

O.lM

0.

22:

0.33

1 0.

44r

0.%

X 0.

55:

O.G6

( 0.

77;

0.8%

1.

19;

0.1

M g

lucc

.se

A liq

uid

Shift

ju

nctio

n co

T-

pote

n-

tial

rcxte

d --

ma.

m

l1

+0.4

0.

0443

+0

.7

0.04

41

t-1.2

0.

0398

f1

.3

0.03

58

+1.4

0.

0340

+1

.7

0.03

32

+1.6

0.

0304

+2

.1

f2.0

0.

0133

+2

.1

0.01

14

+1.9

0.

0102

+1.5

0.

0076

+1

.2

0.00

55

+3.5

*0.0

163

+7.o

to.o

287~

Hase

fra

c-

PII

tion

-- 0.09

5 11

.15

0.19

11

.48

0.37

11

.85

0.53

12

.09

0.61

12

.16

0.68

12

.26

0.77

12

.39

0.94

12

.94

1.20

13

.14

1.45

13

.32

1.42

13

.40

1.29

13

.56

1.22

* 13

.45’

1.34

t 13

.46.

PKI

12.1

: 12

.1

12.0

1 12

.0,

- L j

.- 3 1 3 4 -

L liq

uid

unct

ior

%I- m

s.

+0.5

+O

.S

f1.4

+1

.6

+1.9

+2

.1

+2.6

+2

.8

+3.2

+3.0

+3

.1

+3.2

Y-

Shift

:o

rrect

i

Dol

l

0.06

29

0.06

18

0.05

89

0.05

56

0.05

19

0.05

01

0.04

14

0.03

28

0.02

75

-- T

Base

frn

c-

tion

PII

PKI

--

0.05

0.

11

0.21

0.

31

0.41

0.

48

0.67

0.

80

1.10

;;2gl

“,;;

:t ;z

tia

l rc

cted

tion

~ --

mo.

ao

IL

10.8

3 12

.11

+0.5

0.

0828

0.

03

11.1

8 12

.09

$0.8

0.

0844

0.

06

11.5

4 12

.11

+1.5

0.

0817

0.

11

11.7

6 12

.11

+1.8

0.

0800

0.

17

11.9

5 12

.11

12.0

6 12

.09

+2.4

0.

0720

0.

26

12.3

6 $3

.2

0.07

04

0.39

12

.63

$3.7

0.

0642

0.

51

12.8

6 +5

.1

0.04

23

0.90

1.11

13

.23

1.27

13

.31

1.51

13

.40

1.58

13

.57

$4.5

0.

0324

1.

00

+4.5

0.

0247

1.

03

+4.3

0.

0235

1.

17

+4.8

0.

0210

1.

24

+4,.9

0.

0181

1.

51

PII

PKI

--

r

10.5

0 12

.01

c 10

.80

11.9

9 11

.14

12.0

5 3

11.3

3 12

.02

E i a 11

.68

12.1

3 w

11.8

7 12

.06

’ 12

.10

12.0

8 .?

12

.76

g

13.0

3 e

13.2

5 13

.34

13.4

1 13

.63

* 0.

3 Y

gluco

se.

t 0.

6 M

gluc

ose.

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708 Acidic Property of Sugars

TABLE IV

Glucose at SY’

JiaOH No. SaOH Shift

corrected BafFZ

fraction

(1) (2)

M

20 0.095 21 0.190 22 0.285 23 0.380 24 0.570

A liquid junction potential

(25’) (4) (3) (5) (6)

M volt 0.2 0.0537 0.2 0.0365 0.2 0.0273 0.2 0.0211 0.2 0.0144

mv.

+1.7 +2.6 $2.8 $2.9 +3.1

0.42 0.73 0.95 1.09 1.25

PH PIG

(7) 03 __-

12.20 12.34 12.79 13.14 13.374 13.64

TABLE V

Fructose

5 8

12 14

5 8

10 12 14

12

12

-i- .+I

0.083 0.166 0.500 0.666

0.083 0.166 0.333 0.500 0.666

0.500

0.500

M

0.1 0.1 0.1 0.1

0.2 0.2 0.2 0.2 0.2

0.5

1.0

25” - - Wm. 2.01t

+1.6 0.0434 +2.1 0 0280 +2.4 0.0140 +2.5 0.0119

0.68 12.00 1.11 12.60 2.10 13.30 2.48 13.46

f1.7 0.0682 0.39 11.60 f3.0 0.0579 0.75 12.08 +3.6 0.0332 1.21 12.79 $3.7 0.0237 1.51 13.13 +3.5 0.0179 1.68 13.36

+5.0

+7.0

4O

0.0659 0.92 12.42

0.1176 0.49 11.55 -

-

-

- 0.008 0.02 0.0228 0.22 12.05 0.030 0.02 0.0142 0.66 12.84 0.083 0.02 0.0069 1.04 13.40 0.166 0.02 0.0049 1.56 13.75 0.008 0.04 0.0398 0.17 11.72 0.030 0.04 0.0315 0.57 12.53

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F. Urban and I?. A. Shaffer

II to V. It should be noted in these tables that the figures in columns headed “A liquid junction potential” are differences be- tween the calculated junction potentials in the presence and ab- sence of sugar, the sign in all cases being such that the correction increases the observed Ez - El. The figures in columns headed “shift corrected” include the liquid junction correction. From these values the corrected Eh for sugar solutions, from which pH was calculated, may be obtained (if desired for checking calcula- tions) by subtracting the “shift” from the value of Eh for the corresponding NaOH solution given in Table I. The values for equivalents of base bound at different pH levels are plotted in Figs. 1 to 5.2

2.0

15

1.0

0.5

I

FIG. 1. Amount of base bound by sucrose at various pH levels, at 25”. The ordinate represents the base fraction; the abscissa, the pH.

Sucrose-The polybasic character of the sugars is most simply presented by comparing the amounm of base bound at given pH levels with the theoretical amounts bound by a monobasic acid having the same dissociation constant as that found for the first acidic group of the sugar. The data for sucrose will il1ustrat.e this. Values for pKr’ calculated from the results within the lower pH range, shown in Column 8 of Table II, average 12.54.

2 One of us (U.) has since been able to confirm the values for equivalents of base bound at different pH levels by glucose, using a glass electrode and, thereby, eliminating liquid junction potentials. Results will be published later. Regarding the method see Urban, F., and Steiner, A., J. Physic. Chem.. 36, 3058 (1931).

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710 Acidic Property of Sugars

Curve HA in Fig. 1 represents the theoretical fraction present as salt (i.e., the amount of base bound per mol) of a monobasic acid3 having a dissociation constant, pK’ = 12.54. It will be seen that .this curve approximately fits the experimental points only below about 0.4 salt fraction. Above this sucrose binds more base, due to a second acidic group. The points locate a line which, instead of curving toward the horizontal, runs for a distance approximately straight. The slope of the straight line indicates the ratio of the first to second constants, as made clear by the theoretical treatment of the behavior of dibasic acids by Van Slyke (1922), Hirsch (1924), Michaelis (1922), and others.

If we similarly calculate the fractions of a dibasic acid present in the form of mono- and the dibasic salt4 at different pH levels, the sum of HBA + 2BzA represents the total base bound by the two acidic groups. Curves are drawn on Fig. 1 to represent these fractions and the sum of base bound. It will be seen that the last named theoretical curve fits the somewhat scattered experimental points fairly well up to a total salt fraction (base equivalents bound) about 1. The values of the constants from which the curves shown are calculated, and which best fit these data, are pK1 = 12.60, a = 13.06, and pKz’ = 13.52. The corresponding values reported by Hirsch and Schlags are 12.51, 13.02, and 13.52.

The points at highest pH indicate more base to be bound than corresponds to two acidic groups and hence suggest a third, which begins to function perceptibly above pH about 13. The upper dotted curve as drawn represents the theoretical total base bound if the third const.ant be taken as 14.20. This value roughly fits the data. There are, however, large errors in these results at high pH which make the value for pKs’ only a rough approximation. The errors in this region are not alone experimental but are due chiefly to the mathematical relation which at increasing pH exaggerates increasingly the effect of small experimental errors on

3 Data for this curve are calculated from the equations log R = pH - R

PI<‘, and salt, = - D-41

R + 1’ in which R = - and salt, is the fraction of the

[HAI acid present as salt.

4 The mono- and dibasic salt fractions are calculated from the equation HA- K1. H+ -=

s - in which S is the sum, HQA + HA- + A’.

KIH++H+*+KI-Kz

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F. Urban and P. A. Shaffer 711

the calculated results. For this reason the accuracy attainable by this method of determining dissociation constants declines greatly with the magnitude of the constants. The existence of the third constant for sucrose is based on four points with “shifts” of 6.9 to 18 mv. of which 1.7 to 3.2 is the difference in liquid junction potential. If the shifts were about 2 mv. less the points would fall near the line for a dibasic acid. There is, however, no reason to suppose that the values for the shift are too high, and we, t,here- fore, tentatively conclude t.hat a third acidic group exists, the con- stant for which is of the order given.

Glucose-From what has been said above concerning sucrose, the significance of Figs. 2 and 3 for glucose will perhaps be evident by inspection. The experiments at 25” are quite consistent in the

I!5

a5

FIG. 2. Amount of base bound by glucose at various pH levels, at 25’. The ordinate represents the base fraction; the abscissa, the pH.

lower pH range, and yield values for pK1’ shown in Table III, the best value being 12.09. Considering the wide range of sugar as well as alkali concentrations used, the results seem quite satis- factory. The amounts of base bound above pH about 12.1 give evidence again of a straight line instead of the curve required for a monobasic acid. The value for a is 12.97 and for pKz’, 13.85. The corresponding values of Hirsch and Schlags are pK,, 12.11 (a = 12.96) and pKz, 13.81. Curves showing the theoretical mono- and dibasic salt fractions corresponding to our constants, and Dhe sum of base in both, are drawn on Fig. 2. It will be seen that although the experimental points scatter considerably in the upper range, the theoretical curve for total base appears to repre- sent the mean. Above pH about 13 there appears to be evidence

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712 Acidic Property of Sugars

of a third acidic group, though the points are too scattered to per- mit satisfactory estimate of the value of its constant. For the present we merely suggest its existence.

Fig. 3 shows a few results with glucose at 17”. The values of the constants which appear to fit the data best are pK1’, 12.34; pKz’, 14.10. It is of interest to note that the change of the constants wit,h temperature between 25O and 17” is not very different from the change oi pKnzo. (pK,‘, 12.34 - 12.09 = 0.25; pKHzO, 14.26 - 13.99 = 0.27.)

Fructose-The results for fructose are rather less consistent t,han those for sucrose and glucose perhaps due in part to its more rapid decomposition in alkaline solution. The data for 25” are plotted

FIG. 3. Amount of base bound by glucose at various pH levels, at 17”. The ordinate represents the base fraction; the abscissa, the pH.

in Fig. 4. The values of the constants which seem best to fit the data are for 25”, pKl’, 11.68; pK’2, 13.24. The first is close t,o Hirsch and Schlags value, 11.69, while their values for pKz’ is 13.81, the same as found by bhem for the second constant of glucose. In view of the close structural relations it is attractive t’o suppose that the second acidic groups may be the same in both sugars and may have the same const,ants. But our data do not permit selection of pH 12.75 as the a point, which is necessary to give the Hirsch and Schlag value for ~Kz’. The discrepancy in the two values for pK2’ will require further work to explain.

Again with fructose the points at the highest pH indicate over- lapping of a third acidic group, though because of the scattering of

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F. Urban and P. A. Shaffer 713

the points and the reasons already menConed, it is not possible to locate the line definitely nor to estimate the value of the hypo- thetical constant. In the hope of getting more reliable data with fruct,ose at high alkalinity, a series of measuremenm were made at

FIG. 4. Amount of base bound by fructose at various pH levels, at 25”. The ordinate represents the base fraction; the abscissa, the pH.

o./ Il.4 z.5 a0 0.5

FIG. 5. Amount of base bound by fructose at various pH levels, at 4’. The ordinate represents the base fraction; the abscissa., the pH.

4” with lower sugar and NaOH concentrations. The results are shown in Fig. 5. The points are not very consistent. The best values for the constams appear to be, pK1’, 12.50; a, 13.34; pKz’, 14.18. In 0.166 N NaOH and 0.02 M fructose there is again evi- dence of a third acidic group, though the shift, is so small (4.9 mv.)

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714 Acidic Property of Sugars

that allowing a small error would so decrease the base bound as to make assumption of the third group unnecessary. The constants obtained for the three sugars together with the values of Hirsch and S&lag are brought together in Table VI.

TABLE VI

Apparent Dissociation Constants

pK1’ PKI’ (a)

~-

Glucose 25’ Hirsch-Schlags 12.107 13.813 12.96 25’ Authors 12.09 13.85 12.97 17O “ (12.34)* (14.10)* 13.22

Fructose 25’ Hirsch-Schlags 11.693 13.807 12.75 25’ Authors 11.68 13.24 12.46 4O “ 12.50 14.18 13.34

Sucrose 25’ Hirsch-Schlags 12.513 13.523 13.011 25’ Authors 12.60 13.52 13.06

* Based on a single determination of pKr.

SUMMARY

K x 10’3

8.1 0.14 (4.6)* (0.08)*

20.3 0.156 130 20.9 0.58 36

3.2 0.066 48

3.1 0.30 10 2.5 0.30 8.3

The amounts of NaCH neutralized by glucose (25’ and 17”), by fructose (25” and 4”), and by sucrose (25”) were determined by means of the hydrogen electrode. All three sugars behave as dibasic acids. The following values were obtained for the disso- ciation constants at 25”: glucose, pK1’ 12.09, ~Kz’ 13.85; fructose, pK1’ 11.68, pKn’ 13.24; sucrose, pK1’ 12.60, pKz’ 13.52.

The data appear to indicate that with each of these sugars a third acidic group begins to function at high alkalinity; but because of large errors in this region the existence of the third group must be regarded as uncertain.

BIBLIOGRAPHY

Clark, IV. M., The determination of hydrogen ions, Baltimore, 3rd edition (1928).

Cumming, A. C., Tr. Faraday Xoc., 8,86 (1912). Harried, H. S., J. Am. Chem. Sot., 47,676 (1925). Hirsch, P., Biochem. Z., 147,433 (1924).

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F. Urban and P. A. Shaffer 715

Hirsch, P., and Schlags, R., 2. physik. Chem., 141,387 (1929). Michaelis, L., Die Wasserstoffionenkonzentration, Berlin, 2nd edition,

23 (1922). Shaffer, P. A., and Friedemann, T. E., J. Biol. Chem., 86,345 (1930). Sdrensen, S. P. L., Biochem. Z., 21,168 (1909). Van Slyke, D. D., J. Biol. Chem., 62,525 (1922).

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Frank Urban and Philip A. ShafferTHE ACIDIC PROPERTY OF SUGARS

1932, 94:697-715.J. Biol. Chem. 

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