Partitioning of Cefazolin in Biocompatible Aqueous Biphasic Systems

Aug 20, 2013 - In this research, promising aqueous two-phase systems (ATPS) containing polyoxyethylene (20) sorbitan monolaurate (Tween 20) as a quite...
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Partitioning of Cefazolin in Biocompatible Aqueous Biphasic Systems Based on Surfactant Babak Madadi, Gholamreza Pazuki,* and Bahram Nasernejad Chemical Engineering Department, Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran ABSTRACT: In this research, promising aqueous two-phase systems (ATPS) containing polyoxyethylene (20) sorbitan monolaurate (Tween 20) as a quite biocompatible nonionic surfactant and different salts (K 2 HPO 4 , MgSO 4 , and Na3Citrate) were prepared and their extractive potential was evaluated. These ATPS possess top and bottom phases wellstocked in surfactant and salt respectively which can offer appealing characteristics such as low interfacial tension, low volatility, and ease of scaling-up. Ternary phase diagrams of ATPS consisting of Tween 20 and three different salts were obtained and the results were successfully fitted to the Merchuk correlation. The salting-out ability of salts were assessed. Also, the tie-line data were correlated with the Othmer-Tobias equation. Afterward, the partitioning behavior of cefazolin as a broad-spectrum antibiotic in the proposed ATPS was studied at three temperatures (298.15, 302.15, and 306.15 K) and the influences of Na3Citrate and MgSO4 on partition coefficients were justified by Hofmeister series. Furthermore, the effect of tie-line length on partitioning of cefazolin in ATPS was investigated. The experimental data of cefazolin partition coefficients indicated the proper potential of the Tween 20−Na3Citrate−water biphasic system for the bioseparation process.



INTRODUCTION Separation processes involved in biotechnological and pharmaceutical productions are crucial factors that undeniably determine the cost of the end product as well as its purity. Extraction with an organic solvent is a classic unit operation among separation stages. Using organic solvent in the field of bioseparation often causes denaturation of biomolecules such as proteins, cell components, pharmaceuticals, etc. Aqueous twophase systems (ATPS) are promising bioseparation techniques providing a mild and biocompatible media (due to high content of water and low interfacial tension) for selective extraction of biomolecules. Also, the resemblance between the data taken in tubes and a two liter tank (200-fold increase), make these mild ATPS very suitable for scaling-up.1 In 1896, Beijerinck reported a two-phase system consisting of an aqueous solution of gelatin and agar.2 Albertsson accidentally discovered the extractive capability of PEG (poly ethylene glycol) + dextran + water biphasic system.3 In the late 1950s, he established comprehensive experiments to consider the feasibility of two-phase formation with several pairs of polymer and salt aqueous solutions. He also studied the partitioning of biomolecules in newly arrived biphasic systems.4 Nowadays, four types of ATPS have been introduced including (polymer + polymer), (polymer + salt), surfactant-based, and ionic liquid-based. The (polymer + polymer)5 and (polymer + salt)6 systems have been the most prevalent ATPS in the past decades, however, in recent years various biphasic systems with different characteristics have been introduced. Ionic liquids (ILs) have a great potential to form low viscosity, but relatively high cost,7 two-phase systems. ILs have shown a favorable potential in both (IL + polymer)8 and, more common, (IL + salt)9,10 biphasic © XXXX American Chemical Society

systems. Also, it can be used as an adjuvant extractive agent in a (polymer + salt) two-phase system.11 Surfactants (surface active agents) are other interesting biocompatible substances applied in a wide variety of biotechnological fields.12,13 The term surfactant designates a substance which exhibits some superficial activity. Its molecules have an amphiphilic quality, i.e. a hydrophobic part such as a hydrocarbon chain of the alkyl or alkylbenzene type, and a hydrophilic tail (usually polar) which contains functional groups such as alcohol, ester, acid, sulfonate, etc.14 Polysorbates (also known as Tweens) are nonionic surfactants belonging to the sorbitan derivatives of the polyyol family.15 Study results of the polysorbates, showed no carcinogenicity and genotoxicity.16 These high usage surfactants have been utilized in several areas of bioprocesses such as the carbon source in culture broths,17 a solubilizing agent of membrane proteins,18 coating materials in drug delivery fields,19 biosensors,20 and as a stabilizing agent of emulsions and suspensions in pharmaceutical applications.21,22 The surfactant-based biphasic systems are categorized into four divisions with respect to their constituent components: temperature induced micellar biphasic systems, systems based on two incompatible surfactants, (surfactant + polymer) biphasic systems, and (surfactant + salt) biphasic systems. In the first case, the surfactant(s) exist above CMC (critical micelle concentration) in water to form micelles and then by increasing (or decreasing23) the temperature, a two-phase system would be achieved at a certain temperature called cloud point. Each cloud point corresponds to its CMC. The nonionic surfactants are Received: May 16, 2013 Accepted: August 1, 2013

A

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biphasic system can be exploited for enzymatic synthesis of cefazolin, through a process called bioconversion, similar to the enzymatic production of cephalexin.52,53 In this study, the binodal curve of three biphasic systems comprising (Tween 20 + sodium citrate), (Tween 20 + magnesium sulfate), and (Tween 20 + dipotassium hydrogen phosphate) have been obtained. These data were also fitted into a semiempirical equation, i.e., the Merchuk correlation,1 and the tie-lines were correlated with the Othmer−Tobias54 relation. The partition coefficients of cefazolin in the aforementioned biphasic systems, including magnesium sulfate and sodium citrate, were obtained and the influence of salt types was investigated. Finally, by the application of Hofmeister series the values of partition coefficients were justified.

generally used since they provide a mild and nondenaturing environment for biomolecules especially proteins;23,24 yet systems containing zwitterionic surfactant25 or a mixture of two differently charged surfactants have been developed.25,26 The second case is composed of (nonionic + ionic) surfactants27 or two ionic surfactants with opposite charges (i.e., cationic + anionic).28,29 In the latter, three mechanisms bring about a twophase formation. One is the entanglement of thread-like micelles, another is the formation of vesicles with distinct sizes, and the third is the formation of lamellar structures.30 The studies on the third type of surfactant-based biphasic systems (i.e., surfactant + polymer) are less interesting maybe due to their relatively high viscosity and complex phase diagrams.31 The phase behavior of the (Brij58 (polyoxyethylene cetyl ether) + PEG) system and the salt effect on that has been investigated.32 Also, prefractionation of membrane protein in the similar biphasic system was successfully carried out.33 (Surfactant + salt) biphasic systems, the fourth kind of surfactant-based systems as mentioned above, have promising capability for separation and purification of biomolecules. The formation of these biphasic systems, unlike the micellar systems, is not temperature-dependent, as well as their relatively low viscosity making these systems more conducive for bioseparation purposes. According to our literature review, Xie et al.,34 for the first time, introduced the (surfactant + salt) system (containing Triton X-100 and Sodium Carbonate) and modeled the partitioning of the membrane protein and phase equilibrium in that biphasic system using a virial coefficient model. A couple of similar reports35−38 were made by Foroutan et al. using Brij 58 and Triton X-100 accompanied by several different salts. The partition coefficients of three different kinds of amino acids in (Triton X-100 + magnesium sulfate) and (Triton X-100 + sodium citrate) biphasic systems have been examined by Salabat et al.39 Recently, Á lvarez et al.40 have worked on the phase behavior and the role of several anions in biphasic systems including Tween 20 (and 80) and potassium inorganic salts. Additionally, Ulloa et al.41 have considered the salting out effect of sodium salts on aqueous solutions containing Tween 20 and Triton X-102. ATPS have been a powerful tool for recovery of antibiotics from the spent medium, compared to the organic solvents that commonly have deleterious effects. These benign ATPS enhance the separation yield, especially in the case of hydrophilic pharmaceuticals. The partition coefficient of cefazolin, with IUPAC name (6R, 7R)-3-{[(5-methyl-1,3,4-thiadiazol-2-yl)thio]methyl}-8-oxo-7-[(1H-tetrazol-1-ylacetyl)amino]-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, has been investigated in our research. The conventional methods currently used to extract cephalosporin antibiotics include solid-phase adsorption, liquid−liquid extraction, liquid membrane extraction and membrane (synthetic) separation.42 The reasons for picking cefazolin as the partitioned subject are as follows. As far as we know, there is no report on partitioning of cefazolin using the (surfactant + salt) two phase system; however, there are numerous works applying other types of ATPS in the field dealing with pharmaceuticals such as penicillin’s and cephalosporin’s family.43−49 Cefazolin belongs to the first-generation of cephalosporin semisynthetic antibiotics and it has a clinical importance for treatment of various infections including lungs, skin, bones, stomach, etc.50 Cefazolin has a beta-lactam ring, and plays an impressive role against bacterial pathogens especially gram-negative ones.51 Besides the studying of distributive functioning of cefazolin in the (surfactant + salt) system, this



EXPERIMENTAL SECTION Materials. The chemical formula, molar mass, supplier and purity of compounds used in this study are shown in Table 1. All Table 1. Properties of Compoundsa chemical name

chemical formula

Tween 20

C58H114O26

Sodium Citrate Magnesium Sulfate Potassium Hydrogen Phosphate Cefazolin sodium

Na3C6H5O7 0.5,5H2O MgSO4 0.7H2O

a

K2HPO4 0.3H2O C14H13N8NaO4S3

source

M/g·mol−1

Merck Millipore Merck Millipore Merck Millipore Merck Millipore

1227.72

Jaber Ebne Hayyan Company

mass fraction purity

357.16

0.99

246.48

0.99

228.23

0.99

476.49

0.99

M indicates the molar mass.

materials were utilized without additional purification. The CMC and HLB (hydrophilic lipophilic balance) of Tween 20 are 60 mg/L and 16.7 respectively. Double distilled water was used to prepare the solutions in all of the experiments. The chemical structures of Tween 20 and cefazolin sodium are shown in Figure 1. Methods. Constructing Coexistence Curves. A cloud point method was employed at 298.15 K to determine the phase diagrams.55 First, a stock salt solution was prepared (wsalt = 0.25 to 0.35 depending on the solubility of salt). A certain amount of (Tween 20 + water) mixture, with wTween = 0.7, was put into a

Figure 1. Chemical structure of (a) cefazolin sodium and (b) Tween 20. B

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Table 2. Experimental Tie-Lines Compositions, Tie-Lines Length (TLL), and Slope of Tie-Lines (STL) for {Tween 20 (1) + Na3Citrate (2) + H2O (3)} Biphasic Systemsa feed composition

top-phase composition

bottom-phase composition

T/K

set no.

w1

w2

w1

w2

w1

w2

TLL

STL

Kcef

298.15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

0.250 0.250 0.250 0.250 0.180 0.200 0.220 0.240 0.250 0.250 0.250 0.250 0.180 0.200 0.220 0.240 0.250 0.250 0.250 0.250 0.180 0.200 0.220 0.240

0.120 0.130 0.140 0.150 0.140 0.140 0.140 0.140 0.120 0.130 0.140 0.150 0.140 0.140 0.140 0.140 0.120 0.130 0.140 0.150 0.140 0.140 0.140 0.140

0.468 0.500 0.536 0.544 0.455 0.486 0.506 0.525 0.461 0.511 0.554 0.581 0.493 0.504 0.513 0.536 0.515 0.542 0.565 0.582 0.518 0.518 0.542 0.568

0.028 0.025 0.019 0.027 0.037 0.026 0.026 0.024 0.040 0.036 0.026 0.020 0.035 0.032 0.030 0.026 0.017 0.015 0.017 0.012 0.021 0.029 0.016 0.015

0.086 0.082 0.070 0.069 0.074 0.071 0.070 0.068 0.094 0.085 0.092 0.089 0.075 0.078 0.075 0.073 0.087 0.081 0.086 0.080 0.069 0.070 0.073 0.076

0.180 0.194 0.218 0.231 0.173 0.185 0.199 0.215 0.170 0.191 0.204 0.219 0.168 0.178 0.190 0.207 0.168 0.186 0.198 0.214 0.169 0.180 0.190 0.199

0.411 0.451 0.507 0.517 0.405 0.444 0.469 0.495 0.389 0.453 0.495 0.531 0.439 0.450 0.466 0.497 0.454 0.492 0.512 0.541 0.473 0.473 0.500 0.525

−2.51 −2.48 −2.35 −2.32 −2.81 −2.61 −2.51 −2.39 −2.82 −2.75 −2.59 −2.48 −3.14 −2.92 −2.74 −2.56 −2.83 −2.69 −2.64 −2.49 −3.03 −2.96 −2.70 −2.68

2.86 3.55 4.33 6.12 2.93 3.38 3.40 4.46 1.30 5.50 4.71 6.35 3.50 4.35 5.14 6.66 5.08 4.89 6.19 8.73 4.10 4.80 5.44 5.25

302.15

306.15

a

T, w1, w2, and Kcef indicate the temperature, the mass fraction of Tween 20, the mass fraction of Na3Citrate, and the partition coefficient of cefazolin, respectively. Standard uncertainty u for the surfactant and salt mass fraction is u(w) = 0.03, and for the cefazolin mass fraction is u(wcef) = 0.07. Also, u(T) = 0.1 K.

stirred for 30 min and were finally put into another test tube. Obviously the cefazolin has a concentration of 7 × 10−4 mol/kg in all feed samples. The latter test tube accompanied by the cefazolin-free (blank) test tube were placed into an incubator (Memmert, Germany) at 298.15 K and were allowed to settle for 24 h until two segregated clear phases were achieved. The incubator is supplied with a temperature control system which has an accuracy of ± 0.01 K. Other biphasic sets were made in a way similar to the above procedure. The same protocol was carried out for two supplementary temperatures of (302.15 and 306.15) K. In the last stage, samples of the top phases were meticulously removed by pipettes right above the interface and those of bottom phases were taken out by a plastic syringe with an attachable long needle. All the samples were properly diluted so that the concentration would fall within the range calibrated and then the concentration of cefazolin in each phase was determined by using a UV−vis spectrophotometer (M501, from CamSpec, U.K.) at a wavelength of 272 nm. To prevent the interference of surfactant and salt in estimation of cefazolin concentration, the measurements of cefazolin absorbance in studied samples was carried out against the blanks with the same composition as the samples. Flame photometry (Sherwood Model 410, U.K.) was used for measuring the mass fraction of Na3Citrate through analysis of sodium ion. The concentrations of MgSO4 were found out by magnesium analysis using atomic absorption spectroscopy (Varian, AA240, USA) at a wavelength of 202.6 nm. The concentration of surfactant was evaluated by refractive index measurements using a refractometer (Shimadzu, Japan). Once

glass beaker. Both aforementioned solutions were transparent after complete dissolution. Then the salt solution was added dropwise to the glass beaker while the mixture was being agitated by a magnetic stirrer. Continuing this procedure, there is a drop that causes the mixture not to be transparent any longer. This point (drop) is called the first sign of turbidity which is a criterion for two-phase formation. Once the mixture turns cloudy, the addition of salt solution will be stopped. The composition of the mixture was noted. Then double distilled water was added in drops until a clear monophasic mixture was gained and the above approach was repeated to reach the next point. Alternatively, an inverse procedure could be applied (i.e., Tween 20, as stock solution, would be added to (salt + water) mixture), to verify the initial data points. These points construct a coexistence curve which is called the binodal curve distinguishing between singlephase and two-phase regions. Partitioning of Cefazolin. Eight feed samples were prepared with two different kinds of salt: Na3Citrate and MgSO4 (set numbers 1−8, as shown in Tables 2 and 3). Sets 1 through 4 are the systems that have a fixed concentration of Tween 20 in feed samples (i.e., wTween = 0.25), whereas sets 5 through 8 have a fixed concentration of salt in feed samples (i.e., wsalt = 0.14). These following steps were carried out to achieve the partition coefficient: First of all, the constituents of feed sample (surfactant + salt + water) were weighed and put into a 100 cm3 glass beaker. The various compositions of feed samples have a total weight of 100 g. Afterward, the mixture was agitated for 20 min by means of a magnetic stirrer. Then half of the mixture (i.e., 30 cm3) was directly poured into a test tube; 0.01 g of cefazolin was added to the rest of the mixture and the compounds were thoroughly C

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Table 3. Experimental Tie-Lines Compositions, Tie-Lines Length (TLL) and Slope of Tie-Lines (STL) for {Tween 20 (1) + MgSO4 (2) + H2O (3)} Biphasic Systemsa feed composition

top-phase composition

bottom-phase composition

T/K

set no.

w1

w2

w1

w2

w1

w2

TLL

STL

Kcef

298.15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

0.250 0.250 0.250 0.250 0.180 0.200 0.220 0.240 0.250 0.250 0.250 0.252 0.180 0.200 0.220 0.240 0.250 0.250 0.250 0.250 0.180 0.200 0.220 0.240

0.120 0.130 0.140 0.150 0.140 0.140 0.140 0.140 0.120 0.130 0.140 0.151 0.140 0.140 0.140 0.140 0.120 0.130 0.140 0.150 0.140 0.140 0.140 0.140

0.451 0.478 0.509 0.549 0.436 0.495 0.506 0.498 0.446 0.467 0.496 0.500 0.443 0.468 0.490 0.497 0.461 0.475 0.498 0.520 0.462 0.476 0.487 0.512

0.011 0.010 0.007 0.005 0.012 0.007 0.007 0.008 0.024 0.029 0.026 0.029 0.012 0.010 0.009 0.008 0.030 0.033 0.026 0.019 0.014 0.010 0.008 0.008

0.063 0.058 0.062 0.054 0.042 0.045 0.043 0.050 0.064 0.058 0.061 0.059 0.054 0.050 0.054 0.052 0.079 0.064 0.065 0.066 0.055 0.052 0.052 0.053

0.217 0.233 0.244 0.259 0.215 0.227 0.242 0.254 0.210 0.229 0.241 0.255 0.204 0.220 0.235 0.248 0.201 0.219 0.235 0.247 0.191 0.213 0.226 0.238

0.440 0.476 0.506 0.556 0.443 0.501 0.519 0.511 0.425 0.455 0.486 0.496 0.433 0.468 0.491 0.506 0.419 0.451 0.481 0.508 0.443 0.470 0.487 0.514

−1.89 −1.88 −1.89 −1.94 −1.95 −2.04 −1.97 −1.83 −2.05 −2.05 −2.03 −1.95 −2.02 −1.99 −1.93 −1.86 −2.24 −2.20 −2.07 −1.99 −2.30 −2.09 −2.00 −1.99

2.34 2.97 1.88 2.42 2.00 2.33 2.62 3.05 1.98 1.83 3.09 2.08 1.8 2.17 2.42 2.56 2.25 2.27 2.52 2.38 2.40 1.51 2.31 2.72

302.15

306.15

a

T, w1, w2, and Kcef indicate the temperature, the mass fraction of Tween 20, the mass fraction of MgSO4, and the partition coefficient of cefazolin, respectively. Standard uncertainty u for the surfactant and salt mass fraction is u(w) = 0.03, and for the cefazolin mass fraction is u(wcef) = 0.07. Also, u(T) = 0.1 K.

Y = A exp{(BX 0.5) − (CX3)}

the salt concentration is known, the surfactant concentration will be calculated from the following relation:56 nD = n0 + a1w1 + a 2w2 (1)

where Y and X are the surfactant and salt mass fraction, respectively. A, B, and C are also the adjustable parameters. This semiempirical corelarion was derived from (polymer + salt) systems, yet it applies for miscellaneous kinds of ATPS such as IL-based10 and surfactant-based.41 Our data have become wellfitted to Merchuk’s equation (eq 1). The adjustable parameters for each of the biphasic systems are given in Table 5. The coefficient of determination (R2), as a criterion of goodness of fit, was also calculated by applying the following expression:57

where nD, w1, and w2 are the refractive index, the mass fraction of surfactant (Tween 20), and the mass fraction of salt, respectively. The adjustable parameters based on our data (n0, a1, and a2) are shown in Table 4 (n0 is the refractive index of pure water which is Table 4. Refractive Index Calibration constants component

n0

water Tween 20 Na3Citrate MgSO4

1.3315

a1

a2

R2

0.130 0.100

0.99 0.99 0.99

(2)

n

0.141

2

R =1−

∑1 (Yexp − Ymod)2 n

∑1 (Yexp − Y ̅ )2

(3)

Where the experimental and the adjustable binodal values are denoted by Yexp and Ymod and n is the number of the experimental data and Y̅ is the mean of experimental data.

set to 1.3315 at 298.15 K). According to the concentrations of surfactant and salt in the top and bottom phases the tie-lines can be established.



n ⎛ ∑ Yexp ⎞ ⎜⎜Y ̅ = 1 ⎟⎟ n ⎠ ⎝

RESULTS AND DISCUSSIONS Binodal Curves and Correlation. The data for three biphasic systems (Tween 20 + Na3C6H5O7, Tween 20 + K2HPO4, and Tween 20 + MgSO4) at the temperature of 298.15 K are shown in a triangular chart on the basis of mass fraction of components (Figure 2). It can be inferred from the chart that water is the staple of the aqueous two-phase system. Merchuk et al. proposed a mathematical representation in obtaining the binodal curve:1

(4)

R2 would be in the range of 0−1, and its high value in Table 5 suggests that the above-mentioned equation is proper to satisfactorily reproduce the binodal curves of the studied systems. As it can be seen in Figure 2, the extent of the twophase area follows the descending order: K2HPO4> MgSO4 > Na3C6H5O7 (namely, a less amount of K2HPO4 is required to salt out Tween 20 as compared with MgSO4 or Na3C6H5O7). Our D

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Figure 2. Phase border experimental data at 298.15 K on triangular chart. ○ (Tween 20 + K2HPO4 + H2O); Δ (Tween 20 + MgSO4 + H2O); ▽ (Tween 20 + Na3Citrate + H2O).

namics consistency of obtained tie-lines data from experimental analysis. a, b, and R2 of the contrived systems with their corresponding temperatures are also reported in Table 6. With respect to regression coefficients, a reasonable consistency in tielines data can be concluded.

Table 5. Parameters of Merchuk’s Equation and Determination Coefficient (R2)a Nd

biphasic system Tween 20 + MgSO4 + H2O Tween 20 + Na3Citrate + H2O Tween 20 + K2HPO4 + H2O a

A

B

R2

C −4

35

77.207

−0.267

3.05 × 10

0.99

22

92.790

−0.301

3.07 × 10−4

0.99

−0.386

−3

23

90.001

1.48 × 10

Table 6. Parameters of Othmer-Tobias Equation and Determination Coefficient (R2)a

0.99

Nd indicates the number of data points.

results are consistent with the other results of (Triton X-100 + salt) ATPS obtained by Salabat et al.38 Tie-Lines and Correlation. The tie-lines were obtained by the top and bottom phases mass fractions and are reported in Tables 2 and 3 for biphasic systems containing (Tween 20 + Na3C6H5O7) and (Tween 20 + MgSO4) respectively at three temperatures: 298.15 K, 302.15 K, and 306.15 K. The tie-lines length (TLL) and slope of tie-lines (STL) were calculated using the following relationships: (w1t

TLL = STL =



w1b)2

+

(w2t



w2b)2

a

(5)

(6)

where the subscripts 1 and 2 stand for surfactant and salt as well as the superscripts t and b indicating the top and bottom phases, respectively. To confirm the validity of the empirical tie-lines data, the Othmer-Tobias54 correlation was used: ⎛ 100 − ln⎜ w1t ⎝

w1t

⎛ 100 − w b ⎞ ⎞ 2 ⎟ = a + b ln⎜⎜ ⎟⎟ ⎠ w2b ⎝ ⎠

T/K

a

b

R2

Tween 20 + Na3Citrate + H2O Tween 20 + Na3Citrate + H2O Tween 20 + Na3Citrate + H2O Tween 20 + MgSO4 + H2O Tween 20 + MgSO4 + H2O Tween 20 + MgSO4 + H2O

298.15 302.15 306.15 298.15 302.15 302.15

−1.398 −2.030 −1.556 −1.945 −1.002 −1.068

0.996 1.357 0.936 1.673 0.904 0.898

0.97 0.92 0.97 0.93 0.95 0.94

T indicates the temperature.

Moreover, it could be inferred from the STL data placed in Tables 2 and 3 that the slopes of tie-lines increase with temperature. Voros et al. also came up with similar results for (polymer + salt) ATPS.58 Partitioning of Cefazolin in ATPS. The values of partition coefficient corresponding to each biphasic system set were reported in Tables 2 and 3. The partition coefficient of cefazolin can be obtained from the following equation:

w1t − w1b w2t − w2b

biphasic system

Kcef =

t wcef b wcef

(8)

Kcef, wtcef,

wbcef

where and point to partition coefficient, mass fractions of cefazolin in the top phase, and mass fractions of cefazolin in the bottom phases, respectively. Partition coefficient variations are due to many factors, such as interactions of biomolecules with the surrounding molecules (e.g., ionic and

(7)

where a and b are regressed parameters. wt1 and wb2 are also similar to the earlier definition. This equation assesses the thermodyE

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Figure 3. Effect of salt type on partition coefficient. □ (Tween 20 + Na3Citrate + H2O); ■ (Tween 20 + MgSO4 + H2O).

Figure 4. Effect of TLL on partition coefficient at 302.15 K. −,• (Tween 20 + Na3Citrate + H2O);▼,− (Tween 20 + MgSO4 + H2O).

salting-in effect.62 Citrate and sulfate ions are kosmotropes with almost equal B coefficients (+0.27·10−3 m3/mol for Citrate and +0.206·10−3 m3/mol for Sulfate);60,63 hence, both have similar salting-out strength. Despite having positive B coefficients, the striking difference between Na+ and Mg2+ B-coefficients (+0.085· 10−3 m3/mol for Na+ and +0.385·10−3 m3/mol for Mg2+) implies that Na+ is not as kosmotropic as Mg2+ and can partly interact with biomolecules, which consequently lessen the solubility of cefazolin in the bottom phase. As a result, the higher values of partition coefficients are obtained for the ATPS consisting of (Tween 20 + Na3Citrate). This feature along with its nontoxic biodegradable characteristic makes citrate more attractive for use in aqueous two-phase extraction. The temperature has contrasting effects on partition coefficients of cefazolin in ATPS including Na3Citrate and MgSO4. Considering our data it can be deducted that the favorable temperature for partitioning of cefazolin in {Tween 20 + MgSO4 + H2O} biphasic system is 298.15 K whereas a temperature of 306.15 K is more efficient for partitioning in the {Tween 20 + Na3Citrate + H2O} biphasic system. The influence of TLL on partition coefficients is shown in Figure 4 which confirms the findings of other authors.47,64 The increase of TLL implies a more extended two-phase region and richer phases which can enhance the partition coefficient.

hydrophobic interactions and special affinity with one component) together with other weak forces. Considering the values of partition coefficient in Tables 2 and 3, it can be deduced that the cefazolin molecule has an affinity with the top phase, i.e. the phase abounding with Tween 20. The hydrophobic interaction between micelles and cefazolin molecules can improve the partition coefficient. The biomolecule may be trapped in hydrophobic parts of surfactant micelles accumulated in the top phase. The effect of salt types on partition coefficient values of cefazolin is shown in Figure 3 for all data sets. It is clearly visible that the partition coefficients are bigger in ATPS comprising Na3Citrate compared with those having MgSO4. This behavior is in agreement with the salting-out order of ions proposed by Hofmeister series.59 Both Citrate3‑ and Na+ ions have larger salting-out effects than SO42‑ and Mg2+ respectively. Thus, in the bottom phase, a lower concentration of cefazolin in the case of Na3Citrate can be expected. These results are similar with those of Yang et al.’s study concerning cephalosporin C in (PEG + salt) ATPS.45 This phenomenon can be justified by the kosmotropic and chaotropic properties of ions in solutions. To establish a quantitative scale for these properties in Hofmeister series, the viscosity B coefficient60,61 for different ions can be used. Kosmotropic anions and chaotropic cations have a salting-out effect, while chaotropic anions and kosmotropic cations have a F

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CONCLUSION The ATPS composed of Tween 20 surfactant and three different salts (K2HPO4, Na3Citrate, and MgSO4) and the binodal curve of each system were obtained at 298.15 K. The experimental data for the two-phase boundary were successfully fitted into the three-parameter Merchuk correlation and using the OthmerTobias equation verifies a reasonable consistency in tie-line results. Also, it is found that by increasing the temperature, the slope of the tie-line was increased. Afterward, the partition coefficient of cefazolin in {Tween 20 + Na3Citrate + H2O} and {Tween 20 + MgSO4 + H2O} biphasic systems were examined and larger values of partition coefficients were obtained in ATPS containing Na3Citrate which was justified by Hofmeister series and kosmotropic/chaotropic characteristics of ions. In addition, the effect of tie-line length on partition coefficients was studied. In light of the high values of partition coefficients in Tween− Na3Citrate ATPS, it can be offered as a biocompatible system to processes involving separation and purification of biological products.



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The authors declare no competing financial interest.



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