glucosa fructosa

7/23/2019 glucosa fructosa

1/7

Photometr ic Determinat ion of Glucose in Presence of Fructose

FRED STITT, STANLEY FRIEDLANDER', HAROLD J. LEWIS2, and FRANK E. Y OU N G

West e rn U t i l i za t i o n Resea rch B ranch , Ag r i cu l tu ra l Resea rch Se rv i ce ,

U.

S. D e p a r t m e n t o f A g r i c u lt u r e , A l b a n y 6 Ca l i f .

A

method has been developed for measuring

0.05

to 0.5

of glucose in fructose. Sodium chlorite solution

buffered

to

pH 4.0 is used to oxidize glucose at

a

much

faster rate than fructose. Chlorine dioxide, a product

of the reaction, is measured with a spectrophotometer

or colorimeter. The difference in chlorine dioxide pro-

duced by oxidation of the test sample and of a suitable

reference fructose sample is a measure of the glucose

con tent . Recrystallization of fructose as dihydrate is

shown to produce a suitable aldose-free reference ma-

terial. The method can be used over the entire range of

composition of glucose-fru ctose mixtures and it appears

to be generally applicable to the measurement of

aldoses

in

the presence of ketoses. Analyses by the

spectrophotometric procedure of glucose-fructose

samples of known composition showed standard dsvia-

tions of about 0.003% glucose for samples containing

less than

0.5%

glucose, about

0.03%

glucose for samples

containing

0.5

to

5%

glucose, and about

1%

of the glu-

cose content for samples containing

over 5%

glucose.

The corresponding standard deviation for results by

the colorimetric procedure were greater by a factor of

1.5 to 2. These samples covered a range from 0 to 100

mg.

of

fructose and

0.05

to

1

mg. of glucose per m l. of

the test solution.

URIXG

an investigat ion of t he use of fruc tose dihydrate in

D thepurificationof fructose(I6, 7),aneed arose for amethod

by which small amounts of t he principal impurities, glucose and

mannose, could be determined in the presence of fructose. The

usual methods

3,

5 ,

7 )

acked the desired sensitivity and selec-

tiv ity and, moreover, employed alkaline reagents which caused a

portion of th e fructose being analyzed to transform t o glucose and

mannose during the analysis

3,

) .

Notatin (glucose oxidase),

which catalyzes the oxidat ion of glucose in acid solution, is said to

be specific for glucose

(IO),

but it does not catalyze the oxidation

of mannose ( I ) and appears to be slightly less sensitive th an th e

method reported here. Jeanes and Isbell

(9)

observed th at chlor-

ous acid oxidizes aldoses much more rapidly tha n ketoses, forming

chlorine dioxide as one of the produc ts. Launer, Wilson, and

Flynn ( I S )confirmed this observation and used chlorous acid as an

oxidizing agent in developing both iodometric and photometric

methods for determining glucose in the absence of ketoses. The

authors have adapted their photometric method to th e determi-

nation

of

smalI amount s of glucose in fructose. Th e glucose

content is measured by the difference in chlorine dioxide produced

in a definite period of time in a reaction mixture containing th e

sample of interes t and in a similar reaction mixture containing

stan dard reference fructose. Th e present work was primarily

concerned with glucose, because mannose is likely to be present

a t much lower concentration (14). Mannose, when present , wiII

be included with glucose as aldohexose.

CHOICE OF REACTION CONDITIONS

When sodium chlorite, glucose, and fructose are brough t

to-

gether in acidic aqueous solution, at least three simultaneous

reactions occur which consume chlorite and produce chlorine

1 Present address, Applied Research Laboratories, Glendale, Calif.

Present address, University of Minnesota, Minneapolis, Minn.

dioxide-via., disproportionation of chlorous acid 2 , 9, 13, IS,

I6), oxidation of glucose by chlorous acid (9, 13, IS), and oxida-

tion of fructose by chlorous acid. Because th e stoichiometry

and kinetics of reac tions involving chlorous acid have been found

to vary with conditions

2, ; ) ,

kinetic studies n-ere made

for

the

temperature and concentration ranges of interest to provide an

adeq uate basis for choosing suitab le reaction conditions for an

analytical procedure. Th e results are briefly summarized here

in so far as they supplement earlier kinetic ao rk.

Reactions were followed by photometric measurement of t he

rat e of formation of chlorine dioyide with either a colorimeter

or a recording spectrophotometer at fixed wave length. Except

where otherwise noted, reactions were carried out a t

25.0

C.

in

acetic acid-sodium acetate buffer solutions ivith tota l acetate

concentration of 1.5 X. Because th e acid dissociation constan t

of chlorous acid is abou t a t

25 C.

2 ) ,most of the chlorite

is present as salt in the p H range of

3.7

to

1.7.

The differencr in

the rates of formation of chlorine dioxide in two reaction mixtures

initia lly identical, except for the presence of sugar in one and

not in the other, was used in studying the kinetics of oxidation of

glucose and fructose by chlorous acid.

Disproportionation

of

Chlorous Acid. This reaction is complex,

but under conditions used in this investigation can probably be

most simply approximated by the stoichiometric equation 2 ) :

At pH 4 .3 the order

of

the reaction forming chlorine dioxide was

found to be

1.9

with respect to t otal chlorite concentration when

the latter was varied between

0.006

and

0 2 5 X .

Slightly lower

values were found a t p H 3.7 and pH

4.7,

but these results are in

essential agreement with second-order dependence found by

Barnett

(g)

and by Launer, Wilson, and Flynn (IS). On the

other hand th e formation of chlorine dioxide appeared t o be

nearer to first order with respect to [H

+],

hydrogen ion concen-

trat ion , than second order, the numerical values falling between

1.0

and 1.2. Experiments at an initial chlorite concentration of

C, =

0.075Mshowed approximately the same rate and dependence

on pH when

1.534

citrate buffer was used in place of acet ate buf-

fer. Although a t fixed pH of 4.0 and

4.2 ,

a change in acetate

buffer s trength produced marked changes in r ate in t he same direc-

tion, addition of sodium chloride 1 . O M ) or sodium sulfate

( 0 . 5 M )

to increase the ionic strength

of 1.5L1f

acetate buffer (pH 4.2)

produced no rate changes not accounted for by t he accompanying

shift in pH. Rate data obtained at 19.9 ,

25.0,

and

40.0

C.,

at pH

4.2,

and C, of 0.075 and

0.025M

gave an app arent activa-

tion energy of 20,000 calories per mole and confirmed th e second-

order dependence on total chlorite concentration. Because this

apparen t activation energy is calculated from data for constan t

tot al chlorite concentration, it is a combination of the heat of

dissociation of chlorous acid and the apparen t activation energy

for the reaction involving chlorous acid as a reactant.

Jeanes and Isbell (9) suggested the

following equation for the oxidation of glucose by chlorous acid

:

Oxidation

of

Glucose.

Th e validity of this equation is established by th e authors' results

and by those

of

Launer

t

al.

( I d , IS).

The reaction was studied

over the same range of variables as the disproportionation reac-

tion, The rate of formation

of

chlorine dioxide due to oxidation


2/7

V O L U M E 2 6 , NO.

9,

S E P T E M B E R 1 9 5 4

of glucose a t pH of 3.7, 4.3, and 4.7, showed good agreement with

first-order dependence on to tal chlorite, 0.006 to 0.25M, and on

glucose concentrations, the values of the latter being 0.5,

1,

and

2 mg. per ml.

On the other hand, the dependence on

[H+]

appeared to be definitely less than first order, the numerical values

ranging from slightly less than

0.7

to nearly 0.8. The effect

of

changing buffer strength, adding salts to the buffer, or changing

from acetate to citrate buffer were qualitatively the same as

for the disproportionation reaction. An apparent activation

energy of 16,500 calories per mole was found.

Oxidation of Fructose. The stoichiometry of the oxidation of

fructose by chlorous acid was not investigated, but approximately

3000 times as much fructose was required in a reaction mixture

at 25'

C.

to produce chlorine dioxide at the same rate as a given

concentration of glucose. The comparatively little kinetic dat a

which were obtained on the fructose reaction showed approxi-

mately first-order dependence on fructose and total chlorite

concentrations, the same dependence

on

pH as t he oxidation of

glucose, and an apparent activation energy

of

the order of 23,000

calories per mole.

1479

325

3 1 5 495

WAVE LENGTH p

Figure 1. Absorption Spectra

A .

B

0.06M NaClOz

C. No. 42 filter

D.

0.0015M

ClOz

in

water solution

0.00073iMKzCrOa in

0.05.M

NaOH)

Choice of Reaction Conditions. By varying temperature, pH,

chlorite concentration, buffer strength, and reaction time, the

relative amounts of chlorine dioxide produced by each of the three

reactions discussed can be varied over wide limits. A reaction

temperature of 25O C. was chosen because the advantages of work-

ing near room temperature outweigh the relatively small improve-

ments obtainable at other temperatures in the proportion of

chlorine dioxide produced by oxidation of glucose. Because the

chlorite dependence of the rat e of th e disproportionation reaction

is higher than t ha t of t he rates

of

sugar oxidation reactions, chlc-

rite concentration was chosen as low as seemed consistent with

other requirements. A convenient reaction period corresponding

to practically complete oxidation of glucose, ample buffer capac-

ity, suitable excess chlorite capacity, and chlorine dioxide con-

centrations suitable for photometric measurement are the other

criteria which were applied in picking 25.0' C., 1.5M acetate

buffer

of

pH 4.00, C, = 0.060M sodium chlorite, and reaction

time

of 18

hours for standard reaction conditions. Under these

conditions the disproportionation reaction produces chlorine

dioxide at an initial rate of about 0.000143M per hour, the period

for Soyo oxidation of glucose is approximately 2 hours, and 100

mg. per ml. of fructose produces chlorine dioxide at an initial net

rate

of

about 0.000132-W per hour. This corresponds to produc-

tion in the 18-hour reaction period of about 500 times as much

chlorine dioxide due to oxidation of glucose as to oxidation

of

fructose for the same initial concentrations of the two sugars.

ANALYTICAL METHOD

Reagents. Sodium chlorite solution, 0.60M,

is

prepared by

dissolving the calculated amount of Mathieson analytical grade

salt, allowing

for

the stated purity. The solution can be stand-

ardized precisely by iodometric titration, but need be within only

about

1%

of the nominal value.

It

is filtered through sintered

glass, if necessary, and stored in the dark.

Acetate buffer, 6M, is prepared by dissolving 590 grams of

99.5% (glacial) acetic acid and 302 grams of sodium aceta te tri-

hydra te in water and diluting to 2 liters. The pH should be

4.00 .t

0.05.

Because slightly different oxidation and dispro-

portionation rates have been observed with different buffers of t he

same concentration and p H, it is advisable where possible to pre-

pare successive buffer solutions from the same stock reagents and

to use the same buffer solution for all samples and controls in any

one series

of

analyses.

Size

of

Sample. The size of the sample is chosen

so

that the

photometric measurement of the chlorine dioxide concentration

can be made with reasonable accuracy. For samples containing

less than 0.57' glucose, concentrations of 100 mg. per ml. are

recommended for both the sample in the test solution and the

reference fructose in the control solution. Thrse concentra tions

are each made 10 mg. per ml.

if

the glucose content is between

0.5 and 5%. No fructose is used in the control and 0.6 mg. of

sample per ml.

of

test solution is recommended for samples con-

taining over 5 glucose. These glucose contents refer t o solid

samples on the dry basis.

-

Procedure. To 12.5 ml. of 6 M aceta te buffer in a 50-ml. volu-

metric flask are added the sugar sample and water to a total

volume of about 40 ml. When the reaction is to

be

started,

5.00

ml. of 0.60M sodium chlorite are added, the mixture is diluted

to 50 ml. and well mixed. React ion tubes for replicate specimens

are filled immediately and placed in the dark in a constant tem-

perature bath a t 25

C.

(Colorimeter tubes, 15 nun. in diameter,

modified to accept standard taper glass stoppers, are used as

reaction tubes. Results are not affected if the tubes are not com-

pletely filled). The control solution is prepared in the same way

except that the recommended amount of reference fructose is

added instead of test sample. After the 18-hour reaction period,

the chlorine dioxide concentrations are measured photometrically

without opening the reaction tubes and with minimum exposure

to light. Blthough results are very insensitive to changes of an

hour or two in reaction period; this period must be the qame

within a few minutes for each test solution and its associated

control.

Spectrophotometric Measurement of Chlorine Dioxide Con-

centration. A Beckman Model DU spectrophotometer was used

in this investigation. Aqueous solutions

of

chlorine dioxide show

an absorption maximum a t 358 mp

A ,

Figure I ) , but because of

the absorption

of

sodium chlorite

( B ,

Figure

1)

in this region,

a

higher wave length should be used for spectrophotometric meas-

urement

of

chlorine dioxide in reaction mixtures In order to

eliminate errors in wave length calibration and shift in wave length

a t a fixed wave length dial position due to changes in tempera-

ture of t he the monochromator, much of the development work

was done with the 435

8

or 404.7 mp lines of a mercury arc source

such as is available for wave length calibration of the inst rument .

However, the convenience of t he tungsten source, resulting from

its greater stability, led

to

its use a t 436 mp in later

work.

When

the tungsten source is used, the calibration should be checked

against the mercury arc, and the wave length dial should always

be set precisely and from the same direction. Use of either the

435.8 mp mercury line

or

the tungsten source

of

436.0 mp is

recommended. A t these wave lengths, slit widths up to 1.0 mm.

for the mercury line

or

up to 0.5 uith the tungsten source can

be used without introducing errors in the absorbance measure-

ments.

The absorption coefficientsof chlorine dioxide in water solution

at 25

O C. were

determined a t various wave length. and concen-

trations and were in accord with Beer's law. Values of 113.5,

115.3, and 495 liter per mole-cm. were found for the molar ab-

sorption coefficients at 436.0, 435.8, and 404.7 mp, respectively.


3/7

1480

A N A L Y T I C A L C H E M I S T R

in the reaction tube a nd in m absorption cell of known thicknes

For all tubes used in this study p was approximately 1.29 cm

The tubes should be matched in both blank reading and i

path length

or

suitable corrections applied.

Colorimetric Measurement of Chlorine Dioxide Concentra

tions. A Klet&Summerson colorimeter was used in this stud

but other instruments should be satisfactory if properly calibrate

A

No.

42

filter

is used, the absorbance

of

which is shown by cwv

C of Figure 1. The instrument is operated from a constm

voltage transformer to eliminate detectable changes in readin

due to line voltage changes.

Th e calibration curves for oolorimeter reading versus chlorin

dioxide concentration in aqueous solutions, illustrated in Figur

3, should he determined for the instrument used. These curv

can be obtained

by

iodometric titrat ion of chlorine dioxide solu

tions, or indirectly, by measuring the same solutions on the colo

imeter and

a

spectrophotometer, obtaining concentrations fro

the spectrophotometric data.

Because the readings of the colorimeter may vary with th

refmotive index of the solution measured (with round tubes

it is necessary to determine calihration curves in the presence o

buffer and fructose ooncentrations typical of the controls to b

used. Th e magnitude of this effeot s indicated by the

separatio

of th e curve8

of

Figure 3.

A

correction, ahou t three scale uni

on the instrument used here, for the absorption of sodium chlo

rite should also be determined and added to the scale readings

plotting the calibration curves. Check measurements wit

potassium chromate as the ahsorbing medium showed that th

correction is nearly additive in scale units over the scale rang

used.

Because th e calibration curves of Figure

3

deviate markedl

from Beers law, the tes t and control solutions are each measure

separately against

a

reference containing water. If the tubes ar

not matched in hlmk readings or path lengths, corrections fo

these should he applied by the following equation:

R: = g A R z

- 8,

4

where Ri and

R

are the corrected and observed readings

or

Figure

2.

Tube Compartment for Spectrophotometer

The second

of

these values agrees reasonably

well

with the value

found hy Launer

et

al. 18) who prepared chlorine dioxide by

acidifying sodium chlorite solution.

The authors chlorine dioxide solutions were prepared by the

method

of Brw ( 4 )

n which

150

Erama of oxalic acid,

40 mama

Ihlorine, thr oigh glass wool to remove spray, and into ice water

containing

a small

amount of acetic acid. The appara tus should

be all glass. The chlorine dioxide content is determined iodo-

metrically.

In order to permit measurement

of

chlorine dioxide roncentra-

tions in the reaction tubes,

a

vertical light-tight extension of the

1-cm. cell compartment was constructed for the spectrophotom-

eter. This is fastened by screws

to

the regular com-

part ment of the instrument and accommodates the

stan dard cover (Figure 2). The reaction tubes fit

into

t he

four

positions

of the I-em. square cell carrier

so that test and control reitation tubes can be

pared readily either with each other or with

erenee tube containing water.

~

Because precision of the net chlorine dioxide

urement is increased by differential absalpurvu

measurements between the test and control solutions,

this procedure is recommended. I n addition, th e ab-

sorbance of th e control solution

is

measured

as a

eheok on gross deviations from stan dard condit

such as major temperature fluctuations-durii

reaction period.

If

the chlorine dioxide cone

tions of th e test and control solutions are d ew mu

by

B ,

and

B,,

then the difference in the

tions ( the net chlorine dioxide concentra

ions- 5

ig the

b

entra- 2

-

x

se coneentrar

POO

tion) is

(At - Ae )

CP

Bt -

Be =

(3)

where A , and

A ,

are the absorbances of th e test and

control solutions,

t

is the

molar

absorption coefficient

of chlorine dioxide a t th e wave length used and

p

is

the effective path length in centimeters

of

the reac-

tion tube&

0

0


4/7

V O L U M E

2 6 ,

NO.

9,

S E P T E M B E R 1 9 5 4 1481

Table

I .

Analyses of Prepared Mixtures of Glucose and Fructose

Concentrations Present in

Glucose Found

Error

in Mean

Standard Deviation

Sample Fructose Glucose .\Ig./Ml.

Glucose (Mg./Ml. Glucose)

Set mg./ml. lIg,/ ml. b 18 hr.C 19 hr.d

18 hr,c 19 hr.d 18 hr C 19 hr.d

Test Samples (Mea n Value), Value&, Mg./Ml. from Mean

A e 100.0 0 050 0 . 0 5 0 0 049 0.0 49 -0.0002

-0.0001

0.001 0 002

0.100 0.100 0 , l O O ~ 0 . 1 0 0 ; 0.0000

+0.0003

0 . 0 0 3 0 . 0 0 5

0 , 330 0 . 329 0 , 330 0 , 331 0 , 0 0 0

f 0 .001

0 , 003 0 . 0 0 5

1,000

0.991

0.966

0.969 -0 ,034 -0.031

0 010 0.015

B I 10.0 0 , 0 5 0 0 , 0 5 0 0,0492 0 . 0 5 0 2 -0.0008 + 0 . 0 0 0 2 0 . 003 0 . 0 0 5

0 , 100 0 . 99 0 . 1 0 0 0 0.1016 0.0000 + 0 . 0 0 1 6 0.003

0.003

0.330

3 . 2

0.330 0.328

0.000 - 0 . 0 0 2 0.001 0 . 0 0 5

1 . 000 9 . 1 0 . 981

0.979

-0,019 -0.021

0,005

0.009

C3 6 . 0 0.300

4 . 8

0 300h 0 . 2 9 8 h

0.000h -0.002h

0.001 0.004

3 . 0

0 , 3 0 0

Q . 1 0.301h 0.303h

t 0 . 0 0 1 h

+O.O03h

0.001

0 , 003

1.0

0.300 2 3 . 1 0 299h

0.299h -0.001h

-0.001h 0.003

0.003

0 . 0

0 , 300

100.0 0.301

0 , 302

f O . O O 1

+ 0 . 0 0 2 0.002

0.002

a

Glucose found minus glucose present.

b Percentag e of sug ar content which was glucose.

C

Spectrophotometer.

d

Colorimeter.

e Controls containing 100 mg./ml. fructose sho ved 426

X

10-534 ClOz at

18

hours, 442 X 10-5.11 ClOz a t 19

hours.

Controls containing 10 nig./ml. fructose shon ed 239 X lo-5.M ClOz a t

18

hours 250

X

10-6.M C1Oz at 19 hours.

0

Controls containing no fructose showed 226

X 10-634

ClOz a t

18 hours,

237

X IO-5.M

ClOz a t

19

hours.

h

Corrected

for

C102 evolred by fructose in sample.

solution in tube X ;

S ,

is the reading of tube X filled with water,

and

g.

is an effective path length correction factor given by

(5)

where K O nd

K ,

are readings for the reference tube and tube

X

when filled with 0.00200M potassium chromate solution. The

corrected colorimeter readings for the test and control solutions

Rt' and Rd, are converted into the corresponding chlorine dioxide

concentrations,

Bt

and

B,,

by use of the appropria te calihration

curve for the colorimeter.

Because the use of Equat ion 4

or

making colorimeter tube cor-

rections may be open to question, g

was

shoiw to be essentially

constant by de termining it (Equation 5 ) with various concentra-

tions of potassium chromate solution in several poorly matched

tubes. Potass ium chromate solution was used here for effrctive

path-length measurements because of the similarity

of

its ab-

sorption curve to that of chlorine dioxide in the pertinent spectra l

region D, igure

1).

I t has been extensively studied as a spec-

trophotometric standard solution (6).

A

tube filled with

0.00200.W potassium chromate solution Tvas found useful as a

rapid check on the constancy of the colorimeter scale calibration.

Calculation o Glucose Concentration. The difference in

chlorine dioxide concentrat ions of t he tes t and control solutions

must be corrected for two effects before it represents t he amount

of chlorine dioxide produced by the oxidation of glucose. One

of these is

a

slow reaction consuming chlorine dioxide 13),pre-

sumably hydrolytic disproportionation 4). This effect was

estimated by measuring the rate at which chlorine dioxide disap-

peared in 1. 5X aceta te buffer solutions of p H 4.00 at 25 C.

when initially present in amounts varying from 150

to

1200 x

l O - 5 M . A loss

of

7.8

=I=

.9% (mean deviation) was found in

16.5 hours for nine samples with no significant dependence on

initial concentration. Corresponding figures for 22

5

and 90

hours were 8.9 .7 and 14.9

=t

.5%, respectively. Because no

chlorine dioxide is initially present in reaction mixtures, a correc-

tion

of

about 4.5% is estimated

as

applicable to net chlorine

dioxide concentrations for the authors' standard reaction condi-

tions.

The other correction is required because the chlorite concen-

tration is not the same in test and control solutions

as

the reac-

tions proceed. Launer

12,

I S ) has derived a simple and appar-

ently adequate expression t o correct for this effect for te st solu-

tions containing glucose and control solutions conta ining no sugar.

Th e derivation of t he corresponding expression for the case

where fructose

is

present at the same concentration in both test

and control solutions is given below. This expression may be

written

as

a correction factor by which the net chlorine dioxide

concentration should be multiplied-namely

CO

C,

-

1.5 qBc

where C, is the initial total chlorite concentration, B ,

is

the chlo-

rine dioxide concentration in the control solution, and q is a factor

which reduces to unity for no fructose present and which is

somewhat less than unity for appreciable amounts of fructose.

Values calculated for expression

(6)

corresponding

t o

observed

values of

B ,

of Table I11 for an 18-hour reaction period for con-

trols containing 0, 10, and 100 mg. per

ml. of

fructose are 1.017,

1.017, and 1.057, respectively. Combining these figures with a

4 . 5%

correction for loss of chlorine dioxide formed gives corre-

sponding over-all correction factors , d, to t he observed net chlorine

dioxide concentration of 1.063, 1.063, and 1.105. These are in

good agreement with the experimentally determined values 1.064,

1.073, and 1.108 based

on

stoichiometry of Equation 2.

The concentration

of

glucose in the tes t solution is then 90.1

d

( B ,- Bc mg. per ml., where ( B t- B,) is the observed net chlo-

rine dioxide concentration and

d

is the appropria te theoretical cor-

rection factor just discussed. iin additional correction is made

for impurity of the fructose used in the control solution

if

the

latt er is not glucose-free.

Correction factor for difference in

rate of consumption

of

chlorite in test and control solutions con-

taining fructose. Assume

Derivation

of

Correction.

where B

=

chlorine dioxide concentration,

C

= total chlorite

concentration,

G

=

glucose concentration,

F

= fructose concen-

tration,

a , g,

f = rat e constants for the respective reactions: dis-

proportionation of chlorous acid, oxidation of glucose, oxidation

of fructose,

a , y ,

4 = number of moles of chlorine dioxide pro-

duced per mole of chlorite consumed in the respect ive reactions.

y

= 2/3. Further, assume at time

t

chosen large enough so that

oxidation of glucose is virtually complete

B, = aaCoCet + 1/2

4fF(CO

+ C,) t

Bt = a~CoCt t

+

I/2 +fF Co

+

Ct) t + 2Go

11)

111)

where subscripts

0,

, t

refer to initial value, control solution, and

test solution, respectively.

When

t

is eliminated in combining I1 and 111,we can write the

result as


5/7

1482

A N A L Y T I C A L C H E M I S T R Y

agreement with the earlier work (9, 13). The disaccharides

lactose and maltose do not appear to be significantly hydrolyzed

under the conditions of th e analysis. Thus the chlorine dioxide

evolved by these two sugars per milligram is half of that evolved

by the aldohexoses.

REFERENCE FRUCTOSE

Fructose esssentially free of glucose can be prepared by crys-

tallization as dihydrate. Material prepared in this manner can

be used as a reference standard in cont,rol solutions for determin-

ing the aldose (glucose-equivalent) content of a good grade of

commercial fructose. The latter can then be used in controls

for other analyses.

Preparation.

A

cold

(0 C.)

aqueous solution (about 65% by

weight,) of the best fructose commercially available is seeded with

fructose dihydrate crystals

16)

which are crushed in the solution

and well dispersed. After crystallization at

0

for 12 hours or

more, the crystals ar e freed of mother liquor

so

far as possible by

use of a Biichner funnel (without paper), a sintered glass filter,

or

a centrifuge. The crystals arc then washed by intimate mixing

with eit,her undersaturated solution of previously purified fructose

or

cold water , in the la tte r case using about one fift,h the volume

of the original solution. The n-ashed crystals are separated as

before. The product may be air dried at 0 C. or stored below

10'

C.

as

wet crystals (about 80% fructose),

or

as a sirup (diluted

to

about,

70 ).

Fructose content can be found from refractive

index measurement

(8)

or

by dichromate oxidation

(11).

Table 11. Response

of

Various Sugars and Other

Substances to Chlorous .4cid Oxidation Procedure

Substance

Glucose

Galactose

Mannose

Lactose monohydrate

Maltose monohydrate

Fructose

Sorbose

Sucrose

Raffinose penta hydrate

Ethyl alcohol

Isobutyraldehyde

Betaine hvdrochloride

Lysozymeb

Galacturonic acid monohydrate

Moles of

ClOn

Concentration, per

Mole

of

Substances

g. M1.

0.1

0 . 1

0 . 1

0 . 1

0 . 1

100

50

50

50

50

0.158

5

2

0.2

2 00

1

90

1 98

1 .9 2

1 . 9 8

0

0034

0 0035

0

0038

0 0047

0 00019

1 44

0 013

9 5

2 01

a

Corrected

for

hydro lytic loss of chlorine dioxide and difference in chlo-

rite concentration in test and control. Except for glucose and fructose these

figures are the mea n results

of

duplicate determinstiona.

b

Molecular weight

14,600.

For standard reaction conditions, identical values of

q

were found

fo r the two assumed stoichiometries-via.,

Y = 1/2,

= 1/2 and a = 1/2, J

=

2/3.

RESULTS WITH PREPARED SAMPLES

The analytical method applied to glucose-fructose samples of

known composition gave the results shown in Table I. In this

series of analyses, chlorine dioxide concentrations were measured

with the spectrophotometer at 18-and 20-hours reaction time and

with the colorimeter at

19

hours. The 20-hour data are not in-

cluded as they are almost identical with those a t 18 hours. The

samples were prepared from stock solutions of National Bureau of

Standards dextrose (Standard Sample No. 41) and of fructose

dihydra te containing 0.021 glucose-equivalent impurity oxi-

dizable under the present analytical conditions. Tes t solutions

in each set and the appropriate control were run in duplicate

simultaneously. The mean of the

controls was then used for

calculating the glucose content of each test solution. Because

duplicate analyses for each set were repeated on two additional

occasions, each value in Table I is the mean of s ix analytical re

sults. The average values found for glucose concentrations in

the test solutions are seen to be in error by less tha n 2% of t he

glucose present except for the test solutions which contained

1

mg. per ml. of glucose.

For

the latter solutions, average values

found are 2 to 3% low and t he standard deviations from the means

are larger, This effect is apparently caused by the excessive con-

sumption

of

chlorite due to the high glucose concentration and

can be avoided by the use of

a

smaller sample when the glucose

content is high.

INTERFERING SUBSTANCES

The behavior of

a

number of substances added as sample

impurities in test solutions in the analytical procedure is indicated

by t he exploratory results shown in Table 11. The almost com-

plete oxidation of t he aldoses and the similarity in'lthe behavior

of fructose, sorbose, sucrose, and raffinose indicate tha t themet hod

can easily be adapted to serve as a general method for the de-

termina tion of aldoses in the presence of ketoses, as well as su-

crose, raffinose, and other similar sugars. These results are in

0.00

0

50

100

150

TIME, MINUTES

Dioxide

Figure

4.

Initial Rates

of

Formation

of

Chlorine

I.

11.

Control solution,

0.0600.M

NaClOz, 1.5M acet ate buffer

Same as I, with

100

mg. per ml. aldose-free fructose

(pH 3.97), 25O C.

artrled

_ _ _ _

111.

IV .

Curve calculated for

0.02170

glucose impurity added

to

Same a8 I, with 100mg. per

ml.

fructose (preparation

the fructose of I1

D) added

Purity. To obtain a fructose assumed to be completely free

of

aldose, fructose dihydrate was crystallized from fructose solution

which had been subjected to oxidation by chlorous acid under

conditions somewhat more drastic than employed in the analytica l

procedure-via., 24-hour reaction time a t 25

C.,

pH

3.7 to

4.0,

Co =

0.1M sodium chlorite. After recrystallization, this mater ial

was used as an aldose-free standard n-ith which were compared

other dihydrate preparations. Column 4 of Table I11 shows

the aldose contents of various fructose preparations as determined

by the spectrophotometric procedure referred to the chlorite-

treated preparation as aldose-free fructose. The glucose-equiva-

lent contents of the commercial materials were reduced from

values as high as 0.7 to 0.03%

or

less by the dihydrate crystal-

lization procedurk.


6/7

V O L U M E

2 6 ,

NO. 9, S E P T E M B E R

1 9 5 4

Some of the prepa rations of Tab le I11 were also compared with

the chlorite-treated preparation with reppect to the initial rate

a t which chlorine dioxide is formed in a te st solution containing

fructose under reaction conditions of the analytical procedure.

Figure 4 hows typical results obtained using 1-cm. spectropho-

tometer cells as reaction vessels. Th e pronounced curvature of

curve IV for the tes t solution containing preparation D contrasts

with t he negligible initial curva ture of curve I1 for the test solu-

tion containing the chlorite-treated fructose preparation. Th at

this curvature cannot be accounted for as due to glucose

impurity is seen by comparison

of

curve IV with curve 111. The

la tter curve would have been obtained if all

of

the oxidizable

impurity, found for preparation D by the analytical procedure,

were glucose. Th e much greater initial curvatu re of curve IV

over that of I11 is interpreted as evidence for the presence of

nonglucose impurity which is oxidized much more rapidly than

glucose. As a rough measure of th e amount of such impuri ty the

author s have used the zero-time intercep t of the straigh t line de-

termined by the points of curve IV a t times greater than

60

min-

utes.

Thus, this intercept exceeds that for the control solution

(curve I ) by an amount equivalent to the chlorine dioxide ex-

pected for the oxidation

of

0.024% of glucose. Est imates by this

intercept method of th e content of impurities oxidized more

rapidly than glucose are included

as

the last column of Table

111.

From these figures it was concluded tha t most of t he oxi-

dizable impurity in preparation

D

was not glucose.

1483

Table 111.

Oxidizable Im puri t ie s

in

S i x Purified Fructose

Samples

Oxidizable Impuritiesa

Source

of

Original fructose, Purified Fruct ose

Original photometric

Photometric Rate curve

Preparationb Fructose method

method intercept

C

A

B

C

Df

E

F

X d

Xd

Y

1

z

Z

0 10

0 10

0

18

0

18

0 70

0 70

0 004

o

005

0.008

0

021

0 031

0 039

6

c

0 009

0

024

0 016

0 019

a

Calculated as per cent glucose in anhy drous fructose.

b

Preparations

4 ,

,

C,

and

F

were each washed twice with cold water,

The volumes of water

C Interpreted as nonglucose oxidizable impurity.

d Different lots from same comiriercial source.

and

E once.

used

in washing E and

F

were relatively small.

D wns washed with fructose solution.

DISCUSSION AND CONCLUSIONS

The analytical method presented has the advantages and dis-

advantages characteristic of most difference methods. Thus

about as much chlorine dioxide is formed in the control solution

containing no fructose as

is

produced by t he oxidation of 0.2

mg. per ml. of glucose. Similarly, the chlorine dioxide produced

in control solutions containing

100

mg. per ml. of fructose is ap-

proximately equivalent to that resulting from

0.4

of glucose

impuri ty in a fructose sample. This emphasizes the importance

of obtaining maximum precision in the photometric measure-

ments, especially if a colorimetric procedure is used where th e

difference in chlorine dioxide concentrations of t he te st a nd con-

trol solutions cannot be measured directly in a single measure-

ment.

Th e comparison of test and control solutions prepared from

the same reagents and subject to the same temperature history

practically eliminates,

as

sources of error, the slight differences

in absolute reaction rates observed for different buffer prepara-

tions, th e slight differences in the small amount of chlor ine

dioxide produced immediately upon mixing the chlorite and buf-

fer solutions, and minor fluctuations of temperat ure of t he solu-

tions during th e reaction period.

Minimum exposure of t he reaction mixtures t o light is recom-

mended, because chlorine dioxide solutions are known to be photo-

sensitive, but t he brief exposure incident to transfer of tubes

from the bat h a nd photometric measurement was found to pro-

duce no significant change. Erro rs due to light exposure or other

procedural details can be detected by check analyses using

Sa-

tional Bureau of Standards dextrose as a test sam ple.

The sensitivity of the method for measuring glucose

as

an im-

pur ity in fructose is ultimately limited by the reproducibility of

replicate control and replicate test solutions run simultaneously.

It is seen from Table I that a sta ndard deviation of about 0.003

mg. per ml. of glucose is to be expected if the chlorine dioxide is

measured spec trophotometrically, indicating tha t glucose im-

purity as low as 0.01% could probably be detected and estimated

with considerable uncerta inty provided a suitable fructose stand-

ard were available. Suitahle aldose-free standa rd fructose can

be prepared by recrystallization of fructose dihydrate without

resort to chlorite treatment a s shown by the fact that preparations

A

and

B

of Table I11 did not differ significantly from chlorite-

treated fructose in their yields of chlorine dioxide produced in

the analy tical procedure. Fructose, purified by recrystalliza-

tion as the dihydrate to the point where successive crystalliza-

tions produce no change in response t o th e analytica l procedure,

is accordingly thought to be equivalent to fructose freed from

aldose by treatm ent with chlorous acid. Th e results of Table

I11 further show that reference fructose containing less than

0.03% of glucose-equivalent can be prepared by a single recrys-

tallization of commercially available mater ial as dihydrate pro-

vided the product is adequately washed.

Although thi s work has not been extensive enough t o evaluate

the method fully, the results of Table I indicate that i t can be

used to determine glucose in mixtures of glucose and fructose

over the entir e range of composition. For the spectrophoto-

metric procedure, approximate standard deviations in the per-

centage glucose are 0.003, for samples containing less tha n

0.5%

glucose, 0.03 for samples containing from

0.5

to

5

glucose, and

about 1 of the glucose content for samples containing more than

5

glucose. For the colorimeter procedure the corresponding

standa rd deviations are larger by a factor 1.5 t o 2. The accuracy

of the results for glucose contents below a few per cent is de-

termined b y the accuracy with which the glucose content of the

reference fructose employed in the method is known.

ACKNOWLEDGMENT

The authors wish to thank H. F. Launer for suggesting the

uee

of

sodium chlorite in acid solution as a suitable oxidizing

agent and for making his manuscript ( I S ) available in advance

of publication. Th e authors also benefited from discussions

with H.

F.

Launer a nd Yoshia Tomimatsu, who have recently ex-

tended the sensitivity of determining glucose alone by oxidation

with sodium chlorite in acid solution ( I d ) .

LITERATURE CITED

(1) Baldwin, R. W., Campbell, H. A. , Thiessen, R., Jr., and Lorant,

(2) Barnett, Benjamin, Ph.D. dissertation, University

of

California.

G.

J.,

Food Technol., 7,275 (1953).

Berkeley, 1935.

208 (1942).

(3)

Bates,

F . J.,

and associates, Natl. Bur. Standards, Circ . C440,

4) Bray, William,

2.

hysik. Chem.,

54,

569 (1906).

(6) Browne, C. A. , and Zerban, F.

W.,

Physical and Chemical

Methods of Sugar Analysis, 3rd ed.,

p.

895, New York, John

Waey Sons, 1941.

(6) Haupt, G. W., J . Research Natl. B u r . S t a n d a r d s , 48, 414 (1952)

(RP 331).

7)

Hodge, J. E., and Davis, H.

A.,

U.

S.

Dept.

Agr.,

Bur. Agr. Ind.

Chem.,

AIC

333, 42 46 (1952.)

( 8 )

Jackson, R. F., and Mathews, J. A. , J .

Research

Nat l .

B u r .

S t a n d a r d s ,

8 ,

412

(1932) (RP

426).


7/7

1484 A N A L Y T I C A L

C H E M I S T R Y

Jeanes, Allene,

urds,

27,

125

Keilin,

D.,

and

Launer, H. F.,

and Isbell, H. S., J .

Research Natl.

Bur.

Stand-

(1941) (RP

1408).

Hartree, E. F.,

Biochem.

J . , 42,

21 (1948).

and Tomimatsu,

Y.,

NAL.CHEM. , 5, 1767

(12) Launer, H. F., and Tomimatsu, Y., J .

Am . Chem.

SO C. , 6, 2591

(1953).

(1954).

(13) Launer,. F., Wilson,

W.

K., and Flynn, J. H., J .

Research

(14) Sowden,

J. C.,

and Schaffer,

R.,

J . Am. Chem.

Soc., 74,

499

Natl.

Bur.

Standards,

51,

237 (1953)

(RP 2456).

(1952).

Chem.,

34, 82 (1942).

(15)

White, J.

F.,

Taylor,

M .

C.,

and Vincent,

G .

P.,

Ind. Eng.

(16) Young, F. E., and Jones, F. T., U. S. Patent 2,588,449 (March

(17) Young, F. E., Jones, F. T., and

Lewis,

H. J., J . Phys. Chem.,56 ,

1 1 , 1952).

738 (1952).

RECEIVEDor review December 21, 1953.

Accepted June

7 , 1954.

Presented

before the joint sessions

of

the Divisions

of

Analytical and Carbohydrate

Chemistry. Symposium on Analytical Methods and Instrumentation Applied

to Sugars and Other Carbohydrates a t the 124th RIeeting of the AMERICAN

CHEMICALOCIETY,hicago, Ill. Mention of products by specific manu-

facturers does not imply that they are endorsed

or

recommended by the

Department

of

Agriculture over others of a similar nature not menti oned .

Conductometric Standardization of Solutions of Common

Divalent

Metallic

Ions

Using Disodium Salt

of

Ethylenediami netetraacetic Acid

JAMES

L.

HALL JOHN A.

GIBSON

JR. PAUL

W e s t V i r g i n i a U n i v er s i t y, M o r g a n t o w n ,

W Va.

An effort has been made to evaluate the use of conducto-

metric methods for end-point determinations in the

titration of solutions of the disodium salt of ethylene-

diaminetetraacetic acid and divalent metallic ions.

Conductance methods may be used for accurate stand-

ardization of solution s of copper(II), zinc, lead, nicltel-

(11), cobalt, calcium, barium, strontium, magnesium,

manganese, cadmium, iron(IL), and mercury(I1) in the

concentration range from 0,001 to 0.5M, before dilution

in the titration vessel.

EC ENTLY the disodium salt of ethylenediaminetetra-

R acetic acid (Versenate, Sequestrene, Complexone 111)

has been proposed as a standard for establishing the concentra-

tions of solutions of certa in divalent cations

2 ) .

The stability

constants of t he complexes are great enough t o make precise

end-point determinations possible 1 7 ,

18, 20).

The stoichio-

metric relations

for

the reactions between the metallic ions and

the reagent have already been determined by several methods

with reported accuracies within 0.05 to 2.0 . Metal ion con-

centrations have been determined potentiometrically

1, 10,

11,

19),by use of indicators ( I 4,5 ,9 ,16 ,19),spectrophotometrically

12, I S ,

22 ,

2 3 ) ,polarographically (6,

14 ,1 .5 ,21 ) ,

and b y a special-

ized high-frequency technique 3 ) .

The present work shows that conventional conductometric

methods may be used for the standardiza tion of solutions of sev-

eral common cations. The accuracy compares favorably with

the best previously described methods.

REAGEVTS

Disodium Versenate.

Standard solutions of the reagent were

prepared from the analytical reagent (disodium Versenate di-

hydrate, manufactured by the Bersworth Chemical Co.) and

fro m Versenate purified by the method of Blaedel and Knight (2).

All solutions mere standard ized with e lectroly tic copper, dissolved

in a minimum amount of 6 S nitric acid. The end points were

determined conductometrically as described below. Titr ation in

either acidic or basic solution yielded the same molarity. Solu-

tions O.lOOlM, 0.04724M, O.O1001M, and 0.001004.11were pre-

pnred.

In weighing the copper and disodium Versenate for these solu-

tions, th e weight of the Iersenate was corrected for the difference

in density between the Versenate and the brass weights. Rascd

on a density of 1.8 or the Versenate, this correc tion was 0.06

relative to the metallic copper.

Cation Solutions. Solutions of cupric nitra te, cupric per-

R. WILKINSON and HAROLD 0 HILLIPS

chlorate, nickel nitrate, cobalt nitrate, lead nitrate, zinc sulfate,

manganese sulfate, cadmium chloride, ferrous sulfate, magnesium

sulfate, strontium nitrate, calcium chloride, barium nitrate,

mercuric acetate, lanthanum nitrate, and cerium nitrate were

prepared a t various concentrations from Bakers analyzed

or

Mallinckrodt reagent grade chemicals. I n addition, copper, zinc,

and nickel nitr ate s were prepared by dissolving metal of known

purity in 6n nitric acid. Konconductometric standardizations

were made for most of the solutions; the purity of the calcium

carbonate from which the calcium chloride solution w s made, the

strontium nitrate, and the barium nitrate was established by

gravimetric analyses

2 4 ) .

The normality

of

the manganese(

11),

cadmium, lead, zinc, magnesium, and mercury(I1) salt solutions

was determined with Versenate using the indicator method of

Schwarzenbach

1

. The solutions Ivere made in concentrations

from 0.001 to 0.2;2f.

Where ammonia was required, the

C.P.

product

proved to be satisfactory for solutions of metal ion concentrations

of 0.1.V or greater. At lower concentrations, errors introduced

by impurities became appreciable and distilled ammonia was

necessary. Th e ammonia was distilled into conductivity water

to

a

concent ration of 3M and was stored in polyethylene bottles.

Water. Whenever the available distilled water was used, a cor-

rection equivalent to 0.4 ml. of 0.01M Versenate per 1000 ml. of

aater

was

required . Twice distilled water was preferable for all

solutions

0.lM

or less.

Acid Buffer. Twenty-five grams of Bakers analyzed sodium

hydroxide and 65 ml. of glacial acetic acid were dissolved in water,

mixed, and diluted to 250 ml. Th e pH of this buffer was

5.1.

KO ifference in end-point ratio was found for 0.01M copper(I1)

solution titr ated with and without this buffer. Th e use of U.S.P.

sodium acetate for the buffer yielded a result

295

in error.

Ammonia.

APPARATUS

Th e most precise measurements were made a t 2000 cycles using

a Leeds Xorthrup Type 1553 ratio box and Type

4754

decade

resistance with recommended oscillator and amplifier. A

50-

ppf. variable capacitor, and decade capacitors to provide a total

caparitance up to 1uf.,were connected in parallel with the known

resistance. The null point was determined by observing the

output wave on an oscillograph. Used in this way, the apparatus

has a range of 0.01 to 10,000 ohms with a maximum error of

0.03yo

at 10 ohms or greater.

A

dip-type conduc tivitv cell with

platinized platinum electrodes and a cell constant of

0.0964 wa8

used. Titr ations werr made at room temperature.

Additional conventional conductance measurements were made

using a Model

RCZI15

Serfase direct-reading conductance bridge.

This instrument gave satisfactory results for work at concentra-

tions of 0.01.11 or less.

Many of the determinations were also performed using two

high-frequency instruments 7,

8).

Thefie instruments were satis-

factory for routine work in the more dilute solutions bu t did no t

contribute any new or more useful results. Kumerical dat a are

not included for these high-frequency determinations.

Documents

glucosa fructosa