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Article
Fourier Transform Infrared Spectroscopy (FT-IR)
and Simple Algorithm Analysis for Rapid and
Non-Destructive Assessment of Developmental
Cotton Fibers
Yongliang Liu 1, * and Hee-Jin Kim 2
1 Cotton Structure & Quality Research Unit, United States Department of Agriculture (USDA),
Agricultural Research Service (ARS), New Orleans, LA 70124, USA
2 Cotton Fiber Bioscience Research Unit, United States Department of Agriculture (USDA),
Agricultural Research Service (ARS), New Orleans, LA 70124, USA; HeeJin.kim@ars.usda.gov
* Correspondence: Yongliang.liu@ars.usda.gov; Tel.: +1-504-286-4445

Received: 15 May 2017; Accepted: 20 June 2017; Published: 22 June 2017

Abstract: With cotton fiber growth or maturation, cellulose content in cotton fibers markedly
increases. Traditional chemical methods have been developed to determine cellulose content, but
it is time-consuming and labor-intensive, mostly owing to the slow hydrolysis process of fiber
cellulose components. As one approach, the attenuated total reflection Fourier transform infrared
(ATR FT-IR) spectroscopy technique has also been utilized to monitor cotton cellulose formation, by
implementing various spectral interpretation strategies of both multivariate principal component
analysis (PCA) and 1-, 2- or 3-band/-variable intensity or intensity ratios. The main objective of this
study was to compare the correlations between cellulose content determined by chemical analysis
and ATR FT-IR spectral indices acquired by the reported procedures, among developmental Texas
Marker-1 (TM-1) and immature fiber (im) mutant cotton fibers. It was observed that the R value,
CIIR , and the integrated intensity of the 895 cm−1 band exhibited strong and linear relationships with
cellulose content. The results have demonstrated the suitability and utility of ATR FT-IR spectroscopy,
combined with a simple algorithm analysis, in assessing cotton fiber cellulose content, maturity, and
crystallinity in a manner which is rapid, routine, and non-destructive.

Keywords: Fourier transform infrared spectroscopy; attenuated total reflection; cellulose; fiber
secondary cell wall biosynthesis; principal component analysis; algorithm analysis

1. Introduction
As one of the most important and widely grown crops in the world, cotton is a well-traded
agricultural commodity primarily for its naturally produced textile fiber [1]. Commercial cotton fibers
are harvested from cotton plants. In biological terms, cotton fibers are the dried cell walls of formerly
living cells. They initiate from an ovary of the flower and grow into a mature seed-containing cotton
boll within approximately 1.5~2 months. Fiber growth consists of four overlapping but distinctive
phases: initiation, primary cell wall (PCW) formation for fiber elongation, secondary cell wall (SCW)
biosynthesis for cellulose deposition and cell wall thickening, and maturation [2,3]. The day of
flowering is referred to as anthesis, and the term “days post anthesis” (DPA) is commonly used to
describe the cotton fiber growth (Figure 1). The fiber cells initiate at 0 DPA and then elongate to reach
a fiber length of 22~35 mm within 20 to 25 DPA. The secondary cell wall synthesis starts around
15 to 22 DPA and continues for an additional 30 to 40 days until the maturation phase, when the fibers
dehydrate and collapse into flattened and twisted ribbons. The apparent cellulose amount in cotton
fibers increases with cotton fiber growth, leading to significant differences in chemical, physical, and

Sensors 2017, 17, 1469; doi:10.3390/s17071469 www.mdpi.com/journal/sensors


Sensors 2017, 17, 1469 2 of 13

Sensors 2017, 17, 1469 2 of 13


ribbons. The apparent cellulose amount in cotton fibers increases with cotton fiber growth, leading
to significant differences in chemical, physical, and compositional attributes during cotton fiber
development. Over the during
compositional attributes years, cotton
diversified and well-defined
fiber development. Over fiber testing
the years, methods,
diversified andsuch as high
well-defined
volume instrument
fiber testing (HVI)
methods, suchand advanced
as high volume fiber information
instrument (HVI)system (AFIS), have
and advanced been developed
fiber information to
system
reflect
(AFIS),these
havechanges routinelytoinreflect
been developed the cotton
theseindustry
changes [4].
routinely in the cotton industry [4].

Schematicof
Figure1.1.Schematic
Figure ofgeneral
generalcotton
cottonfiber
fibergrowth
growthat
atvarious
variousdays
dayspost
postanthesis
anthesis (DPA).
(DPA).

Mature
Mature fibers
fibers areare composed
composed mostly mostly ofof cellulose
cellulose (88.0–96.5%),
(88.0–96.5%), followed
followed by by such
such non-cellulosic
non-cellulosic
constituents
constituents as proteins (1.0–1.9%), waxes (0.4–1.2%), pectins (0.4–1.2%), inorganics (0.7–1.6%), and
as proteins (1.0–1.9%), waxes (0.4–1.2%), pectins (0.4–1.2%), inorganics (0.7–1.6%), and
other
other substances
substances(0.5–8.0%)
(0.5–8.0%)[5]. [5].Analysis
Analysisof ofcellcellwall
wallcompositions
compositionsof offibers
fibersfromfrom thethe early
early stages
stages of of
elongation through the period of the SCW formation was achieved
elongation through the period of the SCW formation was achieved by a set of extraction, separation, by a set of extraction, separation,
and
and isolation
isolation steps,
steps, prior
prior to to chemical
chemical and and instrumental
instrumental determination
determination of of targeted
targeted components
components [6–9]. [6–9].
Cotton
Cotton fiber
fiber cellulose
cellulose isis not not easily
easily dissolved
dissolved in in most
most solvent,
solvent, andand thethe extraction
extraction and and separation
separation
process
process in in conventional
conventional methods methods experience
experience significant
significant drawbacks
drawbacks that that include
includethe thetedious
tediousprocedures
procedures
of
of optimal solvent and temperature selection as well as extracted specimen identification. As
optimal solvent and temperature selection as well as extracted specimen identification. As the
the need
need
for
for more
more rapid
rapid approaches
approaches inceases,inceases, aa number
number of of available
available analytical
analytical techniques,
techniques, including
including Fourier
Fourier
transform
transform infrared
infrared (FT-IR)
(FT-IR) spectroscopy,
spectroscopy, differential
differential scanning
scanning calorimeter
calorimeter (DSC),(DSC), thermogravimetric
thermogravimetric
analysis
analysis (TGA),
(TGA), and and pyrolysis-gas
pyrolysis-gas chromatography/mass
chromatography/mass spectroscopy spectroscopy (GC/MS) (GC/MS)methods methods[10–15],
[10–15],
have been applied to identify cellulose and non-cellulose components
have been applied to identify cellulose and non-cellulose components in cottons. These measurements in cottons. These measurements
provide
provide clear
clearevidence
evidenceofofvarious various components
components in developing
in developing cotton fibersfibers
cotton through the onset
through of SCW
the onset of
synthesis.
SCW synthesis.For example, the appearance
For example, of FT-IR bands
the appearance at 1733bands
of FT-IR cm (C=O
−1
at 1733 stretching
cm − 1 originating
(C=O from
stretching
esters or amides)
originating fromand esters1534 orcm −1 (NH2 deformation −
amides) and 1534 cmcorresponding
1 (NH deformation
2
to proteins or amino acids)
corresponding reflects
to proteins
the presence of such non-cellulosic components as esters and proteins
or amino acids) reflects the presence of such non-cellulosic components as esters and proteins in cotton in cotton fibers [11,13]. Among
these
fiberstechniques,
[11,13]. Among the ATR these FT-IR spectroscopy
techniques, the ATR method FT-IRhasspectroscopy
evolved as an important
method alternative
has evolved to
as an
examine cotton fiber development, because it requires minimal
important alternative to examine cotton fiber development, because it requires minimal sample sample preparation by ATR sampling
on a small by
preparation bundle of cotton on
ATR sampling fibers as bundle
a small little asof0.5 mg, fibers
cotton permits routine
as little as 0.5analysis rapidly
mg, permits and
routine
non-destructively, and is easy to operate [10,11,13–20].
analysis rapidly and non-destructively, and is easy to operate [10,11,13–20].
Different
Different spectral
spectral interpretation
interpretation approachesapproaches have have been
been applied
applied to to acquire
acquire useful
useful information
information
from
from FT-IR
FT-IR measurement.
measurement. ItIt includesincludes the the direct
direct use useof of1-band
1-bandintensity,
intensity, thethe estimation
estimation of of 2-
2- or
or 3-band
3-band
intensity ratios, and the implementation of a chemometrical or multivariate
intensity ratios, and the implementation of a chemometrical or multivariate tool known as principal tool known as principal
component
component analysis
analysis (PCA)
(PCA) [10,11,13–21].
[10,11,13–21]. The Theresults
resultshave
haveindicated
indicated that that both
both simple
simple algorithms
algorithms and and
PCA
PCA patterns
patternscan canbe beused
usedto tomonitor
monitorthe thetransition
transitionfrom fromPCWPCWto toSCW
SCWbiosyntheses
biosynthesesin incotton
cottonfibers.
fibers.
Interestingly,
Interestingly, simple
simple algorithms
algorithms were were observed
observed to to have
have the
the ability
ability toto detect
detect thethe subtle
subtle discrepancies
discrepancies
in
in fibers
fibers older
older than
than25 25DPA
DPA amongamong respective
respective fibers
fibers grown
grown in in planta
planta or or in
in culture
culture [17].
[17]. In
In the
the current
current
work, we performed the PCA and simple algorithm examination
work, we performed the PCA and simple algorithm examination of ATR FT-IR spectra representing the of ATR FT-IR spectra representing
the developmental
developmental immature
immature fiberfiber
(im) (im)
mutantmutant
fibersfibers
and itsand its near-isogenic
near-isogenic wildTexas
wild type type Marker-1
Texas Marker-1(TM-1)
(TM-1) fibers,
fibers, and and compared
compared the correlations
the correlations betweenbetween
cellulosecellulose content determined
content determined by conventional
by conventional chemical
chemical
analysis and analysis and respective
respective ATR FT-IRATR FT-IR
spectral spectralacquired
responses responses acquiredspectral
by various by various spectral
interpretation
interpretation strategies. Notably, simple algorithm analysis
strategies. Notably, simple algorithm analysis of ATR FT-IR spectra has been developed to estimateof ATR FT-IR spectra has been
developed
fiber cellulose to maturity
estimate and fibercrystallinity
cellulose maturity
simultaneously and crystallinity
in developing simultaneously
cottons [14–17]. in developing
cottons [14–17].
Sensors 2017, 17, 1469 3 of 13

2. Materials and Methods

2.1. Texas Marker-1 (TM-1) and Immature Fiber (im) Mutant Fibers
Two cotton near-isogenic lines (NILs), TM-1 and im, were grown side by side in a field of
USDA-ARS (New Orleans, LA, USA) in 2011. At day of post anthesis (DPA), cotton flowers were
tagged. Two biological replicates of cotton bolls were taken from different cotton plants at 10, 17, 24, 28,
33, 37 and 44 DPA. Fibers at each DPA were collected from 10 to 30 bolls in fifty plants for each biological
replication by manually removing the seeds, prior to drying in 40 ◦ C incubator. The soil type was
Aquent dredged over alluvium in an elevated location to provide adequate drainage. Harvested fibers
were kept in a dark storage room with a constant temperature (23 ± 1 ◦ C) and relative humidity
(50 ± 10%), and their ATR FT-IR spectra were collected in March 2014. The ~2.5 years gap between the
harvest of fibers and the collection of FT-IR spectra was due to the beginning of collaboration between
two authors in March 2014.

2.2. Cellulose Content


Fiber cellulose content at each developmental stage was measured by the modified Updegraff
method [22]. Briefly, 10 mg of cut fibers were placed into 5 mL of reacti-vials. Non-cellulosic materials
in fibers were hydrolyzed with acetic-nitric reagent (a mixture of 73% acetic acid, 9% nitric acid and
18% water). The remaining cellulose was hydrolyzed with 67% sulfuric acid (v/v) and measured by a
colorimetric assay with anthrone and by the use of Avicel PH-101 (FMC, Rockland, ME, USA) as a
cellulose standard. The average cellulose content for each fiber was obtained from three replications.
It took at least 2 days to measure cellulose content for each sample mostly due to slow hydrolysis
process of fiber cellulose component.

2.3. ATR FT-IR Spectral Collection and Data Analysis


All fibers were scanned by an FTS 3000MX FT-IR spectrometer (Varian Instruments, Randolph,
MA, USA) equipped with a ceramic source, KBr beam splitter, and deuterated triglycine sulfate
(DTGS) detector and attenuated total reflection (ATR) attachment. The ATR sampling device utilized a
DuraSamplIR single-pass diamond-coated internal reflection accessory (Smiths Detection, Danbury,
CT, USA), and a consistent contact pressure was applied by way of a stainless steel rod and an electronic
load display. During the data collection, cautions were taken to make sure that the window (2 mm
in diameter) of the ATR sampling devise was covered completely by fiber samples. At least five
measurements for individual fiber samples, by re-sampling at different locations across entire sample,
were collected over the range of 4000–600 cm−1 at 4 cm−1 and 16 co-added scans. All spectra were
given in absorbance units and no ATR baseline correction was applied.
Importing the spectra to the GRAMS IQ application in Grams/AI (Version 9.1, Thermo Fisher
Scientific, Waltham, MA, USA), the mean spectrum was taken for each sample and then was smoothed
with a Savitzky–Golay function (polynomial = 2 and points = 11). The spectra were normalized by
dividing the intensity of the individual band in the 1800–600 cm−1 region with the average intensity in
this 1800–600 cm−1 region, and subsequent PCA characterization was performed in the 1800–600 cm−1
IR region, with mean centering (MC) and Savitzky–Golay first-derivative (2 degrees and 13 points)
spectral pretreatment, as well as with the leave-one-out cross-validation method. With the use of
the Grams/AI program, integrated intensities of 4 bands at 2900, 1372, 895, and 664 cm−1 were
estimated in the respective ranges of 3000 to 2800, 1410 to 1290, 910 to 875, and 684 to 650 cm−1 from
normalized spectra. Separately, the spectral set was loaded into Microsoft Excel 2007 to execute simple
algorithm analysis.
Sensors 2017, 17, 1469 4 of 13
Sensors 2017, 17, 1469 4 of 13

3. Results
3. Results and
and Discussion
Discussion

3.1. DPA-Dependent
3.1. DPA-DependentCellulose
CelluloseContent
ContentofofDevelopmental
DevelopmentalFibers
Fibers
The DPA-dependent
The DPA-dependentcellulose
cellulose content
content between
between the
the two
two sets
sets of
of developmental
developmental fibers
fibers in
in Figure
Figure 22
shows aa similar
shows similar increasing
increasing pattern
pattern between
between the
the two
two types
types of fibers at
at various
various developmental
developmental stages.
For either
For either TM-1 or im im fiber,
fiber, cellulose
cellulosecontent
contentincreases
increasesrapidly
rapidlyand
andlinearly from
linearly from10 10
to to
37 37
DPA,
DPA,as
expected.
as expected.

100

● TM-1
80 ○ im
Cellulose Content (%)

60

40

20

0
5 10 15 20 25 30 35 40 45 50
DPA (days)

Figure 2.
Figure 2. Cellulose
Cellulose content
content against
against DPA
DPA for
for developmental
developmental Texas
TexasMarker-1
Marker-1(TM-1)
(TM-1)((●) and immature
) and immature
fiber (im) (○) fibers by the modified Updegraff method [22].
fiber (im) (#) fibers by the modified Updegraff method [22].

3.2. ATR
3.2. ATR FT-IR
FT-IRSpectral
SpectralCharacteristics
CharacteristicsofofDevelopmental
DevelopmentalTM-1
TM-1Cotton
CottonFibers
Fibers
With fiber
With fiberDPA
DPAprogressing
progressing(Figure(Figure1), 1),apparent
apparentspectral
spectralintensity
intensity increases
increases oror decreases
decreases in in the
the
mid infrared
mid infrared(mid-IR)
(mid-IR)region
regionof of1800–600
1800–600cm cm−1 are
−1 anticipated for
are anticipated for these
these developmental
developmental TM-1 TM-1 cotton
cotton
fibers (Figure 3). A thorough examination of ATR FT-IR spectral feature
fibers (Figure 3). A thorough examination of ATR FT-IR spectral feature reveals that TM-1 fiber exhibits reveals that TM-1 fiber
aexhibits a nearly spectral
nearly identical identicalpattern
spectraltopattern
im fibertoatim fiberdevelopmental
same at same developmental
point. TM-1point.
fibersTM-1
andfibers and
im fibers,
im fibers, alike, demonstrate growth results in the dominant production
alike, demonstrate growth results in the dominant production of major common chemical component of major common chemical
component
in cotton fibers,in cotton fibers,
cellulose. cellulose.
Spectral Spectral
intensity intensity
changes changes
in Figure in Figure
3 are in good3 are in good with
agreement agreement
those
reported earlier [11,13,17], and characteristic band assignments have been summarized in Table in
with those reported earlier [11,13,17], and characteristic band assignments have been summarized 1.
Table 1.the
Briefly, Briefly, the vibration
vibration at 1740 cm at−1740 cm−1 is assigned
1 is assigned to the C=O to the C=O stretching
stretching mode ofmode of carbonyl
carbonyl groups groups
due to
due to and
lipids, lipids, and a band
a broad broadcentered
band centered at 1620atcm 1620
−1 cm −1 is mainly attributed to the OH bending mode
is mainly attributed to the OH bending mode of
of adsorbed
adsorbed water.
water. Bands
Bands in in
thethe region
region of of 1500–1200
1500–1200 cmcm representthe
−1−1represent thecontributions
contributionsof ofboth
bothCH CH2
2
deformations and C–O–H bending vibrations, and those bands in
deformations and C–O–H bending vibrations, and those bands in the 1200–900 cm region originate the 1200–900 cm − −1 region originate
1
fromthe
from thecoupling
couplingmodesmodesof ofC–O
C–Oand andC–CC–Cvibrations.
vibrations.The Thebands
bandsbetween
between800 800and
and700700cm cm −−1 are likely
1 are likely
due to two crystal forms (I α and Iβ) of cotton cellulose. In addition, there are intense absorptions
due to two crystal forms (Iα and Iβ ) of cotton cellulose. In addition, there are intense absorptions
betweenthe
between the3600
3600andand 27502750
cm−cm
−1 regions that are assignable to the O–H and C–H stretching
1 regions that are assignable to the O–H and C–H stretching vibrations
vibrations
(not shown). (not shown).
A comparison
A comparison of of the
theincreasing
increasingorordecreasing
decreasingintensity
intensity of of
these
thesebands between
bands between shorter DPADPA
shorter (for
example, 10 DPA) and longer DPA (for example, 37 DPA) fibers is tabulated
(for example, 10 DPA) and longer DPA (for example, 37 DPA) fibers is tabulated in Table 1. In general, in Table 1. In general,
intensities of
intensities ofthe
thebands
bandsatat1740,
1740,1620,
1620,1545,
1545,1405,
1405,andand1236 1236cmcm aswell
−1−1as wellasasthose
thoseininthethe850–750
850–750cm cm −−1
1
region decrease, while those at 1425, 1365, 1335, 1315, 1200, 1158, 1104,
region decrease, while those at 1425, 1365, 1335, 1315, 1200, 1158, 1104, 1055, 1028, 985, 895 and 1055, 1028, 985, 895 and 662
cm−1cm
662 increase.
−1 increase.Subjective interpretation
Subjective interpretationof these spectra
of these in Figure
spectra in Figure3 cannot be be
3 cannot applied
applied to compare
to compare or
assess the degree of fiber secondary wall biosynthesis in a semi-quantitative
or assess the degree of fiber secondary wall biosynthesis in a semi-quantitative way. way.
Sensors 2017, 17, 1469 5 of 13
Sensors 2017, 17, 1469 5 of 13

Figure 3.3. Attenuated


Attenuatedtotal
totalreflection
reflectionFourier
Fourier transform
transform infrared
infrared (ATR
(ATR FT-IR)
FT-IR) spectral
spectral response
response of
of TM-1
TM-1
fibers fibers to various
to various DPA. DPA.

Table
Table 1.
1. Characteristic
Characteristic ATR
ATR FT-IR spectral bands of shorter and longer DPA
DPA fibers
fibers [11,13,17]
[11,13,17] *.*.

10 DPA Fiber 37 DPA Fiber Band Assignment


10 DPA Fiber 37 DPA Fiber Band Assignment
1740 (s) 1740 (w) C=O stretching
1740 (s) 1740 (w) C=O stretching
1620 (s) HOH bending of adsorbed water + amide I
1620 (s) HOH bending of adsorbed water + amide I
16201620
(w) (w) HOHHOH bending
bendingof adsorbed
of adsorbedwater
water
1545
1545(s)(s) 15451545
(w) (w) AmideAmide II II
1425(w)
1425 (w) 14251425
(s) (s) CHCH 2 scissoring
2 scissoring
1405 (s) 1405 (w) O–H deformation
1405 (s) 1405 (w) O–H deformation
1365 (w) 1365 (s) C–H bending
1365 (w)
1335 (w) 1365 (s)
1335 (s) C–H CHbending
2 wagging
1335
1315(w)(w) 13351315
(s) (s) CHCH 2 wagging
2 wagging
1236
1315 (w)(s) 13151236
(s) (w) O–H deformation
CH2 wagging or N–H deformation
1200(s)
1236 (w) 1236 1200
(w) (s) O–H deformation C–O orstretching
N–H deformation
1158 (w) 1158 (s) C–O–C stretching
1200 (w)
1104 (w)
12001104
(s) (s) C–OC–O stretching
stretching
1158 (w)
1055 (w) 1158 (s)
1055 (s) C–O–C C–O stretching
stretching
1104
1028(w)(w) 11041028
(s) (s) C–OC–O stretching
stretching
985(w)
1055 (w) 1055 985
(s) (s) C–OC–O stretching
stretching
895 (w) 895 (s) β-glycosidic linkage
1028 (w) 1028 (s) C–O stretching
662 (w) 662 (s) O–H out-of-plane bending
985 (w) 985 (s) C–O stretching
* Parentheses indicate the relative ATR FT-IR spectral intensities between 10 and 37 DPA fibers: s = strong, w = weak.
895 (w) 895 (s) β-glycosidic linkage
662 (w) 662 (s) O–H out-of-plane bending
3.3. Correlation between Cellulose Content and ATR FT-IR Spectral Response of Developmental Fibers
* Parentheses indicate the relative ATR FT-IR spectral intensities between 10 and 37 DPA fibers:
3.3.1.s PC1
= strong,
Scorew and
= weak.
the Correlation with Cellulose Content
As usual, PCA
3.3. Correlation were
between performed
Cellulose to understand
Content the similarity
and ATR FT-IR or dissimilarity
Spectral Response of ATR FT-IR
of Developmental Fibersspectra
that are indicative of fiber growth for TM-1 and im fibers. The plot of the first principal component
(PC1)PC1
3.3.1. score vs. and
Score DPAthe
in Correlation
Figure 4 provides a good Content
with Cellulose visualization of the sample distribution among
two sets of fibers. The first two PCs accounted for 89.5% of the total variation, with the PC1 explaining
As usual, PCA were performed to understand the similarity or dissimilarity of ATR FT-IR
80.4% of the variation. For both TM-1 and im fibers, PC1 scores increase rapidly between 10 and 24 DPA
spectra that are indicative of fiber growth for TM-1 and im fibers. The plot of the first principal
before reaching the relatively constant PC1 score. It implies that the 10 DPA fibers are composed
component (PC1) score vs. DPA in Figure 4 provides a good visualization of the sample distribution
of PCW constituents, whereas the 24 DPA fibers consist of SCW components. The observation is
among two sets of fibers. The first two PCs accounted for 89.5% of the total variation, with the PC1
consistent with accumulated knowledge suggesting that elongating fibers at 10 DPA contain no SCW
explaining 80.4% of the variation. For both TM-1 and im fibers, PC1 scores increase rapidly between
cellulose whereas thickening fibers at 24 DPA are composed of more SCW cellulose. Notably, the PC1
10 and 24 DPA before reaching the relatively constant PC1 score. It implies that the 10 DPA fibers are
scores are nearly independent of fiber DPA when DPA is more than 25 days, suggesting that the overall
composed of PCW constituents, whereas the 24 DPA fibers consist of SCW components. The
contributions from spectral intensity variations in the 1800–600 cm−1 region during this period of fiber
observation is consistent with accumulated knowledge suggesting that elongating fibers at 10 DPA
maturation are probably insignificant.
contain no SCW cellulose whereas thickening fibers at 24 DPA are composed of more SCW cellulose.
Notably, the PC1 scores are nearly independent of fiber DPA when DPA is more than 25 days,
Sensors 2017, 17, 1469 6 of 13

suggesting that the overall contributions from spectral intensity variations in the 1800–600 cm−1
Sensors 2017, 17, 1469 6 of 13
region during this period of fiber maturation are probably insignificant.

0.4

● TM-1
0.2 ○ im

PC1 Score
0.0

-0.2

-0.4

-0.6
5 10 15 20 25 30 35 40 45 50
DPA (days)

Figure4.4.First
Figure First principal
principal component
component (PC1)against
(PC1) score score DPA
against
fromDPA from spectra
ATR FT-IR ATR FT-IR spectra of
of developmental
developmental TM-1 (●)
TM-1 ( ) and im (#) fibers. and im (○) fibers.

The plot of relating cellulose content to PC1 score for developmental TM-1 and im fibers in
The plot of relating cellulose content to PC1 score for developmental TM-1 and im fibers in
Figure 5 offers a better visualization of relationship between two parameters. In general, cellulose
Figure 5 offers a better visualization of relationship between two parameters. In general, cellulose
content increases with PC1 score up to 24 DPA by referring to the Figure 4. Remarkably, the
content increases with PC1 score up to 24 DPA by referring to the Figure 4. Remarkably, the correlation
correlation between the two parameters could be described more accurately by a polynomial (with
between the two parameters could be described more accurately by a polynomial (with the order of 2)
the order of 2) regression than by a linear regression, as shown in Figure 5. It implies that cellulose
regression than by a linear regression, as shown in Figure 5. It implies that cellulose content has a
content has a weak linear relationship with PC1 score.
weak linear relationship with PC1 score.

0.3
Polynomial regression
0.2 y = -9E-05x2 + 0.016x - 0.527
R² = 0.92
0.1

0.0
PC1 Score

-0.1
Linear regression
-0.2 y = 0.0062x - 0.384
R² = 0.82
-0.3

-0.4

-0.5

-0.6
0 10 20 30 40 50 60 70 80 90 100
Cellulose Content (%)

Figure5.5.PC1
Figure PC1 score
score vs. cellulose
vs. cellulose content
content of developmental
of developmental TM-1 and TM-1 and Linear
im fibers. im fibers. Linear and
and polynomial
polynomial
(with (with
the order the
of 2) order of 2)were
regressions regressions
appliedwere
to allapplied to all fiber samples.
fiber samples.

3.3.2. IR
3.3.2. IR maturity
maturity and
and Crystallinity
Crystallinity and
andthe
theCorrelation
Correlationwith
withCellulose
CelluloseContent
Content
Compared to
Compared tothe
thePCA
PCAapproach
approachthat
thatutilized
utilizedaanumber
numberof of624
624datapoints
datapointsor orvariables
variables(from
(fromthe
the
1800to
1800 to600
600cm cm IR
−−1
1 IR region with 1.949
region with 1.949 cm
cm interval
−1
− 1 interval in
in this
this study),
study), previously proposed 3-band
previously proposed 3-band based
based
ratios or algorithms were assessed on the same spectral dataset. The three bands
ratios or algorithms were assessed on the same spectral dataset. The three bands at 1800, 1315, and at 1800, 1315, and
1236 cm −−11 were used to assess the ratio R values and these values were found to be related with fiber
1236 cm were used to assess the ratio R values and these values were found to be related with fiber
growth[17].
growth [17].The
Theratio
ratioR Rvalues
values were
were assessed
assessed by the
by the algorithm
algorithm of Rof R 1315
= (A = (A− A−1800
1315 A1800 )/(A
)/(A 1236 − A1800),
1236 − A1800 ),
where A1800 , A1315 , and A1236 represent the respective band intensities centered at 1800, 1315, and
Sensors
Sensors 2017,
2017, 17,
17, 1469
1469 77of
of13
13
Sensors 2017, 17, 1469 7 of 13

where A1800, A1315, and A1236 represent the respective band intensities centered at 1800, 1315, and 1236
where A1800 , A1315, and−1A1236 represent the respective band intensities centered at 1800, 1315, and 1236
cm
1236 cm−The
−1 [17]. 1 [17].
1800
Thecm1800band −1 band
cmwas offsetwas
to be zeroto
offset inbe
intensity
zero insubjectively. The 1315 cm
intensity subjectively. The −1 band cm−1
1315arises
cm−1 [17]. The 1800 cm−1 band was offset to be zero in intensity subjectively. The 1315 cm−1 band arises
from
band the CH
arises 2 wagging
from the CH2mode thatmode
wagging is increasing in intensity
that is increasing with DPA,
in intensity withwhile
DPA, the 1236
while the cm cm−1
−1 band
1236
from the CH2 wagging mode that is increasing in intensity with DPA, while the 1236 cm−1 band
assigned to O–H/N–H
band assigned to O–H/N–H deformation decreases
deformation in intensity.
decreases As As
in intensity. shown in in
shown Figure
Figure 6, 6,the
theRR value
value
assigned to O–H/N–H deformation decreases in intensity. As shown in Figure 6, the R value
increases
increases along with the DPA, DPA, both
both directly
directlyand
andobviously.
obviously.The Thepattern
patternsuggests
suggeststhat
thatthis
thisalgorithm
algorithm is
increases along with the DPA, both directly and obviously. The pattern suggests that this algorithm
is capable
capable ofof reflecting
reflecting thethe continuous
continuous cellulose
cellulose production
production in fibers
in fibers older
older than
than 24 DPA,
24 DPA, which which
waswas
not
is capable of reflecting the continuous cellulose production in fibers older than 24 DPA, which was
not apparent
apparent from from
the the
PC1PC1
scorescore of PCA
of the the PCA result
result in Figure
in Figure 4. 4.
not apparent from the PC1 score of the PCA result in Figure 4.
2.5
2.5
2.3 ● TM-1
2.3
2.1 ○● im
TM-1
2.1 ○ im
1.9
Values

1.9
Values

1.7
1.7
1.5
RR

1.5
1.3
1.3
1.1
1.1
0.9
0.9
0.7
0.7 5 10 15 20 25 30 35 40 45 50
5 10 15 20 25 30 35 40 45 50
DPA (days)
DPA (days)
Figure
Figure6.6.RRvalue
valueagainst
againstDPA
DPA from
from ATR
ATR FT-IR spectra of developmental TM-1 ((●)) and im (#)
(○) fibers.
fibers.
Figure 6. R value against DPA from ATR FT-IR spectra of developmental TM-1 (●) and im (○) fibers.
Unlike
Unlike the trend in Figure 5, there is no visual disparity in Figure 7 between aa polynomial
Unlike the
the trend
trend inin Figure
Figure 5,
5, there
there is is no
no visual
visual disparity
disparity inin Figure
Figure 77 between
between a polynomial
polynomial
regression
regression and aalinear
and linearregression.
regression.AA strong
strong linear
linear correlation
correlation (R 2 (R
=
2 = 0.96)
0.96) is is within
within the the expectation
expectation since
regression and a linear regression. A strong linear correlation (R = 0.96) is within the expectation
2
since
two two independent
independent methods
methods should
should be be consistent
consistent in in representing
representing cottonfiber
cotton fibergrowth.
growth. This
This result
result
since two independent methods should be consistent in representing cotton fiber growth. This result
demonstrates
demonstrates that
that the
the ATR FT-IR technique, along with this simple algorithm, can be used for
demonstrates that the ATR
ATR FT-IR
FT-IR technique,
technique, along
along with
with this
this simple
simple algorithm,
algorithm, cancan be
be used
used for
for
monitoring
monitoring fiber
fiber cellulose
cellulose content
content and
and fiber
fiber maturity,
maturity, rapidly
rapidly and
and non-destructively.
non-destructively.
monitoring fiber cellulose content and fiber maturity, rapidly and non-destructively.
2.5
2.5
2.3
2.3 Polynomial regression
2.1 y = Polynomial regression
-1E-05x2 + 0.015x + 0.852
y = -1E-05x 2 + 0.015x + 0.852
2.1 R² = 0.96
R² = 0.96
1.9
1.9
Values

1.7
Values

1.7
1.5
Linear regression
RR

1.5
yLinear regression
= 0.014x + 0.872
1.3
y = 0.014x + 0.872
R² = 0.96
1.3
R² = 0.96
1.1
1.1
0.9
0.9
0.7
0.7 0 10 20 30 40 50 60 70 80 90 100
0 10 20 30 40 50 60 70 80 90 100
Cellulsoe Content (%)
Cellulsoe Content (%)
Figure 7. R value vs. cellulose content of developmental TM-1 and im fibers. Linear and polynomial
7. R
Figure 7. R value vs. cellulose content of developmental TM-1 and im
value vs. im fibers.
fibers. Linear and polynomial
polynomial
(with the order of 2) regressions were applied to all fiber samples.
(with the order of 2) regressions were applied to all fiber samples.
(with the order of 2) regressions were applied to all fiber samples.
A further interest was to estimate IR crystallinity (CIIR) by using the respective IR bands at 708,
A further
A further interest
interest was
was toto estimate IR
IR crystallinity
crystallinity (CI
(CIIR)) by using
using the respective IR bands at
at 708,
730, and 800 cm−1 [15–17]. Twoestimate
bands at 708 and 730 cm−1 IR areby theto
assignable respective IRin
Iβ cellulose bands 708,
crystalline
730, and
730, and 800 cm−−11 [15–17]. Two bands at 708 and 730 cm− −11 are assignable to Iβ cellulose in crystalline
[15–17]. Two bands at 708 and 730 cm are assignable to Iβ cellulose
part and 800
to Icm
α cellulose in amorphous part, respectively, whereas the 800 cm
in crystalline
−1 band was used to
part and to Iα cellulose in amorphous part, respectively, whereas the 800 cm−1 band was used to
Sensors 2017, 17, 1469 8 of 13

Sensors 2017, 17, 1469 8 of 13


Sensors 2017, 17, 1469 8 of 13
part and to Iα cellulose in amorphous part, respectively, whereas the 800 cm−1 band was used to offset
offset
in in intensity
intensity subjectively.
subjectively. Plot ofPlot
CI of CIIR against
against DPA in DPA in Figure
Figure 8 reveals
8 reveals a steadya steady CIIR increase
CIIR increase for
for each
offset in intensity subjectively. PlotIRof CIIR against DPA in Figure 8 reveals a steady CIIR increase for
each set,
fiber fiberasset, as anticipated.
anticipated. The general
The general trendtrend in Figure
in Figure 8 is similar
8 is similar to in
to that that in Figure
Figure 6 more6 more so than
so than that
each fiber set, as anticipated. The general trend in Figure 8 is similar to that in Figure 6 more so than
that which
which is in Figure
is in Figure 4, when
4, when examining
examining the CIthe CI
values
IR values
for for
the the
fibersfibers
olderolder
than than
24 24
DPA.
IR CIIR values for the fibers older than 24 DPA. DPA.
that which is in Figure 4, when examining the
100
100

● TM -1
● TM -1
80 ○ im
80 ○ im
(CI
Crystallinity(CI ))
IRIR
60
60
IRIRCrystallinity

40
40

20
20

0
0
5 10 15 20 25 30 35 40 45 50
5 10 15 20 25 30 35 40 45 50
DPA (days)
DPA (days)

Figure 8.8. IR crystallinity


crystallinity (CIIR)) against
against DPA from ATR
ATR FT-IR spectra
spectra of developmental
developmental TM-1 ((●) and
Figure 8. IR
Figure IR crystallinity (CI
(CIIR
IR) against DPA from
DPA from ATR FT-IR
FT-IR spectra of
of developmental TM-1
TM-1 (●)) and
and
im
im (○) fibers.
im (#)
(○) fibers.
fibers.

Figure 9 shows a high linear relationship (R222 = 0.92) between the degree of CIIR and cellulose
Figure 99 shows
Figure shows aahigh
highlinear
linearrelationship
relationship(R (R == 0.92)
0.92) between
between the
the degree
degreeof ofCI
CIIRIR and
and cellulose
cellulose
content. There is an insignificant difference in Figure 9 between a polynomial regression and a linear
content. There is
content. is an
an insignificant
insignificant difference
difference in in Figure
Figure 99 between
between aa polynomial
polynomial regression
regression and and aa linear
linear
regression, and the pattern in Figure 9 is much closer to that in Figure 7, than that which is in Figure 5.
regression, and the pattern in Figure
regression, and the pattern in Figure 9 is much closer to
9 is much closer to thatthat in Figure 7, than that which is in Figure 5.
A strong linear correlation (R222 = 0.92) is much expected, because cotton fiber crystallinity increases
A strong
A stronglinear
linearcorrelation
correlation(R(R == 0.92)
0.92) isis much
much expected,
expected, because
because cotton
cotton fiber
fiber crystallinity
crystallinity increases
increases
with cotton fiber growth [14,16–18]. This result verifies that the ATR FT-IR technique, along with this
with cotton fiber growth [14,16–18]. This result verifies that the ATR FT-IR technique,
with cotton fiber growth [14,16–18]. This result verifies that the ATR FT-IR technique, along with along with this
this
simple algorithm, can also be used for assessing fiber crystallinity, rapidly and non-destructively.
simple algorithm,
simple algorithm,cancanalso
alsobe
beused
usedfor
forassessing
assessingfiber
fibercrystallinity,
crystallinity,rapidly
rapidlyandandnon-destructively.
non-destructively.

100
100

Polynomial regression
80 Polynomial regression
80 y = -0.0025x22 + 0.838x + 10.91
y = -0.0025x + 0.838x + 10.91
))
IRIR

R² = 0.93
(CI

R² = 0.93
Crystallinity(CI

60
IRIRCrystallinity

60

40
40
Linear regression
Linear regression
y = 0.592x + 14.62
y = 0.592x + 14.62
20 R² = 0.92
20 R² = 0.92

0
0
0 10 20 30 40 50 60 70 80 90 100
0 10 20 30 40 50 60 70 80 90 100
Cellulose Content (%)
Cellulose Content (%)
Figure9.
9. IR crystallinity
crystallinity (CIIR
IR) vs. cellulose
cellulose content of developmental
developmental TM-1 and and im fibers.
fibers. Linear and
and
Figure 9. IR
Figure IR crystallinity(CI
(CIIR)) vs.
vs. cellulose content
content of
of developmental TM-1TM-1 and im
im fibers. Linear
Linear and
polynomial
polynomial (with the order of 2) regressions were applied to all fiber samples.
polynomial(with
(withthe
theorder
orderof of2)2)regressions
regressionswere
wereapplied
appliedtotoall
allfiber
fibersamples.
samples.

3.3.3. The 1372 and 2900 cm−1 Band Based IR Crystallinity and the Correlation with Cellulose Content
3.3.3. The 1372 and 2900 cm−1 Band Based IR Crystallinity and the Correlation with Cellulose Content
Two bands at 1372 and 2900 cm−1 based IR Ratio 1372/2900 have been reported to indirectly
Two bands at 1372 and 2900 cm−1 based IR Ratio 1372/2900 have been reported to indirectly
estimate the percentage crystallinity in cellulose materials [14,21]. The 1372 cm−1 band is attributed to
estimate the percentage crystallinity in cellulose materials [14,21]. The 1372 cm−1 band is attributed to
the C–H bending vibration, while the 2900 cm−1 band is assigned to the C–H stretching mode. As
the C–H bending vibration, while the 2900 cm−1 band is assigned to the C–H stretching mode. As
shown in Figure 10, the IR ratio 1372/2900 elevates rapidly from 10 to 28 DPA, and is consistent with
shown in Figure 10, the IR ratio 1372/2900 elevates rapidly from 10 to 28 DPA, and is consistent with
Sensors 2017, 17, 1469 9 of 13

3.3.3. The 1372 and 2900 cm−1 Band Based IR Crystallinity and the Correlation with Cellulose Content
Two bands at 1372 and 2900 cm−1 based IR Ratio 1372/2900 have been reported to indirectly
estimate the percentage crystallinity in cellulose materials [14,21]. The 1372 cm−1 band is attributed
to the2017,
Sensors C–H17,bending
1469 vibration, while the 2900 cm−1 band is assigned to the C–H stretching mode. 9 of 13
As shown
Sensors 2017, in
17, Figure
1469 10, the IR ratio 1372/2900 elevates rapidly from 10 to 28 DPA, and is consistent 9 of 13
with different cotton varieties reported by other researchers [14]. The pattern in Figure
different cotton varieties reported by other researchers [14]. The pattern in Figure 10 resembles the10 resembles
different
the
PC1 PC1
score cotton
score
of PCAof varieties
PCA reportedinby
representation
representation inother
4,researchers
Figure
Figure 4, other
other [14].
thanthan The
those
those pattern
in 6 in
Figures
in Figures 6Figure
and 8. 8.10 resembles the
and
PC1 score of PCA representation in Figure 4, other than those in Figures 6 and 8.
1.0
1.0
● TM-1
0.8 ○● im
TM-1
0.8 ○ im
1372/2900
1372/2900

0.6
0.6
Ratio

0.4
Ratio

0.4
IR IR

0.2
0.2

0.0
0.0 5 10 15 20 25 30 35 40 45 50
5 10 15 20DPA
25 (Days)
30 35 40 45 50
DPA (Days)
Figure
Figure 10.
10. IR
IR Ratio
Ratio 1372/2900
1372/2900against
againstDPA
DPAfrom
fromATR
ATR FT-IR
FT-IR spectra
spectra of
of developmental
developmental TM-1
TM-1 (●)
( ) and
and imim
(#) fibers. The ratios were calculated from integrated intensities of two bands at 1372 and 2900 and
Figure 10. IR Ratio 1372/2900 against DPA from ATR FT-IR spectra of developmental TM-1 (●)
(○) fibers. The ratios were calculated from integrated intensities of two bands at 1372 and 2900 cm −1. −im
1.
cm
(○) fibers. The ratios were calculated from integrated intensities of two bands at 1372 and 2900 cm−1.
Much like the tendency in Figure 5, cellulose content increases with the IR Ratio 1372/2900 in
Much
Much like
likethe tendency ininFigure 5, cellulose content increases with the the
IR Ratio 1372/2900 in the
the manner of a the tendencycurve
polynomial Figure
over a5,linear
cellulose
curvecontent
(Figureincreases
11). with IR Ratio 1372/2900 in
manner of a polynomial curve over a linear curve (Figure 11).
the manner of a polynomial curve over a linear curve (Figure 11).
1.0
1.0 Polynomial regression
y = -8E-05x 2 + 0.0158x + 0.0401
Polynomial regression
0.8 y = -8E-05x2 +=0.0158x
R² 0.98 + 0.0401
(172/2900)

0.8 R² = 0.98
(172/2900)

0.6
Ratio

0.6
Ratio

0.4 Linear regression


Band

0.4 yLinear
= 0.0076x + 0.163
regression
Band

R² = 0.92
y = 0.0076x + 0.163
IR IR

0.2 R² = 0.92
0.2

0.0
0.0 0 10 20 30 40 50 60 70 80 90 100
0 10 20 30 40 Content
Cellulose 50 60 (%)
70 80 90 100

Cellulose Content (%)


Figure 11. IR Ratio 1372/2900 vs. cellulose content of developmental TM-1 and im fibers. Linear and
Figure 11.
Figure 11. IR
polynomial IR Ratio
Ratio
(with the 1372/2900
1372/2900 vs.regressions
order of 2) cellulose content of developmental
were applied fibers. Linear and
TM-1 and im fibers.
to all fiber samples. and
polynomial (with
polynomial (with the
the order
order of
of2)
2)regressions
regressionswere
wereapplied
appliedtotoall
allfiber
fibersamples.
samples.
3.3.4. Integrated Intensity of the 895 cm−1 Band and the Correlation with Cellulose Content
3.3.4. Integrated Intensity of the 895 cm−1 Band and the Correlation with Cellulose Content
The band at 895 cm−1 has been assigned to the β-glycosidic linkage in cellulose, and its
The band
integrated at 895
intensity wascm −1 has been assigned to the β-glycosidic linkage in cellulose, and its
found to be linearly correlated with the percentage of cellulose [13] and of
integrated intensity
crystallinity was found
[14]. Slightly to from
differing be linearly correlated in
the relationship with the percentage
a previous of cellulose
report [13], Figure 12[13] and of
indicates
crystallinity [14]. Slightly differing from the relationship in a previous
an obvious linear response of integrated intensity of the 895 cm band to fiber growth.
−1 report [13], Figure 12 indicates
an obvious
A greatlinear
linearresponse of integrated
relationship (R2 = 0.95) intensity
is observedof the 895 cm
between the−1 band to fiber growth.
integrated intensity of the 895 cm−1
A great linear relationship (R 2 = 0.95) is observed between
band and cellulose content (Figure 13). The pattern in Figure 13 is identicalthe integrated intensity
to those of the 7895
in Figures andcm
9.−1
band and cellulose content (Figure 13). The pattern in Figure 13 is identical to those in Figures 7 and 9.
Sensors 2017, 17, 1469 10 of 13

3.3.4. Integrated Intensity of the 895 cm−1 Band and the Correlation with Cellulose Content
The band at 895 cm−1 has been assigned to the β-glycosidic linkage in cellulose, and its
integrated intensity was found to be linearly correlated with the percentage of cellulose [13] and
of crystallinity [14]. Slightly differing from the relationship in a previous report [13], Figure 12
indicates an obvious linear response of integrated intensity of the 895 cm−1 band to fiber growth.
Sensors
A2017, 1469 relationship (R2 = 0.95) is observed between the integrated intensity of the 89510
great17,linear cmof−13
1
Sensors 2017, 17, 1469 10 of 13
band and cellulose content (Figure 13). The pattern in Figure 13 is identical to those in Figures 7 and 9.
0.004
0.004
● TM-1
-1) -1
)


○ TM-1
im
cmcm

0.003
○ im
(895

0.003
(895
Intensity
Intensity

0.002
0.002
Intergrated
Intergrated

0.001
0.001

0.000
0.000 5 10 15 20 25 30 35 40 45 50
5 10 15 20 25 30 35 40 45 50
DPA (days)
DPA (days)
Figure 12. Integrated intensity of the 895 cm−1 band against DPA from normalized ATR FT-IR spectra
Figure 12.
12. Integrated
Integrated intensity
intensity of
of the
the 895 cm−−11band against DPA from normalized ATR FT-IR
895 cm FT-IR spectra
spectra
Figure
of developmental TM-1 (●) and im (○) fibers. band against DPA from
of developmental
of developmental TM-1
TM-1 ((●) and im
) and im (#)
(○) fibers.

0.004
0.004
)

Polynomial regression
-1) -1
cmcm

0.003
y =Polynomial regression
2E-08x2 + 4E-05x + 0.0008
2 + 4E-05x + 0.0008
y = 2E-08xR²
(895

0.003 = 0.88
(895

R² = 0.88
Intensity
Intensity

0.002
0.002
Integrated

Linear regression
Integrated

yLinear regression
= 3E-05x + 0.0002
0.001 y = 3E-05x + 0.0002
R² = 0.95
0.001
R² = 0.95

0.000
0.000 0 10 20 30 40 50 60 70 80 90 100
30 40
Cellulose 50 0
60 (%)
Content 70 80 10
90 100 20
Cellulose Content (%)
Figure 13.
Figure 13. Integrated
Integratedintensity
intensityof
ofthe
the895 cm−−11 band
895cm band vs.
vs. cellulose
cellulose content of developmental
content of developmental TM-1TM-1 and
and
Figure 13. Integrated intensity of the 895 cm−1 band vs. cellulose content of developmental TM-1 and
im fibers.
im fibers. Linear
Linear and
and polynomial
polynomial(with
(withthe
theorder
orderofof2)
2)regressions
regressionswere
wereapplied
appliedtotoall
allfiber
fibersamples.
samples.
im fibers. Linear and polynomial (with the order of 2) regressions were applied to all fiber samples.
3.3.5. Integrated
3.3.5. Integrated Intensity
Intensity of of the
the 664
664 cm
cm−−11Band
Bandand andthe
theCorrelation
Correlationwith
withCellulose
CelluloseContent
Content
3.3.5. Integrated Intensity of the 664 cm−1 Band and the Correlation with Cellulose Content
The band
The bandat at664
664cm cm−−11 has
has been
been ascribed
ascribed to to OH
OH out-of-plane bending mode
out-of-plane bending mode [13,14].
[13,14]. Its
Its integrated
integrated
The band at 664 cm−1 has been ascribed to OH out-of-plane bending mode [13,14]. Its integrated
intensity had
intensity had aa good
good linear
linear relationship
relationship with
with cellulose
cellulose content
content [13]
[13] and
and the
the percentage
percentage of
of crystallinity
crystallinity
intensity had a good linear relationship with cellulose content [13] and the percentage of crystallinity
in cotton
in cotton fibers
fibers [14].
[14]. The
The trend
trend in in Figure
Figure 1414 is
is much
much similar
similar to
to the
the PC1
PC1 score
score of
of PCA
PCA result
result inin Figure
Figure 44
in cotton fibers [14]. The trend in Figure 14 is much similar to the PC1 score of PCA result in Figure 4
and the IR Ratio 1372/2900 in Figure 10, and also agrees with that for other cotton
and the IR Ratio 1372/2900 in Figure 10, and also agrees with that for other cotton varieties [13]. varieties [13].
and the IR Ratio 1372/2900 in Figure 10, and also agrees with that for other cotton varieties [13].
Figure 15, being dissimilar from the patterns in Figures 7, 9 and 13, reveals a similar tendency to
Figure 15, being dissimilar from the patterns in Figures 7, 9 and 13, reveals a similar tendency to
that which is in Figures 5 and 11, when relating the integrated intensity of the 664 cm−1 band to
that which is in Figures 5 and 11, when relating the integrated intensity of the 664 cm−1 band to
cellulose content.
cellulose content.
Sensors 2017, 17, 1469 11 of 13

Figure 15, being dissimilar from the patterns in Figures 7, 9 and 13, reveals a similar tendency
to that
Sensors which1469is in Figures 5 and 11, when relating the integrated intensity of the 664 cm−1 band to
Sensors 2017,
2017, 17,
17, 1469 11
11 of
of 13
13
cellulose content.
0.010
0.010

cm-1-1))
●● TM-1
TM-1

(664cm
0.008
0.008 ○○ im
im
Intensity(664
IntergratedIntensity
0.006
0.006

0.004
0.004
Intergrated

0.002
0.002

0.000
0.000
55 10
10 15
15 20
20 25
25 30
30 35
35 40
40 45
45 50
50
DPA
DPA (days)
(days)

Figure 14.
14. Integrated
Figure14. intensity
Integratedintensity of
intensityof the
ofthe 664
the664
664cm cm
cm−−11band
band against DPA from normalized ATR
ATR FT-IR spectra
−1
Figure Integrated band against
against DPA
DPA from
from normalized
normalized ATR FT-IR spectra
FT-IR spectra
of developmental
ofdevelopmental TM-1
developmentalTM-1 (●)
TM-1((●) and im
andim (○)
im(#) fibers.
(○) fibers.
fibers.
of ) and

0.010
0.010
Polynomial
Polynomial regression
regression
cm-1-1))

yy == 2E-08x 2
2E-08x2 ++ 4E-05x
4E-05x ++ 0.0008
0.0008
(664cm

0.008
0.008 R²
R² == 0.88
0.88
Intensity(664
IntegratedIntensity

0.006
0.006

0.004
Linear
Linear regression
regression
0.004
yy == 8E-05x
8E-05x ++ 0.0013
Integrated

0.0013

R² == 0.90
0.90
0.002
0.002

0.000
0.000
00 20
20 40
40 60
60 80
80 100
100
Cellulose
Cellulose Content
Content (%)
(%)

Figure 15. Integrated intensity of the 664 cm−−11 band


cm band vs.
vs. cellulose
cellulose content
content of
of developmental TM-1 and
−1
Figure 15. Integrated
Figure15. Integratedintensity
intensityof
ofthe
the664
664cm developmental
developmental TM-1 TM-1and
and
im fibers. Linear and polynomial (with
im fibers. Linear and polynomial (withthe the order
theorder
orderofof 2)
of2) regressions
2)regressions were
regressionswere applied
wereapplied to all
appliedtotoall fiber
allfiber samples.
fibersamples.
samples.

4.
4. Conclusions
4. Conclusions
Conclusions
The present
The present
The study
present study demonstrates
demonstrates the
study demonstrates the great
the great potential
great potential of
potential of ATR
of FT-IR
ATR FT-IR
ATR spectroscopy,
FT-IR spectroscopy,
spectroscopy,in in conjunction
inconjunction
conjunction
with
with simple
simple algorithm
algorithm analysis,
analysis, in
in sensing
sensing the
the cellulose
cellulose
with simple algorithm analysis, in sensing the cellulose deposition deposition
deposition during
during cotton
cotton fiber
fiber development.
development.
Upon comparing
Upon comparing
Upon comparing thethe correlations
the correlations between
correlations between cellulose
between cellulose content
cellulose content determined
content determined
determined byby assay
by assay analysis
assay analysis and
analysis and the
and the
the
ATR FT-IR
ATRFT-IR
ATR spectral
FT-IRspectral response
spectralresponse acquired
responseacquired
acquiredbyby multivariate
bymultivariate
multivariatePCA PCA
PCAandand the
andthe proposed
theproposed simple
proposedsimple algorithms,
simplealgorithms,
algorithms, ititit
has
has become
become evident
evident that
that the
the R
R value,
value, CI
CIIR ,, and
and the
the integrated
integrated intensity
intensity of
of the
the
has become evident that the R value, CIIR , and the integrated intensity of the 895 cm band can be
IR 895
895 cm
cm−−1
1
−1 band
band can
can be
be
utilized to
utilized to
utilized estimate
to estimate cotton
estimate cotton fiber
cotton fiber cellulose
fiber cellulose content,
cellulose content, maturity
maturity and
content, maturity and crystallinity,
and in
in aaa rapid,
crystallinity, in
crystallinity, rapid, routine,
rapid, routine, and
routine, and
and
non-destructive
non-destructive manner.
manner.
non-destructive manner.

Acknowledgments:
Acknowledgments:The
Acknowledgments: The authors
Theauthors
authors thank
thank Tracy
Tracy
thank Condon
Condon
Tracy of
of USDA-ARS-SRRC
of USDA-ARS-SRRC
Condon for the for
USDA-ARS-SRRC for the
the technical
technical assistanceassistance
technical in
in collecting
assistance in
the experimental
collecting
collecting the samples. Mention
the experimental
experimental of a product
samples.
samples. Mention
Mentionor of
specific equipment
of aa product
product or does notequipment
or specific
specific constitute adoes
equipment guarantee
does not or warrantyaa
not constitute
constitute
guarantee
guarantee or
or warranty
warranty byby the
the U.S.
U.S. Department
Department ofof Agriculture
Agriculture and
and does
does not
not imply
imply its
its approval
approval toto the
the exclusion
exclusion
of
of other
other products
products that
that may
may also
also be
be suitable.
suitable.
Author
Author Contributions:
Contributions: Y.Y. Liu
Liu conceived
conceived the
the research,
research, collected
collected the
the ATR
ATR FT-IR
FT-IR spectra,
spectra, interpreted
interpreted the
the results,
results,
and
and wrote the manuscript. H.J. Kim conceived the research, coordinated the fiber chemical/property analysis,
wrote the manuscript. H.J. Kim conceived the research, coordinated the fiber chemical/property analysis,
analyzed
analyzed the
the results
results and
and co-wrote
co-wrote the
the manuscript.
manuscript. All
All authors
authors read
read and
and approved
approved the
the final
final manuscript.
manuscript.
Sensors 2017, 17, 1469 12 of 13

by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that
may also be suitable.
Author Contributions: Y. Liu conceived the research, collected the ATR FT-IR spectra, interpreted the results,
and wrote the manuscript. H.J. Kim conceived the research, coordinated the fiber chemical/property analysis,
analyzed the results and co-wrote the manuscript. All authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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