http://www.revistadechimie.ro REV.CHIM.(Bucharest) ♦67♦No.1♦2016 84Thermal Characterization of Cholesterol in Air vs. Nitrogen Atmosphere… [631788]

http://www.revistadechimie.ro REV.CHIM.(Bucharest) ♦67♦No.1♦2016 84Thermal Characterization of Cholesterol in Air
vs. Nitrogen Atmosphere
LENUTA-MARIA SUTA1, GABRIELA VLASE2, TITUS VLASE2, GERMAINE SAVOIU-BALINT1, TUDOR OLARIU1, IONELA BELU3,
ADRIANA LEDETI1*, MARIUS-SORIN MURARIU4, LAVINIA STELEA5, IONUT LEDETI1
1 University of Medicine and Pharmacy Victor Babes, Faculty of Pharmacy, 2 Eftimie Murgu Sq., 300041, Timisoara, Romania
2 West University of Timisoara, Research Centre for Thermal Analysis in Environmental Problems, 16 Pestalozzi Str., 300115,
Timisoara, Romania
3 University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Technology, 2-4 Petru Rares Str.,
200349, Craiova, Romania
4 University of Medicine and Pharmacy Victor Babes, Faculty of Medicine, Department of Surgery II, First Surgical Clinic,
2 Eftimie Murgu Sq., 300041, Timisoara, Romania
5 University of Medicine and Pharmacy Victor Babes, Faculty of Medicine, Department of Obstetrics and Gynecology, III Clinic,
Eftimie Murgu Sq., 300041, Timisoara, Romania
In this paper, we describe a preliminary study regarding the thermal behavior and solid-state characterization
of pure cholesterol (CH) samples in oxidative air atmosphere vs. nitrogen atmosphere. The study was also
completed by the analysis of CH samples heated at 50, 100, 150, 200, 250 and 300 °C in air atmosphere,
employing FT-IR spectroscopy.
Keywords: cholesterol, thermal analysis, FTIR, stability study, oxidative and inert atmosphere
Cholesterol (abbreviated CH, fig. 1) continues to attract
the attention of physicians, cell biologists and biochemists
due to its importance not only in the human body’sphysiology but in its pathology as well [1]. It plays a
fundamental role in the structure of the cell membrane,
the reproduction process, regulating cellular functions, saltand water balance and the absorption of nutrients.
Moreover, it is also a precursor of vitamin D, bile acids and
steroid hormones. All this considering, it is only natural thatthere are over 100 genes devoted to its synthesis, transport,
metabolism and regulation [2-3].
The membrane of every human cell contains cholesterol
molecules that are required to bond with chains of
phospholipid fatty-acids. This interaction increases
membrane packing which in its turn reduces membranefluidity. The plasma membrane permeability to protons,
sodium ions and neutral solutes it is also reduced by the
presence of CH due to the bonds that form between thepolar ends of sphingolipids and phospholipids and the
hydroxyl group of CH molecules [4].
HOH
HHFig.1. Structural formula
of cholesterol (CH)
* email: [anonimizat] through the bile duct when a meal is consumed to
stimulate solubilization and absorption of dietary lipids and
fat soluble vitamins [6]. A very large part of the bile acids isreabsorbed, transported back to the liver where after the
CH molecules are recuperated, new bile acids are formed
(enterohepatic circulation) [7].
Instrumental analysis, consisting in different techniques
like FTIR spectroscopy [8], thermal analysis [9-11] and
PXRD [12-14] are important tools in characterization ofbiological active molecules [15-18] or potential bioactive
ones [19-20], but as well of great importance in
preformulation studies in pharmaceutical technology [21-22].
Following these considerations regarding the importance
of CH in anatomy and physiology, and the corroboration ofthis study with our previous reported ones in the field of
analyzing gallbladder stones [23] and other solid human
concretions, we set our goal in this paper in thecomparative analysis of thermal stability of CH in air
vs.
nitrogen atmosphere, under dynamic heating. Also, the
study was completed with spectroscopic analysis (FTIR)for CH samples subjected for established period of times
(5 or 15 min) to different temperatures (from 50 to 300 °C,
with 50°C heating step), in air atmosphere.
Experimental part
Materials and methods
Pure sample of Cholesterol (3 β-Hydroxy-5-cholestene,
5-Cholesten-3 β-ol, CAS 57-88-5) was obtained from Sigma
(C8667) and used as received, without further purification(purity >99%, melting point 147-149 °C, boiling point 360
°C). The sample was kept in sealed vial under ambiental
temperature until use.
The thermoanalytical TG/DTG/DTA curves were drawn
up in a dynamic air or nitrogen atmosphere under non-
isothermal conditions at a heating rate β=10 °C·min
-1 using
a Perkin-Elmer DIAMOND equipment. Samples of mass inCH synthesis takes place in the liver, it is transported in
the blood stream with carrier lipoproteins due to its
liposolubility and after fulfilling its role, it is recycled. From
a total content of 35 g, the human body produces about 1 gof CH daily. This lipid suffers an important transformation
in the liver, converting into bile acids, which are then stored
in the gallbladder [5]. This stage in the CH’ metabolism isconsidered a major route for its removal from the body.
After the bile acids are synthesized, they enter the intestinal

REV.CHIM.(Bucharest) ♦67♦No.1♦2016 http://www.revistadechimie.ro 85the range of 4-5 mg were put into aluminum crucibles and
heated by increasing temperature from ambient up to 500
°C (TG analysis). In order to evaluate the accuracy of themeasurements, two repetitions were realised with this
experimental protocol for the samples and the obtained
results were practically identical.
The thermal treatment of CH samples at different
temperatures was carried out using a Perkin-Elmer
DIAMOND equipment and was realised as follows: thesamples were put into aluminum crucibles, heated with
10°C·min
-1 up to desired temperature (i.e. 50 , 100 150,
200, 250 and 300 °C) and maintained in isothermallyconditions for 5 min. A CH sample heated at 300 °C were
also kept in isothermally conditions for 15 min.
The FTIR spectra of the solid samples were obtained on
Perkin Elmer SPECTRUM 100 device using the U-ATR
technique, without further preparation of the samples.
Reesults and discussions
FTIR spectroscopy
Cholesterol samples were analyzed by UATR-FTIR
Spectroscopy technique, in order to determine any
structural modification of the molecule under thermal
treatment. All the discussions are carried out in comparisonwith the FTIR spectra of pure CH, thermally untreated.
Literature data [23-24] presents the main FTIR bands
identified in the spectrum of CH, as well their attributionsto functional moieties: large band between 3600-3200
cm
-1 is due to stretching vibrations of the O-H bond (peak
at 3412 cm-1). This band is also present in the spectrum of
thermally treated cholesterol (up to 300°C), suggesting that
the analysed sample consist in anhydrous CH, without any
adsorbed or crystallization water. These results are also ingood agreement with the ones obtained by thermal
analysis, since no mass loss occurs up to 235 °C.
Other bands were observed, as follows: 2930 and 2901
cm
-1 (CH2 and CH3 asymmetric stretching), 2867 and 2848
cm-1 (CH2 and CH3 symmetric stretching), 1464 and 1436
cm-1 (CH2 and CH3 bending) and 1050 cm-1 (C-C stretching).
Also, the recorded FTIR spectra at the CH samples
subjected to selected temperatures (fig.2), suggest thatthermal treatment in isothermal conditions, for 5 min, up
to 300 °C doesn’t drastically modify the composition of the
sample.
However, the CH sample subjected to 300 °C for 15 min
is different in both aspect (brown powder), but as well as
spectroscopic behavior. This observation is in goodagreement with the thermoanalytical data, when a
heterogeneous degradation of CH occurs.For the CH sample treated at 300 °C for 15 min, the FTIR
spectrum is modified: the broad band between 3600-3200
cm
-1 is dramatically diminished, suggesting that the
degradative process occur involve the destruction of the
CH skeleton near this functional moiety. Two of the four
sharp peaks between 2930-2848 cm-1 associated with
stretching of methyl and methylene groups disappeared
from the spectrum, so only the bands around 2930 and
2867 cm-1 are still present in the spectrum. These
observations are the proof that the degradation of the CH
skeleton occurs in the lateral aliphatic moiety, as well.
Comparative thermal analysis
TG Analysis
The superimposed TG curves for pure CH in both
dynamic oxidative (air) atmosphere and inert (nitrogen)
are presented in figure 3. CH is thermally stable up to 228
°C in air and 197 °C in nitrogen, respectively. Up to thesetemperatures, no mass losses occur. With the increasing
of temperature, the rapid mass loss takes place up to 367
°C in air ( ∆m
air=85.1%) and 344 °C in nitrogen
(∆mnitrogen=97.5%), respectively. In the temperature ranges
367-500 °C in air, respectively 351-500 °C in nitrogen, the
mass loss is considerable lower ( ∆mair=4.6%,
∆mnitrogen=2.4%).
Fig.2. UATR-FTIR spectra of thermally treated CH samples (sample
abbreviation as follows: CH_x_y’, where x=temperature in °C and
y-time in min) vs. untreated sample (CH)
Fig.3. The superimposed TG curves obtained for CH in dynamic
air vs. nitrogen atmosphere
The mass losses suggest that the degradative
mechanism is different and dependent of surrounding
atmosphere, fact that was expected, since in air, the mostprobable mechanism is an oxidative one. However, the TG
data are better understood after corroborating the
information with the ones from DTG curve, where theprocesses are better individualized and separated.
DTG Analysis
DTG curve recorded at a heating rate β=10 °C·min 1
suggest a thermal stability up to 222°C in air atmosphere
and 200°C in nitrogen atmosphere, respectively (fig.4). Thefirst degradative process in air presents a maximum at
331 °C in air, respectively 333 °C in nitrogen. The processes
are well individualized. The main degradative process thatoccur in air atmosphere is followed by another one, with
an considerable smaller amplitude in the temperature
range 381-500 °C, with a maximum of 418°C. This processis not observed when the heating takes place in nitrogen.
DTA Analysis
Anhydrous cholesterol undergoes a polymorphic
crystalline transition at 39°C (endothermal event), which
is in god agreement with already reported data of Loomis,Shipley and Small [25], as presented in figure 5. This
polymorphic transition takes place in nitrogen atmosphere

http://www.revistadechimie.ro REV.CHIM.(Bucharest) ♦67♦No.1♦2016 86too, the DTAmax at 42 °C. However, this polymorphic
transition is not influencing the FTIR spectra of the sample.The DTA curve in air shows the melting of CH at 148°C,
which is in good agreement with the data suggested by
the MSDS of the supplier. The melting is independent of thesurrounding atmosphere. The DTA curve obtained in
oxidative atmosphere is more complex that the one
recorded in nitrogen, due to multiple oxidations andskeleton breakdowns. The DTA pattern is complex,
showing overlapped processes, with maximums at 288
and 357°C. An endothermic event is observed, with amaximum at 407°C, which cannot be associated with a
clear physical process. Even if the MSDS of the supplier
suggest that the boiling of CH occurs at 360°C, our studyproves that isothermal heating for 15 min at 300°C
determines a considerable breakdown of the molecule,
and this process can be associated with evaporation ofsmaller moieties and not of the entire CH structure.
However, the DTA curve recorded in nitrogen
atmosphere suggest that the thermodegradative processesdo not occur, the pattern being more simplistic, suggesting
solely the melting (as previously mentioned) and the boiling
at 340 °C, other thermal events weren’t noticed.
Conclusions
In this study, we set our goal in investigating the thermal
behavior of cholesterol in both dynamic oxidative
atmosphere (air) and inert one (nitrogen), in order to
determine the stability. It was shown that CH is thermallystable up to considerable high temperatures in both
surrounding atmospheres, but the mass loss occurs through
different mechanisms: in air, the main processes are apolymorphic transitions around 40 °C, followed by phase
transition (solid to liquid), and decomposition, while in
nitrogen, solely physical processes are involved in the massloss: melting around 148 °C, followed by evaporation and
boiling at 340 °C.
Also, FTIR spectroscopy was used as complementary
instrumental technique in the analysis of CH samples
treated in isothermally conditions at 50, 100, 150, 200, 250
and 300 °C. It was shown that polymorphic transition thatoccurs at 39 °C does not influence the aspect of the FTIR
spectra. Only the isothermal treatment of CH at 300 °C for
15’ determines a considerable modification ofcomposition, as suggested by the spectroscopic
investigation.
Acknowledgement: This work was supported by a grant financed by
the University of Medicine and Pharmacy “Victor Babes” Timisoara
(Grant PIII-C3-PCFI-2016/2017, acronym STONES to L.-M.S, A.L., M.-
S.M. and I.L.).References
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Manuscript received: 15.11.2015Fig.5. The superimposed
DTG curves obtained for
CH in dynamic air vs.
nitrogen atmosphere
Fig.4. The superimposed
DTG curves obtained for
CH in dynamic air vs.
nitrogen atmosphere