Miller 1959. Use of DinitrosaIicyIic Acid Reagent for Determination of Reducing Sugar.

Table I.
Reduction of Thallium(lll) to Thallium(1)
K O . of
Detns.
Reductor
Cadmium
5
5
7
a
Bismuth
4
4
4
4
4
3
2
5
Silver
mium rod (Fisher Scientific Co.) were
washed with lil' hydrochloric acid and
then with 1N sulfuric acid.
Bismuth. The chunk metal (Fisher scientific Co.) was crushed and the coarse
pieces were sorted out.
Silver. Granular silver (G. Frederick
Smith Chemical Co.).
The columns were washed nith 1K
sulfuric acid and kept filled n i t h 0.01N
sulfuric acid when not in use.
PROCEDURE
Measured aliquots of the standard
thallium solution were treated to give
sulfuric acid concentrations from 0.1 to
l.O*V and total volumes of 50 to 100 ml.
Each solution was uassed through the
\Tit11 jo-ml. portions of 0.1N sulfuric
acid. To the solution and washings
T1, Mg.
Found, av.
31.9
5'2.9
106,3
212.7
32.0
53.1
106.4
213.1
32.0
53.0
106.5
106.5
212.8
213.1
Taken
32.0
53.1
106.4
212.8
32.0
53.1
106.5
212.8
32.0
53.1
Std.
Dev.
0.22
0.33
0.20
0.40
0.10
0.17
0.25
0.39
0.11
0.10
0.14
0.44
was added 40 nil. of 6-Y hydrochloric
acid. The thallium(1) lyas titrated
potentiometrically with 0.1000A7 potassium bromate using the Fisher Titrimeter with a platinum-calomel electrode
pair*
Blanks were run on the reductors
with the above procedure, but substituting 50 nil, of l,oLvsulfuric acid for the
thallium solution. The correction, in
terms of milliliters of 0.1s potassium
bromate, was 0.02 ml. for the bismuth,
0.03 ml. for the silver, and 0.05 ml. for
the cadmium reductor.
After the blank correction was deducted, the results shown in Table I
were obtained.
small but may vary from lot to lot,
depending upon the purity.
The results obtained with silver compared favorably n ith those obtained
a i t h bismuth or cadmium. Upon addition of hydrochloric acid, a precipitate of silver chloride is obtained. Although the results do not indicate any
loss of thallium chloride by coprecipitation with the silver chloride, the use
of the bismuth or cadmium reductors
is considered more convenient. K h e n
bismuth or cadmium is used, thallium(1)
chloride, formed upon addition of hydrochloric acid, disappears as the titration proceeds. I n the ease of silver,
the silver chloride remains, and there is
no visible rridence that the thallium
chloride has been completely oxidized.
This is not a criticism of the reduction
by silver, but rather a limitation of the
bromate method, which requires the
addition of hydrochloric acid.
Other reductors studied were lead,
aluminum, zinc, amalgams of zinc,
cadmium, lead, and bismuth. S o n e of
these proved satisfactory. Sickel reduced the thallium quantitatively but
the blanks nere high and erratic. A
better grade of nickel metal nould
probably serve satisfactorily.
LITERATURE CITED
DISCUSSION
(1) Anderson, J. R. A,, rlx.4~.CHEBI.2 5 ,
108 (1953).
(2) Zintl, E., Rienacker, G., 2. anorg
Chem. 153, 276 (1926).
bismuth, cadmium, or silver. The
blank corrections for these metals are
RECEIVEDfor revieTY January 17, 1958.
Accepted October 27, 1958.
Use of DinitrosaIicyIic Acid Reagent
for Determination of Reducing Sugar
GAIL LORENZ MILLER
Pioneering Research Division, Quarfermasfer Research and Engineering Center, Natick, Mass.
b Rochelle salt, normally present in
the dinitrosalicylic acid reagent for
reducing sugar, interferes with the
protective action of the sulfite, but i s
essential to color stability. The difficulty may be resolved either b y
eliminating Rochelle salt from the reagent and adding it to the mixture of
reducing sugar and reagent after the
color i s developed, or b y adding
known amounts of glucose to the
samples of reducing sugar to compensate for the losses sustained in the
presence of the Rochelle salt. The
optimal composition of a modified
dinitrosalicylic acid reagent is given.
426
ANALYTICAL CHEMISTRY
*T
HE D I N T R O S A L I C Y L I C ACID REAGENT,
developed b y Sumner and coworker (11-14 for the determination
of reducing sugar, is composed of dinitrosalicylic acid, Rochelle salt, phenol,
sodium bisulfite, and sodium hydroxide.
According to the authors of the test,
Rochelle salt is introduced to prevent
the reagent from dissolving oxygen (12);
phenol, to increase the amount of color
produced and to balance the effect of
phenol encountered in urine ( I S ) ; and
bisulfite, to stabilize the color obtained
in the prescnce of the phenol ( I S ) .
The alkali is required for the reducing
action of glucosc on dinitrosalicylic acid.
The major defect in the test is in the
loss of part of the reducing sugar being
analyzed. This was pointed out by
Sumner (12, 1 4 , was referred to by
Brodersen and Ricketts (Z), and has
been observed repeatedly in this laboratory (6, 8, 9). Evidence of loss of
sugar is also given by the data of Hostettler, Borel, and Deuel (4) and of
Bell, hlanners, and Palmer ( 1 ) . As
this defect appears never to have been
fully remedied, the present study was
carried out to investigate the different
factors which might cause it. I n the
course of the investigation, the effects
of varying the concentrations of the
different components of the reagent
also were determined. The findings
which resulted have led to the development of a modified reagent and procedure.
IROCH.
TR€AT€D
SALTS^ I
63
METHOD
The color tests were made with 3-nil.
aliquots of reagent added to 3-ml.
aliquots of glucose solution in 14-mm.
tubes. The mixtures were heated for
5 minutes in a boiling water bath and
then cooled under running t a p n-ater
adjusted to ambient temperature. Cooling to ambient temperature was made
necessary by the effect of temperature
on the absorbance of the colored reaction product (w), a n effect confirmed
by the present studies. The color intensities nere measured in a Beckman
llodel DL spectrophotometer a t 575
nip n ith a slit n idth of 0.06 mm.
The reagent of Suniner and Sisler (14)
and a modified reagent w r e used in
the tests. The former contained approximately 0.63Yc dinitrodicylic acid,
18.2% Rochelle salts, 0.57, phenol,
0.5% sodium biqulfitc, and 2.14%
sodium hydrouide; the modified reagent coiitained 1% dinitrosalicylic acid,
0.2% phenol, 0.057c sodium sulfite,
and 1% sodium hydroxide.
For certain tests the modified reagent
included varying concentrations of Rochelle salt. The composition chosen
for the modified reagent n-as based on
the resultq of preliminary tests n hich
indicated that such a reagent was
optimal and ~ o u l ds e n e best as the
basis of rrference for testing effects of
variation 111 composition. I n the abqcnce of Rochelle salt, the color obtained n ith the modified reagent n as
unstable. T o stabilize the color under
these conditions, 1 ml. of a 40% solution
of the salt was added to the mixture of
reactants subsequrnt to the developnicnt of the color and prior to cooling.
The modified reagent n as prepared
by placing all solid components in a
container and dissolving them simultaneously by stirring with the required
volume of sodium hydroxide solution.
This was much simder than other mocedures (3, 14).
The modified reagent produced the
same color with glucose from day to
day, thus proving more stable in this
respect then the reagent of Brodersen
and Ricketts ( 2 ) . Modified reagent to
which Rochelle salt vias added also did
not change from day to day in this
respect. Depending upon storage conditions, however, the modified reagent
tended eventually to deteriorate from
atmospheric oxidation of the sulfite
present. Deteriorated reagent was rejuveiiated by the addition of fresh
sulfite. The danger attendant upon
oxidation of sulfite could be avoided by
preparing the reagent in large batches
without sulfite, the sulfite being added
to aliquots just prior to the time when
the reagent \vas to be used.
STUDY OF VARIABLES
Removal of Dissolved Oxygen.
OF KITROGEN.Sumner
WITHSTREAM
0
0.1
0.2
0
fbl
0.3
06
0
fc)
03
06
fdJ
03
CITRATE
0
0.3
(e/
0.6
0
03
06
ffJ
MILLIGRAMS
0
03
fgl
06
0
0.3
fhl
06
GLUCOSE
Figure 1. Effect of variables on color produced with glucose and dinitrosalicylic
acid reagent
a.
b.
C.
d.
e.
f.
9.
h.
Stream of nitrogen passed through mixture; Sumner’s reagent
Sodium sulfite treatment piior to addition of Suniner’s reagent
Rochelle salt concentration; modified reagent
Sulfite concentration; modified reagent
Podium hydroxide concentration; modified reagent
Phenol concentration; modified reagent
Dinitrosalicylic acid concentration; modified reagent
Carbox) methl ]cellulose, citrate, and miitures of both present; modified reagent
and Sisler (14) indicated t h a t t h e
loss of glucose with t h e dinitrosalicylic
acid reagent was due t o destruction
b y oxidation, and based their statement on unreported results of experiments in which a stream of nitrogen was passed through t h e reactants.
An attempt to confirm this observation
in the present nork indicated that
passing a stream of nitrogen through a
mixture of Sumner‘s reagent and glucose for 2 minutes prior to the development of color largely eliminated the
destruction of glucose (Figure 1, a ) .
WITH STLFITE. As sulfite had previously been used successfully for removing dissolved oxygen from aqueous
solutions (b), it was surprising that the
sulfite present in Sumner and Sisler’s
reagent failed to accomplish this purpose. T o test whether this failure of the
sulfite may have been due to interfereiice
by other components of the reagent,
sulfite a t a level of 0.1% n-as added to
glucose samples prior to mixing them
with the reagent. This procedure reduced the destruction of glucose by
about 707,. The rewlts, shown in
Figure 1, b, thus provided strong
evidence for the suspected interference
by the other components of the reagent
under the usual conditions of the test.
INTERFEREWE
OF ROCHELLESALT.
Comparative tests were next carried
out with the modified dinitrosalicylic
acid reagent to M hich varying amounts
of Rochelle salt were added. The
results, s h o r n in Figure 1, c, clearly
implicated Rochelle salt as the major
factor involved in the interference with
the removal of oxygen by sulfite,
because, in the absence of the salt, the
sulfite appeared able to remove the
dissolved oxygen and thereby to protect the glucose. iiside from contributing to the loss of a portion of the
glucose, the Rochelle salt caused an
enhancement of the color due to the
remaining glucose.
Sulfite Concentration. T h e effect
of different concentrations of sulfite
in the modified reagent (Figure I , d )
indicated t h a t a maximum color intensity n a s obtained at 0.057,sulfite.
I n experiments not shown, essentially
t h e same results were obtained a t
0.025 and 0.1% sulfite as a t t h e
0.057, level. Low concentrations
caused a lack of linearity, nhile
both high and low Concentrations
caused a depression in color intensity
and a loss of glucose.
Sodium Hydroxide Concentration.
T h e effect of different concentrations
of sodium hydroxide is shown in
Figure 1, e . High concentrations of
sodium hydroxide led t o enhanced
color development, b u t a t t h e same
time contributed t o a loss of glucose.
T h e level of 1% sodium hydroxide
appeared t o be t h e most suitable, as
it produced t h e maximum color inVOL. 31, NO. 3, MARCH 1959
* 427
tensity possible without concomitant
loss of glucose.
Phenol Concentration. Maximum
color development was approached
a t a concentration of about 0.2%
phenol (Figure 1, f ) . I n experiments not shown in t h e figure, the
same results were obtained with 0.5q.’,
phenol as at t h e 0.2% level. Low
concentrations resulted in a lack of
linearity. T h e intensity obtained in
t h e presence of 0.2% phenol was
about 5 times that obtained in the abscence of phenol. Over the range
tested, the phenol had no effect on the
loss of glucose.
Dinitrosalicylic Acid Concentration.
When t h e amounts of dinitrosalicylic
acid Fyere varied, t h e color intensity
approached a maximum at a concentration of 1% (Figure 1, 9). T h e
dinitrosalicylic acid, like t h e phenol,
had no effect on t h e loss of glucose
over t h e range tested.
Other Substances. T h e results of
the preceding tests suggested the possibility t h a t other substances might
affect t h e dinitrosalicylic acid test.
For example, it was of interest t o
asceitain whether, in using t h e test
for the measurement of cellulase
activity (8, IO), the presence of carboxymethylcellulose and citrate buffer
a t p H 5 might cause interference.
K i t h amounts of carboxymethylcellulose and citrate buffer corresponding to
those used in the cellulase measurement, the effects shown in Figure 1, h,
were produced. The carboxymethylcellulose caused an enhancement in
color, whereas the citrate caused a
depression. A mixture of the substances approximately neutralized the
t n o opposite effects.
To determine whether the effect of
the citrate may have been a consequence of its buffering action, tests
ere made with acetate buffer of p H 5
a t an equivalent concentration. The
acetate did not, however, affect the
test.
FINAL M E T H O D
Khen pure reducing sugar solutions
are involved or when any contaminants
which may be present are knorvn not
to affect the color development or to
cause any loss of reducing sugar, the
modified reagent in the absence of
Rochelle salt is used. For stabilization
of the color produced under such conditions, Rochelle salt is added to the
mixture immediately after the development of the color and before the mixture
is cooled. The time of heating is increased to 15 minutes because the 5-
428
ANALYTICAL CHEMISTRY
minute period, adequate for the original
procedure, does not produce complete
color development in the modified
procedure. By this method linearity of
data, protection of glucose, and stability of color are realized.
If interfering substances occur in
unknown samples, special controls are
run. Such controls consist of standard
reducing sugar solutions to n hich appropriate amounts of the interfering
substances are nddrd. K h e n the interfering substanctis bring about a loss
of reducing sugar, and particularly
when the amounts of reducing sugar
to be measured in unknown samples
are equal to or smaller than the amount
lost, known amounts of glucose are
added to both the unknown samples
and the standards.
The procedure of adding glucose can
also be applied advantageously to compensate for the loss of reducing sugar
incurred nhen Rochelle salt is incorporated in the dinitrosalicylic acid reagent. For example, it is convenient
in the cellulase test (IO) to introduce
glucose into the carboxymethyl-cellulose-citrate substrate and to use modified
dinitrosalicylic acid reagent containing
20% Rochelle salt (8). Under these
conditions the separate addition of
Rochelle salt to the reaction mixture
after color dcrelopment is omitted.
The controls for such tests consist of a
blank and standard glucose solutions,
each containing carboxymethylcellulose,
citrate, and compensatory glucose.
DISCUSSION
The chemistrv of the dinitrosalicylic
acid test for reducing sugar has been
clarified previously, a t least in part.
The 3,5-dinitrosalicylic acid is reduced
to 3-amino-5-nitrosalicylic acid while,
in the simplest instances, the aldehyde
groups appear to be oxidized to carboxyl groups (4). The facts, however,
that the equivalence between aminonitrosalicylic acid produced and sugar
is not exact (4) and that different sugars
yield different amounts of color ( 1 , 4 , 7),
suggest that the chemistry of the test
may actually hc appreciably more complicated.
Such complications could conceivably be associated with the various
decomposition reactions of sugars in
alkaline solution ( 3 ) . If this explanation is correct, the reaction of the sugar
aldehyde grouping with dinitrosalicylic
acid could be viewed as competing with
side reactions involving decomposition
of the sugar. The effects of different
concentrations of the various constitu-
ents of the dinitrosalicylic acid reagent,
and also of extraneous substances such
as carboxymethylcellulose or citrate
buffer, upon the amount of color produced and upon the destruction of
glucose, as shown in the present study,
could similarly be interpreted as effects
upon the nature and degree of side
reactions.
The dinitrosalicylic acid reagent, in
a form consisting only of dinitrosalicylic
acid dissolved in strong alkali, has been
used with apparent success for molecular
n-eight measurement of starch breakdown products (?‘). This method depends upon the assumption that all
higher oligosaccharides of the homologous series starting with maltose
would produce equivalent amounts of
color with the reagent. Actual studies
of the reactions of members of homologous series with the dinitrosalicylic
acid reagent, starting with the disaccharide, have not been reported, but
u-ould be of considerable interest in
the present connection.
The principal virtue of the dinitrosalicylic acid test for reducing sugar
lies in its great convenience compared
to most other sugar tests, particularly
u-hen large numbers of tests must be
carried out. However, the factors discussed above must be given due consideration to avoid misinterpretation
of results.
LITERATURE CITED
(1) Bell, D. J., Manners, D. J., Palmer, A,,
J . Chem. Soc. 1952, 3760.
(2) Brodersen, R., Ricketts, H. T., J .
Lab. Clzn. Med. 34, 1447 (1949).
(3) Gilman, H., “Organic Chemistry,
Advanced Trratise,” 2nd ed., Vol. 2 ,
p. 1640, Wiley, Xew York, 1943.
(4) Hostettler, F., Borel, E., Deuel, H.,
Helv. Chznz. d c t a 34, 2132 (1951).
(5) Kolthoff, I. PII., Lingane, J. J.,
“Polarography,” Interscience, Kew
York, 1946.
(6) hlandels, G. R , Quartermaster Re
search and Engineering Center, Natick,
Mass., private communication.
( 7 ) bleyer, K. H., van der Wyk, A. J. A.,
Peng, C., Helv. Chznz. Acta 37, 1619
(1954).
(8) XIiller, G. L., Blum, R., Glennon,
W. E., Quartermaster Research and
Engineering Center, Xatick, Mass.,
unpublished data.
(9) Reese, E. T., Quartermaster Research
and Engineering Center, Satick, illass.,
private communication.
(10) Reese, E. T., Siu, R. G. H., Levinson,
H. S., J . Bacterid. 59, 485 (1950).
(11) Sumner, J. B., J . B i d . Chem. 47,
5 (1931).
(12) Zbid.,62, 287 (1924-25).
(13) Ibzd., 65, 393 (1925).
(14) Sumner. J. B.. Sisler.’ E. B.. Arch.
‘ Bzochem. 4: 333 (1944).
RECEIVED
for review Sovember 4, 1957.
Accepted September 23, 1958.