SAA-15710; No of Pages 4

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2017) xxx–xxx

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Spectrochimica Acta Part A: Molecular and Biomolecular

Spectroscopy
journal homepage: www.elsevier.com/locate/saa

Hydrogen/deuterium (H/D) exchange of gelatinized starch studied by

two-dimensional (2D) near-infrared (NIR) correlation spectroscopy
Hideyuki Shinzawa ⁎, Junji Mizukado
National Institute of Advanced Industrial Science and Technology (AIST), Japan

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

Article history: Hydrogen/deuterium (H/D) exchange of gelatinized starch was probed by in-situ near-infrared (NIR) monitoring
Received 17 October 2017 coupled with two-dimensional (2D) correlation spectroscopy. Gelatinized starch undergoes spontaneous H/D
Received in revised form 19 December 2017 exchange in D2O. During the substitution, the exchange rate essentially becomes different depending on solvent
Accepted 21 December 2017
accessibility of various parts of the molecule. Thus, by analyzing the change in the NIR feature observed during
Available online xxxx
the substitution, it becomes possible to sort out local structure and dynamics of the system. 2D correlation anal-
Keywords:
ysis of the time-dependent NIR spectra reveals the presence of different local structure of the starch, each having
Near-infrared (NIR) spectroscopy different solvent accessibility. For example, during the H/D exchange, the D2O is first absorbed by starch mole-
Two-dimensional (2D) correlation spectroscopy cules especially around the surface area between the starch and water, where the water molecules are weakly
Hydrogen/deuterium (H/D) exchange interacted with the starch molecules. This absorption is quickly followed by the development of HDO species.
Starch Further absorption of the D2O results in the penetration of the molecules inside the starch and eventually de-
velops the relatively strong interaction between the HDO and starch molecules because of the presence of dom-
inant starch molecules.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction Starch granules in an aqueous suspension with sufficient heat can

Water is involved in many kinds of substances, and the water con-
tent and structure of water in them are often key factors governing
functions. Interaction between water and substance has always been a
matter of great interest because of its potential importance in terms of
basic science as well as wide range of applications. Near-infrared
(NIR) spectroscopy has been employed to investigate the water content,
hydrogen bonds of water and hydration in various fields such as food
and polymer industries [1]. The change in hydrogen bonding often re-
sults in the variation of the NIR spectral feature. For example, NIR spec-
tra of water are very sensitive to various cluster structures consisting of
multiple water molecules [2]. In addition, the utilization of the NIR re-
gion with much less absorption enables the light to penetrate much far-
ther into a sample. In fact, the penetration depth of NIR beam can be on
the scale of centimeters and this is particularly suited for probing bulk
material with a high water content [3]. Thus, by analyzing the NIR spec-
tral feature of the system of interest, it becomes possible to sort out the
chemically meaningful information associated with water.
⁎ Corresponding author.
E-mail address: h-shinzawa@aist.go.jp (H. Shinzawa).
weak hydrogen bonding between the starch molecules, followed by
the penetration of water molecules [4,5]. The absorption of water then
leads to swelling in the amorphous regions of the granule, loss of the
crystalline order, and, finally, starch gelatinization [4,5]. This feature
has been utilized for starch plasticization as well as thickening or gelling
agent while the details of the gelatinization phenomena is not fully un-
derstood yet [6,7]. In this work, we report the NIR spectroscopy based
on real-time in-situ monitoring of hydrogen/deuterium (H/D) exchange
of gelatinized starch. H/D exchange, a form of chemical exchange, has
long been used as a method for studying the secondary and tertiary
structure of peptides and proteins using mass spectrometry, NMR, IR
spectroscopy [8–10]. When a molecule is diluted into a solution of
D2O, hydrogen atoms gradually exchange with deuterium. The ex-
change rate essentially becomes different depending on solvent accessi-
bility of various parts of the molecule. By following the substitution
using NIR spectroscopy, it is possible to probe local structure and dy-
namics of the system. Thus, in-situ NIR monitoring of gelatinized starch
undergoing the H/D exchange may provide an interesting opportunity
to probe the dynamics of water absorption as well as determine acces-
sible regions of the gelatinized system.
The time-dependent NIR spectra collected during the H/D exchange
of the gelatinized starch are then subjected to two-dimensional (2D)

https://doi.org/10.1016/j.saa.2017.12.065
1386-1425/© 2017 Elsevier B.V. All rights reserved.

Please cite this article as: H. Shinzawa, J. Mizukado, Hydrogen/deuterium (H/D) exchange of gelatinized starch studied by two-dimensional (2D)
near-infrared (NIR)..., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2017), https://doi.org/10.1016/j.saa.2017.12.065
2 H. Shinzawa, J. Mizukado / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2017) xxx–xxx

correlation spectroscopy [11–13]. The 2D correlation analysis of the

time-dependent NIR spectra reveals significant changes in the spectral
intensity. Of note is that the result clearly derived the detailed picture
of the H/D exchange of the gelatinized starch.

2. Methods

2.1. 2D Correlation Spectroscopy

The basic idea of generalized 2D correlation spectroscopy is the anal-

ysis of the dynamic response of system stimulated by various perturba-
tions [11–13].
Let us consider analytical signal trace y(v,t). The variable v is the
index variable to represent any types of signal responses induced by
the perturbation variable t. Analytical signal trace y(v,t) at m evenly
spaced points in time t between Tmin and Tmax are represented as


y j ðvÞ ¼ y v; t j ; j ¼ 1; 2; ⋯; m: ð1Þ

A set of dynamic spectra is given by


~ðv; t Þ ¼ y v; t j −yðvÞ
y ð2Þ
Fig. 1. Geometry of NIR measurement of gelatinized starch soaked in D2O.

where yðvÞ denotes the reference spectrum, typically the average spec-
Pm 4. Results and Discussion
trum given by yðvÞ ¼ m 1
j¼1 yðv; t j Þ. Synchronous correlation intensity
can be directly calculated from the dynamic spectra as
4.1. Time-dependent NIR Spectra of Gelatinized Starch

1 X m Fig. 2(A) represents time-dependent NIR spectra of gelatinized

Φðv1 ; v2 Þ ¼ y ~ j ðv2 Þ
~ ðv1 Þ  y ð3Þ
m−1 j¼1 j starch undergoing H/D exchange. Special attention was paid to the spec-
tral features observed in wavelength region over 1200–1800 nm. Pure
spectra of H2O, D2O and starch granules are provided as Fig. 2(B), re-
Asynchronous correlation can be obtained by spectively, for reference. The main feature of the initial NIR spectra is
mainly dominated with the signal contribution from those components.
Peaks appearing at 1410 nm and 1650 nm are mostly ascribed to HDO-
1 X m Xm
specific band, where a part of H atom of water molecule is replaced by
Ψðv1 ; v2 Þ ¼ ~ ðv1 Þ 
y ~ j ðv2 Þ
Njk  y ð4Þ
m−1 j¼1 j k¼1 deuterium [14]. The increase of these NIR bands can be interpreted as
the gradual absorption of the deuterium and subsequent substitution,
reflecting the progress of the H/D exchange. It is also noted in
The term Njk corresponds to the i-th column and k-th raw element of Fig. 2(A) that the intensity of the NIR band at 1460 nm steadily increases
the so-called discrete Hilbert-Noda transformation matrix defined as as the H/D exchange proceeds. Unfortunately, the origin of this band is
not fully understood yet, however the gradual increase in the spectral
 intensity suggests that this is not related to the \\OH group of water
0 if j ¼ k
Njk ¼ ð5Þ or starch. For instance, this mixture system consists of H2O D2O and
1=πðk−jÞ otherwise
starch. So the development of NIR peak can be observed only when
the concentration of the complement is increased. Obviously, there is
The intensity of a synchronous correlation spectrum Φ(v1, v2) repre- no way too see the increase in\\OH group concentration as H2O grad-
sents simultaneous changes of two spectral intensity variations mea- ually undergoes the H/D exchange which eventually leads to the de-
sured at v1 and v2 during the interval between Tmin and Tmax [11–13]. crease its concentration. In other words, the emergence of the peaks
In contrast, an asynchronous correlation spectrum Ψ(v1, v2) means the observed in Fig. 2(A) indicates the development of new species pro-
out-of-phase or sequential changes of spectral intensity measured at duced by the H/D exchange, namely HDO. In addition, it is apparent
v1 and v2 [11–13]. that\\OD group of D2O does not provide any specific peak in this NIR
region [14]. This band is, hence, probably associated with the HDO-
3. Experimental species, which is expected to appear during the H/D exchange. While,
the detailed assignment of the NIR band at 1460 nm is not clear at this
Starch from wheat was provided by Wako Pure Chemical Industries, moment, it is obvious that the variation of this band becomes a useful
Ltd. Starch granules of 3 g were dissolved in 30 ml distilled water and empirical inference to sort out the progress of the H/D exchange caused
heated at 90 °C under mixing at 200 rpm for 10 min. The sample was by the penetration of deuterated water molecules. In fact, the assign-
then cooled to room temperature to fully undergo gelatinization. The ment of this band becomes even more explicit with an aid of 2D corre-
gelatinized sample of 0.3 g was soaked with 0.8 ml D2O in a glass cell lation analysis as will be shown later.
and NIR transmittance spectra were measured with a NIR spectrometer Although many of the peaks observed in the NIR spectra are specific
based on acousto-optic tunable filter (Systems Engineering Inc., Tokyo) to different components, it is also clear that most peaks do have sub-
by co-adding 64 scans every 30 s for 10 h. The geometry of the measure- stantial tail overlap with each other to make the completely isolated
ment is illustrated in Fig. 1. analysis of each component somewhat difficult in the mixture. Hence,

Please cite this article as: H. Shinzawa, J. Mizukado, Hydrogen/deuterium (H/D) exchange of gelatinized starch studied by two-dimensional (2D)
near-infrared (NIR)..., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2017), https://doi.org/10.1016/j.saa.2017.12.065
 H. Shinzawa, J. Mizukado / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2017) xxx–xxx 3

Fig. 2. (A) Time-dependent NIR spectra of gelatinized starch soaked in D2O. (B) Pure Fig. 3. (A) synchronous and (B) asynchronous correlation spectra derived from time-
spectra of starch granules, H2O and D2O. dependent NIR spectra of gelatinized starch.

the task of enhancing the spectral resolution as well as determine the
sequential order of intensity variations by taking the advantage of 2D
correlation analysis becomes important to provide penetrating insight
into this system.
4.2. 2D Correlation Spectra
Fig. 3 shows (A) synchronous and (B) asynchronous correlation
spectra derived from the time-dependent NIR spectra of the gelatinized
starch. The spectra were subjected to linear baseline correction prior to
the 2D correlation analysis. The time-averaged spectrum is placed as a
reference at the top and side of the 2D spectrum. By convention, nega-
tive asynchronous peaks are all marked by shading. The synchronous
spectrum is dominated by an intense autopeak on the main diagonal
around 1460 nm, corresponding to the steady increase in the NIR spec-
tral intensities in this NIR region. Along the 1460 nm coordinate of the
synchronous correlation spectrum, a positive cross-peaks is clearly ob-
servable at 1650 nm. The cross-peak indicates that NIR intensities at
the coordinate are changing in the same directions during the reaction,
reflecting the fact that the H/D exchange of the sample induces the in-
crease in the HDO species in the system. One of the noteworthy obser-
vations is the developments of some cross peaks in the asynchronous
correlation spectrum. The peaks appearing at the coordinate (1460,
1410 nm) and (1460, 1650 nm) indicate that the intensity change orig-
inating from the HDO species occurs predominantly before the change
at 1460 nm. If these three bands arise from spectral signals from same
origin, it should result in the simultaneous development of the compo-
nents without any sequential or successive changes in them. In other
words, it should only show synchronous correlation but no asynchro-
nous correlation. Thus the development of the correlation peak be-
comes especially important. The intensity change at 1460 nm seems
to occur at a later time than that for the rapid increase in the deuterium
content characterized by the intensity at 1410 and 1650 nm. Such se-
quence implies the presence of a specific HDO species, existing in differ-
ent molecular environment to induce obvious delay in the development
of the D-substituted molecules. Consequently, the band at 1460 nm is
possibly associated with the HDO species having relatively stronger in-
teraction with surrounding molecules, e.g. starch.
The result sorted out by the 2D correlation analysis suggests an in-
triguing possibility of the existence of different local structures, each
having different solvent accessibility. The sequential order mostly indi-
cates that H2O molecules around the interfacial area develop weaker
hydrogen bonding compared to the other parts of the system, resulting
in dominant substitution at the onset of the H/D exchange. Actually this
agrees with the fact the interaction between starch and water molecules
is essentially influenced by the relative concentration of starch or water

Please cite this article as: H. Shinzawa, J. Mizukado, Hydrogen/deuterium (H/D) exchange of gelatinized starch studied by two-dimensional (2D)
near-infrared (NIR)..., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2017), https://doi.org/10.1016/j.saa.2017.12.065
4 H. Shinzawa, J. Mizukado / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2017) xxx–xxx
in the system [4,5,7]. On the other hand, water and starch in the other
parts (probably inside the gelatinized sample) substantially develop rel-
atively stronger interaction between the molecules due to the presence
of dominant starch molecules, which it, in turn, restricts the deuterium
penetration and subsequent substitution. Now it is possible to derive a
clear picture of this system. The D2O is first absorbed by starch mole-
cules especially around the surface area between the starch and water
to induce the development of the HDO. Further absorption of the D2O
results in the penetration of the deuterated molecules inside the starch
followed by the development of the interaction between the HDO and
starch molecules.
Consequently, the application of 2D NIR correlation spectroscopy to
examine the H/D exchange process of the gelatinized starch had dem-
onstrated several advantages. Although the system studied here is sim-
ple, spectroscopic evidence clearly shows a substantial level of
difference in solvent accessibility depending on the various parts of
the gelatinized starch molecules. For the analysis of the NIR spectral in-
tensity variation during the H/D exchange, 2D correlation analysis may
be a tool of the choice. The technique should further assist NIR studies of
the related systems.
5. Conclusion
In-situ NIR monitoring of gelatinized starch undergoing hydrogen/
deuterium (H/D) exchange was studied. The time-dependent NIR of
the gelatinized starch are subjected to 2D correlation spectroscopy to
sort out the subtle but important change of spectral intensities. The 2D
correlation spectra revealed the predominant increase in the spectral
intensity arising from the HDO-species existing in a specific are occurs
before that in the other areas, revealing the presence of different solvent
accessibility. For example, at the onset of the H/D exchange, absorption
of the D2O mainly occurs around the surface area between the starch
and water, where the water molecules are weakly interacted with the
starch molecules. Further absorption of the D2O results in the
Please cite this article as: H. Shinzawa, J. Mizukado, Hydrogen/deuterium (H/D) exchange of gelatinized starch studied by two-dimensional (2D)
near-infrared (NIR)..., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2017), https://doi.org/10.1016/j.saa.2017.12.065
penetration of the molecules inside the starch and eventually develops
the relatively strong interaction between the HDO and starch molecules
because of the presence of dominant starch molecules.
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