SCIENTIFIC RESEARCH SUBMITTED BY :DARYLL JAY

MAGHINAY
AUTHOR:CAIL LORENZ MILLER (STUDENT)BS.MEDICAL
TECHNOLOGY 1 -B
DATE PUBLISHED:MAY 8 2000

SUBMITTED TO :ALEXANDER URBANO(PROFESSOR) REFERENCE : UCPI CHEMISTRY

LABORATORY

Use of Dinitrosalicylic Acid Reagent

for Determination of Reducing
Sugar
CAIL LORENZ MILLER
Pioneering Research Division, Quartermaster Research and Engineering Center,
Natick, Mass.
Rochelle salt, normally present in 426 • ANALYTICAL CHEMISTRY The major defect in the test is in
the dinitrosalicylic acid reagent for HE DINITROSALICYLIC ACID the loss of part of the reducing sugar
reducing sugar, interferes with the REAGENT, developed by Sumner and being analyzed. This was pointed out
protective action of the sulfite, but is coworker (11—14) for the determination by Sumner (12, 14), was referred to
essential to color stability. The of reducing sugar, is composed of by Brodersen and Ricketts (2), and
difficulty may be resolved either by dinitrosalicylic acid, Rochelle salt, has been observed repeatedly in this
eliminating Rochelle salt from the phenol, sodium bisulfite, and sodium laboratory (6, 8, 9). Evidence of loss
reagent and adding it to the mixture hydroxide. According to the authors of of sugar is also given by the data of
of reducing sugar and reagent after the test, Rochelle salt is introduced to Hostettler, Borel, and Deuel (4) and
the color is developed, or by adding prevent the reagent from dissolving - of Bell, Manners, and Palmer (1).
known amounts of glucose to the oxygen (12) ; phenol, to increase the As this defect appears never to have
samples of reducing sugar to amount of color produced and to balance
the effect of phenol encountered in urine
been fully remedied, the present
compensate for the losses sustained study was carried out to investigate
in the presence of the Rochelle salt. (13) ; and bisulfite, to stabilize the color
obtained in the presence of the phenol the different factors which might
The optimal composition of a (13). The alkali is required for the cause it. In the course of the
modified dinitrosalicylic acid reducing action of glucose on investigation, the effects of varying
reagent is given. dinitrosalieylic acid. the concentrations of the
different components of the reagent Beckman Model DU spectrophotometer at 575 mg with a slit width of 0.06 mm.
also were determined. The findings The reagent of Sumner and Sisler (14) and a modified reagent were used in
which resulted have led to the the tests. The former contained approximately 0.63% dinitrosalicylic acid, 18.2
c
development of a modified reagent c Rochelle salts, 0.5% phenol, 0.5% sodium bisulfite, and 2.14% sodium
and procedure. hvdroxide; the modified reagent contained 1 c-c dinitrosalicylic acid, 0.2%
METHOD phenol, 0.05% sodium sulfite, and 1% sodium hydroxide.
For certain tests the modified reagent includecl varying concentrations of
The color tests were made with 3- Rochelle salt. The composition chosen for the modified reagent was based on
ml. aliquots of reagent added to 3-ml. the results of preliminary tests which indicatecl that such a reagent was optimal
aliquots of glucose solution in 14-mm. and would serve best as the basis of reference for testing effects of variation in
tubes. The mixtures were heated for 5 composition. In the absence of Rochelle salt, the color obtained with the
minutes in a boiling water bath and modified reagent was unstable. To stabilize the color under these conditions, 1
then cooled under running tap water ml. of a 40% solution of the salt was added to the mixture of reactants
adjusted to ambient temperature. subsequent to the development of the color and prior to cooling.
Cooling to ambient temperature was The modified reagent was prepared by placing all solid components in a
made necessary by the effect of container and dissolving them simultaneously by stirring with the required
temperature on the absorbance of the volume of sodium hvdroxide solution. This was much simpler than other
colored reaction product (2), an effect procedures (2, 14).
confirmed by the present studies. The
color intensities were measured in a
 The modified reagent produced the a. Stream of nitrogen passed through mixture; Sumner's reagent
same color with glucose from day to b. sulfite treatment prior to addition of Sumner's reagent
day, thus proving more stable in this c. Rochelle salt concentration; modified reagent
respect then the reagent of Brodersen d. Sulfite concentration; modified reagent
and Ricketts (2). Xlodified reagent to e. Sodium hydroxide concentration; modified reagent
which Rochelle salt was added also
did not change Sodium from day to f. Phenol concentration; modified reagent
day in this respect. Depending upon g. Dinitrosalicylic acid concentration; modified reagent
storage con ditions, however, the h. Carboxymethylcellulose, citrate, and mixtures of both present; modified reagent
modified reagent tended
eventuallv to cleteriorate from and Sisler (14) indicated that the loss of Rochelle salt were added. The
atmospheric oxidation of the sulfite of glucose with the dinitrosalicylic results, shown in Figure 1, c, clearly
present. Deteriorated reagent was re acid reagent was due to destruction implicated Rochelle salt as the major
juvenated bv the addition of fresh by oxidation, and based their factor involved in the interference
sulfite. The danger attendant upon statement on unreported results of with the removal of oxygen by sulfite,
oxidation of sulfite could be avoided experiments in which a stream of because, in the absence of the salt, the
bv nitrogen was passed through the sulfite appeared able to remove the
reactants. An attempt to confirm this dissolved oxygen and thereby to
preparing the reagent in large observation in the present work protect the glucose. Aside from
batches without sulfite, the sulfite indicated that passing a stream of contributing to the loss of a portion
being added to aliquots just prior to nitrogen through a mixture of of the glucose, the Rochelle salt
the time when the reagent was to be Sumner's reagent and glucose for 2 caused an enhancement of the color
used. minutes prior to the development of due to the remaining glucose.
color largely eliminated the Sulfite Concentration. The effect
destruction of glucose (Figure 1, a). of different concentrations of sulfite
STUDY OF VARIABLES
SULFITE. As sulfite had in the modified reagent (Figure 1, d)
Removal of Dissolved Oxygen. WITH indicated that a maximum color
STREAM OF NITROGEN. Sumner previouslv been used successfullv for
intensity was obtained at 0.05%
removing dissolved oxygen from sulfite. In experiments not shown,
aqueous solutions (5), it was essentially the same results were
surprising that the sulfite present in obtained at 0.025 and 0.1% sulfite as
Sumner and Sisler's reagent failed to at the 0.05% level. Low
accomplish this purpose. To test concentrations caused a lack of
whether this failure of the sulfite may linearitv, while both high and low
have been due to interference by concentrations caused a depression
other components of the reagent, in color intensity and a loss of
sulfite at a level of 0.1% was added to glucose.
glucose samples prior to mixing them Sodium Hydroxide Concentration.
with the reagent. This procedure The effect of different concentrations
reduced the destruction of glucose by of sodium hydroxide is shown in
about 70%. The results, shown in Figure 1, e. High concentrations of
Figure 1, b, thus provided strong sodium hvdroxide led to enhanced
evidence for the suspected color development, but at the same
( ( ( (
interference by the other time contributed to a loss of glucose.
components of the reagent under the The level of sodium hydroxide
usual conditions of the test. appeared to be the most suitable, as
INTERFERENCE OF ROCHELLE it produced the maximum color in-
SALT. Comparative tests were next
carried out with the modified
clinitrosalicvlic acid reagent to which
varying amounts
U/LL/GRAMS VOL. 31, NO. 3, MARCH 1959 , 427
GLUCOSE

Figure 1 . Effect of variables

on color produced with glucose and
dinitrosalicylic acid reagent
tensity possible without concomitant concentrations resulted in a lack of dinitrosalicylic acid were varied, the
loss of glucose. linearity. The intensity obtained in color intensity approached a
Phenol Concentration. Maximum the presence of 0.2% phenol was maximum at a concentration of 1%
color development was approached about 5 times that obtained in the (Figure 1, g). The dinitrosalicylic
at a concentration of about 0.2% abscence of phenol. Over the range acid, like the phenol, had no effect on
phenol (Figure 1, f). In experiments tested, the phenol had no effect on the the loss of glucose over the range
not shown in the figure, the same loss of glucose. tested.
results were obtained with 0.5% Dinitrosalicylic Acid Other Substances. The results of
phenol as at the 0.2% level. Low Concentration. When the amounts of the preceding tests suggested the
possibility that other substances which appropriate amounts of the as effects upon the nature and degree
might affect the dinitrosalicylic acid interfering substances are added. of side reactions.
test. For example, it was of interest to M*hen the interfering substances The dinitrosalicylic acid reagent, in a
ascel tain whether, in using the test bring about a loss of reducing sugar, form consisting only of dinitrosalicylic
for the measurement of cellulase and particularly when the amounts acid dissolved in strong alkali, has been
used with apparent success for molecular
activity (8, 10), the presence of of reducing sugar to be measured in weight measurement of starch
carboxymethylcellulose and citrate unknown samples are equal to or breakdown products (7). This method
buffer at pH 5 might cause smaller than the amount lost, known depends upon the assumption that all
interference. M*ith arnounts of amounts of glucose are added to both higher oligosaccharides of the
carboxymethylcellulose and citrate the unknown samples and the homologous series starting with maltose
buffer corresponding to those used in standards. would produce equivalent amounts of
the cellulase measurement, the The procedure of adding glucose color with the reagent. Actual studies of
effects shown in Figure 1, h, were can also be applied advantageously the reactions of members of homologous
series with the dinitrosalicylic acid
produced. The to compensate for the loss of reagent, starting with the disaccharide,
carboxymethylcellulose caused an reducing sugar incurred when have not been reported, but would be of
enhancement in color, whereas the Rochelle salt is incorporated in the considerable interest in the present
citrate caused a depression. A dinitrosalicylic acid reagent. For connection.
mixture of the substances example, it is convenient in the The principal virtue of the
approximately neutralized the two cellulase test (10) to introduce dinitrosalicylic acid test for reducing
opposite effects. glucose into the carboxymethyl- sugar lies in its great convenience
To determine whether the effect of cellulose-citrate substrate and to use compared to most other sugar tests,
the citrate may have been a modified dinitrosalicylic acid reagent particularly when large numbers of
consequence of its buffering action, containing 20% Rochelle salt (8). tests must be carried out. However,
tests were made with acetate buffer Under these conditions the separate the factors discussed above must be
of pH 5 at an equivalent addition of Rochelle salt to the given due consideration to avoid
concentration. The acetate did not, reaction mixture after color misinterpretation of results.
however, affect the test. development is omitted. The controls
FINAL METHOD for such tests consist of a blank and (1)
standard glucose solutions, each RECEIVED for review November 4,
When pure reducing sugar containing carboxymethylcellulose, 1957.
solutions are involved or when any citrate, and compensatory glucose. Accepted September 23, 1958.
contaminants which may be present
are known not to affect the color D'SCUSSION
development or to cause any loss of
reducing sugar, the modified reagent The chemistry of the dinitrosalicylic
in the absence of Rochelle salt is used. acid test for reducing sugar has been
For stabilization of the color clarified previously, at least in part. The
3,5-dinitrosalicylic acid is reduced to 3-
produced under such conditions, amino-5-nitrosalicylic acid while, in the
Rochelle salt is addecl to the mixture simplest instances, the aldehyde groups
iminediately after the developnnent appear to be oxidized to carboxyl groups
of the color and before the mixture is (4). The facts, however, that the
cooled. The time of heating is equivalence between aminonitrosalieylic
increased to 15 minutes because the acid produced and sugar is not exact (4)
5- and that different sugars yield different
amounts of color (1 , 4, 7) , suggest that
the chemistry of the test may actually be
appreciably more complicated.
Such complications could conceivably
be associated with the various
decomposition reactions of sugars in
alkaline solution If this explanation is
correct, the reaction of the sugar
428 ANALYTICAL CHEMISTRY aldehyde grouping with dinitrosalicylic
acid could be viewed as competing with
minute period, adequate for the side reactions involving decomposition of
original procedure, does not produce the sugar. The effects of different
complete color developinent in the concentrations of the various constitu-
modified procedure. By this method ents of the dinitrosalicylic acid
linearity of data, protection of reagent) and also of extraneous
glucose, and stability of color are substances such as
realized. carboxymethylcellulose or citrate
If interfering substances occur in buffer, upon the amount of color
unknown samples, special controls produced and upon the destruction
are run. Such controls consist of of glucose, as shown in the present
standard reducing sugar solutions to study, could similarly be interpreted