Attenuated Total Reflectance/Fourier Transform Infrared

Studies on the Phase-Separation Process of Aqueous

Solutions of Poly(N-isopropylacrylamide)

ORY RAMON,1 ELLINA KESSELMAN,1 RONEN BERKOVICI,2 YACHIN COHEN,2 YARON PAZ2
1
Department of Food Engineering and Biotechnology, Technion, Haifa 32000 Israel

2
Department of Chemical Engineering, Technion, Haifa 32000 Israel

Received 6 February 2001; revised 9 April 2001; accepted 27 April 2001

Published online 00 Month 2001

ABSTRACT: Temperature-induced phase separation of poly(N-isopropylacrylamide) in

aqueous solutions was studied by attenuated total reflectance (ATR)/Fourier transform
infrared spectroscopy. The main objectives of the study were to understand, on a
molecular level, the role of hydrogen bonding and hydrophobic effects below and above
the phase-separation temperature and to derive the scenario leading to this process.
Understanding the behavior of this particular system could be quite relevant to many
biological phenomena, such as protein denaturation. The temperature-induced phase
transition was easily detected by the ATR method. A sharp increase in the peaks of both
hydrophobic and hydrophilic groups of the polymer and a decrease in the water-related
signals could be explained in terms of the formation of a polymer-enriched film near the
ATR crystal. Deconvolution of the amide I and amide II peaks and the OOH stretch
envelope of water revealed that the phase-separation scenario could be divided, below
the phase-separation temperature, into two steps. The first step consisted of the
breaking of intermolecular hydrogen bonds between the amide groups of the polymer
and the solvent and the formation of free amide groups, and the second step consisted
of an increase in intramolecular hydrogen bonding, which induced a coil– globule
transition. No changes in the hydrophobic signals below the separation temperature
could be observed, suggesting that hydrophobic interactions played a dominant role
during the aggregation of the collapsed chains but not before. © 2001 John Wiley & Sons,
Inc. J Polym Sci Part B: Polym Phys 39: 1665–1677, 2001
Keywords: Fourier transform infrared (FTIR); hydrogen bonding; poly(N-isopro-
pylacrylamide); phase separation

INTRODUCTION mational changes such as coil– globule, coil– helix,

The solution properties of many nonionic water-
soluble polymers are temperature-dependent and
exhibit phase separation of the lower critical so-
lution temperature (LCST) type. During the pro-
cess, the macromolecules exhibit dramatic confor-
Correspondence to: Y. Paz (E-mail: paz@tx.technion.ac.il)
Journal of Polymer Science: Part B: Polymer Physics, Vol. 39, 1665–1677 (2001)
© 2001 John Wiley & Sons, Inc.
and association to supramolecular assembly.1 In
a coil– globule transition (CGT), an expanded flex-
ible polymer chain turns into a collapsed globule2
that precipitates on aggregation. The CGT phe-
nomenon has been studied extensively both theo-
retically and experimentally.3 Several studies
concentrated on polystyrene in various organic
solvents,4 determining the molecular parameters
related to the initial and final states, without
clarifying in detail the crucial role of intermolec-
1665
1666 RAMON ET AL.
ular and intramolecular interactions in polymer–
solvent systems.
An investigation of CGT in water-soluble syn-
thetic polymers can be very useful in elucidating
the mechanisms governing denaturation (folding)
of large proteins.5 A common model polymer for
such studies is poly(N-isopropylacrylamide)
(PNIPA), a polyvinyl polymer, containing hydro-
philic amide groups and hydrophobic isopropyl
groups.6 Because of its side chains, the polymer is
soluble in polar solvents (e.g., water) and low-
polarity solvents such as tetrahydrofuran.5 The
crosslinked PNIPA gel in water undergoes a re-
versible volume-phase transition due to changes
in temperature and solvent composition.7 These
thermoreversible phase-separated systems were
studied for their potential use as intelligent ma-
terials in controlled drug delivery, enzyme immo-
bilization, and separation processes.8 It has been
proposed9 that phase separation in PNIPA solu-
tions is a two-step mechanism, initiated by the
collapse of single chains and advanced by aggre-
gation. According to this explanation, PNIPA
chains in aqueous solutions form expanded coils
at room temperature that undergo a CGT when
the temperature is increased to 32–35 °C (ca. the
␪ temperature). The relative roles of hydrogen
bonding10 and hydrophobic interactions11 in pro-
moting the phase transition are a matter of some
debate.
Although the gross scenario of PNIPA solution
phase separation is well understood, the detailed
scenario of the process is still under debate. More
specifically, the role of water–PNIPA hydrogen
bonding versus the role of intramolecular hydro-
gen bonding is not yet fully understood. Many
methods, including cloud-point determination,12
light scattering,13 thermal analysis, differential
scanning calorimetry (DSC) and microcalorim-
etry,14 rheology,15 fluorescence,16 and NMR,17
have been used to study the phase-separation
phenomena. However, these methods lack the
ability to provide detailed information on a mo-
lecular level. Pioneering studies of aqueous poly-
mer solutions by direct absorption IR spectrosco-
py18,19 suggest that the water structure around a
solute is different than that in bulk water. How-
ever, such measurements are hampered at the
fundamental transitions by the exceptionally
high absorbency of water. Implementation of the
attenuated total reflectance (ATR) setup has
given a way to overcome this obstacle. Quantita-
tive analyses of changes in these spectra with the
temperature or Hydrogen/Deuterium (H/D) isoto-
pic ratio have led to a novel description of the
structure and dynamic of water at a molecular
level.20 The same technique was found to be ben-
eficial for extracting information on polymers,
proteins, and their interactions with water.21 In
two pioneering works, ATR/Fourier transform in-
frared (FTIR) was used in studies related to the
temperature-dependent phase separation of
PNIPA gels22 or PNIPA–water solutions.23 In the
latter study, relevant information at a molecular
level was derived on the role of intermolecular
and intramolecular hydrogen bonding and the en-
hancement of the hydrophobic interactions in this
system.
In this study, the ATR technique was used to
investigate the phase separation of well-defined
aqueous PNIPA solutions. The main objective
was to clarify the mechanisms leading to the
phase-separation phenomenon by interpretation,
on a molecular level, of the role of hydrogen bond-
ing and hydrophobic effects below and above the
phase-separation point and by elucidation of the
role of water in the phase-transition process.
EXPERIMENTAL
Preparation of PNIPA
PNIPA samples were prepared with a redox poly-
merization in an aqueous solution at 4 °C with a
protocol similar to that used by Otake et al.24 The
reagents were N-isopropylacrylamide (NIPA)
monomer (Aldrich Chemical Co., United States),
ammonium persulfate (APS; Merck, Darmstadt,
Germany), and N,N,N⬘,N⬘-tetramethylethyl-
enediamine (TEMED; Aldrich Chemical). The
NIPA monomer was recrystallized from a 1:1 tol-
uene/petrol-ether mixture as described else-
where.25 The polymerization was conducted ac-
cording to the following procedure: 0.689 mL of
NIPA monomer was dissolved in doubly distilled
water, and 2 mL of 4% APS was dissolved sepa-
rately. Then, the two solutions were mixed and
cooled to 4 °C, and nitrogen was bubbled over the
solution to remove oxygen. At that stage, 0.48 mL
of TEMED was added, and polymerization contin-
ued for 3 h under a nitrogen atmosphere. The
polymer solution was then dialyzed for 6 days at 4
°C [the molecular cutoff was a weight-average
molecular weight (Mw) of 14,000] to remove non-
reacted monomers, initiator, and catalyst, and
then the unfractionated polymer was freeze-dried
(Martin Christ, Alpha 1, Germany). The molecu-

Figure 1. DSC peak of an aqueous solution of PNIPA (2 wt %).
lar weight (7 ⫾ 1 MDa) was evaluated from the
determination of the intrinsic viscosity of the
PNIPA aqueous solutions with an Ubbelohde cap-
illary viscometer (at 20 °C) and the expression [␩]
⫽ 0.112 Mw0.51 (cm3/g), developed by Kubota et
al.5,7
Determination of the Phase-Transition
Temperature
The phase-transition temperature was deter-
mined by two thermal analysis techniques. The
first, DSC, was done with a PerkinElmer DSC-7
apparatus at a scanning rate of 5 °C/min. Figure
1 presents the thermogram of a 2% (w/w) aqueous
solution of PNIPA. The figure shows an endother-
mic peak (0.81 J/g) at 36.2 °C, with an onset at
35.1 °C. The second technique, microcalorimetry,
was performed with a VP-DSC microcalorimeter
(Micro-Cal Inc., Northampton, MS). Solutions of
0.5 wt % PNIPA in water were degassed and
transferred to a sample cell (0.5 mL) with a cali-
brated syringe. The thermograms were obtained
at heating rates of 1.5 °C/min. Polymer-free solu-
tions (water) were used in the reference cell.
ATR Spectroscopy Studies
ATR/FTIR measurements of PNIPA aqueous so-
lutions (1–3.5 wt %) were performed with a
ATR/FTIR STUDIES OF PNIPA 1667
Bruker IFS55 machine equipped with a temper-
ature-controlled circle cell accessory [circle cell
attenuated total reflection (CCATR)], as shown in
Figure 2. The accessory was made of a ZnSe cy-
lindrical crystal (75 mm, 6-mm diameter, 45° edg-
es), fixed by a leak-proof O-ring at the center of a
temperature-controlled stainless steel vessel (60
mm long, 15-mm inner diameter) containing the
PNIPA solution. To increase the signal/noise ra-
tio, the IR beam was first decollimated and then
redirected onto the crystal edges in a circular
manner that enabled the probing of the entire
circumference of the embedded crystal, with a
reflecting angle of 45°. The IR beam that im-
pinged on the edges of the crystal, outside of the
vessel, propagated through the crystal and exited
through the other end, where it was redirected by
another set of mirrors toward the IR detector. The
short distance from which the signal was collected
by this method (a few micrometers) minimized
problems related to the high absorption coeffi-
cient of water. The temperature of the sample
solution was monitored by a thermocouple in-
serted into the vessel. The ATR/FTIR spectra
were taken in the range of 20 – 48 °C with a res-
olution of 1 °C. The circle cell was equipped with
a transparent poly(methyl methacrylate) cover,
allowing visual observation of the temperature-
induced transition from a clear solution to a tur-
bid solution.
1668 RAMON ET AL.

Figure 2. CCATR/FTIR setup.

All measurements were taken with the empty band peaking at 2125 cm⫺1, a peak that is known
circle cell as a background. Because the IR spec- to be a good indication for water quantity.27 An-
trum of water is known to change as a function of other reason for using this peak as a subtraction
temperature, mainly because of the breaking of
hydrogen bonds,26 care was taken to record a set
of temperature-dependent spectra of water
(20 – 48 °C), later to be subtracted from the tem-
perature-dependent spectra of the aqueous poly-
mer solutions.

RESULTS

Phase-Separation Effect as Probed by the PNIPA IR

Peaks
Figure 3 presents the ATR raw spectra of a 2.5%
aqueous solution of PNIPA at 22, 34, 35, 36, 37,
and 45 °C. As mentioned in the Experimental
section, the background here was an empty cell,
so the spectra are dominated by the water signals
and, more specifically, the OOH stretch envelope
of the solvent at 3000 –3700 cm⫺1, the HOH bend-
ing mode at 1635 cm⫺1, and the wide combination
band peaking at 2125 cm⫺1. At temperatures
greater than 35 °C (the phase-separation temper-
ature as measured by DSC; Fig. 1), a series of
peaks seem to emerge in the spectra. These peaks
could easily be identified as those of the polymer.
To obtain better insight into the temperature-
induced changes in the spectra, we subtracted the
spectra of water at the corresponding tempera-
tures from that of the raw data. To account for the
effects of dilution, changes in penetration depth, Figure 3. Spectra of a 2.5% PNIPA solution at six
and interaction between the solvent and the poly- different temperatures. The polymer signals can hardly
mer, we fitted a multiplication constant to the be resolved because of the dominance of the water-
water spectra. Fitting was based on obtaining a related peaks. Note the difference between the 35 °C
perfect elimination of the water wide combination spectrum and the 36 °C spectrum.
 ATR/FTIR STUDIES OF PNIPA 1669

2882 to 2878 cm⫺1, the CH2(a) peak shifted from

2939 cm⫺1 to 2936, and the CH3(a) peak shifted
from 2982 to 2975 cm⫺1. Line-shape fitting of
these peaks revealed a gradual decrease in their
width below the phase-transition point, followed
by an increase in their width at the phase-sepa-
ration temperature. Above the phase-separation
point and after oversubtraction of water, another
three weak peaks could be resolved. These were
the amide II overtone at 3070 cm⫺1, the hydro-
gen-bonded NOH stretch at 3310 cm⫺1 and an-
other peak at 3550 cm⫺1 (free NOH stretch?)
with intensities of 5, 10, and 7% of that of the
amide II, respectively.
The qualitative description of Figure 4 is given
in a more quantitative manner in Figure 5. Here,
the absorbance, integrated over the envelope of
each peak, is presented as a function of tempera-
ture. According to the figure, the temperature
dependency of the integrated absorbance of all
peaks could be divided into four regions:

1. Below 31 °C, a region of constant intensity.

Figure 4. Subtraction spectra of a 2.5% PNIPA solu-

tion at six different temperatures obtained by the sub-
traction of the water contribution from the crude spec-
tra presented in Figure 3.

guide was the fact that this spectral range is clear

of polymer-related peaks, unlike, for example, the
HOH bending peak that partially overlaps the
amide I peak of PNIPA. For a 2.5% solution, the
multiplication factor below the phase-transition
temperature ranged between 0.98 and 0.99,
whereas above the phase-separation point, the
value of this factor was no more than 0.94 – 0.95.
The subtraction spectra are presented in Figure
4. The phase transition is clearly indicated by the
intensity enhancement in the amide I (mostly
CAO stretch) band and amide II (mostly NOH
in-plane deformation) band at 1628 and 1556
cm⫺1, respectively,28 and in the coupled band of
COH and CH3 at 1460 cm⫺1. In parallel, an in-
crease in the signal of the isopropyl group22 at
1355–1400 cm⫺1 was observed, together with that
Figure 5. Changes in the integrated intensity of the
of the CH2 antisymmetric mode and the CH3 sym- PNIPA peaks as a function of temperature: (■) amide I
metric and antisymmetric stretching modes. (1628 cm⫺1), (Œ) amide II (1556 cm⫺1), (F) CH3 and
Here, a sharp change in the location of the peaks CH2 stretch envelope (2850 –3000 cm⫺1), (䊐) isopropyl
toward lower frequencies was observed with the I (1418 –1484 cm⫺1), and (E) isopropyl II (1358 –1418
phase transition: the CH3(s) peak shifted from cm⫺1).
1670 RAMON ET AL.
Figure 6. Deconvoluted spectra of the amide I and amide II peaks at (A) 25 and (B)
40 °C, showing the intermolecular, intramolecular, and free components of each band.
2. At 31–35 °C, a pretransition region in
which the intensity of some of the peaks is
reduced slightly, whereas the intensity of
others (such as the amide I and II peaks) is
enhanced slightly.
3. At 35–37 °C, a phase-transition region, in-
dicated by a sharp increase in the absor-
bance of all the PNIPA-related peaks.
4. Above 37 °C, a posttransition region, man-
ifested by a very gradual increase of all
peaks.
It is well known that the solubility of PNIPA in
water is due to the hydrophilic amide groups that
possess the capability of hydrogen bonding either
with the surrounding water molecules or with
other amide groups. It is further known that, for
polyamides, the band contour of the amide I and
amide II peaks are conformationally sensitive.21(a)
In agreement with the treatment of Lin et al.,23
the amide I peak (mostly CAO stretch) in the
subtraction spectra was deconvoluted into three
subbands: an intramolecular hydrogen-bonded
subband at 1630 cm⫺1, an intermolecular hydro-
gen-bonded subband at 1620 cm⫺1, and a free
form of non-hydrogen-bonded subband at 1643
cm⫺1. Likewise, the amide II band (mostly NOH
deformation) was deconvoluted into an intramo-
lecular hydrogen-bonded subband at 1551 cm⫺1,
an intermolecular hydrogen-bonded subband at
1565 cm⫺1, and a free form of non-hydrogen-
bonded subband at 1535 cm⫺1. These wave num-
bers are in line with the notion that hydrogen
bonding tends to make steeper the potential sur-
face of deformational modes while flattening the
potential surface of stretching modes.29 In the
deconvolution, peak positions and peak shapes
(20% Lorentzian and 80% Gaussian) were taken
as constant parameters, whereas the intensity
and width of the amide I and II subbands were
regarded as free variables.
Figure 6 presents the deconvoluted IR spectra
of amide I and amide II peaks of a 2.5% PNIPA
solution at 25 and 40 °C. A difference in the peak
shapes is clearly observed. A detailed, quantita-
tive examination of the temperature-dependent
relative contribution of each of the amide II com-
ponents is presented in Figure 7. The figure re-
veals that changes in the relative intensity of the
subbands exist at temperatures lower than the
phase-transition temperature and may provide a
hint about the collapse scenario. Four different
regions can be observed:
1. Below 31 °C, increasing temperature
causes a moderate decrease in the intermo-
lecular hydrogen bonds and a moderate in-
crease in the free amide population. The
intramolecular hydrogen-bond population
hardly changes.
2. At 32–35 °C, there is an abrupt decrease in
the intermolecular hydrogen-bond popula-

Figure 7. Changes in the three subbands of the amide II peak as a function of
temperature for a 2.5% PNIPA solution: (Œ) intermolecular hydrogen-bonding subband,
(F) free amide groups, and (䊐) intramolecular hydrogen-bonding subband).
tion and an abrupt increase in the intramo-
lecular hydrogen bond and the free amide
populations. This region is characterized
also by an abrupt increase in the width of
the free amide subband.
3. At 36 –39 °C, there is an increase in the
relative amount of the intermolecular hy-
drogen-bond population and a decrease in
the integrated absorbance and width of the
free amide subband.
4. Above 40 °C, there is a moderate increase
in the relative population of the free amide,
coupled with a moderate decrease in the
intramolecular hydrogen-bond and inter-
molecular hydrogen-bond populations.
The same trends were observed in an analysis
of the temperature-dependence graphs of the in-
tegrated intensity of the amide I subbands, al-
though amide I graphs were found to be less
smooth than the amide II graphs, probably the
result of some remaining overlap with the HOH
bending. The same four regions were also found in
aqueous solutions containing PNIPA at other con-
centrations.
Although the relative integrated absorbance
should not be taken as representing the relative
quantities of the different populations (because of
a possible difference in the transition moment of
the three populations), it is still possible to obtain
some quantitative information. For example, the
ATR/FTIR STUDIES OF PNIPA 1671
fact that the relative integral of the intramolecu-
lar hydrogen-bonding subband increased four
times from about 7% to about 28% implies that at
low temperatures the percentage of amide groups
engaged in intramolecular hydrogen bonding does
not exceed 25% of the total number of amide
groups.
Phase-Separation Effect as Manifested by the
Water-Related IR Peaks
In the mid-IR range, water has three bands: an
HOH bending mode at 1640 cm⫺1, a wide combi-
nation band (1800 –2500 cm⫺1) peaking at 2125
cm⫺1, and a strong OOH stretch band at 3000 –
3700 cm⫺1. The IR spectrum of water is known to
change with increasing temperature because of
the reduced extent of hydrogen bonding. The
main changes are the shifting of the OOH enve-
lope toward higher frequencies and the narrowing
of the HOH bending peak, accompanied by a
small change in this peak toward lower frequen-
cies. It was already mentioned that the 2125-
cm⫺1 peak was used for internal calibration to get
an accurate subtraction of the water spectra from
that of the PNIPA solution. On the basis of this
analysis, an apparent sharp decrease in the water
signal was observed with the phase transition.
This complementary section discusses changes in
the temperature-induced behavior of water due to
interaction with solvated PNIPA.
1672 RAMON ET AL.
Figure 8. Integrated intensity of water-related peaks
in (E,‚) pure water and (F, Œ) a 2.5% PNIPA solution:
(A) HOH bending mode (1635 cm⫺1) and (B) OOH
stretch envelope (3000 –3700 cm⫺1).
The integrated absorbance of the HOH bending
mode (1640 cm⫺1) of water and water containing
dissolved PNIPA is presented in Figure 8(A) as a
function of temperature. We calculated the area
of the HOH peak of the PNIPA solution by sub-
tracting the area of the amide signals from the
overall area of the peak. As shown in the figure,
the integrated intensity of pure water increased
slightly with temperature. In contrast, the inte-
grated intensity of this peak in the PNIPA solu-
tion exhibited a maximum at the phase-transition
temperature and decreased at temperatures
higher than the LCST. As shown in Figure 8(B),
the area of the coupled intramolecular and inter-
molecular OOH stretch band (3000 –3700 cm⫺1)
in pure water decreased as a function of temper-
ature because of the breaking of hydrogen bonds.
Such monotonic behavior was observed also in the
PNIPA solution, although in the latter case, a
sharp decrease at the phase-transition tempera-
ture was observed. Furthermore, a change in the
slope of the graph, to a more pronounced de-
crease, was observed at temperatures above the
transition point.
In accordance with the procedure of Sammon
et al.,30 the OOH stretch band of pure water and
water containing PNIPA was deconvoluted into
four subbands, reflecting four distinct types of
water environments, that differed by the degree
of hydrogen bonding (Fig. 9). Although this clas-
sification is somehow artificial, it does follow ex-
perimental evidence showing that strong hydro-
gen bonding skews the 3000 –3700-cm⫺1 envelope
toward lower wavenumbers, whereas a lesser ex-
tent of hydrogen bonding skews that envelope
toward higher wavenumbers. In the deconvolu-
tion, the subbands were (a) 3223 cm⫺1, strong
hydrogen bonding; (b) 3395 cm⫺1, medium-
strength hydrogen bond; (c) 3525 cm⫺1, weak hy-
drogen bond; and (d) 3612 cm⫺1, free water. In a
fashion similar to the procedure taken during the
deconvolution of the amide I and amide II peaks,
the peak positions and peak shapes (20% Lorent-
zian and 80% Gaussian) were taken as constant
parameters, whereas the intensity and width of
the subbands were regarded as free variables. To
verify that the NH stretch bands of the PNIPA
and its amide II overtone did not alter the anal-
ysis, we took care to include these peaks as well,
but their contribution was found to be negligible,
compared with the water signal.
The relative areas of the OH stretch subbands
of water and the relative areas of the OH stretch
subbands of water containing 2.5% PNIPA are
presented, as a function of temperature, in Figure
10. As shown in the figure, at a temperature of 25
°C, the area of subband a was close to 55% of the
total area of the peak, whereas the areas of sub-
bands b– d were 33, 10, and 2%, respectively. Wa-
ter in the PNIPA solution was found to differ from
pure water by a lower extent of strong hydrogen
bonds and a higher extent of medium-strength
hydrogen bonds. In contrast, hardly any differ-
ence could be observed in the extent of weak hy-
drogen bonds or free water. The lower amount of
strong hydrogen-bonding subbands found for the
PNIPA solution correlates well with the claim
that PNIPA behaves like a structure breaker, be-
cause the water must reorient around the nonpo-

Figure 9. Deconvoluted spectrum of the OH stretch envelope of water, showing the
four types of populations.
lar regions of the solute.31 At low temperatures,
the differences between pure water and PNIPA
solutions can hardly be observed by an inspection
of the crude data (Fig. 3) because of the small
concentration of solute. However, these differ-
ences are easy to observe if one inspects the sub-
traction spectra (Fig. 4), where a negative peak is
observed at the locus of the strong hydrogen-
bonding subband.
As the temperature of the liquid is increased, a
decrease in the 3223-cm⫺1 subband of pure water
could be observed, together with an increase in
the 3395-cm⫺1 subband. Slight changes were
found also in the other two subbands (the 3525-
cm⫺1 subband decreased, and the 3612-cm⫺1 sub-
band increased); however, these changes were
smaller than the deconvolution error. A similar
behavior was found also for water containing
PNIPA. At the phase-transition point, however,
an abrupt decrease in the relative amounts of the
strong hydrogen-bonding subband and weak hy-
drogen-bonding subband coupled with an abrupt
increase in the relative amount of medium-
strength hydrogen-bonding subband was ob-
served. At higher temperatures, a subtle increase
in the slope of the temperature-dependent rela-
tive areas could be observed.
Effect of PNIPA Concentration on Phase-Transition
Phenomena as Measured by ATR
As already mentioned, the measurements were
performed at various concentrations of PNIPA
ATR/FTIR STUDIES OF PNIPA 1673
(1–3.5%). The phase-transition temperature was
found to be the same, within an experimental
error of 1 °C, for all solutions. Qualitatively, the
spectral changes were similar, regardless of poly-
mer concentration. These changes included an
increase in the PNIPA IR signals, a decrease in
water-related signals, and a similar behavior for
deconvoluted amide peaks. An interesting point
was the increase of the PNIPA peaks with the
phase transition. Below the transition tempera-
ture, the integrated absorbance of the PNIPA
peaks increased, more or less, linearly with the
concentration, as could be expected. In contrast,
the integrated absorbance at higher tempera-
tures was almost constant, regardless of the
PNIPA concentration. This constant absorbance
as a function of concentration occurred for all the
PNIPA-related peaks.
DISCUSSION
Reduction in Water Concentration near the ATR
Crystal
As described in the previous sections, the phase
transition was characterized by a sharp increase
in the ATR signal of all the PNIPA-related peaks
(Fig. 4). This increase at the phase-transition
temperature was coupled with a decrease in all
the water-related peaks: the HOH bending at
1640 cm⫺1 [Fig. 8(A)], the OH stretch envelope at
3000 –3700 cm⫺1 [Fig. 8(B)], and the wide combi-
1674 RAMON ET AL.
Figure 10. Relative area of the four subbands in the
OH stretch envelope for pure water (empty symbols)
and a 2.5% PNIPA solution (filled symbols): (䊐,■)
strong hydrogen-bonding subband (3223 cm⫺1), (‚,Œ)
medium-strength hydrogen-bonding subband (3395
cm⫺1), (〫,⽧) weak hydrogen-bonding subband (3525
cm⫺1), and (E,F) free-water subband (3612 cm⫺1).
nation band at 2125 cm⫺1 (see the description on
the subtraction procedure). The aggregation of
molecules is known to have strong effect on band
intensity. However, in this case it is expected that
each band will respond to aggregation in a differ-
ent manner, according to the change in its in-
duced dipole moment. In particular, it could be
expected that the extent of the increase would
differ significantly between NH-related peaks
and COH-related peaks, or between stretch
modes and deformation modes. The fact that the
area of all PNIPA peaks, including those related
to the hydrophobic groups, were found to increase
at the transition approximately by the same fac-
tor (for a given concentration) suggests that the
observed increase in the signal was mostly due to
an increase in the average concentration of the
polymer close to the crystal surface. This sugges-
tion is supported by the decrease in all the water-
related signals at the phase-transition tempera-
ture. Furthermore, the fact that the area of the
PNIPA peaks was, above the transition tempera-
ture, independent of polymer concentration in the
solution may suggest that a film with a constant
PNIPA concentration is formed with the phase
transition. If indeed this is the case, the average
PNIPA concentration near the crystal (at a dis-
tance of a few micrometers) should be 7–10%,
based on calibration according to the IR signals of
the PNIPA bands as measured below LCST. This
value is further supported by the water peak
data.
Phase-Transition Scenario
The temperature-induced changes in the relative
areas of the three subbands in the amide II peak
(Fig. 7) imply a four-stage process of phase sepa-
ration in the PNIPA solution, which will be
termed the early (A), intermediate (B), aggrega-
tion (transition temperature), relaxation (C), and
postrelaxation (D) stages.
The early stage, below 31 °C, is characterized
by the domination of the water–PNIPA intermo-
lecular hydrogen bonding, which contributes to
approximately 60% of the overall amide II signal
measured at this stage. As the temperature is
raised, the relative contribution of this subband is
gradually reduced, whereas that of the subband
corresponding to non-hydrogen-bonded amides
(free amides) is increased. Because of the in-
creased number of free amides, the movement of
the side chains becomes less restricted, a factor
that may have an influence on the flexibility of
the backbone of the polymer. The required in-
crease in entropy during this stage could result
from a decrease in the number of ordered water
molecules due to the breaking of the thin shell of
ordered molecules around the polymer. However,
it is possible that during this pretransition stage,
the required entropy comes from structural
changes in the backbone of this relatively flexi-
ble32 polymer, as water, considered to be a good
solvent at low temperatures, becomes a ␪ solvent
at ⬃32 °C. Indeed, both the radius of gyration and
the hydrodynamic radius are known to decrease
moderately at this stage,33 approaching maximal
entropy at the ␪ temperature.

The intermediate stage (31–35 °C) is charac-
terized by a sharp decrease in the relative inten-
sity of the intermolecular hydrogen-bonding sub-
band. This decrease is coupled with a sharp in-
crease in the amide–amide hydrogen-bond
population in the lower temperatures of this stage
and an increase in the population of the free
amide groups at the higher temperatures of this
stage. The phase separation, as indicated by DSC
measurements and visual inspection, occurred
quite abruptly at the end of this stage, that is, at
35 °C. The behavior below the phase-separation
temperature suggests that the phase-separation
process is triggered by the formation of free amide
groups. Once a critical number of side chains are
free to change their conformation, they begin to
form amide–amide hydrogen bonds. It is likely
that at this stage the amide–amide bonds are
intramolecular because, otherwise, one could
have expected this scenario to be concentration-
dependent. The structural changes involved in
the formation of the intramolecular hydrogen
bonds trigger a very fast decrease in the intermo-
lecular water–amide bonds, thus further increas-
ing the amount of free amides. At this point, the
polymer side chains are expected to be very flex-
ible, thereby able to form hydrophobic interac-
tions between their isopropyl groups and/or their
backbone. Because in PNIPA the hydrophobic iso-
propyl groups are located adjacent to the amide
groups, one could assume that the formation of
the intramolecular hydrogen bonding at 33 °C
should be coupled with the formation of hydro-
phobic interactions, however, no change in the
intensity or vibration frequency of methyl or
methylene groups could be observed at tempera-
tures below the transition temperature.
Phase separation (i.e., aggregation of the col-
lapsed chains) occurred at a point where the
amount of free amide groups was at its maximum.
This point was characterized also by a shift in the
vibrational band of the hydrophobic groups, prob-
ably reflecting the formation of intermolecular
hydrophobic interactions. As can be deduced from
analyzing the water-related peaks, this aggrega-
tion stage is characterized also by the repulsion of
water from the vicinity of the chains and the
formation of polymer-rich regions on the surface
of the ZnSe crystal. This scenario is in line with
claims made by other groups, according to which
the PNIPA–water system is thermodynamically
stable at low temperatures, and it becomes unsta-
ble at the onset of the phase-separation stage.34 It
has been suggested that at this stage the col-
ATR/FTIR STUDIES OF PNIPA 1675
lapsed globules contain less water22,34and expose
their isopropyl groups to the solvent, which be-
comes less polar in the vicinity of the polymer, as
demonstrated by Winnik.35 This hydrophobically
driven aggregation mechanism does not contra-
dict the mechanism suggested by us but rather is
complementary to the one presented here.
Above the transition temperature and up to 40
°C, a decrease in the amount of free amide groups,
coupled with an increase in the intermolecular
component (i.e., water–amide hydrogen bonding),
could be observed. It is believed that at this re-
laxation stage hydrogen bonds are formed be-
tween water molecules that were trapped inside
the polymer globules and amide groups that were
free. Above the transition temperature, the rela-
tive amount of water molecules belonging to the
weak hydrogen-bonding population is decreased,
whereas that of the medium-strength hydrogen-
bonding population is increased (Fig. 10). It is not
clear whether this difference is due to water
trapped within the globules or water molecules at
the interface between bulk water and polymer
aggregates. A true equilibrium is achieved only
above 40 °C, as demonstrated in Figure 7. Hence,
it can be said that although phase separation in
PNIPA is well defined and abrupt, the scenario of
this process can be quite complex and occurs over
a wide range of temperatures.
It is interesting to compare these findings with
those of other groups who used different tech-
niques. On the basis of an analysis of the temper-
ature dependence of the ratio between the radius
of gyration and the hydrodynamic radius, as ob-
tained from light scattering experiments, it has
been claimed that, before aggregation, the chains
go through a four-stage scenario where the chains
contract, crumple, rearrange, and collapse.33,34
CONCLUSIONS
This article has demonstrated that the ATR/FTIR
technique can provide detailed information on the
process of temperature-induced phase separation
in aqueous solutions of hydrophobic polymers,
such as PNIPA. Based on the ATR/FTIR results,
the following scenario for the temperature-
induced collapse of PNIPA is proposed.
At low temperatures, the polymer is solvated in
such a manner that the hydrophilic groups form
relatively strong hydrogen bonds with water. As
the temperature is raised, more and more hydro-
gen bonds between amide groups and water are
1676 RAMON ET AL.
broken. On average, the free amides do not form
new intramolecular hydrogen bonds as long as
their number is low. Once a critical number of
bonds are broken, intramolecular hydrogen bonds
are formed, at the expense of the free amide group
population and the intermolecular hydrogen-bond
population. As a result, the polymer begins to
transform itself to a more compact conformation.
This promotes the breaking of more intermolecu-
lar hydrogen bonds, which eventually causes the
CGT of the chain. The exposed hydrophobic moi-
eties induce aggregation due to hydrophobic in-
teractions. The collapsed, aggregated polymer
tends to adhere to the ATR crystal, so an increase
in the intensity of all polymer-related peaks is
observed, together with a decrease in the inten-
sity of all water-related peaks. On the basis of the
decrease in the free amide signal and the increase
in the signal of the intermolecular hydrogen
bonds above the phase-separation temperature, it
is believed that, with phase separation, water
molecules that were entrapped within the col-
lapsed areas form hydrogen bonds with free
amide moieties.
The detailed role of water in the phase-transi-
tion process of PNIPA solutions has been revealed
(to our knowledge) for the first time on a molecu-
lar level by the CCATR/FTIR technique. At
present, this technique is used to study the effect
of low molecular weight solutes (salts) on that
process. A forthcoming article on that subject is
now under preparation.
This work was performed under contract number
205-97 of the Israel Science Foundation. The authors
thank Professor Rolfe Herber (Hebrew University) for
lending the circle cell and for a critical reading of the
manuscript.
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