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Pitfalls in the 3, 5-dinitrosalicylic acid DNS assay for the reducing sugars:
Interference of furfural and 5-hydroxymethylfurfural

Article  in  International Journal of Biological Macromolecules · April 2020

DOI: 10.1016/j.ijbiomac.2020.04.045

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International Journal of Biological Macromolecules

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Pitfalls in the 3, 5-dinitrosalicylic acid (DNS) assay for the reducing

sugars: Interference of furfural and 5-hydroxymethylfurfural
Narendra Naik Deshavath a, Gourab Mukherjee b, Vaibhav V. Goud a,c,
Venkata Dasu Veeranki a,d,⁎, Chivukula V. Sastri a,b,⁎⁎
a
Centre for the Environment, Indian Institute of Technology Guwahati, 781039, Assam, India
b
Department of Chemistry, Indian Institute of Technology Guwahati, 781039, Assam, India
c
Department of Chemical Engineering, Indian Institute of Technology Guwahati, 781039, Assam, India
d
Department of Bioscience and Bioengineering, Indian Institute of Technology Guwahati, 781039, Assam, India

a r t i c l e i n f o a b s t r a c t

Article history: Transformation of renewable biomass into value-added chemicals and biofuels has evolved to be a vital field of
Received 28 February 2020 research in recent years. Accurate estimation of reducing sugars post pretreatment of lignocellulosic biomass
Received in revised form 21 March 2020 has been very inconsistent. For a few decades, 3,5-dinitrosalicylic acid (DNS) assay has been widely employed
Accepted 3 April 2020
for the estimation of reducing sugars derived from pretreatment of lignocellulosic biomass. This assay tests for
Available online 11 April 2020
the presence of free carbonyl group (C=O), the so-called reducing sugars. This involves the oxidation of the al-
Keywords:
dehyde functional group present to the corresponding acid while DNS is simultaneously reduced to 3-amino-
Lignocellulosic biomass 5-nitrosalicylic acid under alkaline conditions. However, the presence of other active carbonyl groups can poten-
Pretreatment tially also react with DNS leading to incorrect yields of reducing sugars. Therefore, a detailed study has been car-
Reducing sugars ried out to evaluate the influence of active carbonyl compounds like furfural and 5-hydroxymethylfurfural (5-
Furans HMF) in the overall estimation of reducing sugars (glucose, xylose and arabinose) by DNS assay. In addition to
DNS assay this, reducing sugars estimation in the presence of furans were also investigated, it reveals that reducing sugars
HPLC estimation was found to be 68% higher than actual sugars. Therefore, current findings strongly indicate that the
employment of DNS assay for quantifying the reducing sugars in the presence of furans is not appropriate.
© 2020 Published by Elsevier B.V.

1. Introduction
Different colorimetric methods have been well established for the
estimation of reducing sugars that includes DNS [1] and Nelson –
Somogyi [2,3]. Among them, DNS assay has been the most widely
used method for reducing sugars estimation, which is easy to perform
and rapidly quantifies a greater number of samples in a shorter period
of time. But, the major problem that impede the DNS assay is its inaccu-
racy, where the different reducing sugars yield dissimilar colour intensi-
ties [4,5]. In 1986, Deschatelets and Ernest reported that DNS assay is
not suitable for quantifying single sugar in the presence of other mix-
ture of sugars [6]. Moreover, the presence of unwanted impurities
such as phenol in DNS reagent preparation increases the colour inten-
sity of a sample which leads to overestimation of reducing sugars
⁎ Correspondence to: V. Dasu, Department of Bioscience and Bioengineering, Indian
Institute of Technology Guwahati, 781039, Assam, India.
⁎⁎ Correspondence to: C. V. Sastri, Department of Chemistry, Indian Institute of
Technology Guwahati, 781039, Assam, India.
E-mail addresses: veeranki@iitg.ac.in (V.D. Veeranki), sastricv@iitg.ac.in (C.V. Sastri).
concentration [7]. Therefore, phenol-free DNS reagent has been imple-
mented to correctly estimate the reducing sugars [7–9]. DNS method es-
pecially designed for the quantification of single reducing sugar, have
also been adopted for quantifying the mixture of reducing sugars (pen-
tose, hexose and oligosaccharides) which are the common products of
lignocellulosic biomass hydrolysis [5,6,10].
Although, DNS method is not suitable for quantifying the mixture of
sugars but it is still being used for the total reducing sugars estimation
[9,11]. Since a few decades, DNS assay has been the most widely used
method for the quantification of reducing sugars derived from lignocel-
lulosic biomass pretreatment [12–19]. In general, the basic structure of
lignocellulosic biomass constitutes cellulose, hemicellulose and lignin
[20]. Hemicellulose and cellulose are the polymeric carbohydrates;
upon hydrolysis, hemicellulose yields pentose sugars (xylose and arab-
inose) and cellulose yields glucose [21,22]. Whereas, lignin is a complex
aromatic heteropolymer usually found as a hydrophobic matrix around
the polysaccharide components of plant cell walls [23,24]. Its three-
dimensional structure provides decay-resistance, which thereby re-
stricts the dilute acid hydrolysis (generally known as dilute acid
pretreatment) process of the polymeric carbohydrates. It is mainly

https://doi.org/10.1016/j.ijbiomac.2020.04.045
0141-8130/© 2020 Published by Elsevier B.V.

Please cite this article as: N.N. Deshavath, G. Mukherjee, V.V. Goud, et al., Pitfalls in the 3, 5-dinitrosalicylic acid (DNS) assay for the reducing
sugars: Interference of furf..., https://doi.org/10.1016/j.ijbiomac.2020.04.045
2 N.N. Deshavath et al. / International Journal of Biological Macromolecules 156 (2020) xxx
composed of p-hydroxyphenyl (H), guaiacyl (G), and syringyl
(S) monomers that are produced from p-coumaryl alcohol, coniferyl al-
cohol, and sinapyl alcohol, respectively [24]. This lignin and polysaccha-
rides remain covalently linked in the wood and non-wood biomass to
form the lignin-carbohydrate complexes (LCCs) [25]. These LCCs consti-
tute five types of lignin-carbohydrate bonds, viz. phenyl glycosides
(PG), benzyl ethers (BE), γ-esters (GE), ferulate/coumarate esters (FE/
CE) and hemiacetal/acetal linkages [26]. Therefore, due to the rigid na-
ture of lignocellulosic biomass, pretreatment should be carried out at
highly severe conditions such as high temperature and pressure with
dilute acid [27–33] or superheated water (hydrothermal pretreatment)
[18,33,34] which make biomass amenable for enzymatic hydrolysis
[35–37]. During these types of pretreatment processes, lignocellulosic
biomass-derived reducing sugars have been decomposed to form furans
such as furfural (a dehydration product of xylose and arabinose) and 5-
hydroxymethylfurfural (5-HMF) (a dehydration product of glucose)
[28,38]. Xylose, arabinose, glucose, furfural and 5-HMF are leading prod-
ucts of lignocellulosic biomass pretreatment process [31,38,39]. All of
these compounds have a common free carbonyl group. DNS is prone
to reduction to the corresponding 3-amino-5-nitrosalicylic acid thereby
oxidising its carbonyl centre to carboxylic acid [1]. Thus, it is indeed a
redox reaction involving a colour change from yellow to brick red. How-
ever, the origin of the colour change is the reduced form of DNS, irre-
spective of the source of the carbonyl group. Therefore, we predict
that during the estimation of reducing sugars by DNS method, decom-
position products of the reducing sugars such as furfural and 5-HMF
may also interfere to show higher colour intensity than actual sugars
concentration.
It is necessary to provide the technical and experimental reasons to
avoid inaccurate methodologies in the field of scientific research. There-
fore, in the present study, the error of estimation (standard deviation)
between three individual reducing sugars such as glucose, xylose and
arabinose has been investigated through plotting the calibration stan-
dard curve by DNS assay. To avoid inaccuracies in the estimation, the re-
activity of furfural and 5-HMF towards DNS have also been considered.
Moreover, the estimation of reducing sugars mixture has been per-
formed using DNS assay to evaluate the percentage of error against
the calibration plot of single reducing sugar alone. Furthermore, a syn-
thetic hydrolysate has been prepared with respect to the lignocellulosic
biomass pretreatment to investigate the feasibility of using DNS assay
for quantifying the reducing sugars in the presence of furfural and 5-
HMF. Finally, to validate the proposed hypothesis, a thermo-chemical
pretreatment of lignocellulosic biomass has been carried out by using
dilute sulfuric acid as a catalytic agent and the hydrolysate was analysed
by HPLC (to evaluate the concentration of each and individual reducing
sugars and furans) as well as DNS assay for the comprehensive compar-
ison of reducing sugars concentration.
2. Materials and methods
2.1. Chemicals
D-(+)-xylose (99%), L-(+)-arabinose (99%), D-(+)-glucose anhy-
drous (≥99.5%), 5-hydroxymethylfurfural (≥99%) and furfural (99%)
3,5-dinitrosalicyclic acid (98%) were purchased from Sigma-Aldrich,
Bangalore (India). Sodium potassium tartrate (99.98%) and Sulfuric
acid (98%) were purchased from Merck, Pvt., Ltd., India.
2.2. DNS assay
2.2.1. 3, 5-Dinitrosalicyclic acid reagent preparation
The following method was followed for the preparation of 3,5-
dinitrosalicylic acid. [1] 1.6 g NaOH was added to 75 mL of distilled
water under continuous stirring and then 3, 5-Dinitrosalicyclic acid
was added to it. Finally, 3 g of Sodium potassium tartrate was added
and the remaining volume was filled with distilled water to make
Please cite this article as: N.N. Deshavath, G. Mukherjee, V.V. Goud, et al., Pitfalls in the 3, 5-dinitrosalicylic acid (DNS) assay for the reducing
sugars: Interference of furf..., https://doi.org/10.1016/j.ijbiomac.2020.04.045
100 mL of DNS reagent. Phenol was not used in the preparation of
DNS reagent which unnecessarily increases the colour intensities of
the measured concentration range of compounds [7–9].
2.2.2. Preparation of calibration standard curves
A series of calibration standard curves of each and individual reducing
sugars (glucose, xylose and arabinose) and furans (furfural and 5-HMF)
are prepared by DNS assay at a concentration range of 0.4–1 mg/mL
(Table 1).
2.2.3. Preparation of hydrolysates to examine the furans effect on reducing
sugar estimation
A synthetic hydrolysate was prepared which consists of reducing
sugars (glucose, xylose and arabinose, all together 0.3–1.8 mg/mL)
and furans (furfural and 5-HMF, together 0.2–1.2 mg/mL) at concentra-
tion range of 0.5–3 mg/mL (Table 2) and are quantified by DNS assay.
Furthermore, to evaluate the effect and interference of furans in the
DNS assay, a furan free reducing sugars mixture medium (consists of
glucose, xylose and arabinose, all together at a concentration range of
0.3–1.8 mg/mL, listed in Table 2) have been prepared and are quantified
by DNS assay and the results are compared with that of the synthetic
hydrolysate for the clear observation of overestimated results of reduc-
ing sugars in the presence of furans.
Finally, pre-hydrolysate was prepared by conducting the pretreat-
ment of sorghum biomass with 1% sulfuric acid at 121 °C for 120 min,
with 5% (w/v) solid loading. The pretreatment derived hydrolysate (de-
noted as pre-hydrolysate) containing total reducing sugars concentra-
tion was determined by DNS assay and the concentration of each and
individual reducing sugar such as glucose, xylose, arabinose and furans
such as furfural, 5-HMF are quantified by high performance liquid chro-
matography (HPLC) for the comprehensive comparison between DNS
assay and HPLC analysis. The amount of reducing sugars released per
gram of sorghum biomass has been calculated by the following equa-
tions (Eqs. (1) and (2)).
Sugars ðmg=mLÞ
¼ Concentration dectected by DNS assay=HPLC analysis  DF ð1Þ
 
Eq:1  Total Volume of prehydrolysate
Sugars ðmg=g Þ ¼ ð2Þ
Initial weight of biomass
Moreover, bioethanol potential of reducing sugars derived from di-
lute acid pretreatment process has been calculated through the follow-
ing equations (Eqs. (3), (4) and (5)).
C6H12O6→2C2H5OH þ 2CO2 ð3Þ
0:51 kg ethanol
TEY ðkg=tonÞ ¼ CS ðkg=tonÞ  ð4Þ
kg sugar
TEY
V ðL=tonÞ ¼ ð5Þ
ρ
where, Cs is the concentration of sugars (C6 or C5) present in the ligno-
cellulosic biomass, 0.51 (gp/gs) is theoretical ethanol yield TEY constant,
ρ is the ethanol density (0.789 kg/L at 20 °C).
2.2.4. Reducing sugar estimation procedure
DNS assay was carried out by placing the 1 mL of standard or test
sample in a 25 mL test tube and then 3 mL of DNS reagent was added.
Table 1
List of reducing sugars and furans for making of four-pointed calibration curve.
Components Glucose Xylose Arabinose Furfural 5-HMF
Concentration range (mg/mL) 0.4–1 0.4–1 0.4–1 0.4–1 0.4–1
 N.N. Deshavath et al. / International Journal of Biological Macromolecules 156 (2020) xxx
Table 2
Composition of synthetic hydrolysate and mixture of reducing sugars.
S. No Reducing sugars (A) (mg/mL) Furans (B) (mg/mL)
Xylose Glucose Arabinose Furfural 5-HMF
1 0.1 0.1 0.1 0.1 0.1
2 0.2 0.2 0.2 0.2 0.2
3 0.4 0.4 0.4 0.4 0.4
4 0.6 0.6 0.6 0.6 0.6
The reaction mixture was heated in boiling water for 5 min. The absor-
bance spectra of the test samples were measured at 540 nm using 2 mm
path length quartz cuvettes in the UV–vis spectrophotometer (Agilent,
Carry 100, USA). Blank test was performed in 1 mL of distilled water in-
stead of the test sample.
2.3. High performance liquid chromatography
High performance liquid chromatography was used for the quantifi-
cation of individual sugars and furans present in the synthetic hydroly-
sate and pre-hydrolysate. The calibration standards for individual
sugars and furans (concertation range are listed in Table 1) were
made by plotting their area under the curve against concentrations.
The HPLC separation system was equipped with a refractive index (RI)
detector (355 Varian), solvent delivery system (210 Varian) and a com-
puter software-based integration system (Varian, The Netherlands). Re-
ducing sugars such as glucose, xylose, arabinose and furans like 5-
hydroxymethylfurfural (5-HMF), furfural were analysed by using
MetaCarb-87H carbohydrate column (300 × 6.5 mm, particle size
8 μm) at 60 °C. 0.013 N sulfuric acid was used as a mobile phase for
the elution of compounds with 0.5 mL/min flow rate.
3. Result and discussion
The basic principle of DNS assay includes the reduction of one of the
nitro groups in 3,5-dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid
and subsequent oxidation of the aldehyde groups in substrates to car-
boxylic acids. [1] However, with employment of different sugars, the
production of 3-amino-5-nitrosalicylic acid is not uniform [4] and
hence different sugars yield dissimilar colour intensities [5], which sug-
gests that the chemistry of the test demands a detailed scrutiny. Since
the lignocellulosic materials are heterogeneous in nature, consequently
different types of reducing sugar formation can occur during the process
of pretreatment [28]. Reducing sugar decomposition products such as
furfuraldehyde (furfural) and 5-Hydroxymethylfurfuraldehyde (5-
HMF) are also formed as by-products during the pretreatment process
depending upon the reaction temperature, pressure and type of catalyst
used for hydrolysis [14,27,28,35,40]. Xylose, arabinose, glucose (reduc-
ing sugars), cellobiose, xylobiose (disaccharides), acetic acid, gluconic
acid (organic acids), furfural and 5-HMF (reducing sugars decomposi-
tion products) are the general pretreatment products of lignocellulosic
material [28,38]. Apart from the above listed disaccharides and organic
acids, aldehyde is found to be a common functional group present in the
reducing sugars as well as furans. Being reducing in nature, DNS could
also oxidize the aldehyde group in furfural and 5-HMF along with that
in the reducing sugars. Therefore, to discriminate the effect of DNS on
the sugars and the by-products, we performed a thorough analysis. A
typical set of calibration standard curves of glucose, xylose, arabinose,
furfural and 5-HMF were made by using the DNS method (Fig. 1).
As expected, dissimilar colour intensities were observed when DNS
reagent were reacted with different reducing sugars and furans. It is
understood that aldehyde groups of reducing sugars and furans are
oxidized to carboxyl groups, simultaneously 3,5-dinitrosalicylic acid re-
duces to 3-amino-5-nitrosalicylic acid under alkaline conditions and ex-
hibits different colour intensities. The highest error of estimation
(standard deviation) obtained from all the reducing sugars components
Please cite this article as: N.N. Deshavath, G. Mukherjee, V.V. Goud, et al., Pitfalls in the 3, 5-dinitrosalicylic acid (DNS) assay for the reducing
sugars: Interference of furf..., https://doi.org/10.1016/j.ijbiomac.2020.04.045
3
Mixture of reducing sugars (mg/mL) Synthetic hydrolysate (mg/mL)
(A) (A + B)
0.3 0.5
0.6 1
1.2 2
1.8 3
tested was b1.5%. However, for the most practical purposes, this error
margin should be considerable and acceptable for the monitoring of re-
ducing sugars. Based on these results, DNS assay was also examined for
the mixture of reducing sugars (the furans free reducing sugar medium)
and the results were quite in acceptable error range (b1.5%) with corre-
spondence to standard calibration curves of each and individual reduc-
ing sugars (Fig. 2).
Apart from the reducing sugars, we found out that furfural and 5-
HMF also reacted separately with 3,5-dinitrosalicylic acid to yield differ-
ent colour intensities. This encouraged us to investigate the feasibility of
DNS assay for a mixture of reducing sugars in the presence of furans and
thus quantify the exact estimation of sugars. A synthetic hydrolysate
medium was prepared corresponding to the subcritical water or dilute
acid pretreatment at high temperature and pressure. The dehydration
of lignocellulosic biomass derived reducing sugars to form furans is a
common phenomenon in the pretreatment process conducted at high
temperature and pressure with dilute acid or water (at subcritical
state) [41]. However, in enzymatic pretreatment process no formation
of furans is observed. [18,28,31,34,37] In the commercial prospective,
dilute acid pretreatment is well established process and relatively
cheaper than enzymatic pretreatment [42]. Based on the aforemen-
tioned literature, synthetic hydrolysate medium was prepared contain-
ing a mixture of reducing sugars and furans (Table 2). The concentration
ranges of reducing sugars (glucose, xylose and arabinose, all together
0.3–1.8 mg/mL) used in this synthetic hydrolysate medium is similar
to that of the aforementioned mixture of reducing sugars medium (a
furan- free reducing sugars medium) (Table 2). Since the concentration
of the reducing sugars in both the synthetic mediums were kept con-
stant for absorption spectroscopy, the corresponding absorbance for
the two mediums were expected to be similar. However, enhancement
of colour intensity was observed in the furan containing synthetic hy-
drolysate medium (Fig. 2). As evident from the concentration against
absorbance plot in Fig. 2, DNS assay for the furan containing synthetic
hydrolysate gives higher absorbance values at a definite concentration
and that the absorbance increases with increasing concentration. There-
fore, this surge in absorbance in the furans containing synthetic hydro-
lysate can be attributed to the reaction of furfural and 5-HMF with 3,5-
dinitrosalicylic acid. As such, almost 68% enhancement in absorbance
Fig. 1. Absorbance against concentration plot for various aldehyde containing compounds
in DNS assay. All the absorbance values were recorded at 540 nm at room temperature.
4 N.N. Deshavath et al. / International Journal of Biological Macromolecules 156 (2020) xxx
Fig. 2. Absorbance against concentration plot for synthetic hydrolysate (blue line) and
mixture of sugars (red line), keeping sugar concentration constant. All the absorbance
values were recorded at 540 nm at room temperature.
was observed in the quantification of reducing sugars in the presence of
furans. Thus, a detailed analysis of the two synthetic hydrolysate media
infers an overestimation in the quantification of reducing sugars by DNS
assay in the presence of furans. Henceforth, it is suggested that employ-
ment of DNS assay results in imprecise estimation of reducing sugars
present in the pre-hydrolysates of lignocellulosic biomass. Precise
sugar estimation is more important for the prediction of bioethanol
yield for the development of a specific bio-refinery process.
For instance, sorghum biomass pretreatment (at 121 °C, 120 min
with 1% sulfuric acid strength) derived pre-hydrolysate containing re-
ducing sugars were quantified by both DNS assay and HPLC analysis.
For the better reflection of results, SI (standard international) units of
reducing sugars in-terms of mg/mL (mg of reducing sugars per mL of
pre-hydrolysate) is converted into mg/g (mg of reducing sugars per
gram of biomass) by the aforementioned equations Eqs. (1) and (2). Ac-
cording to the DNS assay, per gram of sorghum biomass produces
434.3 mg of reducing sugars which is equivalent to 434.3 kg/ ton of sor-
ghum biomass. Whereas, the actual reducing release (analysed by
HPLC) was found to be 281 mg/g (sum of glucose (45.2 mg), xylose
(219.7 mg) and arabinose (16.1 mg)) or 281 kg/ton of sorghum bio-
mass. Based on the stoichiometric equation (Eq. (3)), the maximum
theoretical yield (gp/gs) of ethanol per gram of substrate (C6 or C5
sugar) is 0.51 g. Therefore, according to the Eq. (4), 281 kg of reducing
sugar (detected by HPLC) can be converted into 143.3 kg of ethanol,
whereas 434.3 kg of reducing sugar (detected by DNS assay) can be con-
verted into 221.4 kg of ethanol. Therefore, a misinterpretation of about
Fig. 3. HPLC chromatogram of synthetic hydrolysate.
Please cite this article as: N.N. Deshavath, G. Mukherjee, V.V. Goud, et al., Pitfalls in the 3, 5-dinitrosalicylic acid (DNS) assay for the reducing
sugars: Interference of furf..., https://doi.org/10.1016/j.ijbiomac.2020.04.045
78.1 kg or 99 L/ton (based on Eq. (5)) has been taking place by
employing the DNS assay for the quantification of reducing sugars de-
rived from pretreatment of lignocellulosic biomass. Hence, wrong inter-
pretation of ethanol yield significantly hampers the techno-economic
viability of a bio-refinery process.
Since past few decades, DNS assay is the most widely used method
to monitor the production of reducing sugars from pretreatment of lig-
nocellulosic biomass. However, the interference of furfural and 5-HMF
in the said process remained unnoticed. Mechanism of the formation
of furfural and 5-HMF from pentose (xylose and/or arabinose) and hex-
ose (glucose and/or fructose) sugars includes dehydration of the reduc-
ing sugars with subsequent loss of three water molecules [43]. It is a
known fact that the decomposition of pentose and hexose sugars were
initiated when the pretreatment reaction has been conducted in a pres-
surized batch reactor at elevated temperatures by using different types
of catalysts [27–29,34,38]. Generally, pretreatment of lignocellulosic
biomass are conducted by either hydrothermal process (where water
act as a catalyst at subcritical sate) [18,33,34,44] or thermochemical
process (where dilute mineral acids act as a catalyst) [27–29,31,40] for
significant hydrolysis of polymeric carbohydrates. The temperature
range for hydrothermal pretreatment and dilute acid pretreatment
would be 160 °C–220 °C and 121 °C–160 °C, respectively with corre-
spondence to the reaction pressure [18,27–29,31,33,34,40]. Therefore,
significant amount of furan formation is expected to occur when the lig-
nocellulosic biomass pretreatment is conducted at these elevated tem-
peratures. On the other hand, degradation of sugars does not occur
upon the pretreatment of lignocellulosic biomass conducted with bio-
logical enzymes, which are generally performed in the temperature
range of 25 °C to 80 °C (thermo-stable enzymes). However, a recent
study conducted by Teixeira et al. 2012, suggested that the measure-
ment of cellulase and xylanase activities by using DNS method show in-
correct (overestimated) results which can be due to the interference of
enzymes that contain amino acids such as cysteine, tyrosine, histidine
and hydroxylproline [7].
Therefore, employment of DNS assay for monitoring the reducing
sugars derived from the hydrolysis of lignocellulosic biomass at high
temperature (121 °C - 160 °C and 160 °C– 220 °C for dilute acid pretreat-
ment and hydrothermal pretreatment, respectively) is not that reliable
and feasible. Indeed, the data obtained from DNS assay was directly cor-
roborated with HPLC analysis of the synthetic hydrolysate. It was ob-
served that upon separation of the individual components in HPLC,
the concentration of sugar remained intact in both mediums (mixture
of reducing sugars and synthetic hydrolysate shown in Table 2), and
that furans (furfural and 5-HMF) have no significant impact on increas-
ing the reducing sugar concentration during the HPLC analysis (see
 N.N. Deshavath et al. / International Journal of Biological Macromolecules 156 (2020) xxx
Fig. 3). As such, DNS assay for reducing sugar analysis is in general not a
true reflection for furan containing samples. A comparison of DNS assay
with HPLC analysis is imperative of the fact that the DNS assay leads to
an inaccurate estimation of reducing sugars derived from pretreatment
of lignocellulosic biomass conducted at elevated temperatures.
4. Conclusion
Overall, the current study reveals the limitation involved with the
DNS assay, in which 3,5-dinitrosalicylic acid is seen to react with the un-
wanted by-products (in the form of furans) along with the reducing
sugars. This leads to incorrect estimation of sugars due to a parallel reac-
tion going on inside the reaction medium. In particular, the presence of
furfural and 5-HMF in the synthetic hydrolysate enhances the reduction
of DNS to 3-amino-5-nitrosalicylic acid. This leads to magnified absorp-
tion values in the visible spectrum. Thus, to infer, we establish that the
estimation of reducing sugars by DNS assay is not a reliable method in
the presence of furfural and 5-HMF.
Acknowledgement
We would like to thank Central Instrumentation Facility (CIF) IIT
Guwahati for providing instrumentation facilities. This research was fi-
nancially supported (Grant No: IITG/EVC-61.16/127/2017-18) by
Indian Institute of Technology Guwahati, India.
Credit author statement
Narendra Naik Deshavath: Conceptualization, Methodology, Inves-
tigation, Writing- Original draft preparation. Gourab Mukherjee: For-
mal analysis, Writing- Original draft preparation. Vaibhav V. Goud:
Visualization, Investigation. V. Venkata Dasu & Chivukula V. Sastri: Su-
pervision, Validation, Writing- Reviewing and Editing.
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Please cite this article as: N.N. Deshavath, G. Mukherjee, V.V. Goud, et al., Pitfalls in the 3, 5-dinitrosalicylic acid (DNS) assay for the reducing
sugars: Interference of furf..., https://doi.org/10.1016/j.ijbiomac.2020.04.045
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