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J. of Supercritical Fluids 84 (2013) 173– 181

Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

jou rn al hom epage: www.elsev ier .com/ locate /supf lu

olubility of thymol in supercritical carbon dioxide and itsmpregnation on cotton gauze

toja Milovanovic, Marko Stamenic, Darka Markovic, Maja Radetic, Irena Zizovic ∗

niversity of Belgrade, Faculty of Technology and Metallurgy, Karnegijeva 4, 11120 Belgrade, Serbia

r t i c l e i n f o

rticle history:eceived 17 July 2013eceived in revised form 11 October 2013ccepted 14 October 2013

eywords:olubility

a b s t r a c t

Through this study, an attempt has been made to evaluate the solubility of thymol in supercritical carbondioxide as well to investigate a prospect of its impregnation on cotton gauze on laboratory scale. Solubilityof thymol in supercritical carbon dioxide was determined at temperatures of 35 ◦C, 40 ◦C and 50 ◦C, andpressures ranging from 7.8 to 25 MPa (CO2 density range 335.89–849.60 kg/m3) using a static method.The solubility data were correlated using semi-empirical equations introduced by Chrastil, Adachi andLu and del Valle and Aguilera. Taking into account obtained results, temperature of 35 ◦C and pressure

mpregnationupercritical carbon dioxidehymolotton gauzentimicrobial activity

of 15.5 MPa were selected as operating conditions for the impregnation process. Impregnation of cottongauze with thymol was performed in a cell using carbon dioxide as a solvent. Kinetics of the processwas determined and modeled. Masses of thymol on cotton gauzes after 2 h and 24 h of impregnationwere 11% and 19.6%, respectively. FT-IR analysis confirmed the presence of thymol on the surface ofthe cotton fibers. The impregnated gauze provided strong antimicrobial activity against tested strains of

occus

Escherichia coli, Staphyloc

. Introduction

Thymol (2-isopropyl-5-methylphenol) is a natural monoter-ene phenol abundantly present in essential oils of thyme, oreganond winter savory [1]. It has been reported that among thedentified natural antimicrobial agents, thymol exhibited sig-ificant antimicrobial activity [2]. Its antimicrobial power wasroven against Gram positive bacteria (e.g. Staphylococcus aureus,taphylococcus epidermidis, Enterococcus faecalis, Listeria monocyto-enes, Bacillus cereus), Gram negative bacteria (e.g. Escherichia coli,almonella typhimurium, Yersinia enterocolitica) and yeasts (e.g.andida albicans, Saccharomyces cerevisiae), which were the mostensitive [3]. Thymol was shown to be efficient even againstethicillin-resistant staphylococci [4] with minimum inhibitory

oncentration values determined by agar dilution method in theange of 0.03–0.06% (v/v). Thymol also acts as a powerful scavengerf reactive oxygen species and therefore has strong antioxidantroperties [5,6]. Recently, it was found that thymol significantly

mproved inflammatory responses and promoted wound healingy reducing the edema and diminishing the influx of leukocyteso the injured area [7]. Therefore, it was suggested that thymol

ould be utilized as a promising compound in the treatment ofnflammatory processes and wound healing.

∗ Corresponding author. Tel.: +381 11 3303 795; fax: +381 11 3303 795.E-mail address: zizovic@tmf.bg.ac.rs (I. Zizovic).

896-8446/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.supflu.2013.10.003

aureus, Bacillus subtilis, Enterococcus faecalis and Candida albicans.© 2013 Elsevier B.V. All rights reserved.

Transdermal systems capable to deliver a bioactive agent intoskin cutaneous/subcutaneous levels are of great interest for ther-apeutic treatments. A wound dressing system should provideflexibility, controlled adherence to the surrounding tissue, gaspermeability and durability/biodegradability. In addition, wounddressing systems should have the capacity to absorb fluids exudedfrom the wounded area and simultaneously to control water loss[8]. The choice of the dressing material is crucial since its interactionwith the wound may significantly influence the healing process.Hence, natural-based biodegradable and biocompatible materialsare gaining increasing attention [9]. Cotton fibers have been inuse for over five thousand years. Cotton gauze is widely used forhygienic purposes because of its natural softness, high hygroscop-icity and heat retaining properties [10]. Therefore, cotton gauze isa natural material, which meets all requirements for a good wounddressing.

Since supercritical carbon dioxide (scCO2) is not only knownas a solvent for valuable compounds but also for its high diffusionability in organic matter, the latter property can be exploited forimpregnation of solid matrices with natural antibacterial agents[11]. There are few studies in literature that combine these pro-cesses [8,11–13]. In order to perform an efficient impregnation,data on solubility of active substance in scCO2 are necessary.

The methods for experimental determination of solubility

in supercritical fluids can be classified into three categories:static, dynamic and chromatographic methods [14]. Direct exper-imental determination of the solubility data at high pressureis time-consuming and it requires costly equipment. Desirable

1 ercritical Fluids 84 (2013) 173– 181

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74 S. Milovanovic et al. / J. of Sup

inimization of the experimental efforts can be obtained by intro-uction of the methods of correlation and prediction. There are twopproaches in the literature dealing with a problem of representa-ion and estimation of high-pressure solubility data: (1) applicationf an expression directly relating the concentration of solute to theensity of supercritical solvent, and (2) application of an equation oftate [15–17]. The equation of state models require many physicalroperties (macroscopic critical properties and sublimation pres-ure are needed for cubic equations of state as well as moleculararameters for perturbed equations), which are estimated by groupontribution methods leading to erroneous correlations. On thether hand, semi-empirical equations, like density based models,equire only available independent variables like pressure, tem-erature and density of pure supercritical fluid instead of solidroperties. They are based on simple error minimization [17].

To the best of our knowledge, there are no data available inhe open literature on solubility of thymol in scCO2 at tempera-ures below 50 ◦C. Therefore, in this study, solubility of thymol incCO2 was determined at different conditions using a static method.n the basis of obtained solubility data, optimum operating condi-

ions for thymol impregnation on cotton gauze using scCO2 wereelected. Kinetics of the process, as well as antimicrobial propertiesf the impregnated cotton gauze, was investigated.

Since semi-empirical density-based models which provide aelationship between the solubility of the solute and the pureensity of the solvent represent an efficient tool for the data extrap-lation to process conditions that have not been acquired by theeasurements, the aim of this work was also to determine which

emi-empirical equation provides the best fit to solubility data ofhymol in scCO2. Density-based models introduced by Chrastil [18],dachi and Lu [15] and del Valle-Aguilera [19] were employed.hose models were selected as the first and the simplest densityased model (Chrastil), improved model regarding density depend-nce of solubility (Adachi-Lu) and improved model regarding thenthalpy of vaporization dependence on temperature (del Valle-guilera).

. Materials and methods

.1. Materials

Thymol (purity >99%) was supplied by Sigma–Aldrich ChemiembH, Germany. Commercial CO2 (purity 99%) was purchased

rom Messer–Tehnogas, Serbia. Sterile cotton gauze with weavingensity of 17 threads/cm2 was produced by Niva, Serbia.

.2. Solubility determination

Solubility of thymol was determined using a static method in high-pressure view chamber (Eurotechnica GmbH) presented inig. 1. The view chamber is set up for observation of multiphasend interfacial behavior at elevated pressures and temperatures.

The chamber has cylindrical interior with internal volume of5 mL. Electrical heating jacket around the cell allows quick andniform heating. Changes inside the cell can be recorded by USB-CD monochrome camera. Solid sample of thymol (1.00 g) in a glassontainer was placed inside the chamber. The stainless steal filteras placed at the top of the container in order to avoid the thy-ol precipitation in the container during the decompression. After

eaching the desired temperature, CO2 was pumped into the sys-em. When the desired pressure was attained, the system has been

ept at the constant temperature and pressure for a certain timentervals. At the end of process, the valve V2 was opened and theressure was slowly lowered to the atmospheric pressure (decom-ression speed was 0.33 MPa/min in all experiments). The mass of

Fig. 1. Schematic presentation of the high-pressure view chamber.

remained thymol in the glass was determined gravimetrically usingan analytical scale with accuracy ±0.0001 g. The mass of dissolvedthymol was subsequently calculated.

Solubility of thymol in scCO2 was experimentally determined attemperatures of 35 ◦C, 40 ◦C and 50 ◦C, and pressures ranging from7.8 to 25 MPa (scCO2 density range 335.89–849.60 kg/m3 [18]). Thetime needed for the system to reach equilibrium was evaluated aswell. All the experiments were performed in triplicate.

2.3. Correlation of solubility data

Chrastil [19] introduced semi-empirical equation for relatingsolubility of the solute and density of supercritical fluids:

c = �e1 exp(

a1 + a2

T

)(1)

where c (kg/m3) is the concentration of a solute in dense gas, �(kg/m3) is density of the gas, T (K) is temperature and e1, a1 and a2are solubility coefficients with following physical meanings: onemolecule of solute is assumed to associate with e1 molecules of thegas; constant a1 depends on the molecular weights of solute andsolvent and also is a function of e1; constant a2 is proportional tothe heat of solvation and heat of vaporization of the solute.

Adachi and Lu modified Chrastil’s equation to improve its capa-bility of data representation [16]. A modification was made byconsidering the quantity e1 to be density dependent:

c = �e1+e2�+e3�2exp

(a1 + a2

T

)(2)

Del Valle and Aguilera [20] improved Chrastil’s equation in a waydemonstrated by Eq. (3). To recompense for the change in theentalpy of vaporization with temperature, they proposed the fol-lowing modification:

c = �e1 exp(

a1 + a2

T+ a3

T2

)(3)

On the basis of the solubility (experimentally determined or corre-lated) mole fraction of thymol in scCO2 can be expressed as [21]:

y = c

c + (M2/M1)�(4)

where M1 and M2 are the molar weights of scCO2 and thymol,respectively.

The model parameters were calculated by minimizing AARDfunction (Eq. (5)) using excel solver tool:

AARD = 1n

n∑i=1

∣∣∣∣yi,exp − yi,cal

yi,exp

∣∣∣∣ × 100% (5)

S. Milovanovic et al. / J. of Supercritical Fluids 84 (2013) 173– 181 175

stat; P

weef

2

tssaMwiwswtatucttadow

(e

wep

Fig. 2. Autoclave engineers screening system: B, CO2 bottle; C, cryo

here n is the number of experimental data points, yi,exp is thexperimental value of the mole fraction of thymol in scCO2 forxperimental point i, and yi,cal is the calculated value of the moleraction of thymol in scCO2 corresponding to point i.

.4. Supercritical impregnation

Impregnation of cotton gauze with thymol was performed inhe apparatus presented in Fig. 2. Autoclave engineers screeningystem is designed for small batch research runs using CO2 as theupercritical medium. Volume of the extractor is 150 mL. Heatersre supplied on the extractor vessel for temperature elevation.aximum allowable working pressure is 413 bar at 238 ◦C. Thymolas placed at the bottom of the vessel and the cotton gauze fitted

n stainless steel supports was placed above it. Gauze/thymol ratioas 0.65 ± 0.05 g/g. In order to prevent splashing of thymol on the

urface of the gauze, porous barrier with pore diameter of 0.09 mmas placed between the gauze and thymol. Operating pressure and

emperature were selected from the obtained solubility data (35 ◦Cnd 15.5 MPa). After putting thymol and the gauze inside the vessel,he temperature was elevated and CO2 was pumped into the systemntil the required pressure was obtained. After reaching the desiredonditions, valve V2 was closed and the pump was turned off. Sys-em was kept at a constant temperature and pressure for desiredime intervals. A slow decompression (0.33 MPa/min) was appliedt the end of the process. The quantity of thymol impregnated wasetermined gravimetrically by measuring the impregnated gauzen an analytical scale with accuracy ±0.0001 g. All experimentsere performed in triplicate.

Chairat et al. [22] and Gamal et al. [23] applied the pseudo firstEq. (6)) and pseudo second order (Eq. (7)) kinetic models on mod-ling of dyes adsorption onto cotton fiber:

dqt

dt= k1(qe − qt) (6)

dqt = k2(qe − qt)2 (7)

dt

here qe is the mass of substrate adsorbed per mass of cotton inquilibrium, qt is the time dependant mass of substrate adsorbeder mass of cotton, t is the impregnation time, and k1 and k2 are

, high pressure liquid pump; E, extractor vessel; S, separator vessel.

the rate constants for the pseudo first-order and pseudo second-order kinetics, respectively. The same equations are applied in thisstudy for modeling of the kinetics of thymol impregnation on cottongauze. However, it should be noticed that according to the staticmethod applied in this study, this process included solubilizationof thymol in scCO2 as well. The best fit is obtained by minimizingthe residual sum of square (RSS), which can be expressed as follows:

RSS =n∑

i=1

[Yi − Ycal]2 (8)

where Yi and Ycal are the values of qt obtained experimentally andby the model, respectively.

2.5. Characterization of the cotton gauze

The surface morphology of cotton gauze fibers was followed byfield emission scanning electron microscopy (FESEM, Mira3 Tes-can). The samples of cotton gauze were coated with a thin layer ofAu/Pd (85/15) prior to analysis.

Fourier transform infrared (FT-IR) spectra of the control cot-ton gauze and the cotton gauze impregnated with thymol wererecorded in the ATR mode using a Nicolet 6700 FT-IR Spectrometer(Thermo Scientific) at 2 cm−1 resolution, in the wavenumber range500–4000 cm−1.

2.6. Antimicrobial activity of impregnated gauze

Antimicrobial activity of the cotton gauze was evaluated againstGram-negative bacteria E. coli ATCC 25922, Gram-positive bacterialstrains S. aureus ATCC 25923, B. subtilis ATCC 6633, E. faecalis ATCC29812 and fungus C. albicans ATCC 24433 using the standard testmethod ASTM E 2149-01 [24]. Microbial inoculum was preparedin the tripton soy broth (Torlak, Serbia), which was used as thegrowth medium for microbes while the potassium hydrogen phos-phate buffer solution (pH 7.2) was used as the testing medium.

Microbes were cultivated in 3 mL of tripton soy broth at 37 ◦C andleft overnight (late exponential stage of growth). 50 mL of sterilepotassium hydrogen phosphate buffer solution (pH 7.2) was inoc-ulated with 0.5 mL of a microbial inoculum. One gram of sterile

176 S. Milovanovic et al. / J. of Supercritical Fluids 84 (2013) 173– 181

Table 1Mole fractions of thymol in scCO2 at 35 ◦C, 40 ◦C and 50 ◦C in scCO2 density range from 335.89 kg/m3 to 849.60 kg/m3.

P (MPa) y × 103 � (kg/m3) P (MPa) y × 103 � (kg/m3) P (MPa) y × 103 � (kg/m3)

35 ◦C 40 ◦C 50 ◦C

7.8 0.900 335.89 8.5 0.837 356.78 10.0 1.88 385.358.0 2.37 426.85 9.0 1.26 487.29 11.0 3.05 504.108.2 4.64 545.32 10.0 4.49 629.75 14.0 7.05 673.588.7 6.12 637.35 13.0 6.77 744.09 16.0 8.15 723.17

12.5 9.27 777.71 16.0 8.65 795.79 25.0 14.7 834.9715.5 11.0 822.08 21.0 12.3 849.60

Table 2Parameters of semi-empirical equations applied.

Eq. e1 e2 e3 a1 a2 a3 T (◦C) AARD (%) AARD%(%)

(1) 3.7876 – – −19.9815 −621.7667 – 35 10.9940 33.64 17.8250 8.85

(2) 5.0732 −1.15E − 03 7.63E − 07 −23.0883 −1427.9593 – 35 16.8440 27.34 16.4550 5.17

(3) 3.7404 – – −19.3191 −754.6777 4079.9090 35 12.1640 31.30 16.27

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0.0126 (at 8.11 MPa), while the values obtained for thymol in thisstudy were in the range from 0.0009 (at 7.8 MPa) to 0.00612 (at8.7 MPa) and 0.011 (at 15.5 MPa).

ause cut into small pieces was put in the flask and shaked for 2 h. mL aliquots from the flask were diluted with physiological salineolution and 0.1 mL of the solution was placed onto a tryptone soygar (Torlak, Serbia). After 24 h of incubation at 37 ◦C, the zero timend two hour counts of viable microbial were made.

The percentage of microbial reduction (R, %) was calculatedsing the following equation:

= C0 − C

C0× 100 (9)

here C0 (CFU – colony forming units) is the number of microbialolonies on the control cotton gauze and C (CFU) is the number oficrobial colonies on the cotton gauze impregnated with thymol.

. Results and discussion

.1. Experimental results and correlation of the solubility data

Thymol was placed in the View Chamber at ambient conditionsn the form of powder, but during the vessel pressurization withO2, its transition into liquid state occurred. Time needed for reach-

ng the equilibrium solubility was investigated first. Dependence ofhymol concentration in scCO2 inside the view chamber on time at0 ◦C and 10 MPa is presented in Fig. 3. As can be seen, the timeeeded for reaching the equilibrium was 24 h, and therefore all theolubility measurements were performed at this exposure time.

Experimentally determined mole fractions of thymol in scCO2t temperatures of 35 ◦C, 40 ◦C and 50 ◦C, and pressures rangingrom 7.8 to 25 MPa (scCO2 density range from 335.89 kg/m3 to49.60 kg/m3) are presented in Table 1 and Figs. 4 and 5. Model-

ng results are presented in Figs. 4 and 5. Parameters of the appliedorrelations are given in Table 2. Generally, Fig. 4 reveals that allhe equations tested can be used to correlate the solubility of thy-

ol in scCO2. According to the AARD% values (Table 2) the highest

ccuracy was obtained in the case of del Valle–Aguilera equation,ollowed by Adachi-Lu and Chrastil equations. Fig. 4 also indicateshat solubilities obtained at 35 ◦C and lower pressures were com-arable to those obtained at 50 ◦C and higher pressures. Thus, the

50 5.35

temperature of 35 ◦C and pressure of 15.5 MPa were selected forthe impregnation process.

Mukhopadhyay and De [25] measured solubilities of thymolin scCO2 at 50 ◦C and 70 ◦C and pressures up to 14 MPa using astatic method. The authors obtained results at 50 ◦C similar to thoseachieved in the current study. They reported thymol mole frac-tion values at 50 ◦C ranging from 0.00083 (at 7.8 MPa) to 0.00721(at 12.7 MPa) while the values obtained in this study for the tem-perature of 50 ◦C ranged from 0.00188 (at 10 MPa) to 0.00705 (at14 MPa) and 0.0147 (at 25 MPa).

Sovova and Jez [21] measured solubilities of menthol (similarto thymol fine chemical) in scCO2 at temperatures in the rangefrom 35 ◦C to 55 ◦C and pressures up to 11.56 MPa using a dynamicmethod in a flow type apparatus. The authors reported mentholmole fraction values at 35 ◦C ranging from 0.00096 (at 7.52 MPa) to

Fig. 3. Thymol concentration in scCO2 in the view chamber vs. time at 40 ◦C and10 MPa.

S. Milovanovic et al. / J. of Supercritical Fluids 84 (2013) 173– 181 177

Fig. 4. Solubility of thymol in scCO2 versus pressure data correlated by: (a) Chrastil equation, (b) Adachi–Lu equation and (c) del Valle–Aguilera equation.

3

tnaaMmfabi

TR

.2. Supercritical carbon dioxide impregnation

The rate of static impregnation process under selected condi-ions (35 ◦C and 15.5 MPa) is presented in Table 3 and Fig. 6. It isoticeable that the adsorption was fast in the first two hours, butfterwards its rate decreased. The masses of impregnated thymolfter 2 and 24 h of impregnation were 11% and 19.6%, respectively.odeling results are depicted in Fig. 6, while the parameters of theodels are presented in Table 4. It is evident, from Fig. 6 as well as

rom the values of RSS, that the pseudo-first order kinetics is more

ppropriate for mathematical description of the process. It shoulde stressed that selected technique of static impregnation also

ncluded solubilization process of thymol in scCO2. Consequently,

able 3ate of thymol impregnation at 35 ◦C and 15.5 MPa.

No. Time (h) Amount ofimpregnatedthymol (%)

1 1 1.742 1.5 4.853 2 11.04 6 13.15 12 18.26 24 19.6

kinetics of the combined solubilization–impregnation process isobtained. Chairat et al. [22] and Gamal et al. [23] reported val-ues of kinetic rate constant k1 in the range from 0.0195 min−1

to 0.0662 min−1 for conventional methods of dyes adsorptiononto cotton fiber. Those values are one order of magnitude lowerthan the value obtained in this study (Table 4) for the thymolimpregnation using scCO2. This is reasonable result due to thehigher diffusivity of scCO2 into fibers comparing to aqueous solu-tions.

3.3. Cotton gauze characterization

The FESEM images of untreated and impregnated cotton gauzesare presented in Fig. 7. Characteristic morphology of cotton fiber

can be observed in Fig. 7a. Although the thymol crystallites onthe surface of cotton fibers after 2 and 24 h long impregnationcannot be observed, the change in fiber morphology is evident.Fine pleats parallel to the cotton fiber axis have been formed and

Table 4Values of the model parameters.

qe k1 (h−1) k2 (h−1) RSS

Pseudo first-order 0.2444 0.199 – 0.00294Pseudo second-order 0.2444 – 1.293 0.00339

178 S. Milovanovic et al. / J. of Supercritical Fluids 84 (2013) 173– 181

Fig. 5. Solubility of thymol in scCO2 versus CO2 density. Data correlated by: (a) Chrastil equation, (b) Adachi–Lu equation and (c) del Valle–Aguilera equation.

sswm(fis

tbFsaCaCtTpaa[Fs[t

2334 cm ) and adsorbed water (at 1650 cm ) was also detected[26–31].

The FT-IR analysis of cotton gauze impregnated with thymol(24 h long impregnation) confirmed the presence of thymol on

pecific surface area was enlarged. Katayama et al. [10] obtainedimilar results in the investigation on the influence of scCO2 andater on the cotton fiber surface morphology. The wrinkles for-ation was explained by the difference in degasification speed

after decompression) between the surface and interior of cottonber, which induced the difference in shrinkage between these twoegments.

Results of the FT-IR analysis of control and impregnated cot-on gauze during 24 h are presented in Fig. 8. Evidently, a broadand in the region between 3500 and 3200 cm−1 is observed in theT-IR spectrum of control cotton fabric, which is assigned to thetretching vibrations of cellulosic OH group [26–29]. In addition,

broad band with a peak centered at 2898 cm−1 corresponds to H asymmetric stretching [26]. The bands at 1429, 1368, 1317nd 1281 cm−1 originate from C H in plane bending vibrations,

H bending (deformation stretch) vibrations, C H wagging vibra-ions and C H deformation stretch vibrations, respectively [26,27].he bends at 1337, 1247 and 1203 cm−1 corresponds to OH in-lane bending vibrations [26]. The bands at 1160 and 1108 cm−1

re assigned to asymmetric bridge C O C [26], whereas the peakt 1053 cm−1 is ascribed to asymmetric in plane ring stretching26]. The peak at 1030 cm−1 is related to C O stretching [26].

inally, the peak corresponding to asymmetric out-of-phase ringtretching at C1 O C4 �-glucosidic bond appears at 900 cm−1

25–29]. Observed bands are characteristic for cotton and fit wello literature data. The presence of CO2 (doublet at 2362 and

−1 −1

Fig. 6. Experimental and modeling results for pseudo first-order (a) and pseudosecond-order (b) kinetics.

S. Milovanovic et al. / J. of Supercritical Fluids 84 (2013) 173– 181 179

Fig. 7. FESEM images of: (a) untreated gauze, (b) 2 h impregnated gauze, (c) 24 h impregnated gauze.

t2i[r1tai[

3

Tatpmttwl

he surface of cotton fibers. The peaks in the FT-IR spectrum at957 and 2866 cm−1 can be assigned to asymmetric C H stretch-

ng vibration and deformation overtones of CH3 group, respectively30,32]. The peaks at 1623 and 1447 cm−1 correspond to aromaticing C C stretching vibrations [29,32]. Bands detected at 1286 and259 cm−1 are assigned to combination of OH deformation vibra-ions and C O stretching vibrations [29]. The peak at 1360 cm−1 isssigned to OH bending of phenolic group [33]. Band at 804 cm−1

s attributed to out-of-plane aromatic C H wagging vibrations34].

.4. Antimicrobial analysis

The results of antimicrobial analysis are presented in Table 5.he samples which were impregnated for 2 and 24 h were evalu-ted. Both samples exhibited strong antimicrobial activity againstested strains of E. coli, S. aureus, B. subtilis, E. faecalis and C. albicansroviding maximum microbial reduction (99.9%) for all testedicroorganisms. The exact mechanism of antimicrobial action of

hymol has not been established yet. Some studies indicated thathymol has an ability to disrupt lipid structure of the bacteria cellall, further leading to a destruction of cell membrane, cytoplasmic

eakage, cell lysis and cell death [35,36].Fig. 8. FT-IR spectra of control and impregnated cotton gauze.

180 S. Milovanovic et al. / J. of Supercritical Fluids 84 (2013) 173– 181

Table 5Antimicrobial activity of impregnated gauze.

Sample Microorganism 2 h impregnation 24 h impregnation

Number of bacterial colonies (CFU) R (%) Number of bacterial colonies (CFU) R (%)

Control gauze E. coli 2.2 × 104 7.0 × 103

Gauze + thymol <10 99.9 <10 99.9Control gauze S. aureus 4.4 × 104 5.0 × 103

Gauze + thymol <10 99.9 <10 99.9Control gauze B. subtilis 1.5 × 105 6.0 × 103

Gauze + thymol <10 99.9 20 99.9Control gauze E. faecalis 5.4 × 105 4.7 × 104

4

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Gauze + thymol <10

Control gauze C. albicans 7.4 × 104

Gauze + thymol <10

. Conclusions

Solubility of thymol in supercritical carbon dioxide waseasured at temperatures of 35 ◦C, 40 ◦C and 50 ◦C, and

ressures ranging from 7.8 to 25 MPa (scCO2 density range35.89–849.60 kg/m3) using a static method. Obtained data wereorrelated by the semi-empirical equations introduced by Chrastil,dachi and Lu and del Valle and Aguilera with the acceptable accu-acy. On the basis of the solubility data, the process of supercriticalmpregnation of cotton gauze with thymol was proposed. Kinet-cs of the process was determined and modeled using pseudo firstrder and pseudo second order kinetic models at selected processonditions of 35 ◦C and 15. 5 MPa. After 2 h of impregnation, mass ofmpregnated thymol was 11%, while its value after 24 h of impreg-ation reached 19.6%. Both samples exhibited strong antimicrobialctivity against tested strains of E. coli, S. aureus, B. subtilis, E. faecalisnd C. albicans providing maximum microbial reduction (99.9%) forll tested microorganisms. Results of the FT-IR analysis confirmedhe presence of thymol on the surface of cotton fibers. Accordingo the results of this study, supercritical impregnation with carbonioxide was indicated as a feasible technique for impregnation ofotton gauze with thymol in order to obtain an efficient woundressing with antibacterial properties. Further investigations onytotoxic effects of the impregnated gauze in vitro on cell lines areeeded.

cknowledgments

Financial support of the Serbian Ministry of Education, Sci-nce and Technology Development (Projects III45017 and 172056)s gratefully acknowledged. We gratefully acknowledge Dr Bojanokic (University of Belgrade, Serbia) for providing FESEM measure-

ents.

eferences

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[2] A. Wattanasatcha, S. Rengpipat, S. Wanichwecharungruang, Thymolnanospheres as an effective anti-bacterial agent, International Journal ofPharmaceutics 434 (2012) 360–365.

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