Journal of Molecular Liquids 174 (2012) 95–99

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Journal of Molecular Liquids

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Measurement of cephalexin partition coefficients in PEG+K2HPO4+H2O aqueoustwo-phase systems at 301.15, 306.15 and 311.15 K

Sara Ghayour Doozandeh a, Gholamreza Pazuki b,⁎, Babak Madadi b, Ali Asghar Rohani a

a Department of Chemical Engineering, Shahrood Branch, Islamic Azad University, Shahrood, Iranb Department of Chemical Engineering, Amirkabir University of Technology, Tehran, Iran

⁎ Corresponding author. Tel.: +98 21 64543159.E-mail address: ghpazuki@aut.ac.ir (G. Pazuki).

0167-7322/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.molliq.2012.07.026

a b s t r a c t

a r t i c l e i n f o

Article history:Received 3 January 2012Received in revised form 18 July 2012Accepted 21 July 2012Available online 2 August 2012

Keywords:LLE dataAqueous two-phase systemsPartitioningPEGK2HPO4

Cephalexin

In this study, the liquid–liquid equilibrium (LLE) data for poly(ethylene glycol) with molecular weights 1500and 6000 — K2HPO4 aqueous two-phase systems containing cephalexin antibiotic have been measured atthree temperatures 301.15, 306.15 and 311.15 K. The effects of temperature, PEG molecular weight and con-centrations of PEG and salt have been studied on the LLE data. Also, the partitioning of cephalexin in PEG–saltaqueous two-phase systems has been estimated using the LLE data. The experimental data showed that theconcentration of salt has a large effect on the partitioning of cephalexin in aqueous two-phase systems.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

There are many processes involved in bioseparation. Aqueoustwo-phase systems (ATPS) are one of the most promising and devel-oping separation techniques used in downstream processing. Thesesystems consist of two immiscible aqueous phases with high watercontent in both phases. Four main types of ATPS, depending ontheir chemical composition, are known: water+polymer+polymer,water+polymer+salt, water+salt+salt and water+surfactant(micellar systems).

The advantages of the ATPS than the other separation methodsare: biocompatibility due to aqueous environment, low viscosity,low interfacial tension between two phases, high selectivity of sep-aration, feasibility of continuous operation, possibility of recyclingphase components and easy scale up from laboratory to industrialscale [1].

Beijerinck in 1896 firstly obtained an ATPS by mixing gelatin andagar [2].

Albertsson used polymer–polymer ATPS for separation and purifica-tion of biomolecules [3]. In the late of 1950s, Albertsson found thatwhen two-water soluble polymers (such as PEG and dextran) dissolvedin water, a two-phase system is formed, in which one-phase was rich inPEG and the other phase, rich in dextran. He evolved ATPSs and appliedthese systems to separation cell organelles [3]. In recent years a large

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number of studies have been performed to develop ATPS's as a benignbioseparation method [4,5].

Also, Grossmann et al. studied the partitioning process of someamino acids such as glycin, L-glutamic acid, L-phenylalanine and L-lysineand some of their lowmolecular weight peptides in the ATPS formed byK2HPO4 and PEG [6].

Salabat et al. measured partition coefficients of some amino acidsin PPG–MgSO4 and PPG–sodium phosphate aqueous two-phase sys-tems at 298.15K [7,8].

Recently, Pazuki and co-workers measured and modeled parti-tion coefficients of amino acid, enzyme and pharmaceutical com-pounds such as penicillin G, cephalexin, L-lysine, α-amylase andβ-amylase in PEG–salt aqueous to-phase systems at different tem-peratures [9–12].

Using ATPSs for purification and recovery of pharmaceuticals is anattractive issue to many researchers [13,14]. Guan et al. studied theapplication of aqueous two-phase system in the recovery of penicillin[15,16] and acetylspiramycin [17]. Also partitioning of antibiotics inATPSs based on ionic liquids as a novel biphasic system has beenreported; Coutinho et al. examined the partition coefficient of cipro-floxacin in [C4mim][CF3SO3]+lysine+water biphasic system [18].Partitioning behavior of penicillin G in [Bmim]Cl+NaH2PO4+waterwas investigated by Liu et al. [19].

Cephalexin is a first generation cephalosporin belonged to beta-lactam antibiotics. It places itself in the cell wall of bacteria and inter-feres with developing of the cell wall and annihilates bacteria [20].The partition coefficient of this antibiotic and behavior of relevant bi-phasic systems are reported by Shahriari et al. [21] and Khederlou

Table 1Parameters of refractive index introduced into Eq. (1).

Component a0 a1 a2

Water 1.3325PEG 1500 0.143PEG 6000 0.1471K2HPO4 0.163

96 S.G. Doozandeh et al. / Journal of Molecular Liquids 174 (2012) 95–99

[11] in ATPS's containing various PEG molecular weights and differentsalts such as K2HPO4 and Na3 citrate.

In this research, liquid–liquid equilibriumdata for PEG+K2HPO4+water+cephalexin at three temperatures 301.15, 306.15 and 311.15 K.Also, the partition coefficients of cephalexin in these systems areobtained.

2. Experimental section

2.1. Materials

Polyethylene glycol with molecular weights of 1500 and 6000,K2HPO4 with purity of 99.5% was purchased from Merck (Germany,Darmstadt). Cephalexin monohydrate with 99.9% was obtained fromJaber-Ebne-Hayyan Company (Tehran, Iran). Double distilled deion-ized water with the conductivity of 0.055 μs/cm was used in all ofthe experiments.

2.2. Methods

The aqueous two-phase systems were prepared by using PEG,K2HPO4, cephalexin and water in a beaker with 100 cm3 volume.

Table 2The mass fractions of PEG1500 (1)+K2HPO4 (2)+cephalexin (3) in the top and bottom ph

T/K Feed Top ph

100w1 100w2 100w3 100w1 100w

301.15 15.2744 13.1305 0.0098 37.4431 3.16301.15 15.0081 14.6609 0.0061 39.5852 2.94301.15 15.7512 10.3549 0.0092 31.3416 4.09301.15 14.8579 13.1971 0.0107 44.0149 2.18306.15 15.2822 13.1285 0.0098 39.8082 3.29306.15 15.2757 13.1308 0.0062 37.8194 3.87306.15 15.7508 10.3564 0.0101 30.7455 4.99306.15 14.8575 13.1986 0.0116 37.5013 3.78311.15 15.2746 13.1260 0.0089 41.4092 2.62311.15 15.2766 13.1345 0.0062 41.7971 2.71311.15 15.7519 10.3556 0.0092 36.9603 3.34311.15 14.8587 13.1970 0.0107 41.2826 2.98

Table 3The mass fractions of PEG6000 (1)+K2HPO4 (2)+cephalexin (3) in the top and bottom ph

T/K Feed Top pha

100w1 100w2 100w3 100w1 100w

301.15 15.2741 13.1291 0.0089 42.2071 2.094301.15 15.2767 13.1337 0.0062 42.5834 1.693301.15 15.7509 10.3556 0.0101 36.8617 2.316301.15 14.8579 13.1971 0.0107 41.3233 2.094306.15 15.2773 13.1299 0.0107 43.4615 2.004306.15 15.2773 13.1343 0.0062 42.0347 2.495306.15 15.7512 10.3549 0.0092 36.5905 2.806306.15 14.8588 13.1962 0.0107 41.5402 2.450311.15 15.2737 13.1315 0.0089 43.0973 2.272311.15 15.2773 13.1306 0.0062 42.3989 2.227311.15 15.7512 10.3549 0.0092 38.2156 2.628311.15 14.8590 13.1964 0.0098 42.5964 2.049

For example one of these biphasic systems consist of 17.070 gPEG1500, 14.674 g K2HPO4 and 0.010 g cephalexin plus 80 mL H2O.The ATPSwas shaken using amagnetic stirrer for 30 min. Then, the solu-tion was set to reach equilibrium for 24 h in an incubator (Memmert,Germany) with an accuracy of ±0.01 °C in order to setting temperatureof aqueous solution. Samples of the top and bottom phases were re-moved by a plastic syringe.

2.3. Analytical methods

The mass fraction of K2HPO4 was estimated by using flame pho-tometry (Sherwood Model 410, UK). The mass fraction of PEG wasobtained by refractive index measurements using Shimadzu, Japan.The relationship between the refractive index (nd) and the mass frac-tions of polymer (wp) and salt (ws) can be correlated as the followingrelation:

nd ¼ a0 þ a1wp þ a2ws: ð1Þ

The parameters of the Eq. (1) and standard deviations for differentmolecular weights of PEG and K2HPO4 are reported in Table 1.

The mass fractions of cephalexin in the top and bottom phaseswere obtained by using a UV/Vis spectrophotometry at 262 nm byusing a spectrophotometer (M501, from CamSpec, England). It isnoted that the aqueous two-phase systems (polymer+salt+water)without cephalexin were as blank solutions.

3. Results and discussion

For two aqueous two-phase systems containing PEG 1500, 6000and K2HPO4 as well as cephalexin antibiotic, the mass fractions data

ases in ATPS as well as the partitioning coefficients of cephalexin in ATPS.

ase Bottom phase Kceph

2 100w3 100w1 100w2 100w3

34 0.0123 8.6729 12.6979 0.0094 2.142306 0.0086 8.2666 13.0544 0.0068 1.615390 0.0075 6.9547 11.6286 0.0094 1.394032 0.0173 9.4953 14.1237 0.0099 2.040270 0.0187 8.3232 12.6979 0.0108 1.343062 0.0040 7.7904 13.4108 0.0060 1.886601 0.0115 7.2712 11.2277 0.0093 0.900371 0.0189 7.1868 13.6336 0.0098 2.873987 0.0193 8.5330 12.6979 0.0090 1.345178 0.0274 6.8829 14.3910 0.0056 1.837416 0.0142 6.1878 13.0989 0.012851 0.0208 7.2958 14.2128 0.0090 1.9215

ases in ATPS as well as the partitioning coefficients of cephalexin in ATPS.

se Bottom phase Kceph

2 100w3 100w1 100w2 100w3

0 0.0203 9.0430 12.6979 0.0094 2.15340 0.0111 6.1172 14.9702 0.0054 2.05058 0.0132 4.5384 13.1435 0.0090 1.46260 0.0202 6.0484 14.4801 0.0096 2.10379 0.0146 8.0912 12.6979 0.0109 1.34540 0.0114 8.5162 11.7623 0.0069 1.65939 0.0084 7.1056 10.8267 0.0093 0.90814 0.0284 8.9427 11.8069 0.0130 2.18662 0.0121 10.606 12.6979 0.0090 1.34557 0.0104 9.1797 13.1880 0.0062 1.69167 7.9473 11.5395 0.01065 0.0174 9.1983 13.2326 0.0098 1.7781

0

10

20

30

40

50

0 2 4 6 8 10 12 14 16

100WSalt

100W

PE

G

T=306.15 K

T=311.15 K

Fig. 1. Effect of temperature on two phase separation in PEG1500 (1)+K2HPO4 (2) aqueoustwo-phase systems.

0

10

20

30

40

50

100W

PE

G

0 2 4 6 8 10 12 14 16

100WSalt

Fig. 3. Phase diagram and tie lines of PEG1500 (1)+K2HPO4 (2) aqueous two phasesystems at 301.15 K.

97S.G. Doozandeh et al. / Journal of Molecular Liquids 174 (2012) 95–99

at three temperatures 301.15, 306.15 and 311.15 K are reported inTables 2 and 3, respectively.

The partition coefficient of cephalexin in aqueous two-phase sys-tems can be defined as the following relation:

Kceph ¼ wIceph

wIIceph

ð2Þ

where in the above equation, Kceph is partition coefficient of cephalex-in, wceph

I and wcephII refer to the concentration of cephalexin in the top

and bottom phases, respectively.As observed from the results reported in Tables 2 and 3, the mass

fraction of cephalexin in the top phase (polymer rich phase) is morethan that of cephalexin in the bottom phase (salt rich phase). Thus,the partition coefficient is higher than unity in all of systems. It is con-cluded that cephalexin prefers to stay in the polymer-rich phase in allof experiments.

Also, from the experimental data expressed in Tables 2 and 3, it canbe concluded that increasing the mass fraction of PEG in feed can de-crease the partition coefficients of cephalexin. The reason for such a de-crease factor is as increasing hydrophobicity of the PEG-rich phase.

It can be found from Tables 2 and 3 that the partition coefficientsof cephalexin increase as PEG molecular weight increases.

The experimental results showed that the influence of tempera-ture on the partition coefficients of cephalexin is small.

PEG 1500

PEG 600

0

10

20

30

40

50

100W

PE

G

0 2 4 6 8 10 12 14 16

100WSalt

Fig. 2. Effect of PEG molecular weight on two phase separation in PEG (1)+K2HPO4 (2).

The effect of temperature on the binodal curve for the PEG1500+K2HPO4+water is shown in Fig. 1. It can be inferred fromFig. 1 that by raising the temperature the two-phase region is ex-panded. The same results are reported by Voros et al. [22]. In theATPS, the entropic contribution causes demixing (phase separation)but the enthalpic contribution favors dissolution (phase integration).A polymer dissolves in water through H bonds (enthalpic attitude)while lessening bonded water molecules causes reduction in entropyand thus the entropy opposed the dissolution process. Since raising oftemperature increases the entropy contribution, two-phase forma-tion at upper temperature is more readily available [23].

Fig. 2 shows the effect of molar mass of PEG on the separation ofaqueous two-phase systems. By increasing the PEG molar mass, thebinodal curve shifts toward a lower mass fraction of PEG and salt,i.e. the increasing of polymer M.W. would result in phase separationat lower concentrations of polymer and salt. The same effect isreported by Pazuki et al. [6] and Shahriari et al. [24]. The effect ofpolymer molecular weight can be justified by the extension of thepolymer hydration shell. Compatibility between the water structuresin the hydration shells may grow if the molecular weight of the poly-mer increases. Also cooperative interaction of hydrophobic and hy-drophilic groups regularly situated in the polymer is a key factor forcompatibility of water structures [1].

The phase diagrams of the PEG 1500+K2HPO4+water at T=301.15 Kand PEG 6000+K2HPO4+water T=311.15 K are shownin Figs. 3 and 4. It can be seen from these figures that the two-phase

0

10

20

30

40

50

100W

PE

G

0 2 4 6 8 10 12 14

100WSalt

Fig. 4. Phase diagram and tie lines of PEG6000 (1)+K2HPO4 (2) aqueous two phasesystems at 311.15 K.

⎟⎟⎠

⎞⎜⎜⎝

⎛ −I

I

w

w

1

11ln

⎟⎟⎠

⎞⎜⎜⎝

⎛ −II

II

w

w

2

21ln

R2 = 0.8979

R2 = 0.8321

R2 = 0.9981

0.2

0.3

0.4

0.5

0.6

1.6 1.7 1.8 1.9 2 2.1 2.2

T=301.15 K

T=306.15 K

T=311.15 K

Fig. 5. Othmer–Tobias plot for PEG1500 (1)+K2HPO4 (2) aqueous two-phase systemsat three temperatures 301.15, 306.15 and 311.15 K.

Fig. 7. The variations of partition coefficients vs. the weight fractions of PEG in feed.

98 S.G. Doozandeh et al. / Journal of Molecular Liquids 174 (2012) 95–99

region for system containing PEG 1500 is greater than the systemcontaining PEG 6000. It should be emphasized that the PEG1500+K2HPO4+water ATPS is the best system for partitioningof cephalexin.

Fig. 6 illustrates the effect of temperature on the slope of tie-line.The slope of tie-line increases as a result of temperature rising. Thisbehavior agrees with previous reports of Voros et al. [10] andShahriari et al. [21].

Effect of PEG fraction in feed on partition coefficient has been indi-cated by Fig. 7. For each of three temperatures, an increase of PEGconcentration in feed causes lower partition coefficient of cephalexin.Similar results are observed by Mokhtarani et al. [25]. The correlationproposed by Othmer and Tobias [26] is used for correlating the massfractions of the tie line compositions:

ln1−wI

1

wI1

!¼ kþ n ln

1−wII2

wII2

!: ð3Þ

In the above equation, k and n are constant. w1I is mass fraction of

polymer in the top phase andw2II is mass fraction of salt in the bottom

phase.An Othmer–Tobias plot is presented in Fig. 5 for the PEG

1500+K2HPO4+water. As can be seen, the correlation factor(R2) at three temperatures is good.

Fig. 6. Effect of temperature on the tie-lines slope in aqueous two-phase systems.

Also, a linear equation between ln Kceph and Δw1 (the differencebetween mass fractions of polymer in the top and bottom phases)proposed by Diamond and Hsu [27] is used for correlating the parti-tion coefficient as:

lnKceph

Δw1¼ Aþ BΔw1 ð4Þ

where A and B are constant. Parameters A and B can be obtained froma linear regression between the experimental partition coefficientand those obtained from Eq. (4). Parameters A and B as well as theroot mean square deviation (rmsd) are reported in Table 4. The resultsshow that the proposed correlation can accurate correlate the parti-tion coefficients of cephalexin in the PEG+K2HPO4+water aqueoustwo-phase systems.

In Table 5, the partition coefficients of cephalexin in aqueoustwo-phase systems are compared with the results obtained byKhederlou et al. at 301.15 K. Our values obviously are in an upperorder that shows the cephalexin molecules prefer the top phase inlower molecular weight (MW) of PEG while they favor bottomphase in higher MW of PEG. This outcome coincides in what hasbeen mentioned by Albertsson [3].

4. Conclusion

In the present study, the liquid–liquid equilibrium data for thePEG+K2HPO4+water+cephalexin is measured at three tempera-tures 301.15, 306.15 and 311.15 K. The partitioning of cephalexin isdepending on temperature, polymer molar mass, and salt and poly-mer concentrations in feed. The Othmer–Tobias and Diamond andHsu correlations are used for the tie line compositions and the parti-tion coefficients. The results of these models showed that these rela-tions can accurately correlate the tie line compositions and partitioncoefficients in polymer–salt aqueous two-phase systems. The experi-mental data indicated that the partitioning of cephalexin is strongly

Table 4Parameters A and B constants reported in Eq. (3) along with the rmsd of the modelfrom the experimental partition coefficient data.

System A B rmsd

PEG 1500+K2HPO4 −7.0712 28.308 0.329PEG 6000+K2HPO4 −7.3335 28.268 0.421

Table 5Comparison of the experimental data of partition coefficients for cephalexin in PEG(1)+K2HPO4 (2)+water (3) aqueous two-phase systems with the results reportedby Khederlou et al. [11].

PEG 1500+K2HPO4+H2O PEG 10000+K2HPO4+H2O 22

100w1 100w2 Kceph 100w1 100w2 Kceph

15.27 13.13 2.142 26.47 11.40 0.96715.75 10.35 1.394 27.06 9.29 0.54614.86 13.20 2.040 16.61 8.89 0.786

99S.G. Doozandeh et al. / Journal of Molecular Liquids 174 (2012) 95–99

depending on concentration of salt in feed and the temperature has asmall effect on partitioning of cephalexin. Also, the partitioning ofcephalexin is increasing with increasing the molar mass of PEG.

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