SUGAR DETERMINATION BY THE FERRICYANIDE ELECTRODE*

BY PHILIP A. SHAFFER AND RAY D. WILLIAMS

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

(Received for publication, July 29, 1935)

Alkali ferricyanide is in some respects an ideal oxidant for the quantitative determination of reducing sugars. Stable in alka- line solution, it is reduced by sugars to a stable soluble reductant (ferrocyanide) which is unaffected by atmospheric oxygen. The quantity reduced may readily be determined in several ways: by iodometric titration (l), by gasometric (2) and calorimetric (3) measurement, and by the time period required for reduction (4). Another simple and convenient method is measurement of the electrical potential of a platinum wire immersed in the solu- tion, the well known ferri-ferrocyanide electrode. If the sum of ferri- and ferrocyanide concentrations is known, and certain constants are established for the particular solutions and condi- tions used, the measured potential indicates the quantity of ferricyanide reduced, from which the quantity of sugar oxidized may be calculated.

This method has several important advantages. No accessory solutions and no further chemical treatment are needed. An electrode and salt bridge are inserted and the potential is read against a reference half-cell by means of a potentiometer and galvanometer. The potentials are exceptionally well poised, are quite reproducible, and may be quickly measured with consider- able accuracy. The values are unaffected by pH change or by the products of the sugar oxidation, and are independent of the volume of solution about the electrode. The potential is definite in quite low ferri-ferrocyanide concentrations, which permits

* Aided by a fund given by the Rockefeller Foundation to Washington University for research in science.

707

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708 Sugar Determination

determination of smaller quantities of sugar than are required for other methods. Amounts of glucose of the order of 0.005 mg. suffice for a determination. Since the composition of. the solution is substantially unaltered by measuring its potential, repeated readings of the same solution may be made, thereby indicating the rate as well as the final result of reaction in a single solution.

The purpose of this paper is to outline the principal considera- tions concerning the application of the ferri-ferrocyanide electrode to sugar analysis and to describe procedures in which it is utilized for the determination of sugar in blood. Applied to blood filtrates prepared by zinc hydroxide precipitation, approximately correct values for “true sugar” are obtained. So small a quantity as 0.02 to 0.05 cc. of blood is enough for an analysis. Applicable also to larger quantities, the sensitiveness of the potentiometric method makes it especially suitable for small samples of cutaneous blood, and for repeated analysis of the blood of small animals without making necessary their sacrifice.

The method is quite suitable for use with pure sugar solutions, and should be applicable to other biological solutions besides blood. But ferricyanide is reduced by many- substances other than sugars and their possible interference must be considered before this oxidant can safely be used with any untried solution. This restriction applies by whatever method the ferricyanide re- duction is measured. The potentiometric method may have an advantage in this respect; no supplementary reagents being used, possible side reactions between these and such other substances as may be present are avoided. In some blood filtrates, for ex- ample, the results calculated from potentials are less than results by iodometric titration, owing apparently to a consumption of iodine in acid solution by substances (glutathione?) not com- pletely oxidized by ferricyanide in alkaline solution. This sort of interference is avoided by the potentiometer method.

The ferricyanide electrode was first used to study the rate of sugar oxidation in 1928 by Ariyama and Shaffer (5) and was the basis of a method for blood sugar analysis, demonstrated (6) in 1929. Details of this work were not published, but much use of the method has since been made in this laboratory. The inde- pendent application of ferri-ferrocyanide electrode potentials to similar problems by Wood (7) prompts us to report a summary

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the cell

Pt

Reference Salt Fe+ + Fe0 electrode bridge reagent + (Fe6 + Fe, sugar Pt reagent + water)

A B c

the observed potential is the algebraic sum of single potentials at R, liquid junctions at A and B, and the electrode C. It is the potential at C which must be measured. A simple and effective way to avoid change of the liquid junction potentials at A and B is to use as the reference electrode R another sample of the Fe;-Fe, sugar reagent diluted with water instead of sugar solution. The solutions at R and C then have the same electrolyte composi- tion, and the junction potentials at A and B are equal and op- posite in sign, and cancel. Under these conditions a change in the cell potential represents the change of potential at C resulting from the decrease of Fe< and increase of Fe, caused by sugar oxidation.

P. A. Shaffer and R. D. Williams 709

of our experience and the procedure used in this laboratory. It should be stated that the potentiometric method is not advocated as a substitute for satisfactory titrimetric or calorimetric proce- dures unless the number of analyses to be made, the volume of blood available, or other circumstances justify its choice. For only occasional analyses, or in the hands of workers who are not somewhat familiar with the technique of potential measurements, it would be less satisfactory than the standard methods now generally used.

Properties of the Ferri-Ferrocyanide Electrode

When an alkaline solution containing both ferri- and ferro- cyanide (the reagent, before or after being heated with reducing sugar) is joined by a salt bridge with a reference electrode, as in

The next step is to relate the potentia1 to concentration of the sugar oxidized. Although this may be done merely by experi- mentally determining the potentials corresponding to different sugar concentrations under defined conditions, it is desirable also to relate the potentials first to Fe: and Fe, concentrations, and

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710 Sugar Determination

these to sugar concentrations, as would be done with chemical methods of analysis. This requires consideration of the behavior of the Fei-Fe, electrode.

The theoretical equation is

RT (FeJ E=&+yIn~

E = & + E In [Feil + R_T In ri F [Fe,1 F y.

where the terms in parentheses represent activities. The equation may be written also in the second form, involving concentrations denoted by brackets and activity coefficients (7).

Study of the electrode by a number of workers (8, 9) shows that the potential of solutions equimolar as to ferri- and ferro- cyanide concentrations varies greatly with their sum and also with the concentration of all other electrolytes. The extent of this effect is illustrated by the data in Table I and by similar data recorded by Michaelis (9). The principal factor appears to be the total cation concentration, an increase of which sup- presses the dissociation of Fe, to a greater degree than that of Fei, thus increasing the activity ratio and giving a more positive potential. The effect is quite complex, owing perhaps to a step- wise dissociation of the salts of polyvalent ions, yielding a mixture of several sets of oxidant-reductant pairs. Whatever its explana- tion, this salt effect must either be avoided or taken into account in attempts to relate potentials to Fei and Fe, ratios and concen- trations. Fortynately it is simple to sidestep this complicating factor by adding to the solutions a large excess of another elec- trolyte. This addition serves also another useful purpose.

In the absence of other electrolyte the potentials become indefi- nite at Fe; + Fe, concentrations below about 0.001 M. But in the presence of 1 or 2 M KC1 the potentials are poised and reproducible at 0.0001 M (Table I). This stabilizing effect of added electrolyte on the potential of quite dilute ferri-ferrocyanide solutions makes possible the potentiometric analysis of similarly dilute sugar solutions, an application which constitutes one of the method’s principal advantages. High salt content also tends

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P. A. Shaffer and R. D. Williams 711

to minimize the effect of electrolytes added in the sugar solution. (This effect of electrolyte added in the sugar solution must, how- ever, receive consideration.)

In any series of solutions, the electrolyte concentration of which is kept constant and high in relation to [Fei + Fe,], it may be assumed that the activity coefficients of Fei and Fe, will be ap- proximately constant. The activity term in Equation 2 may then be regarded as constant and may be combined with the value E. to give Efo, the apparent normal potential for that set of solu-

TAB~LE I

Potentials of Eguimolar Solutions of K,Fe(CN)a and K,Fe(CN)s

Solutions read against a saturated KCl-calomel electrode at 25”, liquid junction potentials being disregarded.

T

[Fe< + Fe,1

M

0.25 0.20 0.10 0.01 0.001 0.0001

0.01

No added salt

volt 0.217* 0.213 0.198 0.158

Indefinite “

M KC1

volt 0.242 0.240 0.235 0.229 0.229 0.229

2aaKCI 2 M N&l

vozt 0.257* 0.255* 0.252* 0.249 0.249 0.250*

vozt 0.257* 0.255* 0.249* 0.244 0.243 0.246*

* We are indebted to Mr. John D. Stull for these values.

tions. When two such similar solutions are combined in a cell, its potential should vary in accordance with the equation

(3) Eobs. = E; + y In E, 1 [ RT [Feil“ - E; + 7 In [~e,l’; 1

We find that under the conditions stated the potentials comply rather accurately with this equation, with variation of the ratio [Fei]/[Fe,] between 20 and 0.05. Illustrative data are given in Tables II and IV. Beyond these extremes of the ratio the devia- tions of measured potential become considerable, though, owing to the mathematical relations, the corresponding error in terms

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712 Sugar Determination

of chemical equivalents is not very serious. The potential change of solutions in which [Fe;] is reduced by sugar complies with the equation beyond the above limits rather better than do mixtures of KzFe(CN)c and K4Fe(CN)6, in which a change of electrolyte concentration is unavoidable. The deviations observed with mixtures are therefore perhaps due in large part to a change of cation concentration, which need not occur with sugar solutions.

Having established the validity of Equation 3, we may use it under the stated conditions to calculate change in concentration

TABLE II Variation of Potential with Ferricyanide to Ferrocyanide Ratio

The reference electrode in each case was a solution of the sameconcen- tration of added salts and [Fei + Fe,] as in the half-cell measured, but Fei = Fe,. Saturated KCl-agar bridge, 25”.

ei + Fe,

M

0.01

0.001

0.0005

F.% Fe 0

10 0.1

10 0.1

10 to 0.1

--

Potential difference

NOW3 M NazSO ___-

volt volt

0.0585 0.0598 0.0585 0.0581

0.0577 0.0552

l These solutions contained0.24

Additional salts I

I - Ml

Theory ~MKCI 2 w N&l Reagent II

volt volt volt aolt

0.0594 0.0593 0.0590* 0.0591 0.0585 0.0583 0.0591* 0.0591

Resgent III

0.0594 0.0592 0.0595* 0.0591

0.0593 0.0597 o.o6Qo* 0.0591

0.1174* 0.1182

Na&Oa, 0.06 MNaHCOs, and 1 MNaCl.

of Fei caused by its reduction by sugar or other reducing sub- stance. Reagents are constructed containing known concentra- tions of Fe; and Fe,, a mixture of carbonate and bicarbonate to provide the desired alkalinity, and a large excess of NaCl or other additional electrolyte. This solution, diluted with an equal volume of water (or with a salt solution of the same composition as contained in the sugar solution), is used as the reference elec- trode. Another portion of the same reagent, similarly diluted with an equal volume of the sugar solution, is heated under con- ditions to accomplish nearly complete oxidation of the sugar and

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P. A. Shaffer and R.. D. Williams 713

reduction of a corresponding quantity of Fe< to Fe,,, without change of concentration by evaporation. The second portion is then joined by a salt bridge with the reference electrode, both at the same known temperature, and the potential of the cell is measured. This procedure, which is in principle the same as that used by Wood (7), avoids the difficulty both of liquid junc- tion potentials and of activity coefficients. Another advantage in using the reagent as the reference electrode is that the sign of potential is always the same, which avoids confusion. It also simplifies calculation of results.

Calculation of equivalents of Fei reduced from the observed potentials is made as follows: Equation 3 may be written for 25” in the following form. (When the potential is read at another temperature, the quantity 0.0591 is substituted by the appro- priate value.)

(4) &bs. = (E; + 0.0591 log R) - (E; + 0.0591 log R’) (4 (B)

Reference electrode, Reagent + sugar reagent + water solution

where R represents the known ratio [Fei]/[Fe,] of the reagent and R’ the unknown ratio after heating reagent and sugar solution. From Equation 4

(5)

and

log R' = log R - &,,./0.0591) (25”)

(6) R'

- = F (fraction of total Fei + Fe, as Fei in B) R' + 1

Subtraction of the molar concentration of Fei in B from that in A (the reagent as prepared) gives the molar concentration of Fei reduced by an equal volume of the sugar solution.

The quantity of ferricyanide reduced by a given quantity of pure reducing sugar thus calculated from the potential agrees well with the results obtained by iodometric titration of residual ferricyanide, with the same reagent, sugar concentrations, and conditions during the heating period. A few results with glucose which illustrate comparison by the two methods of ferricyanide determination are given in Table III. The data show also that

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714 Sugar Determination

the equivalent reduction is fairly constant (for a given reagent), independent of sugar concentration. This confirms the same conclusion by Hawkins and Van Slyke and by Wood.

Composition of Reagents

In deciding upon an optimum composition for a ferricyanide reagent to be used for potentiometric measurement, two consi- derations are involved: those factors which affect the sugar oxi- dation, and those which concern the potential measurement. We

TABLE III

Determination oj Ferricyanide Reduction by Potentials and by Iodometric Titration

5 cc. of glucose solution of the concentrations stated plus 5 cc. of Reagent II, heated 15 minutes. Potentials read at 25” against reagent plus water (unheated).

Glucose concentratior

% :z 0 2

10 20 40 50

TnM mv.

0 2.7 0.2 0.111 10.4 0.86 0.556 31.4 3.50 1.11 50.4 6.84 2.22 84.8 13.14 2.78 115.0 16.14

AE from

reagent

Fe< reduced

Calculated from potential

Corrected

0.66 0.7 5.94 6.30 3.30 3.4 5.94 6.12 6.64 6.7 5.98 6.04

12.94 13.0 5.82 5.86 15.94 15.7 5.74 5.65

By iodo- metric

titration; corrected for blank

d per M glucose oxidized

Potential Titration

shall consider these briefly in order that a composition may be better selected to suit particular circumstances.

The alkalinity of the solution influences the rate of sugar oxi- dation by Fei and the reduction equivalence in the same manner as found by Shaffer and Somogyi with copper solutions (10). Wood (7) has recently studied this question in some detail, using glycocoll-NaOH buffers. Our experiments with Fei in carbonate- bicarbonate solutions, referred to in an earlier paper ((10) p. 700), are in general agreement with his conclusions. The higher the ratio of carbonate to bicarbonate (the higher the pH of the solu-

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P. A. Shaffer and R. D. Williams 715

tion), the more rapid the sugar oxidation at a given temperature, and the less Fe; reduced per mole of sugar. Increasing the total carbonate content has the same effect as lowering the ratio, or the effective alkalinity. The addition of high concentration of of NaCl or other salt (for the purpose of poising the potentials and to minimize the effect on the potential of change of electrolyte concentration) slows the rate of sugar oxidation. The relative rate of oxidation of various sugars is indicated in the paper on copper reagents (10).

To improve its keeping quality as well as to poise the reference electrode potential some Fe, must be included in the reagents. For greater sensitiveness we use such quantities of Fe, as to give an initial ratio of Fe;/Fe, = 9 (instead of 1, adopted by Wood). This allows a potential difference (from reagent) of 0.1128 volt at 25” or 0.1408 volt at loo”, when after reduction by the sugar solution 10 per cent of the total Fei + Fe, is left unreduced. The greater this difference, the smaller will be the percentage error from variations in potential readings.l

Although the relation of E.M.F. to quantity of Fei reduced is obviously not linear, a difference of 1 millivolt corresponds to an average of somewhat more than 0.5 per cent of the total Fe; + Fe,. To attain the highest accuracy in results it is, therefore, necessary that the reagent contain only a small fraction of Fe, and that a large fraction of the Fe; be reduced by the sugar solu- tion. In order that this may be realized the Fei content of the reagent must be related to the sugar concentration. An optimum concentration, when equal volumes of reagent and sugar solution are used, is M Fei = 0.5 X gm. per cent of glucose (the maximum expected in the solutions), M representing molar Fei in the reagent.

1 The precision of potential measurements will depend upon many factors, including the type of apparatus and efficiency of temperature control. Because of greater variations in other steps of the determination, especially during the period of heating reagent and sugar solution, it is not necessary to attain maximum precision in the potential measurement. The degree of over-all reproducibility is indicated by the agreement of potentials of duplicate sugar solutions, usually within 0.5 to 1 millivolt. We have used a Leeds and Northrup type K potentiometer, a high grade galvanometer, and automatic constant temperature (water) bath. Such equipment is, however, not necessary. The less costly “student” type of apparatus used with quinhydrone electrodes is quite satisfactory.

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716 Sugar Determination

Reagents should be constructed with these points in mind. As with copper reagents, no single composition is optimum for all sugars, for all sugar concentrations, periods of heating, or other experimental conditions. We give the composition of three solu- tions which have been found suitable for different concentrations of glucose. Other mixtures might be equally satisfactory, pro- vided a suitable heating period is adopted.

Ferricyanide Reagents

Reagent I-For glucose solutions 0.05 to 0.2 per cent.

KaFe(CN)s. ......... K4Fe(CN)s.3Hz0 .... Na&Oa ............. NaHCOa. ........... NaCl. ...............

Y pm. per 1.

0.09 29.6271 0.01 4.2233 0.47 50.0 0.12 10.0 2.0 117.0

Reagent II-For 1: 10 or 1:20 blood filtrates or for glucose solu- tions 0.001 to 0.05 per cent.

K8Fe(CN)B. . . . . . . . . . KaFe(CN)s*3HzO.. . .

M gm. per 1.

. . . . . . . . . . . 0.018 5.9254

. . . . . . . . . . . 0.002 0.3446

With carbonate, bicarbonate, and chloride content the same as in Rea- gent I.

Reagent III-For blood filtrates of greater dilution or for glu- cose solutions 0.0001 to 0.0025 per cent: K3Fe(CN)6, 0.0009 M,

KJFe(CN)n, 0.0001 M, with carbonate and chloride content as in Reagents I and II. This reagent is unstable and should be pre- pared the day it is used. Measuring 5 cc. of Reagent II into a 100 cc. volumetric flask and diluting to volume with a stock carbonate-chloride solution of the same concentrations as are con- tained in Reagents I and II give Reagent III. The carbonate- chloride solution is made up separately and (after dilution with an equal volume of water) is used also for the salt bridge at 100” as described below.

In preparing the reagents the ferri- and ferrocyanides (reagent

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P. A. Shaff er and R. D. Williams 717

quality) are weighed accurately. It is convenient to dissolve the Na2C03, NaHC03, and NaCl in order and to add the ferro- and ferricyanides last. After dilution to volume Reagents I and II should be filtered through good quality paper, the first portion of filtrate being discarded. The solutions should be kept in stoppered Pyrex bottles or flasks, away from Iight.

Table IV gives observed values of the potentials for these reagents, which serve to check whether a batch has been correctly made. Rechecking the value of the potential of a reagent at intervals under the conditions stated gives a measure of its sta- bility. With pure chemicals the potentials decrease only a few

TABLE IV Potentials of Ferri-Ferrocyanide Reagents

Reagent plus an equal volume of water.

I

II

III

En, 25”

oolt

+0.539

+0.534

+0 ,532

-

-

Fe< + Fe.

M

0.05 0.05 0.05 0.01 0.01 0.01 0.0005 0.0005 o.ooo5

9:l 1:l 1:9 9:l 1:l 1:9 9:l 1:l 1:9

I AE from reagent, 9: 1

-

-

250

Observed

Tim.

56.4 112.7

56.3 112.7

55.9 112.8

- _-

-

Theory Ibserved Theory

n&v. ma. VW.

56.4 112.8

56.4 69.9 70.4 112.8 140.5 140.8

56.4 62.0 70.4 112.8 140.0 140.8

- 100”

-

millivolts in the course of months. If not greater, the change is negligible as affecting the results with sugar solutions, being in large part corrected for by using the reagent as the reference electrode.

The standard of reference for the values of Eh in Table IV is the quinhydrone electrode in 0.05 M acid potassium phthalate, assigning to it, at 25”, the value +0.4643 volt to the normal hydrogen electrode. This corresponds with Clark’s value of 3.974 for the pH of this solution, and 0.6992 for Eo of quinhydrone at 25”. Saturated KCl-3 per cent agar bridges were used and liquid junction potentials are neglected. An independent check

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718 Sugar Determination

is to prepare additional solutions of the same composition as the reagent but having different ratios of ferri- to ferrocyanide, as il- lustrated in Table IV. The potential difference between elec- trodes in similar pairs of solutions joined by a bridge should be within 1 millivolt of that calculated by the electrode equation. Table IV gives values observed under these conditions.

Procedure

5 cc. (or 2 cc. if desired) of the sugar solution (blood filtrate) are measured into a test-tube (25 X 200 mm. for 5 cc.), an equal volume of the proper reagent is added, and the solutions are mixed by shaking. A second tube is prepared with the same quantity of reagent, with water instead of the sugar solution (or with a salt solution of like concentration to that of the sugar solution) for use as the reference electrode. This reference tube is heated in order to correct for the small autoreduction of the re- agent.2 Both tubes are covered by glass bulbs (or clean rubber stoppers bearing a capillary tube) to avoid loss of water by evapo- ration. The rack holding the tubes is placed in a vigorously boiling water bath for 15 minutes (see (10) p. 710).

Either of two procedures may be used for measuring the po- tential. (a) After heating, the tubes are removed, cooled by setting the rack in cold tap water, and brought to a fixed tem- perature (25”) by being placed in a constant temperature bath, in which the potentials are read. (b) The boiling water bath may be used as the constant temperature bath, the potentials being read with the tubes immersed in it. In either case each tube is removed at the end of the boiling period and shaken so as to restore to the solution the water which has been condensed on the walls of the tube. On its return to the bath in which

2 With reagent grade chemicals and good distilled water these blank po- tential changes are fairly constant, for Reagent I about 2 to 3 millivolts; for Reagent II, 3 to 4 millivolts; for Reagent III, 5 to 10 millivolts. Varia- tions within this limit introduce negligible errors, since the heated blank potential decrease is automatically subtracted. Since these corrections enter indirectly into the slope of the calibration curve of the reagent, they should be determined by individual workers. If corrections are much greater than the above values, the reagent should be discarded or a new calibration curve should be made with it by analysis of known amounts of pure glucose.

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potential is to be read, a “seasoned” platinum electrode is inserted in each tube, followed by a salt bridge joining the sugar tube with the reference electrode.

The bridge used at lower temperature is a small bore glass tube bent like a hairpin and filled with saturated KC1 in 3 per cent agar-agar. A new surface is exposed by cutting off a short section

TABLE V

Potentials Corresponding to Glucose Concentrations

5 cc. of reagent plus 5 cc. of glucose solution, heated 15minutes. Poten- tials were read at 25” against reagent plus an equalvolume of water (heated). Saturated KCI-agar bridge. Bureau of Standards glucose was used.

Reagent I Total ferri f ferro = 0.10 M

-7

Glume per co. solution

ma.

0.05 0.10 0.15 0.20 0.30 0.40 0.45 0.60 0.80 1.00 1.20 1.40 1.50 1.60 1.70 1.80 1.60 2.06

Eobs.

0.0045 0.0090 0.0130 0.0165 0.0235 0.0290 0.0315 0.0385 0.0476 0.0562 0.0650 0.0745 0.0800 0.0860 0.0930 0.1010 0.1100 0.1204

Reagent II Total ferri + ferro = 0.02 M

Glucose per cc. solution

mQ.

0.01 0.02 0.03 0.04 0.05 0.08 0.10 0.12 0.14 0.16 0.20 0.25 0.30 0.35 0.40 0.44 0.48 0.50

-7-

- 1:

Eobs.

0.0041 0.0079 0.0114 0.0145 0.0200 0.0249 0.0288 0.0339 0.0374 0.0404 0.0478 0.0554 0.0631 0.0716 0.0821 0.0919 0.1044 0.1124

Reagent III rota1 ferri + ferro = 0.001 I

Glucose per cc. solution

ms.

0.001 0.002 0.003 0.004 0.005 0.006 0.008 0.009 0.010 0.014 0.016 0.018 0.020 0.021 0.022 0.023 0.024 0.025

gobs.

0.0055 0.0115 0.0157 0.0197 0.0230 0.0265 0.0330 0.0360 0.0389 0.0507 0.0565 0.0625 0.0688 0.0725 0.0762 0.0805 0.0850 0.0896

of each arm with a file each time the bridge is used. For the boiling temperature the bridge is a Y-tube, the arms of which are attached by rubber tubing, the ends having capillary tips or being closed by sintered glass powder. The whole tube is filled with the carbonate-NaC1 solution (without Fe,-Fe,) diluted with an equal volume of water. A pinch-clamp at the top, closed when the

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720 Sugar Determination

bridge is in place, is opened after its removal to allow a few drops to flow out before insertion for the next reading.

The potential is then read by potentiometer and galvanometer. The electrode in the sugar tube is always negative to the reference electrode. The electrodes are “seasoned” by being kept, when not in use, in a sample of the reagent solution. They are rinsed and wiped dry each time before use.

The results are most simply calculated by use of curves or tables constructed from such data as are given in Tables V and VI. It will be noted that the data given are applicable only for the sugar

TABLE VI Potentials Corresponding to Glucose Concentrations

Potentials read at 100” f 1”. Reference electrode, reagent plus equal volume of water. Bridge, the (diluted) salt solution. Bureau of Standards glucose. Heating period, 15 minutes. A “student” potentiometer was used.

Reagent II

Glucose per cc. solution

w.

0.02 0.05 0.10 0.15 0.u) 0.30 0.40

Eobs.

0.0095 0.0210 0.0350 0.0475 0.0585 0.0790 0.1030

-

-

-

Reagent III

Glucose per cc. solution h’obs.

mg.

0.002 0.004 0.008 0.01 0 015 0.02 0.025

0.0180 0.0280 0.0450 0.0525 0.0680 0.0880 0.1170

stated (glucose) and when the composition of reagents and all experimental conditions are the same as here described.

Procedure for Determination of Sugar in Blood-Filtrates of 1: 10 or 1:20 dilution, prepared by zinc hydroxide precipitation as described by Somogyi (ll), yield approximately “true sugar” values by the potentiometric method. Filtrates from tungstic acid or iron precipitation yield higher values by ferricyanide methods, both titrimetric and potentiometric, than by copper reagents.

For convenience the composition of the Somogyi solutions for blood precipitation are given below, together with a modified

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P. A. Shaffer and R. D. Williams 721

solution for use with small quantities of blood. For blood pre- cipitation at 1: 10 (or 1:20) dilution: Solution 1. 12.5 gm. of ‘ZnSOa. 7HzO and 125 cc. of 0.25 N H2S04 per liter. Solution 2. 0.75 N NaOH. Lake 1 volume of blood in 8 volumes of Solution 1, add 1 volume of Solution 2, shake well, and centrifuge or filter. To remove the trace of excess zinc salt present in these filtrates add to the filtrate a pinch of solid Na2C03 (about 1 mg. per 10 cc.), dissolve, and again centrifuge or filter. 5 (or 2) cc. of the solution are then added to an equal volume of Reagent II, follow- ing the procedure described above.

Microdetermination-When only small quantities of blood are available, the following procedure is used. The blood is measured in 0.02 (or 0.05) cc. pipettes of capillary tubing such as are used for the determination of hemoglobin. The following solutions are used for the precipitation: Solution A. To 148 cc. of water add 40 cc. of the Somogyi Solution 1 above. Solution B. 0.030 N NaOH. Measure 3 cc. of zinc Solution A into a 15 cc. centri- fuge tube. Into this the blood is discharged, the pipette being rinsed with the solution. The mixture is well stirred by blowing air through the solution by means of the pipette. 2 cc. of Solution B (0.03 N NaOH) are added, and the tube is stoppered, shaken throughly, and centrifuged. Without removing the solution from the tube, the residual zinc is precipitated by adding a pinch (0.5 mg.) of dry Na2C03 which is dissolved by shaking gently so as not to disturb the protein precipitate. The tube is then centri- fuged again. The clear solution may then be poured off; if nec- essary it may be filtered through a small, washed, dry filter paper. (Unless the filter papers have been carefully prepared, filtration is not recommended.) The dilution is 1:251 (or 1:lOl if 0.05 cc. of blood is used). 2 cc. of the solution are used with 2 cc. of Re- agent III, as above described for solutions of higher concentra- tion. Reagent III plus water, heated, is used as the reference electrode.

Table VII shows a comparison of results by the electrometric method with values by the Shaffer-Somogyi copper Reagent 50 (10). Many similar results appear to justify the conclusion that the results by the ferricyanide electrode on zinc filtrates of blood are substantially the same as are obtained with the iodo- metric copper reagents on the same filtrates, and represent ap-

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722 Sugar Determination

proximately “true sugar” values. It will be noted that the results with Reagent III on filtrates of 1:251 dilution, for which only 0.02 cc. of blood was used, do not differ greatly from the values on 1: 10 filtrates.

TABLE VII

Blood Sugar. Comparison of Results on Zinc Filtrates by Iodometric-Copper Reagent and by Fe&cyanide Potentials in Filtrates of Different Dilutions

The data are given in mg. (glucose) per 100 cc. of blood.

I 1: 10 fi1tratas I:251 filtrates 1:251 diluted from 1: 10 filtrates

Blood No.

copper Reagent 50

-

102 108 99 14 20 14 44 44 38 82 86 89

118 122 120 100 108 102 92 100 95

156 158 160 I- I

Potentials measured at 100’

Ferricyanide Ferrioyanide Ferricyanide Reagent II Reagent III Reagent III

Potentials measured at 25’

98 18 38

150 156 159 58 60 63

128 127 134 84 88 loo

216 224 220 242 242 240

SUMMARY

Procedures are described for the determination of reducing sugars by the ferri-ferrocyanide electrode. Provided interfering substances are absent, satisfactory results are easily and con- veniently obtained over a wide range of sugar concentrations. Smaller amounts of sugar are required for the determination than by other methods so far used. With filtrates prepared by zinc hydroxide precipitation of blood, sugar concentration may be

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P. A. Shaffer and R. D. Williams 723

determined on 20 c.mm. of blood. The method is applicable to the analysis of similar quantities of other solutions.

BIBLIOGRAPHY

1. Hagedorn, H. C., and Jensen, B. N., Biochem. Z., 136,46 (1923). 2. Van Slyke, D. D., and Hawkins, J. A., J. Biol. Chem., 79, 739 (1928). 3. Folin, O., J. Biol. Chem., 77, 421 (1928); 81, 231 (1929). Folin, O., and

Malmros, H., J. Biol. Chem., 83, 115 (1929). 4. Hawkins, J. A., J. Biol. Chem., 84,79 (1929). Hawkins, J. A., and Van

Slyke, D. D., J. BioZ. Chem., 81,459 (1929). 5. Ariyama, N., and Shaffer, P. A., J. BioZ. &em., 78, li (1928). 6. Shaffer, P. A., Demonstration at the Thirteenth International Physio-

logical Congress, Boston (1929); Am. J. Physiol., 90, 513 (1929). 7. Wood, W. B., Jr., J. BioZ. Chem., 106, cii (1934); 110, 219 (1935). 8. Fredenhagen, C., 2. anorg. Chem., 29, 396 (1992). Schaum, K., and

Linde, R., 2. Elektrochem., 9,406 (1993). Schoch, E. P., J. Am. Chem. Sot., 26, 1422 (1994). Lewis, G. N., and Sargent, L. W., J. Am. Chem. Sot., 31, 355, 363 (1999). Mtiller, E., 2. phgsik. Chem., 89, 46 (1914). Schoch, E. P., and Felsing, W. A., J. Am. Chem. Sot., 38, 1928 (1916). Linhart, G. A., J. Am. Chem. Sot., 39, 615 (1917). Kol- thoff, I. M., 2. anorg. Chem., 110, 143 (19‘29).

9. Michaelis, L., Oxydations-Reductions Potentiale, Berlin, 2nd edition, 64 (1933).

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Philip A. Shaffer and Ray D. WilliamsFERRICYANIDE ELECTRODE

SUGAR DETERMINATION BY THE

1935, 111:707-723.J. Biol. Chem. 

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