In situ IRRAS studies of molecular recognition of barbituric acid lipids to melamine at the air-water interface

Article abstract of DOI:10.1021/jp207863x

Recognition and detection of melamine are of very significance in food industries. Molecular recognitions of barbituric acid lipids to melamine at the air-water interface have been investigated in detail using in situ infrared reflection absorption spectroscopy (IRRAS). Hydrogen bonding patterns and molecular orientations of the molecular recognitions have been revealed. Prior to molecular recognition, the barbituric acid moieties in the monolayers were hydrogen bonded with a flat-on fashion at the air-water interface, and the alkyl chains were preferentially oriented with their CCC planes perpendicular to the water surface. After molecular recognition, the NH₂ stretching bands of recognized melamine were clearly observed at the air-water interface as well as primary characteristic bands, the barbituric acid moieties underwent a change in orientation with non-hydrogen bonded C4=O bonds almost perpendicular to the water surface and C2=O and C6=O bonds involved in hydrogen bonds with melamine, and the alkyl chains were preferentially oriented with their CCC planes parallel to the water surface. The monolayers of barbituric acid lipids exhibited excellent selectivity for melamine over nucleosides.

Full text of DOI:10.1021/jp207863x

ARTICLE
pubs.acs.org/JPCB
In Situ IRRAS Studies of Molecular Recognition of Barbituric Acid
Lipids to Melamine at the AirÀWater Interface
Xianming Kong and Xuezhong Du*
Key Laboratory of Mesoscopic Chemistry (Ministry of Education), State Key Laboratory of Coordination Chemistry, and School of
Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, People’s Republic of China
S Supporting Information
b
ABSTRACT: Recognition and detection of melamine are of very significance in
food industries. Molecular recognitions of barbituric acid lipids to melamine at the
airÀwater interface have been investigated in detail using in situ infrared reflection
absorption spectroscopy (IRRAS). Hydrogen bonding patterns and molecular orienta-
tions of the molecular recognitions have been revealed. Prior to molecular recognition,
the barbituric acid moieties in the monolayers were hydrogen bonded with a flat-on
fashion at the airÀwater interface, and the alkyl chains were preferentially oriented with
their CCC planes perpendicular to the water surface. After molecular recognition, the
NH2 stretching bands of recognized melamine were clearly observed at the airÀwater
interface as well as primary characteristic bands, the barbituric acid moieties underwent
a change in orientation with non-hydrogen bonded C4dO bonds almost perpendi-
cular to the water surface and C2dO and C6dO bonds involved in hydrogen bonds
with melamine, and the alkyl chains were preferentially oriented with their CCC planes
parallel to the water surface. The monolayers of barbituric acid lipids exhibited excellent selectivity for melamine over nucleosides.
’ INTRODUCTION
orientation and structure of the LB films is not equivalent to that
obtained directly from the monolayers at the airÀwater interface.
Infrared reflection absorption spectroscopy (IRRAS) has been
a leading structural method for the in situ characterization of the
monolayers at the airÀwater interface on the molecular level.21À31
Weck et al.13 used the IRRAS technique in 1997 to study the
hydrogen bonding interactions between barbituric acid lipid
monolayers and melamine from aqueous subphase. However,
in general, the quality of these spectra is not ideal.17 Huo et al.17
used polarization modulatedÀIRRAS (PMÀIRRAS) technique
in 1999 to study the hydrogen bonding interactions between
melamine-derived lipid monolayers and barbituric acid. So far,
few studies have been devoted to the molecular recognitions by
means of hydrogen bonds at the airÀwater interface using the
IRRAS technique.13,17,32,33
In this paper, molecular recognitions between the monolayers
of barbituric acid lipids at the airÀwater interface and melamine
in aqueous subphases have been investigated in detail along with
the selectivity of molecular recognitions in the presence of other
molecules (such as nucleosides) with complementary donating
and accepting groups for probable hydrogen bonding interac-
tions with the barbituric acid moieties, the chemical structures of
which are presented in Figure 1. Some new information on
hydrogen bonding patterns and molecular orientations of the
molecular recognitions has been obtained.
Since 2008, melamine has attracted much attention in the
scandal of illegally adding melamine to dairy products to falsely
increase protein content. This has led to serious kidney damages
in tens of thousands of infants and even several cases of death.1,2
The damaging effect is considered to likely result from the
aggregates of melamine with associated impurities or metabolites
existing in body fluids such as cyanuric acid and uric acid bearing
imide moieties.3,4 It is obvious that recognition and detection of
melamine become more important than ever. Molecular recog-
nition is one of the most important processes for the construc-
tion of biological assemblies. The mutual recognition of comple-
mentary components by means of multiple hydrogen bonds is
not only a vital biological process5 but also the basis for the
construction of a variety of sensors. However, the hydrogen
bonding interactions cannot yield significant driving forces for
molecular recognition in bulk water due to the strong competi-
tive binding of water molecules.6 Langmuir monolayers at the
airÀwater interface, with half a framework structure of cellular
membranes, provide unique microenvironments for molecular
interactions7 and have been developed in the past decades to
mimic molecular recognition.8À10 The molecular recognitions of
barbituric acid and melamine derivatives at the airÀwater inter-
face and in LangmuirÀBlodgett (LB) films have been extensively
studied,11À20 but most of the works are limited to the recogni-
tions of melamine-derived lipids to barbituric acid in aqueous
subphase14À20 because characteristic infrared peaks of barbituric
acid (CdO stretching vibration) can be readily detected from
transferred LB films. However, the information on molecular
Received: August 16, 2011
Revised:
October 2, 2011
Published: October 13, 2011
r
2011 American Chemical Society
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The crude product was further purified by recrystallization from
ethanol for three times to yield white crystal. 1H NMR (500 MHz,
DMSO): δ 11.20 (s, 2H, 2NH); 3.51 (t, 1H, CH), 1.87 (q, 2H,
CH2), 1.26 (m, 32H, 16CH2), 0.86 (t, 3H, CH3). ESI-MS m/z:
calcd for C22H40N2O3, 380.5; found 379.5 (MÀH)À.
Monolayer Isotherms and LB Film Preparation. The spread-
ing and transfer of the monolayers were performed on a Nima
611 Langmuir trough with two barriers. A Wilhelmy plate (a
piece of filter paper) was used as the surface pressure sensor and
situated in the middle of the trough. Monolayers were obtainedby
spreading the chloroform solution of C18BA on aqueous subphases.
The subphase temperature was kept at 22 ꢀC. For the preparation
of LB films, the monolayers were first compressed to a desired
surface pressure of 20 mN/m, and then 30 min was allowed for
the monolayers to reach equilibrium. For CaF2 substrates, 3-layer
LB films (Y type) were transferred on two sides by the vertical
method at a dipping rate of 2.0 mm/min with the transfer ratios of
nearly unity for upstroke and about 0.8 for downstroke. For gold-
coated silicon wafers, first monolayer was deposited by the
vertical method with a transfer ratio of nearly unity, and following
20 layers (Y type) were transferred on one side by the horizontal
method at a dipping rate of 2.0 mm/min with a transfer ratio of
about 2.
Figure 1. Chemical structures of 5-octadecylbarbituric acid (C18BA),
melamine, adenosine, thymidine, and cytidine.
IRRAS Spectrum Measurements. IRRAS spectra of the mono-
layers at the airÀwater interface were recorded on an Equinox 55
FTIR spectrometer (Bruker) connected to an XA-511 external
reflection attachment with a shuttle trough system and a liquid-
nitrogen-cooled MCT detector. Sample (monolayer-covered
surface) and reference (monolayer-free surface) troughs were
fixed on a shuttle device driven by a computer-controlled stepper
motor for allowing collection from the two troughs in an alternate
mode at 22 ꢀC. A KRS-5 polarizer was used to generate p- and
s-polarized lights. The C18BA molecules were spread from their
chloroform solution, and 20 min was allowed for solvent
evaporation. The measurement system was then enclosed for
humidity equilibrium and monolayer relaxation for 4 h prior to
compression. The external reflection absorption spectra of
aqueous solutions were used as references, respectively. The
monolayers were compressed discontinuously to the desired
surface pressure of 20 mN/m from approximately 0 mN/m. After
30 min of relaxation, the two moving barriers were stopped and
the monolayer areas were kept constant. The spectra at different
surface pressures were collected at an incidence angle of 30ꢀ, and
the spectra at 20 mN/m with the incidence angles from 30ꢀ to
65ꢀ to obtain information on molecular orientation. The spectra
were recorded with a resolution of 8 cmÀ1 by coaddition of 1024
scans. A time delay of 30 s was allowed for film equilibrium
between trough movement and data collection. Spectra were
acquired using p-polarized radiation followed by data collection
using s-polarized one. The IRRAS spectra were used without
smoothing or baseline correction.
’ EXPERIMENTAL SECTION
Chemicals. The chemical reagents used in the experiments
were commercially available. 1-Bromooctadecane (98%), diethyl
malonate (99%), urea (99%), sodium (99.7%), melamine (99%),
and ethanol (99%) were purchased from Sinopharm Chemical
Reagent Co., Ltd. Adenosine (97%), thymidine (99%), and cytidine
(99%) were acquired from Fluka. Ethanol was purified to exclude
water by the standard procedures34 prior to use, and other
chemical reagents were used as received. 5-Octadecylbarbituric
acid (C18BA) was synthesized according to the method reported
previously13 with a small modification in amount of reactants and
volume of solvents. The water used was double distilled (pH 5.6,
resistivity of 18.2 MΩ cm, surface tension of 73.06 mN/m at
22 ꢀC) after a deionized exchange.
Preparation of Diethyl β-Octadecylmalonate. Diethyl
malonate of 1.60 g (10 mmol) was dissolved in 10 mL of absolute
ethanol containing 0.23 g (10 mmol) of sodium. The mixture was
heated to reflux for 20 min, and then 3.33 g of 1-bromooctade-
cane (10 mmol) was added dropwise within 2 min. The reflux
continued for 3 h under ambient conditions, and the mixture was
then filtered before cooling. The filtrate was concentrated to
yield colorless oil, and the crude product was further purified by
column chromatography (silica gel, ethyl acetate/hexane, 1:20, v/v)
1
to give diethyl β-octadecylmalonate of colorless oil. H NMR
(500 MHz, CDCl3): δ 4.21 (q, 4H, 2OCH2), 3.33 (t, 1H, CH),
1.90 (q, 2H, CH2), 1.30 (m, 38H, 16CH2 + 2CH3), 0.90 (t, 3H,
CH3).
FTIR Spectrum Measurements. FTIR transmission (TR)
spectra of the LB films on CaF2 substrates were recorded on a
VERTEX 70 V spectrometer (Bruker) with a DTGS detector.
Typically 1024 scans were collected to obtain a satisfactory
signal-to-noise ratio with the resolution 4 cmÀ1. The film spectra
were obtained by subtracting the spectra of blank CaF2 substrates
from the corresponding sample spectra. Reflection absorption
(RA) spectra of the LB films on gold-coated silicon wafers were
recorded on the FTIR spectrometer at an incidence angle of 80ꢀ
with a bare gold-coated wafer as a reference. The spectra were
recorded with a resolution of 4 cmÀ1 by coaddition of 1024 scans.
Synthesis of C18BA. A total of 0.24 g of urea (4 mmol) was
dissolved in 10 mL of absolute ethanol containing 0.28 g (12 mmol)
of sodium. The mixture was heated to reflux for 20 min, and then
1.56 g of diethyl β-octadecylmalonate (3.8 mmol) was added
dropwise within 2 min. The reflux continued for 9 h under
ambient conditions, so that most of the solvent was evaporated
out. Then 40 mL of water was added to the residue, and the pH
was adjusted to 5.0À6.0 with hydrochloric acid. The precipitate
was isolated by suction filtration and washed with water.
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Melamine of low concentrations (0.001 mM) hardly caused a
change in isotherm, and a slight condensation of the isotherm
was observed upon increase of melamine concentration to
0.01 mM. In the case of 0.1 mM melamine, the isotherm was
considerably condensed with a limiting area of 0.363 nm2; however,
an increase in limiting area was followed upon further increase of
melamine concentration to 1 mM. This means that molecular
recognitions between the barbituric acid moieties and melamine
occurred at the relatively high concentrations of melamine, con-
comitant with a change in orientation of barbituric acid planes from
a flat-on fashion to an end-on one followed by a rotation of C2v
symmetry axis of the planes. In the presence of adenosine,
thymidine, and cytidine (0.1 mM) individually in the aqueous
subphases, only a slight change in isotherm occurred in the cases of
adenosine and cytidine, and no change was observed in the case of
thymidine because of the similar chemical structure to barbituric
acid (Figure 2b).
IRRAS Spectra of Monolayers at the AirÀWater Interface.
It is well-known that IRRAS data are defined as plots of reflectanceÀ
absorbance versus wavenumber. ReflectanceÀabsorbance is de-
fined as Àlog(R/R0), where R and R0 are the reflectivities of the
monolayer-covered and monolayer-free surfaces, respectively.
The “metal selection rule” does not apply to the airÀwater inter-
face, because water is a low absorbing substrate and the intensity
of the reflected beam is much lower than the intensity of the
incoming beam. Therefore, all of three electric field components
have a sufficiently high intensity at the water surface to interact
with the monolayers. In the case of s-polarization, the electric
field vector is perpendicular to the plane of incidence, i.e., parallel
to the water surface. The bands are always negative and their
intensities decrease with increasing incidence angle.21,25,27 In the
case of p-polarization, the electric field vector is parallel to the
plane of incidence: (i) for the vibrational transition moments
parallel to the water surface, the bands are initially negative and
their intensities increase with increasing incidence angle and
reach maximum values, then minima in the reflectivity occur at
the Brewster angle, and above the Brewster angle the bands
become positive and their intensities decrease upon further
increase of incidence angle;21,25,27 (ii) for the vibrational transi-
tion moments perpendicular to the water surface, the bands are
first positive and then become negative above the Brewster
angle;21,27,28,36,37 (iii) if the vibrational transition moments are
tilted to the airÀwater interface, the intensities of the bands are
weak and even zero.36
Figure 3 shows p-polarized IRRAS spectra of the monolayers
of C18BA on pure water at various surface pressures. These
spectral behaviors were very similar except for band intensities,
and their band frequencies were almost independent of surface
pressure. The spectral baselines were distorted in the regions
corresponding to the vibrational transitions of water because of
the altered structure of the water adjacent to the headgroups of
the film constituents, and the distortions were maximal for small
incidence angles. The two bands at 2919 and 2852 cmÀ1 were
assigned to the antisymmetric and symmetric CH2 stretching
vibrations [νa(CH2) and νs(CH2)] of alkyl chains,38,39 respec-
tively. It is well-known that the νa(CH2) and νs(CH2) frequen-
cies are sensitive to conformation order of alkyl chains.38,39
Lower wavenumbers are characteristic of all-trans conformations
in highly ordered chains, while higher wavenumbers are indica-
tive of gauche conformations in highly disordered chains.38,39 It is
clear that the alkyl chains in these monolayers were basically
ordered but with a small amount of gauche conformers. It is
Figure 2. Surface pressureÀarea isotherms of the monolayers of C18BA
at the airÀwater interface: (a) on aqueous melamine subphases with
different concentrations and (b) on aqueous subphases containing
nucleosides (0.1 mM).
’ RESULTS AND DISCUSSION
Isotherms of Monolayers at the AirÀWater Interface.
Surface pressureÀarea (πÀA) isotherms of the monolayers of
C18BA on pure water and aqueous melamine subphases are
shown in Figure 2a. C18BA lipids formed a stable monolayer
with a transition from the limiting area of 0.515 to 0.416 nm2
and a collapse pressure of about 45 mN/m. The limiting area
was much greater than the cross-sectional area of hydrocarbon
chains (ca. 0.20 nm2), which indicates that the limiting area of
the lipids was predominantly determined by barbituric acid moie-
ties. The crystal structure of barbituric acid dihydrate, which is
selected for consideration since the lipids were spread at the
airÀwater interface, is determined to be orthohombic with the
unit cell dimensions a = 1.274, b = 0.624, and c = 0.889 nm
containing four molecules.35 The crystal structure consists of
planar layers of barbituric acid and water molecules hydrogen-
bonded into a compact arrangement.35 On the basis of the
dimensions,35 each barbituric acid dihydrate occupied an area
of 0.566 nm2 when lying flat on a plane. It was obvious that
the barbituric acid moieties initially almost adopted a flat-on
orientation on the water surface. This suggested the formation of
intermolecular hydrogen bonds between adjacent barbituric acid
moieties and between barbituric acid and water molecules. In the
presence of melamine in the aqueous subphases, the isotherm
underwent a change dependent on melamine concentration.
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Figure 3. p-Polarized IRRAS spectra of the monolayers of C18BA at the
airÀwater interface on pure water at different surface pressures at an
incidence angle of 30ꢀ.
noted that the νa(CH2) band intensities were nearly twice higher
than the νs(CH2) ones at various surface pressures, which
indicates that the alkyl chains were preferentially oriented with
their CCC planes perpendicular to the water surface. The strong
doublet bands at 1728 and 1709 cmÀ1 were attributed to the
CdO stretching vibrations of the barbituric acid moieties.40 The
frequency of the CdO stretching band is strongly dependent on
hydrogen bonding. The CdO stretching band of barbituric acid
in an argon matrix appears at 1754 cmÀ1 for the monomer and
1732 cmÀ1 for the dimer.40 These peaks shift to 1694 cmÀ1 in the
solid state at 20 K, owing to hydrogen bonding formation
between barbituric acid molecules.40 The formation of the
hydrogen bonds weakens CdO bond strength, and the CdO
stretch occurs at lower vibrational frequency. It is clear that the
intermolecular hydrogen bonds were formed between the ad-
jacent barbituric acid moieties in the monolayers, so that the
planes of barbituric acid preferentially adopted a flat-on orienta-
tion on the water surface.
Figure 4. p-Polarized IRRAS spectra of the monolayers of C18BA at the
airÀwater interface on aqueous melamine subphases with various
concentrations at 20 mM/m at different incidence angles: (a) 30ꢀ and
(b) 60ꢀ, together with a difference spectrum in the presence and absence
of 1 mM melamine.
Figure 4a shows p-polarized IRRAS spectra of the monolayers
of C18BA on aqueous melamine subphases with different con-
centrations at the surface pressure of 20 mN/m at an incidence
angle of 30ꢀ. On the aqueous melamine subphase of 0.01 mM, no
significant spectral change was detected except for a weak
negative peak at 1762 cmÀ1, but remarked spectral features were
observed upon increase of melamine concentration to 0.1 and
1 mM. These new peaks at 3354 (NH2 antisymmetric stretching),
3242 (NH2 symmetric stretching), 1632 (NH2 scissoring), 1539
(quadrant triazine ring stretching),17 1452 (semicircle ring
stretching),17 and 1425 cmÀ1 (semicircle ring stretching)17 re-
sulted from melamine due to the occurrence of molecular
recognition between the barbituric acid moieties and melamine.
The assignments of the bands for the monolayers are listed in
Table 1. We first reported in situ IRRAS detection of NH
stretching vibrational bands from the monolayers at the airÀ
water interface,42 and those NH stretching bands resulted from
amide groups of the host monolayers.42À44 Herein, the detected
NH2 stretching bands were attributed to the vibrational transi-
tions of amino groups of recognized melamine molecules. NH
stretching bands from amino groups at the airÀwater interface
have not been observed using the IRRAS technique before. A
strong CdO stretching band from barbituric acid shifted down
to 1691 cmÀ1, indicating the occurrence of molecular recognition,
meanwhile, a positive peak at 1762 cmÀ1 appeared at the inci-
dence angle of 30ꢀ in the case of 1 mM melamine, and a strong
negative band at 1763 cmÀ1 was clearly observed at the incidence
angle of 60ꢀ (above the Brewster angle, Figure 4b), which
indicates that non-hydrogen bonded CdO groups appeared,
with their vibrational transition moments almost perpendicular
to the water surface. For the monolayers of N-substituted
barbituric acid lipids on aqueous melamine subphases, Weck
et al.13 also observed the shifts to high wavenumbers (although
the quality of the spectra is not ideal) but could not give a
clear explanation. In the alternate LB films of C18BA and N,N-
substituted melamine lipids, free CdO stretching bands at
1755 cmÀ1 were observed at the high surface pressures of 15
and 20 mN/m.18 The free CdO groups were considered not to
interact with any group in the tilted molecule model.18 From
Figure 4b, an obvious negative band was also observed in the
case of 0.1 mM melamine but was weaker in intensity than that in
the case of 1 mM melamine. Considering the smaller molecular
area and higher molecular density in the case of 0.1 mM
melamine (Figure 2a), the free CdO (either C4dO or C6dO)
bonds of barbituric acid moieties in the monolayers
in the case of 1 mM melamine were more perpendicular to
the water surface than those in the case of 0.1 mM melamine.
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Table 1. Absorption Bands and Their Assignments of IRRAS Spectra of the C18BA Monolayers on Different Aqueous Subphases
and Transmission and Reflection Absorption Spectra of the Corresponding LB Films
a
wavenumber (cmÀ1
)
IRRAS/monolayers
TR/LB films
H2O mel
RA/LB films
H2O mel
H2O (30ꢀ)
H2O (60ꢀ)
mel (30ꢀ)
mel (60ꢀ)
orientationb
assignmentc
νa(NH2)18
ν(NH)40
3354 w
3337 m
3341 m
3234 w
3260 w
3238 w
3242 w
3234 m
νs(NH2)
3089 w
2956
ν(NH)40
2959
2958
2956
2956
2960
νa(CH3)
2919 s
2852 m
2918 m
2851 w
2919 s
2851 s
1762 w
1691 s
2916 m
2850 m
1763 s
2918 s
2850 m
2918 s
2851 s
1760 w
2919 s
2851 s
2920 s
2851 m
1762 s
νa(CH2)38,39
νs(CH2)38,39
ν(CdO)18,40
ν(CdO)18,40
ν(CdO)40
ν(CdO)40
δ(NH2)17
ν(Q ring)17
ν(Q ring)17
ν(Q ring)17
δ(CH2)41 + ν(S ring)17
ν(S ring)17
ν(S ring)17
ν(S ring)17
ν(S ring)17
^
^
1728 s
1709 s
1683 m
1735 m
1692 m
1675 m
1739 s
1691 w
1674 w
1704
1687 s
1660 w
1629 w
1666 m
1632 w
1539 s
1626 m
1580 w
1557 m
1538 w
1468 w
1538 s
1540 s
1470 w
1358 w
1468 w
1442 m
1424 m
1471 m
1430 w
1452 s
1425 s
1453 m
1425 m
1395 w
1359 w
1396 m
^
1360 w
1358 w
a s, strong; m, medium; w, weak. , parallel to substrate; ^, perpendicular to substrate. ν, stretching; νa, antisymmetric stretching; νs, symmetric
b
c
stretching; δ, bending; Q, quadrant; S, semicircle.
That means that the C2v symmetry axis of barbituric acid planes
was tilted away from the normal of the monolayer and a relatively
big room was required for the C5-substituted alkyl chains to
orient upward in the case of 1 mM melamine, leading to an
increase in molecular area. The IRRAS data well supported the
behaviors of corresponding isotherms dependent on melamine
concentration. The νs(CH2) band intensities were considerably
increased after molecular recognition, even larger than the
νa(CH2) ones in the case of 0.1 mM melamine at the incidence
angle of 60ꢀ (Figure 4b), which indicates that the CCC planes of
the alkyl chains were preferentially oriented parallel to the water
surface. From the p- and s-polarized IRRAS spectra at different
incidence angles before and after molecular recognition (Figures
S1ÀS3 in the Supporting Information), the νa(CH2)/νs(CH2)
intensity ratio as a function of incidence angle (Figure S4) further
demonstrates that the alkyl chains underwent a change with their
CCC planes from perpendicular to the water surface29 before
molecular recognition to parallel to the water surface28 after
molecular recognition. The illustration of the molecular recogni-
tion between the monolayers of barbituric acid lipids and
melamine was schematically represented in Figure 5 (C4dO
Figure 5. Schematic illustration of hydrogen bonding patterns and
bonds are drawn to be non-hydrogen-bonded). Owing to the
molecular orientations of barbituric acid lipids and melamine at the
inherent weak signals and interference of water vapor at the
airÀwater interface before and after molecular recognition.
incidence angle of 60ꢀ, a difference spectrum in the presence and
absence of 1 mM melamine was presented in Figure 4b for
comparison. A negative dip at 1660 cmÀ1 appeared in the dif-
ference spectrum. It is not unambiguous to determine whether it
was a vibrational band (CdO stretching vibration of barbituric
acid or NH2 scissoring one of melamine) or a false peak. In the
following, a band at 1666 cmÀ1 was obviously observed in the RA
spectrum of the corresponding LB film (Figure 6), which
indicates that the above “negative dip” was a vibrational band.
In the solid state of hydrogen bonded melamine, the NH2
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Figure 7. p-Polarized IRRAS spectra of the monolayers of C18BA at the
airÀwater interface on different aqueous subphases (0.1 mM, the
mixture containing thymidine, cytidine, and melamine) at 20 mM/m
at an incidence angle of 30ꢀ.
that in the RA mode, which suggests that the CCC planes of the
alkyl chains were preferentially oriented perpendicular to the
substrate surface. The CdO stretching bands at 1735, 1692, and
1675 cmÀ1 in the TR mode corresponded to the those at 1739,
1691, and 1674 cmÀ1 in the RA mode. For the LB film from
aqueous melamine subphase, both νa(CH2) and νs(CH2) bands
in the TR and RA modes were also clearly observed, but the
νa(CH2)/νs(CH2) intensity ratio in the TR mode was smaller
than that in the RA mode, which suggests that the CCC planes
of the alkyl chains were preferentially oriented parallel to the sub-
strate surface. After molecular recognition, a weak free ν(CdO)
band at 1760 cmÀ1 in the TR mode corresponded to a strong
band at 1762 cmÀ1 in the RA mode, which indicates that the
C4dO bonds were oriented nearly perpendicular to the sub-
strate surface. A band at 1666 cmÀ1 in the RA mode was clearly
observed, which was the downshift of the ν(CdO) band at
1674 cmÀ1 before molecular recognition. This spectral feature
indicates that the weak C2dO bonds underwent a change in
orientation after molecular recognition. Meanwhile, a new band
at 1396 cmÀ1 due to the semicircle ring stretch of melamine17 in
the RA mode was obviously observed. Compared with Figure 6b,
the negative dip at 1660 cmÀ1 in the difference spectrum of
Figure 4b was owing to the C2dO stretching vibration with the
transition moment almost perpendicular to the water surface.
The observed bands at 1540, 1442, and 1424 cmÀ1 in the TR
mode and the bands at 1580, 1557, 1538, and 1396 cmÀ1 in the
RA mode were attributed to the quadrant and semicircle ring
stretches of melamine17 with most of their transition moments
perpendicular to each other.
Selectivity of Molecular Recognition. Figure 7 compares
p-polarized IRRAS spectra of the monolayers of C18BA on pure
water and aqueous subphases containing adenosine, thymidine,
cytidine, melamine, and their mixtures at 20 mM/m at the
incidence angle of 30ꢀ, respectively. The monolayers on the
aqueous subphases containing nucleosides individually exhibited
slight spectral changes in comparison with that on pure water. A
negative peak at 1762 cmÀ1 was observed in these cases in
contrast to a positive peak in the case of melamine. The most
characteristic absorption bands of the nucleosides should be
apparent in the vicinity of 1700 and/or 1650 cmÀ1, which made it
difficult to distinguish these characteristic bands because of the
Figure 6. FTIR spectra of the LB films of C18BA transferred from pure
water and aqueous melamine subphase (0.1 mM): (a) transmission
(TR) mode and (b) reflection absorption (RA) mode.
scissoring band appeared at 1653 cm ;
À1 17 however, the band ap-
peared at 1627 cmÀ1 in aqueous melamine solution (Figure S5).
At the airÀwater interface, the NH2 scissoring band of melamine
shifted up to 1632 cmÀ1 (Figure 4a) due to the occurrence of the
molecular recognition between barbituric acid moieties and
melamine. It is known that when hydrogen bonds are formed
the stretching vibrations occur with lower frequencies and the
scissoring ones with higher frequencies. It is clear that the peak at
1660 cmÀ1 in the difference spectrum resulted from the down-
shift of the CdO stretching band after molecular recognition,
which was attributed to C2dO bonds of the barbituric acid
moieties with the vibrational transition moments perpendicular
to the water surface to a great extent.
FTIR Spectra of LB Films. In order to give a deep insight into
the molecular structures of the monolayers before and after
molecular recognition, the corresponding LB films were studied
using a combination of TR and RA modes of FTIR spectroscopy
(Figure 6), and the assignments of the absorption bands are also
listed in Table 1. The absorption bands are very strong in the TR
spectra when their transition moments are parallel to the
substrate surface, and the bands are very strong in the RA spectra
when their transition moments are perpendicular to the substrate
surface. For the LB film of barbituric acid lipids from pure water,
both νa(CH2) and νs(CH2) bands in the TR and RA modes were
obviously observed, which indicates that the alkyl chains were
tilted away from the surface normal, moreover the νa(CH2)/
νs(CH2) intensity ratio in the TR mode was much larger than
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The Journal of Physical Chemistry B
ARTICLE
absorption bands of the barbituric acid moieties and distorted
spectral baselines. However, no characteristic band of cytidine
’ ACKNOWLEDGMENT
This work was supported by the National Natural Science
Foundation of China (No. 20873062) and the Fundamental
Research Funds for the Central Universities (1103020503).
(1600À1620 cmÀ1 32,33
)
was clearly observed, which indicates
that cytidine did not efficiently bind to the C18BA monolayer.
Combining with the spectra at the incidence angle of 60ꢀ (Figure S6),
the nucleosides with complementary donating/accepting groups
could not be effectively recognized in the monolayers of C18BA.
On the aqueous subphase containing the mixture of thymidine,
cytidine, and melamine, the monolayer almost displayed the
same spectrum as that in the presence of melamine only. The
barbituric acid moieties exhibited excellent selectivity for mela-
mine over nucleosides. The same conclusion is also obtained
from the FTIR spectra of the corresponding LB films (Figure S7).
From their chemical structures, double hydrogen bonds could be
only formed between the nucleosides and barbituric acid against
the double hydrogen bonds between the barbituric acid moieties,
whereas triple hydrogen bonds could be readily formed between
melamine and barbituric acid.
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’ ASSOCIATED CONTENT
S
Supporting Information. IRRAS spectra (p- and s-polari-
b
zation) of the C18BA monolayers on pure water and aqueous
melamine subphases at the surface pressure of 20 mN/m against
different incidence angles, the νa(CH2)/νs(CH2) intensity ratio
as a function of incidence angle, FTIR spectra of melamine in
powder and in aqueous solution, p-polarized IRRAS spectra of
the C18BA monolayers on pure water and aqueous subphases
containing adenosine, thymidine, cytidine, melamine, and their
mixtures at 20 mM/m at the incidence angle of 60ꢀ, and FTIR
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’ AUTHOR INFORMATION
Corresponding Author
*E-mail: xzdu@nju.edu.cn. Fax: 86-25-83317761.
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