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Spectroscopic Studies of Cholesterol: Fourier Transform Infra-Red and

Vibrational Frequency Analysis

Article · April 2014

DOI: 10.1166/mat.2014.1161

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 Copyright © 2014 by American Scientific Publishers Materials Focus
All rights reserved. Vol. 3, pp. 211–217, 2014
Printed in the United States of America (www.aspbs.com/mat)

Spectroscopic Studies of Cholesterol: Fourier

Transform Infra-Red and Vibrational
Frequency Analysis
Ujval Gupta, Vivek K. Singh, Vinay Kumar, and Yugal Khajuria∗
School of Physics, Shri Mata Vaishno Devi University, Kakryal, Katra 182320, J&K, India

ABSTRACT
The present paper deals with the experimental and theoretical study on the vibrational spectra of cholesterol
molecule. The vibrational and electronic transition analysis of the molecule was performed using Fourier Trans-
form Infra-Red (FT-IR) spectroscopy and UV-Vis spectroscopy. FT-IR spectra were recorded in the spectral
regions of 400–4000 cm−1 and the UV-Vis absorption spectrum of the molecule has been recorded in powder
form using UV-Vis spectrophotometer in the spectral region 190–1400 nm. The optimized geometry with 6-
311G (d, p) basis set is used to determine the vibrational spectra of the molecule using Gaussian 09 software.
The vibrational harmonic frequencies are scaled using a scale factor, yielding a good agreement between the

ARTICLE
experiment and the theory. The Mulliken atomic charges have also been computed. The calculated HOMO and
LUMO energies show that charge transfer occurs within the molecule.
KEYWORDS: Cholesterol, FT-IR Spectroscopy, UV-Vis Spectroscopy, B3LYP, Density Functional Theory (DFT),
Mulliken Charges.
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1. INTRODUCTION Copyright: American Scientific Publishers
the permeability of the plasma membrane to neutral solutes
Cholesterol (Fig. 1) is a most abundant sterol and found protons and sodium ions.7
8
among the lipids in the blood stream of human body. It Cholesterol was first discovered in bile and in gall-
is a ubiquitous component of mammalian cell membranes stones (in solid form) by François Poulletier de la Salle in
and is needed for other functions.1
2 In vertebrates it is 1769.9
10 The chemist Eugène Chevreul rediscovered it in
formed predominantly in the liver. It plays a crucial role 1815 and named it “cholesterine.” Researcher Boudet was
in determining the physical and chemical properties of the the first to find cholesterol in blood in 1833.11 Brown and
membrane and thus regulates membrane fluidity and per- Goldstein described cholesterol as the “the most highly
meability and adjusts the lateral mobility of membrane pro- decorated small molecule in biology.”12 Cholesterol was
teins. Cholesterol thus plays a pivotal role in variety of first isolated in 1769, but productive research on its struc-
functions of the human body. In the liver, it is converted to ture and function did not begin until the 20th century.
bile, which is then stored in the gallbladder. It was found Development of new research equipment has helped the
as one of the principle component of human gallstones researchers to understand the structure of the cholesterol
formed inside the gallbladder of the human body.3
4 It is molecule, as well as its function. Wieland and Windaus
also required to build and maintain membranes and it mod- won the Nobel Prize in chemistry in 1928 for their work,
ulates membrane fluidity over the range of physiological which also included discoveries of the molecular compo-
temperatures. Reinitzer (1888) discovered the molecular sition and structure of cholesterol.13
formula of cholesterol merely by precise elemental anal- The complex structure of cholesterol molecule has
ysis of a series of cholesteryl ester dibromides5 and his created increased interest for its spectroscopic studies.
formula remains unchallenged today. The structure of the It has also been observed that there have been a fewer
tetracyclic ring of cholesterol contributes to the decreased studies on the vibrational spectroscopy of the choles-
fluidity of the cell membrane as the molecule is in a trans terol molecule. Infrared spectrum of cholesterol molecule
conformation making all but the side chain of cholesterol has been reported by several groups of researchers but
rigid and planar.6 In this structural role, cholesterol reduces only fragmentary band assignments are available.3
4
14–17
∗
It is a major constituent of human gallstones and FTIR
Author to whom correspondence should be addressed.
Email: yugal@smvdu.ac.in
spectroscopy has been extensively used to characterize
Received: 20 January 2014 the gallstones and to analyze the cholesterol inside the
Accepted: 8 March 2014 gallstones.3
4
14–17 Previously, Raman spectroscopy has

Mater. Focus 2014, Vol. 3, No. 3 2169-429X/2014/3/211/007 doi:10.1166/mat.2014.1161 211

 Spectroscopic Studies of Cholesterol: FT-IR and Vibrational Frequency Analysis
H understanding the fundamental mode of vibration of the
H H
HO
Fig. 1. Structure of cholesterol.
also been used to study the cholesterol molecule.18 Raman
spectroscopy has also been used to examine the chem-
ical constituents i.e., cholesterol and bilirubin in human
gallstones.19
20 Recently, Kett et al.21 investigated the
spectra of the cholesterol in the C H stretching region
using the surface specific technique of Sum Frequency
ARTICLE
Generation (SFG) vibrational spectroscopy and the com-
plementary technique of Reflection Absorption Infrared
Spectroscopy (RAIRS). Absorption spectrum of choles-
terol has also been studied by few research groups.22
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The assignment of the vibrational IP:spectrum of cholesterol
124.124.47.116 On: Thu, 16 Apr 2015 17:47:38
is surprisingly incomplete for such a fundamental
molecule Scientific
Copyright: American
and hence there is a great need to study the cholesterol
molecule. As Quantum chemical computational methods
have proven to be an essential tool for predicting the vibra-
tional spectra23 and no such studies with higher basis set
have been carried out for the molecule in detail. A sig-
nificant advancement in this area was made by combin-
ing semi-empirical quantum mechanical method, ab-initio
quantum mechanical method and density functional theory
24–29
(DFT), each method having its own advantages.
The main aim of the present work is to study the elec-
tronic as well as vibrational characteristics of cholesterol
molecule. In the present paper, we have recorded the FT-
IR spectrum and electronic absorption spectrum of the
molecule. Assignments of vibrational bands for all possible
modes have been made, the calculated vibrational frequen-
cies has been compared with the experimental FT-IR spec-
trum. The values of energy separation between the HOMO
and LUMO and dipole moment have also been calculated.
2. MATERIALS AND METHODS
Cholesterol sample obtained in powder from Sigma
Aldrich Chemicals Co. Ltd. was used in the experiment.
The FT-IR spectrum of the title compound was recorded
in transmittance mode in the region 4000–400 cm−1 by
dispersing and palletizing in KBr pellets using Perkin-
Elmer IR spectrometer. Electronic absorption spectrum of
cholesterol molecule in powder form was recorded ISR
212
Gupta et al.
assembly attached with Shimadzu UV-2600 Double beam
spectrophotometer in the region 190–1400 nm.
3. COMPUTATIONAL METHOD
The atomic numbering scheme of the cholesterol molecule
is given in Figure 2. The combination of vibrational spec-
tra with quantum chemical calculation is effective for
compound. Calculations of vibrational frequencies of the
cholesterol molecule were carried out using Gaussian 09
program package through density functional theory (DFT)
approach. The optimized geometry is used to calculate
the vibrational frequencies with 6-311G (d, p) basis set.
The cholesterol molecule C27 H46 O consists of 74 atoms
and gives 3N-6 i.e., 216 vibrational levels in the range
32.68–7329.91 cm−1 . It is well known that vibrational
frequencies obtained by quantum chemical calculations are
typically larger than their experimental counterparts, and
thus, empirical scaling factors are used to compare the
results. For the present study a scaling factor of 0.967 has
been used. The scaled vibrational frequencies show good
agreement with the experimental data. The assignments for
the various vibrational bands have been made by a careful
analysis with the help of Gauss-View visualization pro-
gram which is a part of Gaussian 09.30
4. RESULTS
Publishers AND DISCUSSION
4.1. FTIR Spectroscopy and Vibrational Assignments
The goal of the vibrational analysis of the cholesterol
molecule is to find out the vibrational modes con-
nected with specific molecular structures of the compound.
We have calculated the vibrational frequencies for all the
possible 216 modes using the quantum mechanical calcula-
tion based on the density functional theory (DFT). The cal-
culated vibrational frequencies are compared against the
experimental data and these assignments are additionally
checked by comparing with characteristic group frequen-
cies. Table II lists only those calculated vibrational frequen-
cies for which experimental vibrational bands are observed.
The major bands identified for cholesterol molecule
were found at 3400, 2932, 2899, 2866, 1464, 1438,
1378, 1055, 1022, 985, 840, and 800 cm−1 (Fig. 3).
The bands between 2800–3000 cm−1 are characterized
due to asymmetric and symmetric stretching vibrations of
CH2 and CH3 groups.4
17 The observed broad and intense
band (Fig. 3) nearly at 3400 cm−1 is attributed to OH
stretching.14
17 The characteristic strong peak at 2899 cm−1
is due to CH2 symmetric stretching vibration. Cholesterol
has one double band (C C) in the second ring. This was
prominently shown at 1674 cm−1 (Fig. 3). This assignment
of 1674 cm−1 for the double bond in the second ring of
cholesterol is in good agreement with the results reported
by Zheng et al.19 and also with the 1693–1671 cm−1
assignment for cyclopentene.31 The band at 1464 cm−1 is
due to asymmetric stretching vibrations of CH2 and CH3
Mater. Focus, 3, 211–217, 2014
Gupta et al. Spectroscopic Studies of Cholesterol: FT-IR and Vibrational Frequency Analysis

Fig. 2. Numbering scheme of cholesterol molecule.

groups and the band at 1378 cm−1 is attributed to the

Table I. Atomic Mulliken charges of cholesterol molecule.
CH2 and CH3 bending vibration of cholesterol molecule.14

ARTICLE
Atom Mulliken Atom Mulliken The sharp peak at 1055 cm−1 can be attributable to ring
numbering charge numbering charge deformation of cholesterol.14 The band at 840 cm−1 is
C1 0015 H30 0340 due to the C C C stretching of cholesterol molecule.19
C2 −00145 H31 0187 The bands between 900–675 cm−1 are characterized due
C3 0075 Delivered
H32 by Publishing
0326 Technology
the C H to: Guest User
out-of-plane bending which are the characteris-
C4 −0240 IP: H41
124.124.47.116 On: Thu, 16 Apr 2015 17:47:38
0085
Copyright: American tic of the aromatic
Scientific Publishers substitution pattern and mainly deter-
C5 −0083 H42 0066
C6 −0124 H43 0062 mined by the number of adjacent hydrogen atom on the
C9 −0112 H44 0055 ring and not very much affect by the nature of substitu-
C10 −0023 H45 0033 tions normally.24
32
33 The experimentally observed wave
C13 −0020 H46 0052 numbers for C H out-of-plane bending mode in FT-IR
−0133
C14 H47 0048
spectrum are at 700, 739, 800 and 885 cm−1 . We have also
C16 −0475 H48 0053
C22 −0157 H49 0063 observed several weak bands in the experimental FTIR
C23 −0183 H50 0107 spectra of cholesterol molecule between 1689–1994 cm−1
C24 −0399 H51 0286 (at 1689, 1706, 1719, 1739, 1753, 1779, 1799, 1833, 1849,
C25 −0049 H52 0041 1874, 1924, 1945, and 1994 cm−1 ) which may be consid-
C26 −0362 H53 0041 ered as the characteristic overtones and reflect the substi-
C27 −0074 H54 0116
C28 −0016 H55 0117
tution pattern on the ring.33 The vibrational bands at 927
C29 −0365 H56 0116 and 985 cm−1 are characterized mainly due to the C H
C33 −0245 H57 0137 bending vibrations.34 A number of weak bands observed
C34 −0225 H58 0021 in the FT-IR spectrum at frequencies 3653, 3679, 3749,
C35 −0071 H59 0022 3825, 3841, 3856 and 3921 cm−1 that may involve over-
C36 −0520 H60 0281
C37 −0046 H61 0022
tone and combination modes. Table II shows that there is
C38 −0089 H62 0042 a good agreement between calculated and experimentally
C39 −0193 H63 0009 observed values of the vibrational bands discussed above.
C40 −0132 H64 0064 Each vibrational bands present in the IR spectra of the
H7 0034 H65 0055 cholesterol molecule is characteristic of molecular vibra-
H8 0075 H66 0152
tional motions, which, taken all together, are unique for
H11 0196 H67 0116
H12 0058 H68 0063 cholesterol. For example, as discussed above the peaks
H15 0075 H69 0079 at 1378 cm−1 is attributed to the CH2 and CH3 bend-
H17 0259 H70 0065 ing vibration of molecule, the characteristic vibrational
H18 0197 H71 0054 peak at 1440 cm−1 is due to the CH2 and CH3 deforma-
H19 0076 H72 0043
tion vibrations, the peak at 1674 cm−1 is due to C C
O20 −0307 H73 0008
H21 0185 H74 0161 stretching vibrations, and the characteristic strong peak at
2899 cm−1 is due to CH2 symmetric stretchingvibration.

Mater. Focus, 3, 211–217, 2014 213

 Spectroscopic Studies of Cholesterol: FT-IR and Vibrational Frequency Analysis Gupta et al.

Table II. Vibrational assignments of fundamental observed frequencies and calculated frequencies for cholesterol using DFT/B3LYP/6-311G (d, p)
basis set.

Frequency calculated Frequency corrected Frequency of observed Literature value

(cm−1 ) (cm−1 ) bands in FTIR (cm−1 ) (cm−1 )14
17–19
33
34 Assignments

737 713 674 680 C H out-of-plane bending

764 739 700 C H out-of-plane bending
814 787 739 C H out-of-plane bending
828 801 800 C H out-of-plane bending
876 847 840 849 C C C stretching
930 899 885 C H out-of-plane bending
959 927 927 920 C H bending
982 950 956 961 CH CH2 and CH2 waging
1028 994 985 C H bending
1039 1005 1022 1035 in in-plane C H bending
1078 1042 1055 1056 Ring deformation
1142 1104 1108 in-plane C H bending
1175 1136 1131 in-plane C H bending
1218 1178 1170 in-plane C H bending
1225 1185 1191 1191 C C stretching
1287 1245 1236 1255 CH2 deformation
1311 1268 1272 1275 CH2 bending
1351 1306 1317 1305 CH2 waging
1376 1331 1331 1333 CH2 bending
1429 1382 1378 1378 CH2 and CH3 bending
ARTICLE

1438 1440 CH2 and CH3 deformation vibrations

1519 1469 1464 1466 CH2 and CH3 symmetric stretching
1563 1511 1512 1506 C C stretching in aromatic ring
1722 1665 1674 1674 C C stretching
2974 2876 2866 2860 CH2 and CH3 symmetric stretching
Delivered by Publishing
2899 Technology to: Guest
2901 User CH2 symmetric stretching
3058 IP: 124.124.47.116 2932
2957 On: Thu, 16 Apr 2015 17:47:38
2925 CH2 and CH3 asymmetric stretching
Copyright: American
3400 Scientific Publishers
3398 O H stretching

It is concluded that if any sample of biological tissue con-
tains cholesterol, these peaks will be present in its IR spec-
trum. In other words, the molecular structure and com-
position of a material under study is encoded as a set of
frequency in the IR spectrum. Thus, the IR spectroscopy
can provide a ‘fingerprint’ of a substance from which the
molecular composition can be determined.
4.2. UV-Vis Spectroscopy
The UV-Vis absorption spectrum of cholesterol molecule
has been recorded in powder form using ISR assem-
bly attached with Shimadzu UV-2600 Double beam
spectrophotometer in the spectral region 190–1400 nm.
Figure 4 shows the absorption spectra of the cholesterol
molecule in the spectral region 190–350 nm. Figure 5

FTIR spectra of cholesterol molecule

207 nm

100
195 nm

80
4
Absorbance (arb. units)
%Transmittance

60
3

40
2

20
1
239 nm
0 283 nm 294 nm
0
4000 3500 3000 2500 2000 1500 1000 500 200 220 240 260 280 300 320 340
Wave number (cm–1) Wavelength (nm)

Fig. 3. FTIR spectrum of cholesterol. Fig. 4. UV-vis spectrum of Cholesterol.

214 Mater. Focus, 3, 211–217, 2014

Gupta et al. Spectroscopic Studies of Cholesterol: FT-IR and Vibrational Frequency Analysis

0.6 peaks are also observed at 239 nm, 283 nm, and 294 nm
in the spectrum (Fig. 5). The spectra (Figs. 4 and 5)
0.5 239 nm
clearly indicate that cholesterol molecule gave indications
Absorbance (arb. units)

0.4
of an extended region of absorption on the ultra-violet side
(Figs. 4 and 5). No significant observable absorption peaks
0.3 are seen between 350–1400 nm.
271 nm
0.2 283 nm
4.3. Atomic Charges
294 nm
0.1 The bonding capability of a molecule depends on the elec-
tronic charge on the chelating atoms. The atomic charge
0.0
values have been obtained by the Mulliken population
analysis.29 The Mulliken atomic charges of cholesterol
225 250 275 300 325 350
molecule are calculated at B3LYP/6-311G (d, p) levels
Wavelength (nm)
and are listed in Table I. It is observed that the hydro-
Fig. 5. A part of the UV-Vis spectrum of cholesterol in the spectral gen atoms exhibit positive charges. The range of hydro-
region 225–350 nm. gen atom charges is from 0.008 of H73 to 0.340 of H30.
The charge distribution shows that C1 and C3 are posi-
shows a part of the absorption spectra in the spectral region tively charged where as the remaining carbon atoms are
225–350 nm. The strong and broad absorption peaks at negatively charged.
195 nm and 207 nm are seen (Fig. 4). This is in good
agreement with the literature data since the maximum 4.4. Frontier Molecular Orbital Analysis

ARTICLE
absorbance of cholesterol and most oxysterols have been The most important orbitals in a molecule are the fron-
reported below 200 nm.35 Generally, if –C C– bands are tier molecular orbitals, called highest occupied molecular
attached to one or two tertiary carbon atoms, their absorp- orbital (HOMO) and lowest unoccupied molecular orbital
tion occurs at a slightly longer wavelength (red shift) of (LUMO). These orbitals determine the way the molecule
about 200 nm.36 Thus, it may be concluded that the absorp- interacts with
Delivered by Publishing Technology other species.
to: Guest User The HOMO energy is directly
tion peak appeared at 207 nm isIP:due the –C C– band proportional to
124.124.47.116 On: Thu, 16 Apr 2015 17:47:38the ionization potential and the energy of
present in the cholesterol molecule. TheCopyright: American Scientific
other absorption the LUMO Publishers
is directly proportional to the electron affinity.

ELUMO = –6.88 eV

EHOMO-ELUMO = 6.43 eV

EHOMO = –0.45 eV

Fig. 6. Frontier molecular orbital analysis of cholesterol molecule.

Mater. Focus, 3, 211–217, 2014 215

 Spectroscopic Studies of Cholesterol: FT-IR and Vibrational Frequency Analysis
Table III. B3LYP calculated total dipole moment and HOMO/LUMO
energy using 6-311G (d, p) basis set.
Dipole moment values (debye) Calculated Reported value37
X −07715 −07605
Y 13382 13429
Z −04296 −04167
HOMO/LUMO energy gap (eV) 643 7057
In order to evaluate the energetic behavior of the choles-
terol the energies of HOMO, LUMO and their orbital
energy gaps using 6-311G (d, p) basis sets are calculated.
Cholesterol has 344 orbitals, the HOMO/LUMO energy
difference is the difference between the occupied 108th
orbital and the unoccupied 109th orbital. The pictorial
illustration of frontier molecular orbitals namely HOMO,
LUMO are shown in Figure 6. The calculated energy value
of HOMO is − 045 eV and the LUMO is − 688 eV. Thus,
the value of energy separation between the LUMO and
HOMO is 6.43 eV. The maximum absorption wavelength
(max ) corresponds to the electronic transition from the
ARTICLE
38
HOMO to LUMO with major contribution. Using DFT
method, the value of the maximum absorption max , cal-
culated using the relation max = hc/EHOMO–LUMO , comes
out to be 193.3 nm which is very close to the experimen-
Delivered by Publishing
tally recorded absorption bands at 195 nm and 207 nm Technology
IP: 124.124.47.116 On: Thu, 16130231-discovery-cholesterol/#ixzz2efGEIVlO.
(Fig. 4). Copyright: American Scientific
The dipole moment in a molecule is another important
electronic property which results from non-uniform dis-
tribution of charges on the various atoms in a molecule.
The calculated value of the total dipole moment for
the molecule is 1.6407. Table III presents the calculated
HOMO/LUMO energy gap and the total dipole moment
values which are in good agreement with the earlier
reported values Osman et al.37
5. CONCLUSION
The normal mode frequencies and corresponding
vibrational assignments of the cholesterol molecule are
examined theoretically by using Gaussian 09 package.
The harmonic vibrational frequencies of cholesterol
molecule have been determined and analyzed at DFT
level of theory. The calculated frequency values obtained
with 6-311G (d, p) basis set coincide well with the
experimental values. The result presented in this work
for cholesterol indicates that DFT with B3LYP functional
set and 6-311G (d, p) basis set is reliable for the pre-
diction of Infrared spectra, HOMO and LUMO energy
gap and total dipole moment. The energy separation
between the HOMO and LUMO shows that a charge
transfer occurs within the cholesterol molecule. The UV-
Vis spectrum of cholesterol molecule gives the indication
of an extended region of absorption on the ultra-violet
side.
216
Gupta et al.
Acknowledgments: Financial assistance from Science
and Engineering Research Board (SERB), DST, New
Delhi (SR/S2/LOP-0020/2012) is gratefully acknowl-
edged. We are also thankful to Professor S. B. Rai, Depart-
ment of Physics, Banaras Hindu University, Varanasi for
providing FT-IR experimental facility.
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