Volume 57, Number 2, 2003 APPLIED SPECTROSCOPY 139

0003-7028 / 03 / 5702-0139$2. 00 / 0
q 2003 Society for Applied Spectroscopy
Short-Wavelength Near-Infrared Spectra of Sucrose,
Glucose, and Fructose with Respect to Sugar Concentration
and Temperature
MIRTA GOLIC, KERRY WALSH,* and PETER LAWSON
Plant Sciences Group, Central Queensland University, Rockhampton, 4702, Australia
Short-wavelength near-infrared (SW-NIR) (7001100 nm) spectra
of aqueous solutions of sucrose, D-glucose, and D-fructose were mon-
itored with respect to change in temperature and sugar concentra-
tion. Sugar OH and CH related vibrations were identied by anal-
ysis of the spectra of sugar solutions in deuterium oxide (D
2
O), and
of sucrose-d
8
solutions in D
2
O. Absorption spectra were explained
in terms of the second and third overtones of OH stretching vibra-
tions and the third overtone of CH
2
and CH stretchings. In deuter-
ated solutions, CH and CH
2
higher overtone vibration bands be-
came apparent. The major spectral effect of decreased temperature
or increased sugar concentration was a decrease in absorbance at
960 nm and an increase in absorbance at 984 nm, interpreted as an
increase in the degree of H bonding. Partial least-squares (PLS)
calibrations on sugar concentrations (with spectra collected at sev-
eral sample temperatures) relied strongly on the 910 nm sugar CH
related bands, whereas calibrations on temperature depended
equally on all OH associated vibrations (750, 840, 960, and 985 nm).
Index Headings: Band assignment; Chemometric analysis; D-glucose;
D-fructose; Spectroscopy; Sugars; Sucrose; Short-wavelength near-
infrared; SW-NIR; Water.
INTRODUCTION
Electronic transitions are responsible for the strong ab-
sorption of the UV and visible spectral region (200780
nm) by biological material. Molecular vibrations, partic-
ularly by OH and CH bonds, are responsible for strong
absorption bands in the near- (NIR; 11002500 nm) and
mid- (MIR; 250025 000 nm) infrared spectral regions
by biological material, with the absorption bands of water
OH groups dominating in hydrated material. Studies of
aqueous samples therefore require very thin or diluted
samples (most MIR and NIR studies typically use path-
lengths of approximately 15 mm and 0.1 to 1.0 mm, re-
spectively). In contrast, the short-wave near-infrared
(SW-NIR; 7801100 nm) region is weakly absorbed by
biological material, with this absorption typically asso-
ciated with second, third, and fourth overtone vibrations.
Therefore, SW-NIR spectroscopy can be applied to thick-
er samples (typical aqueous cell pathlengths of 1 to 100
mm) and can yield information on the internal attributes
of biological material (e.g., sugar and water content of
fruit, as by Kawano et al.,
15
and blood glucose levels in
humans
6,7
).
Unfortunately, the absorption bands in the SW-NIR re-
gion are typically broad (approximate bandwidth of 50
nm), overlapped, and difcult to assign. It is possible to
differentiate between the SW-NIR spectra of crystalline
sucrose and glucose, but the SW-NIR spectra of sugar
Received 9 November 2001; accepted 27 September 2002.
* Author to whom correspondence should be sent.
solutions are practically identical.
8
In consequence, rela-
tively little attention has been given to the issue of band
assignments in the SW-NIR spectral region, in compari-
son to the 11002500 nm and higher regions. Most re-
ports of calibration of SW-NIR spectra of high-moisture
samples (e.g., sugar and water content of intact fruit)
have adopted a purely statistical approach. However, the
robustness of such calibrations may be poor (e.g., the
robustness of fruit sugar content calibrations across va-
rieties and growing districts was not satisfactory
911
).
Basing the calibration on spectroscopically signicant
wavelengths is a logical approach to improving the ro-
bustness of the calibration, as highlighted by Maeda and
co-workers in their study of hydrogen bonds of water and
alcohols.
12,13
In the current study, we therefore aim to
conrm the assignment of SW-NIR spectral features of
water and the simple sugars (sucrose, glucose, and fruc-
tose), to support calibration development.
The band assignments of the major water and sugar
(OH and CH) vibrations are summarized in Table I. The
chemical environment of each OH and CH bond in water
and sugar molecules is different, so the effective absorp-
tion bands are relatively wide, even at the fundamental
frequency. Spectral features relevant to the SW-NIR in-
clude the second and third overtones of OH vibrations at
around 1000 nm (10 000 cm
21
) and 760 nm (13 167
cm
21
), respectively, and the OH combination bands at
around 1100 and 840 nm, respectively (Table I).
1417
SW-
NIR features relevant to the sugar CH groups include
second overtone stretching bands between 1100 and 1200
nm and a third overtone band at 910 nm (Table I). A CH
2
third overtone is reported at 930 nm.
1520
The SW-NIR spectra of sugars in aqueous solutions are
difcult to interpret due to strong water OH absorption
bands. Further, changes in chemical environment, partic-
ularly with respect to the degree of hydrogen bonding,
can cause shifts in the stretching and bending frequencies
of the OH bonds.
16,21,22
Thus, sugar OH absorption fre-
quencies are sensitive to water concentration, sugar con-
centration, and temperature, but are relatively insensitive
to pH and ionic strength.
2326
Given the importance of an understanding of hydrogen
bonding in the liquid water to the interpretation of spectra
of sugars, a brief summary follows. Most of the numer-
ous published NIR studies on water structure have sub-
scribed to either a mixture or a continuum model for
water structure.
8,12,2736
Mixture models assume the exis-
tence of discrete water species, differentiating in the num-
ber of H bonds per water molecule. Continuum models
assume that the hydrogen bonds are uniformly distributed
140 Volume 57, Number 2, 2003
TABLE I. Literature-reported band assignments for major OH and CH vibrations for simple sugars and water. Vibrational bands of
sugars are better dened in the MIR than in the NIR, with overtone and combination bands largely overlapped, appearing as one broad
band in NIR absorbance spectra. Therefore, the vibrational frequencies in the MIR region are reported as a spectral range, while the
bands in the NIR are reported as a single value (center of the band), except where several values have been reported in the literature. We
report both wavenumber and wavelength values, as these two different scales are traditionally used by MIR and NIR spectroscopists,
respectively.
Tentative assignment Fundamental
Vibrational frequency overtones
1st 2nd 3rd References
OH stretching
OH combinations
CH stretching
nm
cm
21
nm
cm
21
nm
cm
21
28603120
32003500
19202080
48005200
33003470
28803000
14101440
69507100
1100
9090
16001800
55506250
970
10 300
840
11 900
11001230
81009100
738
13 550
910
11 000
8, 16, 44, 45
14, 16
1517
CH combinations
CH
2
stretching
CH
2
combinations
nm
cm
21
nm
cm
21
nm
cm
21
21002352
42504750
34603500
28802910
23102325
43004330
17201765
56705820
1215
8230
930
10 750
16, 18
16, 19, 20
16
OH, CH, and CH
2
deformations nm
cm
21
nm
cm
21
69008330
11 11125 000
12001450
400900
22502320
43104440
24002600
38404170
18502120
47205400
14, 16
and become distorted rather than broken when tempera-
ture increases.
37,38
There is general agreement that there
are two main water species present in liquid water,
8,12,33,3538
although mixture models propose presence of other water
species as well.
8,12,33
One of the major species (S
1
species,
H bound to only one other water molecule in mixture
models;
8,12,33
with deformed hydrogen bonds in continu-
um models
35,36,38
) prevails at higher temperatures, while
other species (with a higher degree of hydrogen bonding
(S
3
);
8,12,33
or more structured hydrogen bonds
35,36,38
) pre-
vail at lower temperatures. The NIR band positions of
these two species are slightly different. Water species pre-
sent at higher temperatures are expected to resonate at
higher energies (shorter wavelengths) due to a decrease
in hydrogen bonding and an increase in the strength of
covalent bonds.
37
Repeated dominance of the lower wavelength peaks at
higher temperatures was reported in an FT-NIR study of
water bands in the spectral region 25008300 nm (tem-
perature range 2080 8C).
36
Further, a combination band
of ice at 1990 nm was reported to shift to 1930 nm with
a temperature change from 270 to 0 8C.
33
The rst over-
tone of OH stretching in water has been reported to shift
from 1450 to 1420 nm, with a residual shoulder at 1450
nm, from 1460 to 1424 nm, and from 1461 to 1418 nm,
with a temperature change from 20 to 80 8C,
39
6 to 80
8C,
37
and 5 to 85 8C,
12
respectively. The bandwidth of the
rst overtone of the OH stretching absorption peak was
also reported to decrease (from 4.8 nm to 2.1 nm) with
extent of hydrogen bonding, as temperature was varied
from 270 to 0 8C.
33
Few published reports deal with the effect of H bond-
ing on SW-NIR spectral features. An exception is the
work of Abe et al., who reported that the absorption peak
of the second overtone of OH stretching vibrations of
sucrose solutions was shifted from 1040 to 970 nm as
temperature increased from 2120 to 75 8C.
8
It is therefore expected that sugar calibrations based on
OH vibration bands will be sensitive to temperature. As
single wavelength calibrations of sugar concentration
(1.560% w/w of individual sugars in aqueous solution)
in the region 15501850 nm
40
are presumably based
around the rst overtone of the CH stretching vibration,
they should be more robust with respect to temperature
than a single wavelength calibration based on a combi-
nation of OH stretching and OH deformation (e.g., 2270
nm,
8
as used by Chang et al.
41
to predict sugar content
of pure sugar solutions and fruit juices; R
2
5 0.982, stan-
dard error of prediction, SEP 5 0.62).
In the current study we conrm the OH and CH band
assignments within the SW-NIR for sugars and water,
with reference to the effect of concentration and temper-
ature on absorption frequency, and relate the weightings
of partial least-squares (PLS) based calibrations to these
assignments.
MATERIALS AND METHODS
Sucrose, D-glucose, and D-fructose (analytical grade;
BDH) were dissolved in RO water to obtain 3% and 5
through 50% in increments of 5% (w/w) sugar solutions.
All solutions were prepared 24 h prior to use to ensure
that an equilibrium of all sugar forms in solutions was
established. Sucrose solutions (10, 20, 30, 40, and 50%
w/w) were also prepared using deuterium oxide (99.93%
isotopic purity; supplied by the Australian Nuclear Sci-
ence and Technology Organisation) as a solvent. To min-
imize hydrogen exchange between the sugar and D
2
O in
this study, spectra were collected within 0.5 h of disso-
lution of sucrose into D
2
O.
Sucrose-d
8
was prepared by lyophilizing sucrose so-
lution in D
2
O (ve fold excess of D
2
O).
42,43
This process
was repeated three times to ensure a complete conversion.
Methanol-d (99.5% isotopic purity; Aldrich) was used to
precipitate a white powder of sucrose-d
8
(m.p. 196 8C;
67% reaction yield, not optimized).
Near-infrared spectra (4002500 nm) were collected in
triplicate for each sample, at each temperature (1550 8C,
with 5 8C increments) using a scanning spectrophotom-
eter (model 6500, FOSS-Pacic), equipped with a trans-
APPLIED SPECTROSCOPY 141
FIG. 1. (a) Absorbance spectra of water (solid line), 50% sucrose in
water (dash-dash), D
2
O (dot-dot), and 50% sucrose in D
2
O (dot-dash).
Absorbance values of D
2
O and 50% sucrose in D
2
O were multiplied by
six to present all spectra on the same y scale; (b) SW-NIR second
derivative of absorbance spectra of water (solid line), 20% w/w sucrose
(dot-dot), glucose (dash-dash), and fructose (dot-dash) solutions; (c)
50% sucrose (dot-dot), glucose (dash-dash), and fructose (dot-dash) so-
lutions.
mittance detector and a transport module with tempera-
ture-controlling unit. This instrumentation records at 2
nm steps, and with a resolution of ;10 nm (FWHM).
Each recorded spectra represented an average of 32 scans.
An in-house-built cuvette with a pathlength of 26 mm
was used throughout this study. An empty cuvette was
used as a reference.
For data collection NSAS v3.30 software was em-
ployed and The Unscrambler v7.6 software package was
used for regression analysis. The optimal number of prin-
cipal components to be used in regression was automat-
icall y determined by The Unscrambler software.
GRAMS32 v5.2 and kg-2d softwares were used for the
production of two-dimensional (2D) correlation spectra,
with the average spectrum used as a reference.
Second-derivative spectra were obtained by using the
SavitzkyGolay second derivative, a gap of 18 nanome-
ters, and a second-order polynomial. Second-derivative
difference spectra of 10, 20, and 50% sucrose-d
8
in D
2
O
were obtained by subtraction of second-derivative D
2
O
spectra multiplied by 0.90, 0.80, and 0.50, respectively,
from the corresponding spectra of deuterated (10, 20, and
50%) sugar solutions.
RESULTS AND DISCUSSION
Absorbance Spectra. The SW-NIR absorbance spec-
tra of water and sugars (sucrose, D-glucose, and D-fruc-
tose) in water solution were relatively similar and fea-
tureless, except for a major absorption band around 970
nm, as shown for the 50% sucrose spectra in Fig. 1a (as
previously reported, Table I). As this band was absent in
spectra of D
2
O, but present in sucrose dissolved in D
2
O
(Fig. 1a), it is associated with OH vibrations. This band
is ascribed to the second overtone of OH stretching vi-
brations of sugars and water.
16,27,44,45
Second Derivative of Absorbance Spectra: Over-
tones of OH Vibrations. Peak resolution improves sig-
nicantly with use of the second derivative of absorbance
data. For example, the dominant band of the SW-NIR
absorbance spectra of water (at 970 nm) can be resolved
as a composite of an absorption peak at 960 nm (negative
peak in second-derivative plot), with a shoulder at 984
nm (Figs. 1b and 2a). The shoulder is presumably due to
the OH stretching vibration of a hydrogen bonded spe-
cies, because it decreased signicantly in favor of the 960
nm peak as temperature was increased from 15 to 50 8C
(Fig. 3a; band assignments summarized in Table III). Rel-
atively minor absorption peaks appeared at 840 nm (OH
combination band) and at 772 and 740 nm (two bands
for the third overtone of OH stretching vibrations) (Figs.
2a and 3a).
27
The dominant band of the SW-NIR second-derivative
absorption spectra of sugar solutions was also located at
960 nm (Fig. 1b; second overtone of a stretching vibra-
tion of OH
27
). Decreasing temperature of sugar solutions
was associated with a decrease in intensity of the 960 nm
peak in favor of the 984 nm peak (Fig. 3b), as noted for
water spectra. The temperature-sensitive spectral regions
of sugar in water, as indicated by the synchronous 2D
correlation spectra of 50% sucrose solution over a tem-
perature range 1550 8C (Fig. 3c), were consistent with
OH-related vibrational bands (750, 840, 960, and 985
nm). The effect of increasing temperature is attributed to
a decrease in hydrogen bonding (sugarwater, waterwa-
ter, and sugarsugar) within solutions.
Increase in sugar concentration was also associated
with a decrease of the band at 960 nm and an accentu-
ation of a shoulder at 984 nm (Figs. 1b, 1c, and 2a).
Indeed, in spectra of 50% sugar solutions, the 984 nm
second-derivative peak was greater (more negative) than
the 960 nm peak (Figs. 1c and 2a). The SW-NIR spectra
of the three sugars differed primarily in terms of the 984
nm peak, with accentuation of the 984 nm band relative
to the 960 nm band with increasing sugar concentration
greater in D-fructose and D-glucose than sucrose (Figs. 1b
and 1c).
These observations are in agreement with the interpre-
tation of the 984 nm band as a composite of the second
overtones of OH stretching vibrations of sugar OH and
142 Volume 57, Number 2, 2003
FIG. 2. The effect of concentration on SW-NIR second derivative of
absorbance spectra of sucrose in (a) H
2
O and in (b) D
2
O. Spectra of
water (solid line in (a)) and D
2
O (solid line in (b)), 5% sucrose (dot-
dash in (a), but hidden by the water spectrum), 20% sucrose (dot-dot),
and 50% sucrose (dash-dash) are presented. Spectra were collected
within 0.5 h of dissolution of sucrose into D
2
O. (c) Second-derivative
difference spectra of 10% (solid line), 20% (dot-dot), and 50% (dash-
dash) sucrose-d
8
in D
2
O. Spectra were obtained by subtraction of D
2
O
spectrum multiplied by 0.90, 0.80, and 0.50, respectively, from the sec-
ond-derivative spectra of deuterated sucrose-d
8
solutions.
FIG. 3. (a) The effect of temperature on SW-NIR second derivative of
absorbance spectra of water. Representative spectra collected at 15 (sol-
id line), 35 (dash-dash), and 50 (dot-dot) 8C are presented. (b) The effect
of temperature on SW-NIR second derivative of absorbance spectra of
20% sucrose. Representative spectra collected at 20 (solid line) and 50
(dot-dot) 8C are presented. (c) Synchronous 2D correlation spectra of
50% sucrose solution over the temperature range 1550 8C, with the
average of these spectra used as the reference.
of water OH hydrogen bonded to sugars and to other
water molecules. The intensity of the band associated
with hydrogen bonding is smaller in sucrose than in glu-
cose and fructose, consistent with the smaller number of
sucrose molecules in the same w/w % solutions and ster-
ical hindrance of sucrose OH groups. The difference in
molecular weight of sucrose (MW 5 342.30) relative to
glucose and fructose (MW 5 180.16) is such that a 50%
w/w solution of sucrose will have 1.89 times fewer mol-
ecules than glucose or fructose, and in total, 1.18 times
fewer OH groups available for H-bonding than 50% glu-
cose and fructose solutions (calculation based on an as-
sumption that most of the glucose and fructose would be
in glucopyranose and fructofuranose forms, respectively).
Steric hindrance of sucrose OH groups occurs because of
free rotation of pyranose and furanose rings around the
glycoside linkage.
The spectral area 730775 nm is confused, as it can
contain peaks for the third overtones of OH stretching
vibrations,
27
fourth overtones of CH stretching vibra-
tions,
16
and combination bands of OH vibrations.
27
With
increase in sucrose concentration, the 740 and 770 nm
peaks followed the same pattern as the 960 and 984 nm
peaks, i.e., the intensity of the 740 peak decreased, while
the intensity of the 770 nm peak increased (Fig. 2a). The
same change in peak intensities as for 960/984 nm was
observed with temperature alterations (Figs. 3a and 3b).
Therefore, we attribute the 740 and 770 nm bands to the
third overtones of OH stretching vibrations (of low and
high H bonded species, respectively) (Table III).
APPLIED SPECTROSCOPY 143
Second Derivative of Absorbance Spectra: Over-
tones of CH Vibrations. An absorption peak at around
910 nm was evident with increase in sugar concentration
to 50% (Figs. 1c and 2a). This band is assigned to the
third overtone of sugar CH stretching vibrations.
16,46,47
An
absorbance band around 930 nm has been ascribed to the
third overtone of CH
2
stretching in sugars.
16,48
However,
in the current study this band was masked by large bands
associated with OH vibrations in spectra of aqueous sugar
solutions.
Deuteration Studies. The OD vibrations occur at
higher wavelengths than the corresponding OH vibrations
due to the mass difference of D compared to H. Thus,
the use of D
2
O as a solvent allows for an assignment of
the second overtones of the OH stretching vibrations of
sucrose and also allows for detection of the sucrose CH
related vibrations, due to the disappearance of the large
water-related 960 nm peak, which masks the CH bands
in aqueous sucrose solutions. Second-derivative spectra
of sucrose in D
2
O solutions were dominated by bands
located at 762, 906, 954, and 976 nm (Fig. 2b). Signal
intensity was stronger as sucrose concentration was in-
creased, except at 762 nm. The bands at 954 and 976 nm
are assigned to the second overtones of sucrose OH
stretching vibrations (low and high H bonded states, re-
spectively), while the 906 nm band is ascribed to a third
overtone of the CH stretching vibrations.
Intensity of the 762 nm peak decreased with addition
of sugar (Fig. 2b), so it is likely to be associated with
D
2
O vibrations. It is most likely one of the OD combi-
nation bands. Bayly et al.
27
reported the second overtone
of the OD stretching vibrations at 1348 nm, and there-
fore, the third overtone would be expected at around 1020
nm and the fourth at about 820 nm. We observed bands
at 1294 and 1330 nm that could represent the second
overtone peaks of OD stretching (two H bonded species)
(spectra not shown). The absorption band at about 1040
nm (Fig. 3c) could represent the third overtone of OD
stretching. However, the band at 762 nm is unlikely to
represent the fourth overtone of OD stretching on the
bases of its intensity relative to that of the 1040 nm band
and as it is expected to appear at a higher wavelength
(about 820 nm). Further, it lacks the double-band feature
(for less and more H bonded species) characteristic of
OD (OH) vibration bands.
The 1040 nm band could also be related to CH and
CH
2
vibrations of sucrose.
16
The band intensity increased
with the addition of sugar (Fig. 2b) and is therefore likely
to represent combination bands of the rst overtone of
CH stretching and rst overtone of CH
2
deformation vi-
brations of sucrose in sugar solutions.
16
Absorption due to the third overtone of CH
2
stretching
in sugars, expected at around 930 nm,
14,16
was not ap-
parent in any of the spectra, including the spectra of deu-
terated sucrose in D
2
O (i.e., in the absence of OH groups)
(Fig. 2c). This band would be of small intensity and could
easily be obscured by other features (e.g., 914 nm CH
band).
In the SW-NIR spectra of sucrose-d
8
dissolved in D
2
O,
two main absorption bands were located at 772 and 914
nm (Fig. 2c). Bands of smaller intensity were placed at
1040 and 742 nm, while the bands belonging to the OH
vibration overtones (strongest intensity bands at 954 and
976 nm in Fig. 2b) were not present. Intensities of all
four bands increased with increase in sugar concentration
(Fig. 2c) and are therefore associated with CH vibrations.
This conclusion is consistent with the assignment of the
band at about 910 nm to the third overtone of the CH
stretching and the assignment of the band at 1040 nm to
combination bands of the rst overtone of CH stretching
and rst overtone of CH
2
deformation vibrations.
16
As noted earlier, assignment of the 772 and 742 nm
bands is difcult, as several vibrations have their over-
tone bands in this spectral region. The band at 772 nm
is assigned to the fourth overtone of CH stretching vi-
bration, due to its increase in intensity with increase in
sucrose-d
8
concentration. The band placed at 742 nm
most likely belongs to the fourth overtone of CH
2
stretch-
ing in the spectra of sucrose-d
8
solutions. We ascribed a
band at 740 nm to the third overtone of OH stretching
(Fig. 2a) in the spectra of sucrose in water. However, in
sucrose-d
8
in D
2
O there are no OH groups present, and
if deuteration was not complete, we would expect a larger
peak at 960 nm. Bands assigned to the fourth overtones
of CH and CH
2
stretching vibrations were not visible in
the spectra of sugar solutions in water (Figs. 1b, 1c, and
2a), due to their close proximity of the third overtone
bands of OH stretching vibrations.
Chemometrics: Concentration Effect. Partial least-
squares calibrations on the concentration of sugar solu-
tions (at 30 8C) using SW-NIR absorbance or the second
derivative of absorbance data (7001050 nm) exhibit
high correlation coefcients (R
2
$ 0.989 in all cases)
(Table IIA). The number of principal components needed
to describe the spectral changes with changes in concen-
trations varied from one to three (the latter only for D-
fructose absorbance spectral data). Root mean square er-
ror of cross validation (RMSECV) for the sucrose solu-
ti ons was t he l owest for second-derivat ive data
(RMSECV 5 0.67, on a population SD 5 13.6) (Table
IIA). For all sugars assessed, the RMSECV values based
on second-derivative data were approximately one half of
those generated using absorbance data (Table IIA).
B-coefcients for the PLS absorbance concentration
calibrations (constant temperature) (Fig. 4a) were strong-
ly and positively weighted at 910 nm for fructose, su-
crose, and, to a lesser extent, glucose solutions. For su-
crose, the coefcients were fairly constant over the spec-
tral window 890 to 920 nm (Fig. 4a). Other positive B-
coefcients were found in the region of 750820 nm for
sucrose, D-glucose, and D-fructose. Negative coefcients
occurred over a window from 840 to 880 nm. The largest
negative B-coefcient for D-fructose was observed at
1012 nm (Fig. 4a). The corresponding coefcient was
placed at 1016 nm for D-glucose and 1022 nm for su-
crose. These weightings are spectroscopically relevant in
terms of the second overtone vibration of CH stretching
at 910 nm and the third overtone of sugar OH stretching
at 760 nm.
Chemometrics: Temperature Effect. Temperature of
the sugar solutions was well correlated with absorbance
features, with PLS R
2
above 0.92 and an RMSECV of
;3 8C (for solutions over the range 1550 8C, SD 12 8C)
(Table IIC). The differences between temperature cali-
brations of the three sugar solutions are not likely to be
signicant, and probably relate more to errors in the mea-
144 Volume 57, Number 2, 2003
TABLE II. Calibration statistics using absorbance and/or second derivative of absorbance (d
2
Absorbance) data over the wavelength range
7001050 nm for (A) sugar concentration (% w/w) at a constant temperature of 30 8C; (B) sugar concentration (% w/w) for spectra collected
at a range of temperatures from 5 to 50 8C, with temperature included as a spectral variable in the calibration data set; and (C) temperature,
using spectra of 20% (w/w) sugar solutions varied in temperature from 15 to 50 8C.
# samples Mean SD # PC R R
2
SEC RMSECV
(A) Sugar concentration, constant temperature
Sucrose
Absorbance
d
2
Absorbance
60
60
25.6 13.6 2
2
0.995
0.999
0.989
0.998
1.30
0.64
1.41
0.67
D-Glucose
Absorbance
d
2
Absorbance
D-Fructose
Absorbance
d
2
Absorbance
36
36
36
36
23.2
23.2
16.6
16.6
2
1
3
1
0.997
0.997
0.998
0.994
0.994
0.994
0.996
0.989
1.13
1.16
0.84
1.66
1.22
1.27
1.00
1.72
(B) Sugar concentration, temperature varied
Sucrose (absorbance)
D-Glucose (absorbance)
D-Fructose (absorbance)
429
362
362
26.5
23.1
23.1
13.5
16.6
16.3
4
3
4
0.997
0.987
0.993
0.993
0.987
0.993
1.10
1.83
1.29
1.12
1.86
1.32
(C) Temperature
Sucrose (absorbance)
D-Glucose (absorbance)
D-Fructose (absorbance)
28
30
30
30.4
30.0
30.0
12.1
11.7
11.7
2
2
2
0.983
0.974
0.962
0.966
0.948
0.926
1.89
2.44
2.66
2.19
2.64
3.15
FIG. 4. PLS1 B-coefcients for calibrations based on absorbance data
for concentration of sucrose (solid line), D-glucose (dot-dot), and D-
fructose (dash-dash) aqueous solutions, collected from solutions at (a)
a constant temperature (30 8C), (b) over a range of temperatures from
15 to 50 8C, and (c) over a range of temperatures from 15 to 50 8C,
with temperature added as an x-data point in the calibration.
surement of sample temperatures lower than room tem-
perature (a feature of the temperature-controlling unit em-
ployed). For example, the fructose solution temperature
calibration improved (R
2
5 0.974; RMSECV 5 2.34)
when measurements at 15 8C were omitted from the cal-
ibration.
The PLS regression B-coefcients for a temperature
calibration on absorbance data of 20% sucrose solutions
consisted of large positive coefcients at 950 and 980
nm, with smaller coefcients at around 750 and 840 nm
(Fig. 4b). The B-coefcients for temperature calibrations
involving D-glucose and D-fructose solutions mirrored
those for sucrose; however, the largest coefcients were
observed at about 920 and 990 nm (Fig. 4b). The weight-
ings at 740, 840, and around 960980 nm are attributable
to changes in the position and intensities of OH stretching
vibration bands, with temperature impacting on the de-
gree of H bonding. A temperature calibration of a D
2
O
solution would be expected to weight around the 1040
and 760 nm regions.
Given the sensitivity of OH stretching vibrations to
temperature, it is to be expected that change in temper-
ature will impact heavily on the performance of a cali-
bration for sugar concentrations developed on samples at
a given temperature. Development of a calibration across
a range of temperatures should result in a de-emphasis of
those areas of the spectrum associated with OH stretch-
ing, favoring those areas associated with other spectral
bands of the sugars (Fig. 4c). The prominent band in this
context is the 910 nm band that corresponds to the third
overtone of CH stretching vibrations. In calibrations de-
veloped across a range of temperatures, the largest pos-
itive B-coefcients were observed at 910 nm (particularly
for D-fructose) (Fig. 4c). Calibration performance on sug-
ar concentration was degraded when spectra were col-
lected across a range of temperatures relative to calibra-
tions developed at a single temperature (data not shown),
but when temperature was included as an x-data point,
calibration performance was similar to that achieved for
spectra collected at a constant temperature (Table IIA, B).
APPLIED SPECTROSCOPY 145
TABLE III. Band assignments for major OH and CH vibrations
for simple sugars and water from the current study.
Tentative assignment Overtone
Molecular environment
Sucrose
in H
2
O
Sucrose
in D
2
O
Sucrose-d
8
in D
2
O
CH/CH
2
combination
OH stretching (more H bonded)
OH stretching
CH stretching
OH combination
. . .
second
second
third
. . .
984
960
910
840
1040
976
954
906
1040
914
CH stretching
OH stretching (more H bonded)
OD combination
CH
2
stretching
OH stretching
fourth
third
. . .
fourth
third
770
740
762
730
772
762
742
CONCLUSION
The major SW-NIR spectral features of sugar solutions
are related to OH and CH vibrations (Table III). The OH
stretching vibration is very sensitive to H bonding and
thus to temperature. To have spectroscopic relevance, cal-
ibrations of sugar content of intact fruit should be based
on OH and CH features, emphasising the CH feature to
be less sensitive to temperature.
ACKNOWLEDGMENTS
Funding support was provided through a CQU merit grant. We thank
Dr. Yan Wang for for providing the kg-2d software. We dedicate this
manuscript to our colleague Peter Lawson, who passed away July 12th,
2001.
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