A UV-Vis spectrophotometric method for the estimation of aldehyde groups in periodate-oxidized polysaccharides using 2,4-dinitrophenyl hydrazine

Article abstract of DOI:10.1080/07328303.2015.1068793

In this study, efforts have been made to develop a simple approach to determine aldehyde content in periodate-oxidized polysaccharides. A UV-Vis analytical technique using 2,4-dinitrophenylhydrazine (DNPH) was employed to calculate the aldehyde content. W

Full text of DOI:10.1080/07328303.2015.1068793

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A UV-Vis Spectrophotometric Method
for the Estimation of Aldehyde Groups
in Periodate-Oxidized Polysaccharides
Using 2,4-Dinitrophenyl Hydrazine
a
a
Mythili Tummalapalli & Bhuvanesh Gupta
a
Bioengineering Lab, Department of Textile Technology, Indian
Institute of Technology, New Delhi 16, India
Published online: 29 Jul 2015.
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To cite this article: Mythili Tummalapalli & Bhuvanesh Gupta (2015) A UV-Vis Spectrophotometric
Method for the Estimation of Aldehyde Groups in Periodate-Oxidized Polysaccharides Using
2
1
,4-Dinitrophenyl Hydrazine, Journal of Carbohydrate Chemistry, 34:6, 338-348, DOI:
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Journal of Carbohydrate Chemistry, 34:338–348, 2015
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2015.1068793
A UV-Vis Spectrophotometric
Method for the Estimation
of Aldehyde Groups
in Periodate-Oxidized
Polysaccharides Using
2,4-Dinitrophenyl Hydrazine
Mythili Tummalapalli and Bhuvanesh Gupta
Bioengineering Lab, Department of Textile Technology, Indian Institute of Technology,
New Delhi 16, India
GRAPHICAL ABSTRACT
In this study, efforts have been made to develop a simple approach to determine alde-
hyde content in periodate-oxidized polysaccharides. A UV-Vis analytical technique us-
ing 2,4-dinitrophenylhydrazine (DNPH) was employed to calculate the aldehyde con-
tent. When tested against compounds like acetaldehyde and formaldehyde with known
aldehyde content, the DNPH method yielded accurate results. The DNPH assay was
Received March 20, 2015; accepted June 29, 2015.
Address correspondence to Bhuvanesh Gupta, Bioengineering Lab, Department of
Textile Technology, Indian Institute of Technology, New Delhi 16, India. E-mail:
bgupta@textile.iitd.ernet.in
Color versions of one or more of the figures in the article can be found online at
www.tandfonline.com/lcar.
3
38

Analysis of Aldehydes in Polysaccharides by UV-Vis
3
39
further utilized to analyze the aldehyde generation during periodate oxidation of pectin,
a naturally occurring plant polysaccharide. It was found that the DNPH assay was sen-
sitive even at low concentrations of aldehyde and yielded accurate results. Therefore,
this method could be used to control and optimize reaction parameters during the peri-
odate oxidation of polysaccharides.
Keywords Aldehyde analysis; Pectin; Dinitrophenyl hydrazine; UV-Vis; Periodate
INTRODUCTION
Functionalization of polymers offers an alternative and economic route for
product development, as compared to polymer synthesis. Today, a lot of re-
search is devoted to product development using naturally available polymers,
[
1–4]
such as polysaccharides and proteins.
Polysaccharides contain a large num-
ber of functional groups on their backbones, such as hydroxyl, carboxyl, amine,
carboxymethyl groups, and so forth, which render them easily susceptible to
functionalization.
In the current article, the emphasis is placed on reaction mechanisms that
lead to the inclusion of aldehyde units in the backbone structure of polysac-
charides. Aldehyde functional groups are highly reactive and may further be
used to form strong covalent linkages between the modified polysaccharide
and other biopolymers, such as proteins. Aldehydes are generated primar-
ily through oxidation of hydroxyl functional groups present on the backbone
of glucopyranose rings in polysaccharides. Two of the most popular meth-
ods used for the oxidation of polysaccharides are the 2,2,6,6-tetramethyl-1-
piperidinyloxy (TEMPO)-mediated oxidation using sodium bromide or sodium
hypochlorite, and periodate oxidation. While both of these methods are highly
selective, the TEMPO-mediated reaction oxidizes primary hydroxyl functional
groups to aldehydes or metal salts, depending on the polysaccharide.[
29,30]
This
methodology cannot be used universally to incorporate aldehyde functional
groups in the polysaccharide structure, since only those materials containing
–CH2OH groups can be converted to aldehydes. Contrastingly, periodate ox-
idation, irrespective of the presence or absence of –CH2OH units, selectively
cleaves the C C bond between the vicinal diols or amino alcohols present on
the C1-C2 or C2-C3 carbons of the polysaccharide chain to yield a dialdehyde
structure.[
31,32]
The reaction mechanism is highly selective, and it is necessary
that the vicinal diols or amino alcohols are oriented in an equatorial-equatorial
or axial-equatorial position. An alternative method for the generation of alde-
hyde units in polysaccharides was put forth by Kulkarni and Mehta.[ In a
manner similar to periodate oxidation, the vicinal diols present on the C2 and
C3 carbons are converted to aldehyde moieties. However, this methodology has
33]
4
+
not been pursued much due to the difficulty in separating leftover Ce from
the reaction medium. On the other hand, periodic acid and its salts are highly
biocompatible and do not pose toxicity issues even when used in close contact

40 M. Tummalapalli and B. Gupta
3
with biological systems. Therefore, periodate oxidation seems the most viable
method to incorporate aldehyde units in polysaccharide structures. Periodate-
oxidized polysaccharides have been used for a variety of biomedical applica-
[
5,6]
[7–9]
[10–12,15]
tions such as wound care,
tissue engineering,
and drug delivery.
Apart from biomedical applications, periodate-oxidized polysaccharide prod-
[
34]
ucts have been used as flocculating agents
and for ion-exchange separa-
tion.[ A recent patent outlines the method for production of nonwoven fi-
brous mats with high wet and dry strength by crosslinking periodate-oxidized
cellulose with transition metal containing crosslinking agents.[ In each case,
the aldehyde content determined the suitability of the material for a given ap-
plication. Additionally, periodate oxidation is concomitantly accompanied by
degradation of the glycosidic linkages. Therefore, to control the loss in molecu-
lar weight and optimize the reaction parameters, a precise analysis regarding
the degree of oxidation is mandatory.
35]
36]
A large number of methods have been reported earlier to determine the
aldehyde content. Maekawa et al. calculated the degree of oxidation in par-
tially oxidized 2,3-dicarboxy cellulose by UV-Vis spectrophotometric analysis of
[
13]
residual periodate ions.
At the end of the oxidation reaction, the absorbance
of the unreacted periodate ions was measured and a calibration equation was
employed to calculate the degree of oxidation. Since oxidation is an instanta-
neous and continuous process, the time gap between the end of the reaction
and measurement plays a crucial role in this process, thus allowing for the
possibility of an error. One of the most popular procedures used for the de-
termination of aldehyde content is the oxime conversion method.[14,16–19] The
aldehyde groups generated are converted to oximes by Schiff base reaction
with hydroxylamine hydrochloride. These were further analyzed by titrimetry,
NMR analysis, elemental analysis, and thermogravimetric analysis. A major
drawback of titrimetry is the ambiguity involved in determining the end point
by the operator. While elemental, NMR, and thermogravimetric analysis yield
accurate aldehyde content values, they are expensive and tedious methods.
Alternatively, a UV-Vis analytical technique using t-butyl carbazate and
trinitrobenzenesulfonic acid was utilized to decipher the degree of oxida-
tion.[
20,21]
Aldehyde moieties on the oxidized polysaccharide backbone re-
acted with t-butyl carbazate to form Schiff bases. Unreacted carbazate units
were then allowed to react with trinitrobenzenesulfonic acid and the col-
ored complex thus formed was quantified spectrophotometrically. In order
to further simplify the aldehyde assay, we designed a one-step procedure
using 2,4-dinitrophenyl hydrazine (DNPH). It has already been established
[
22–24]
that hydrazines react with carbonyl compounds to form hydrazones.
[
25]
DNPH has previously been used to quantify protein carbonyl markers
and
stereoisomers of low-molecular-weight aldehyde compounds[ by HPLC anal-
ysis. In the current study, we developed a new and easier method to analyze
periodate oxidation using DNPH assay spectrophometrically. The DNPH assay
26]

Analysis of Aldehydes in Polysaccharides by UV-Vis
3
41
was initially employed against low molecular weight compounds with known
aldehyde content as the control with a view to check the accuracy of the ob-
tained results. Pectin, a naturally occurring polysaccharide, was subsequently
oxidized by periodic acid and the aldehyde content generated was analyzed
using the DNPH assay.
RESULTS AND DISCUSSION
Pectin is a naturally occurring polysaccharide, poly(1,4-galacturonic acid), ob-
tained from the cell walls of terrestrial plants. Under suitable conditions, the
vicinal diols present on the C2 and C3 carbons of the anhydro-D-glucopyranose
ring undergo oxidation by periodic acid and are converted to aldehyde units.
The periodate oxidation is instantaneous, selective, and highly dependent on
the reaction parameters. Due to the formation of hemiacetals between alde-
hyde units and neighboring hydroxyl groups, complete oxidation is not pos-
sible. In addition, acid-catalyzed cleavage of the β-1,4-glycosidic bonds takes
[
17]
place, leading to degradation.
That being the case, a precise control over
aldehyde generation is important so as to retain the applicability of the poly-
◦
mer. In the current study, pectin was oxidized by 0.5 M periodic acid at 60 C
for different periods of time. The reaction mechanism is shown in Figure 1(a).
While various methods are available in the literature for aldehyde analysis,
most of them are subject to tedious sample preparation and inaccuracy. There-
fore, we have attempted to develop a simple method for detecting and quan-
tifying aldehyde generation in periodate-oxidized polysaccharides by using
DNPH.
The reaction of carbonyl compounds with DNPH forming the correspond-
ing hydrazones is one of the most important qualitative methods in organic
analysis.[ In addition to detecting the presence of carbonyl compounds, the
DNPH assay could also be used to identify the specific aldehyde or ketone. Dur-
ing this addition-elimination reaction, nucleophilic addition of the -NH2 group
to the carbonyl group takes place with the removal of a water molecule. In
the current study, we have harnessed this simple reaction mechanism and de-
veloped a UV-Vis-based analytical technique to quantify the aldehyde content
in periodate-oxidized polysaccharides. Initially, the DNPH UV-Vis methodol-
ogy was tested against low-molecular-weight aldehydes, such as formaldehyde,
glutaraldehyde, and acetaldehyde, to evaluate its accuracy and efficiency. A
representative reaction between formaldehyde and DNPH is depicted in Fig-
ure 1(b). Immediately upon addition of aldehyde to the DNPH solution, a yel-
low precipitate typical of an aliphatic compound is formed. A distinct shift
and loss in intensity of the absorption maxima was observed when the UV-Vis
spectra of the supernatant (unreacted DNPH) fluid were recorded, as shown in
Figure 2. A strong UV band in between 355 and 360 nm was observed. This
is attributed to transitions due to the excited resonance state of DNPH in
26]

42 M. Tummalapalli and B. Gupta
3
Figure 1: (a) Periodate oxidation of pectin. (b) Reaction of formaldehyde with DNPH.
(
c) Hydrazone formation upon reaction of DNPH with periodate-oxidized pectin. Reaction
◦
temperature 25 C; reaction time 1 h.
solution form.[28] While the absorption maximum for DNPH was recorded at
3
60 nm, it shifted to ∼357 nm after reaction with formaldehyde. Uchiyama
[26]
et al. theorized that due to stereoisomerism, the absorption maximum expe-
riences a shift. Therefore, the DNPH UV-Vis assay can also be used to identify
the type of isomer. In order to calculate the aldehyde content, a calibration
equation was formulated at 357 nm from the calibration curve presented in
Figure 3. The theoretical and actual values of aldehyde content in formalde-
hyde, acetaldehyde, and glutaraldehyde at varying concentrations were com-
pared and are found to be in reasonably good agreement, as shown in Figure 4.
Therefore, it was surmised that the DNPH UV-Vis assay gave an accurate mea-
sure of the carbonyl content.
Subsequently, the DNPH assay was utilized to analyze the aldehyde con-
tent generated in periodate-oxidized pectin. The oxidized pectin thus produced
was reacted with DNPH; the reaction mechanism and hydrazone formation
are depicted in Figure 1(c). The reaction between the aldehyde groups in
oxidized pectin and DNPH is instantaneous, resulting in the formation of a
yellow precipitate. Though there is a possibility of hydrogen bond formation
between the unreacted hydroxyl groups of pectin and the amino groups of
DNPH, they are considered to be miniscule. Also, the possible interactions

Analysis of Aldehydes in Polysaccharides by UV-Vis
3
43
Figure 2: UV-Vis spectra of DNPH solutions upon reaction with formaldehyde at varying
concentrations: (a) DNPH alone; (b) with 20% formaldehyde; (c) with 30% formaldehyde;
◦
(
d) with 37% formaldehyde. Reaction temperature 25 C; reaction time 1 h.
between the carboxylic groups of pectin and the amino groups of DNPH are
hindered due to steric factors. The effect of reaction time on the aldehyde con-
tent was monitored and is presented in Figure 5. With an increase in reaction
time, a progressive increase in the aldehyde content was observed. However,
Figure 3: Calibration curve of DNPH in water–ethanol mixture.

44 M. Tummalapalli and B. Gupta
3
Figure 4: Effect of concentration on the aldehyde content of theoretically calculated
(
(
a) glutaraldehyde, (d) acetaldehyde, and (f) formaldehyde, and experimentally observed
b) glutaraldehyde, (c) acetaldehyde, and (e) formaldehyde.
at a reaction time of 3 h, it seems as though the saturation level has been
reached, indicating the highest degree of oxidation. Thus, it could be surmised
◦
that to achieve maximum aldehyde content with 0.5 M periodic acid at 60 C
and solution pH of 3.5, the reaction time should be 3 h.
◦
Figure 5: Effect of oxidation time on the aldehyde content. Oxidation temperature 60 C; pH
3
.5; periodic acid concentration 0.5 M.

Analysis of Aldehydes in Polysaccharides by UV-Vis
3
45
CONCLUSIONS
A facile and economic analytical approach using DNPH and UV-Vis
spectrophotometry has been developed to determine aldehyde content in
periodate-oxidized polysaccharides. DNPH reacts with carbonyl groups to form
the corresponding hydrazones. A close resemblance was obtained between the
theoretical and calculated values of aldehyde content in formaldehyde when
it was analyzed using the DNPH UV-Vis assay. The DNPH UV-Vis assay was
further employed to analyze aldehyde generation during periodate oxidation
of pectin. The results indicate sensitivity and accuracy of the protocol. There-
fore, the DNPH UV-Vis assay could be used to optimize and control reaction
conditions during the periodate oxidation mechanism.
EXPERIMENTAL
Materials
Citrus pectin (Mw∼30,000 g/mol, degree of esterification∼72%) and
DNPH were purchased from CDH Fine Chemicals (India). They were used
as received. Formaldehyde (35%), glutaraldehye (25%), acetaldehyde (25%),
periodic acid, sulphuric acid (95%), absolute ethanol, and isopropanol were ob-
tained from Merck Chemicals (India). All other chemicals used were of analyt-
ical grade. Millipore water was used for all the experiments.
Oxidation of Pectin by Periodic Acid
Periodate oxidation of pectin was carried out according to the procedure re-
[
27]
ported in our earlier work. To a 2% solution of pectin in distilled water, 3 mL
of 0.5 M periodic acid was added. The pH of the medium was maintained at 3.5
using dilute hydrochloric acid and sodium bicarbonate solution. The reaction
was allowed to take place under constant stirring for specific time periods at
◦
6
0 C. The reaction vessel was wrapped in several layers of aluminium foil and
the reaction was carried out in the dark, to prevent autooxidation due to light.
At the end of the reaction, oxidized pectin was precipitated out using excess
◦
isopropanol, separated by vacuum filtration, and dried at 60 C.
Preparation of 2,4-dinitrophenylhydrazine Reagent
4
0 mg of DNPH was dissolved in 0.3 mL of concentrated sulphuric acid.
◦
To this slurry, 3 mL of ethanol was added under continuous stirring at 25 C.
The mixture was allowed to react for 10 min to achieve homogenization. The
volume was then made up to 10 mL with distilled water.

46 M. Tummalapalli and B. Gupta
3
Determination of Aldehyde Content
To 10 mL of freshly prepared DNPH solution, 100 μL of formaldehyde so-
lution (10%–37%), glutaraldehyde (5%–25%), acetaldehyde (5%–25%), or 0.3%
oxidized pectin was added. The reaction mixture was allowed to stand for 1 h
◦
at 25 C and then centrifuged at 10,000 rpm for 10 min. The absorbance of
unreacted DNPH in the supernatant fluid was measured at λ of 357 nm us-
ing a Shimadzu UV 2450 UV-Vis spectrophotometer. The amount of aldehyde
generated was calculated according to:
ꢀ
ꢁ
ꢂ
Reacted DNP (mmol/g)
1
98.14
Aldehyde conc. (mmol/g) =
,
Conc.(%) × 10−
4
where 198.14 is the molecular weight of DNPH.
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