glucosa fructosa
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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
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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.
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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
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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
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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.
-
7/23/2019 glucosa fructosa
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).
-
7/23/2019 glucosa fructosa
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.
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