small but may vary from lot to lot,
Table I. Reduction of Thallium(lll) to Thallium(1) depending upon the purity.
T1, Mg.
K O . of Std.
Reductor Detns. Taken Found, av. Dev.
Cadmium 5 32.0 31.9 0.22
5 53.1 5'2.9 0.33
7 106.4 106,3 0.20
a 212.8 212.7 0.40
Bismuth 4 32.0 32.0 0.10
4 53.1 53.1 0.17
4 106.5 106.4 0.25
4 212.8 213.1 0.39
Silver 4 32.0 32.0 0.11
3 53.1 53.0 0.10
2 106.5 106.5 0.14
5 212.8 213.1 0.44
mium rod (Fisher Scientific Co.) were was added 40 nil. of 6-Y hydrochloric
washed with lil' hydrochloric acid and acid. The thallium(1) lyas titrated
then with 1N sulfuric acid. potentiometrically with 0.1000A7 potas-
Bismuth. The chunk metal (Fisher sci- sium bromate using the Fisher Titrim-
entific Co.) was crushed and the coarse eter with a platinum-calomel electrode
pieces were sorted out.
Silver. Granular silver (G. Frederick pair*
Smith Chemical Co.). Blanks were run on the reductors
with the above procedure, but substitut-
The columns were washed nith 1K ing 50 nil, of l,oLvsulfuric acid for the
sulfuric acid and kept filled n i t h 0.01N thallium solution. The correction, in
sulfuric acid when not in use. terms of milliliters of 0.1s potassium
bromate, was 0.02 ml. for the bismuth,
PROCEDURE 0.03 ml. for the silver, and 0.05 ml. for
the cadmium reductor.
Measured aliquots of the standard After the blank correction was de-
thallium solution were treated to give ducted, the results shown in Table I
sulfuric acid concentrations from 0.1 to were obtained.
l.O*V and total volumes of 50 to 100 ml.
Each solution was uassed through the DISCUSSION
\Tit11 jo-ml. portions of 0.1N sulfuric bismuth, cadmium, or silver. The
acid. To the solution and washings blank corrections for these metals are
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
*T HE D I N T R O S A L I C Y L I C ACID REAGENT,
developed b y Sumner and co-
worker (11-14 for the determination
protective action of the sulfite, but i s of reducing sugar, is composed of di-
essential to color stability. The diffi- nitrosalicylic acid, Rochelle salt, phenol,
culty may be resolved either b y sodium bisulfite, and sodium hydroxide.
eliminating Rochelle salt from the rea- According to the authors of the test,
gent and adding it to the mixture of Rochelle salt is introduced to prevent
reducing sugar and reagent after the the reagent from dissolving oxygen (12);
color i s developed, or b y adding phenol, to increase the amount of color
known amounts of glucose to the produced and to balance the effect of
samples of reducing sugar to com- phenol encountered in urine ( I S ) ; and
pensate for the losses sustained in the bisulfite, to stabilize the color obtained
presence of the Rochelle salt. The in the prescnce of the phenol ( I S ) .
optimal composition of a modified The alkali is required for the reducing
dinitrosalicylic acid reagent is given. action of glucosc on dinitrosalicylic acid.
426 ANALYTICAL CHEMISTRY
The results obtained with silver com-
pared favorably n ith those obtained
a i t h bismuth or cadmium. Upon ad-
dition of hydrochloric acid, a precipi-
tate of silver chloride is obtained. Al-
though the results do not indicate any
loss of thallium chloride by coprecipi-
tation 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 hy-
drochloric acid, disappears as the titra-
tion 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 re-
duced the thallium quantitatively but
the blanks nere high and erratic. A
better grade of nickel metal nould
probably serve satisfactorily.
LITERATURE CITED
(1) Anderson, J. R. A,, rlx.4~.CHEBI.2 5 ,
108 (1953).
(2) Zintl, E., Rienacker, G., 2. anorg
Chem. 153, 276 (1926).
RECEIVEDfor revieTY January 17, 1958.
Accepted October 27, 1958.
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 lab-
oratory (6, 8, 9). Evidence of loss of
sugar is also given by the data of Hos-
tettler, 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 develop-
ment of a modified reagent and pro-
cedure.
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. Cool-
ing to ambient temperature was made
necessary by the effect of temperature
on the absorbance of the colored reac-
tion product (w), a n effect confirmed
by the present studies. The color in-
tensities 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 ap-
proximately 0.63Yc dinitrodicylic acid,
18.2% Rochelle salts, 0.57, phenol,
0.5% sodium biqulfitc, and 2.14%
sodium hydrouide; the modified rea-
gent 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 Ro-
chelle 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 ab-
qcnce of Rochelle salt, the color ob-
tained 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 develop-
nicnt of the color and prior to cooling.
The modified reagent n as prepared
by placing all solid components in a
container and dissolving them simul-
taneously by stirring with the required
volume of sodium hydroxide solution.
This was much simder than other mo-
cedures (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 con-
ditions, however, the modified reagent
tended eventually to deteriorate from
atmospheric oxidation of the sulfite
present. Deteriorated reagent was re-
juveiiated 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.
WITHSTREAMOF KITROGEN.Sumner
TR€AT€D IROCH. SALTS^ I
63
0 0.1 0.2 0 0.3 06 0 03 06
fbl fc) fdJ
03
CITRATE
0 0.3 0.6 0 03 06 0 03 06 0 0.3 06
(e/ ffJ fgl fhl
MILLIGRAMS GLUCOSE
Figure 1. Effect of variables on color produced with glucose and dinitrosalicylic
acid reagent
a. Stream of nitrogen passed through mixture; Sumner’s reagent
b. Sodium sulfite treatment piior to addition of Suniner’s reagent
C. Rochelle salt concentration; modified reagent
d. Sulfite concentration; modified reagent
e. Podium hydroxide concentration; modified reagent
f. Phenol concentration; modified reagent
9. Dinitrosalicylic acid concentration; modified reagent
h. Carbox) methl ]cellulose, citrate, and miitures of both present; modified reagent
and Sisler (14) indicated t h a t t h e of Rochelle salt were added. The
loss of glucose with t h e dinitrosalicylic results, s h o r n in Figure 1, c, clearly
acid reagent was due t o destruction implicated Rochelle salt as the major
b y oxidation, and based their state- factor involved in the interference with
ment on unreported results of ex- the removal of oxygen by sulfite,
periments in which a stream of nitro- because, in the absence of the salt, the
gen was passed through t h e reactants. sulfite appeared able to remove the
An attempt to confirm this observation dissolved oxygen and thereby to pro-
in the present nork indicated that tect the glucose. iiside from con-
passing a stream of nitrogen through a tributing to the loss of a portion of the
mixture of Sumner‘s reagent and glu- glucose, the Rochelle salt caused an
cose for 2 minutes prior to the develop- enhancement of the color due to the
ment of color largely eliminated the remaining glucose.
destruction of glucose (Figure 1, a ) . Sulfite Concentration. T h e effect
WITH STLFITE. As sulfite had pre- of different concentrations of sulfite
viously been used successfully for re- in the modified reagent (Figure I , d )
moving dissolved oxygen from aqueous indicated t h a t a maximum color in-
solutions (b), it was surprising that the tensity n a s obtained at 0.057,sulfite.
sulfite present in Sumner and Sisler’s I n experiments not shown, essentially
reagent failed to accomplish this pur- t h e same results were obtained a t
pose. T o test whether this failure of the 0.025 and 0.1% sulfite as a t t h e
sulfite may have been due to interfereiice 0.057, level. Low concentrations
by other components of the reagent, caused a lack of linearity, nhile
sulfite a t a level of 0.1% n-as added to both high and low Concentrations
glucose samples prior to mixing them caused a depression in color intensity
with the reagent. This procedure re- and a loss of glucose.
duced the destruction of glucose by Sodium Hydroxide Concentration.
about 707,. The rewlts, shown in T h e effect of different concentrations
Figure 1, b, thus provided strong of sodium hydroxide is shown in
evidence for the suspected interference Figure 1, e . High concentrations of
by the other components of the reagent sodium hydroxide led t o enhanced
under the usual conditions of the test. color development, b u t a t t h e same
INTERFEREWE OF ROCHELLESALT. time contributed t o a loss of glucose.
Comparative tests were next carried T h e level of 1% sodium hydroxide
out with the modified dinitrosalicylic appeared t o be t h e most suitable, as
acid reagent to M hich varying amounts it produced t h e maximum color in-
VOL. 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 experi-
ments 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 ab-
scence 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 concen-
tration 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 pos-
sibility 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 car-
boxymethylcellulose and citrate buffer
a t p H 5 might cause interference.
K i t h amounts of carboxymethylcellu-
lose and citrate buffer corresponding to
those used in the cellulase measure-
ment, the effects shown in Figure 1, h,
were produced. The carboxymethyl-
cellulose caused an enhancement in
color, whereas the citrate caused a
depression. A mixture of the sub-
stances approximately neutralized the
t n o opposite effects.
To determine whether the effect of
the citrate may have been a conse-
quence 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 con-
ditions, Rochelle salt is added to the
mixture immediately after the develop-
ment of the color and before the mixture
is cooled. The time of heating is in-
creased 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 sta-
bility 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 ap-
propriate amounts of the interfering
substances are nddrd. K h e n the in-
terfering 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 com-
pensate for the loss of reducing sugar
incurred nhen Rochelle salt is incor-
porated in the dinitrosalicylic acid rea-
gent. For example, it is convenient
in the cellulase test (IO) to introduce
glucose into the carboxymethyl-cellu-
lose-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 car-
boxyl groups (4). The facts, however,
that the equivalence between amino-
nitrosalicylic 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 com-
plicated.
Such complications could conceiv-
ably be associated with the various
decomposition reactions of sugars in
alkaline solution ( 3 ) . If this explana-
tion 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 pro-
duced 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 break-
down products (?‘). This method de-
pends upon the assumption that all
higher oligosaccharides of the homol-
ogous series starting with maltose
would produce equivalent amounts of
color with the reagent. Actual studies
of the reactions of members of homolo-
gous series with the dinitrosalicylic
acid reagent, starting with the disac-
charide, have not been reported, but
u-ould be of considerable interest in
the present connection.
The principal virtue of the dinitro-
salicylic 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 dis-
cussed above must be given due con-
sideration 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.