Effect of Nanoparticle Additives on Partitioning of Cephalexin in

Article pubs.acs.org/jced

Effect of Nanoparticle Additives on Partitioning of Cephalexin in Aqueous Two-Phase Systems Containing Poly(ethylene glycol) and Organic Salts Bahareh Afzal Shoushtari,† Javad Rahbar Shahrouzi,*,† and Gholamreza Pazuki‡ †

Chemical Engineering Faculty, Sahand University of Technology, Sahand, Iran Chemical Engineering Department, Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran

‡

ABSTRACT: In this work, the ability of four nanoparticles including Al2O3, TiO2, graphene, and graphene oxide (GO) as additives for extraction of cephalexin in aqueous two-phase systems composed of polyethylene glycol (PEG) and three sodium-based organic salts was investigated. For this purpose, phase diagrams and the liquid−liquid equilibrium (LLE) data for the {PEG 6000 + sodium citrate, sodium tartrate, sodium succinate + H2O} and partition coefficient for cephalexin were determined. The addition of 0.001 wt % graphene oxide increased the partition coefficient by 9% for the {PEG + sodium citrate + water} system, while other nanoparticles had no effect or decreased the partition coefficient. To investigate the effect of GO concentration on the partition coefficient, various concentrations ranging from 0.005 to 0.04 wt % was examined. It is observed that at a GO concentration equal to 0.01 wt %, the maximum partition coefficient improvement is achievable to be 59%. The highest partition coefficient 5.74 was obtained for the system {PEG6000 19 wt %, sodium citrate 10 wt %, and GO 0.01 wt %}. It can be concluded that a trace amount of nanoparticles can change the partition coefficient either by increasing or decreasing. It seems that the concentration of nanoparticle also has a significant effect on the partition coefficient.

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INTRODUCTION Cephalosporin is most important type of beta-lactam antibiotics. Cephalexin belong to first generation cephalosporin antibiotic and it is produced by an enzymatic method. However, the similarity between cephalexin and substrate may cause an important drawback in the separation process. To overcome this problem, aqueous two-phase systems (ATPS) can been applied for enzymatic synthesis and separation of cephalexin simultaneously.1−3 The ATPS systems are very useful technique for the extraction, separation, and purification of diverse biomolecules. These systems can be more economical than other separation methods such as chromatography and membrane separations. In addition, these systems are biocompatible and provide a gentle environment for biomolecules because of high water content in the systems.4,5 During the past decade, the ATPS systems are used for separation and purification of different biomolecules like antibiotics,6,7 enzymes,8,9 and proteins.10 Furthermore, removing dye from textile wastewaters and separation of nanoparticles using ATPS are also reported.11,12 There are five types of the ATPS: polymer−polymer, polymer−salt, surfactant−salt, ionic liquid−salt and alcohol− salt.5,13,14 Polymer−salt systems are prevalent due to the low cost of the phase-forming component, rapid phase separation, and low viscosity compared to other types of ATPS. It should be noted that the polymer−polymer ATPS are formed based © XXXX American Chemical Society

on polyethylene glycol (PEG) because of its low cost and biocompatibility. PEG and sulfate and phosphate salts form two-phase systems; however, a high concentration of the mentioned salts in the effluent streams leads to environmental problems. Recently, the use of organic salts such as citrate, acetate, and succinate has become attractive because of their biodegradability and nontoxicity. Biodegradable salts can be discharged into biological wastewater treatment plants.15−17 In this regard, Zafarani-Moattar et al.18 and Malpiedi et al.19 have reported liquid−liquid equilibrium (LLE) data for ATPSs containing PEG and organic salt including sodium tartrate and sodium citrate. Biomolecules are partitioned unequally between two phases. The partitioning of biomolecules between the two phases depends on physicochemical properties such as isoelectric point, surface hydrophobicity, polymer molecular weight, polymer/salt concentrations, pH, and temperature. Because of the wide range of affecting parameters on partitioning, the ideal operation condition needs to be determined by various experimental works.5,11,20 The partitioning behavior of biomolecules can be improved by introducing some additives such as neutral salts, polymers, Received: March 28, 2016 Accepted: June 16, 2016

A

DOI: 10.1021/acs.jced.6b00270 J. Chem. Eng. Data XXXX, XXX, XXX−XXX

Journal of Chemical & Engineering Data

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surfactants, and hydrophilic solvents.1,21 Recently, a novel approach toward improvement in biomolecules partitioning by the addition of nanoparticles to ATPS was proposed.22 The aim of this work is to study the effect of a nanoparticle additive on the partitioning of cephalexin in the system of PEG 6000 and three organic salts such as sodium citrate, sodium tartrate, and sodium succinate.

Methods. Determination of Binodal Curves. The binodal curves were determined through the cloud point method at 298 K. This method is a visually determination technique.23 At first, aqueous stock solutions of known concentration of polymer and salt were prepared. After that, a salt solution of known concentration was added dropwise to the PEG solution (or vice versa) until the solution became cloudy which indicates an aqueous biphasic system. Hereby, double distilled water was added dropwise until the solution became clear and a monophasic region was observed. In the next step, the composition of the mixture must be calculated. For this purpose, with knowledge of the initial mixture composition and the amount of added solution, the total system composition was calculated and provided a point on the binodal curve. This procedure was repeated until enough points of the binodal curves were obtained. In all steps, the solution was mixed with a magnetic agitator. To relate the equilibrium concentration, the experimental binodal curves were fitted to the Merchuk equation:24

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MATERIALS AND METHODS Materials. The detailed properties of compounds used in this study are provided in Table 1. Cephalexin monohydrate Table 1. Detailed Properties of Studied Compounds chemical materials

chemical name polyethylene glycol trisodium citrate dehydrate disodium succinate hexahydrate disodium tartrate cephalexin monohydrate

chemical name

chemical formula HO(C2H4O)nH

source

molar mass (g mol−1)

Scharlau (Spain) Scharlau (Spain)

0.99

6000

0.99

294.10

Merck (Germany)

0.99

270.15

Merck (Germany) C16H17N3O4S.H2O Daana Pharma Co. (Iran) nanoparticles

0.99

230.08

0.99

365.41

C6H5Na3O7·2H2O C4H4Na2O4·6H2O C4H4Na2O6·2H2O

chemical formula

titanium dioxide alumina

TiO2

graphene

carbon structure carbon structure

graphene oxide

mass fraction purity

Al2O3

average size (nm)

Degussa (Germany) US Research (USA) synthesized

0.99

20

0.99

20

0.99

5

synthesized

0.99

5

(1)

where Y and X are the PEG and salt weight fraction, respectively. The values of constants A, B, and C can be obtained by using regression of the experimental binodal data. It should be noted that standard uncertainties u(x) were calculated by the following equation: u(x) =

mass fraction purity

source

Y = A exp{(BX 0.5) − (CX3)}

1 n(n − 1)

n

n

∑ (xi − x ̅ )2 i=1

x̅ =

∑i = 1 xi n

(2)

where n is the number of observations. Determination of Tie-Line and Phase Compositions. To determine the tie-line and phase composition, a gravimetric method was applied. This method is based on mass balance, without any requirement of chemical analysis.24 The feed samples at the biphasic region were prepared by mixing appropriate amounts of PEG + salt + water. After sufficient mixing, the samples were placed in a glass decanter and left for 24 h at 298 K in order to reach equilibrium and were separated into two clear phases. After 24 h, two phases were separated and weighed carefully by using an analytical balance with a precision of ±0.1 mg (Mettler Toledo-AE240- Switzerland). To determine the phase composition, the following mass balance equations were applied:

was obtained from the Daana Pharma Company (Iran). The chemical structures of cephalexin are shown in Figure 1. Double distilled water was used in all of the experiments. All materials were used without further purification.

YM 1−α − YB α α

YT = XT =

α=

(3)

XM 1−α − XB α α

(4)

weight of top phase weight of total mixture

(5)

where M, T, and B subscripts represent the mixture, the top phase, and the bottom phase, respectively. α is the weight ratio of the top phase to the total mixture (sum of phases). The phase composition (XT, XB, YT, YB) can be determined by solving the system of four equations including two Merchuk’s equations and two mass balance equations. Tie-line length (TLL) and slop of tie-line (STL) were obtained by the following equations: TLL =

Figure 1. Chemical structure of cephalexin. B

(X T − XB)2 + (YT − YB)2

(6)


Journal of Chemical & Engineering Data YT − YB X T − XB

STL =

Article

where K is the partition coefficient of cephalexin and RV is the phase volume ratio which can be obtained by dividing the volume of the top phase (VT) to that of the bottom phase (VB). Hereafter, the partition coefficients of cephalexin in the presence of nanoparticles are presented by CCeph * to be distinguished from the original solution. To investigate the possible interaction between cephalexin and nanoparticles Fourier transform infrared (FTIR) analysis was carried out (Tensor 27, Bruker, Germany). Also, to characterize the surface charge of nanoparticles, the zeta potential of samples were measured by particle size analyzer (Nanotrac Wave, Microtrac, US).

(7)

Finally, after the separation of two phases, the pH value for top and bottom phases were measured with a pH meter (TajhizatSanjesh, Iran). Standard uncertainties for pH is u(pH) = 0.06. Determination of the Partition Coefficient of Cephalexin. Two samples were prepared with different salts including sodium citrate (PEG + sodium citrate + water) and sodium tartrate (PEG + sodium tartrate + water). For each ATPS sample, four different mixture compositions at the biphasic region were selected and prepared. The following consecutive steps were carried out for sample preparation: at first, 40 g of feed sample was prepared by mixing appropriate amounts of PEG + salt + water. Afterward, mixture was well agitated, and half of the mixture (20 g of the mixture) was put into another glass beaker which contained 0.002 g of cephalexin. The mixture was stirred for 1 h and was poured into a test tube. The rest of the primary mixture was put into glass decanter to act as a blank solution. It should be noted that the sample was taken by pipet during stirring, so the phase was single and homogeneous. Similarly, 20 g of feed with the same initial composition was prepared, but in this step 5 g of total amounts of water (this amount is arbitrary) poured into a test tube which contained nanoparticles, and this mixture was sonicated using ultrasonic (Elmasonic S 15, Germany) for 1 h at 100 W power. The rest of the primary feed was put into another test tube which contained 0.002 g of cephalexin. The mixture was stirred for 1 h. Then nanoparticles suspensions were added dropwise to the mixture which contained cephalexin while the mixture was being agitated by a magnetic stirrer. Finally, test tubes and the decanter were left for 24 h at 298 K to reach the equilibrium. After 24 h, samples of the top and bottom phases were carefully separated using a decanter. To determine the concentration of cephalexin in both phases, 1 cc of each phase were taken, diluted and analyzed using a UV−vis spectrophotometer (Jenway-6705 UV−vis, UK). The analysis were carried out at a wavelength of 262 nm. It is noted that to avoid interference from the phase components, the samples were analyzed against the blank solution which mentioned above. Moreover, after sampling and measuring the concentration of cephalexin by UV−vis spectrophotometer, cephalexin overall mass balance was written in order to reduce experimental error. The partition coefficients of cephalexin, KCeph, were determined according to following:

K Ceph =

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RESULT AND DISCUSSION Phase Diagrams and Tie-Lines. The experimental data for the three aqueous two-phase systems containing PEG 6000 and three organic salts at 298 K are reported in Table 2. The

Table 2. Binodal Data as Mass Percentage for the Three Aqueous Two-Phase Systems Containing PEG 6000 (WP) and Three Organic Salts (Ws) at 298 Ka PEG6000 + sodium citrate

T CCeph B CCeph

a

R B% =

1 R VK

100 1 + R VK

WP

WS

WP

WS

WP

WS

47.59 43.55 42.74 37.59 35.93 34.67 29.41 26.92 25.47 24.55 22. 51 20.57 18.77 13.18 12.58 10.47 9.85 7.77 6.60 5.72 4.36 3.94 1.22 0.93

0.97 1.61 1.77 2.48 2.54 2.96 3.94 4.41 4.77 5.00 5.56 6.01 6.51 8.41 8.65 9.54 9.85 11.00 11.63 12.11 13.00 13.26 15.77 16.30

38.87 34.88 29.82 27.14 26.34 25.92 24.10 23.76 22.90 21.92 20.10 19.94 18.67 17.54 16.54 15.14 14.12 12.95 11.40 10.29 9.74 8.38 5.99 0.41

8.56 9.05 10.13 11.25 11.33 11.48 12.10 12.25 12.64 13.04 13.79 13.86 14.45 14.98 15.45 16.19 16.70 17.38 18.55 19.44 19.79 20.68 22.62 39.69

36.85 35.32 32.24 30.46 28.93 27.00 26.15 25.85 24.34 23.94 21.66 20.72 18.78 17.66 16.42 14.41 13.51 12.81 11.44 10.83 8.75 7.51 6.03 4.32

3.65 4.11 4.85 5.28 5.52 5.92 6.05 6.14 6.44 6.53 7.10 7.38 8.00 8.37 8.83 9.59 9.97 10.28 11.12 11.41 12.42 13.05 13.82 14.88

Standard uncertainties for weight fraction is u(W) = 0.19.

adjustable parameters for the Merchuk equation for each of the three aqueous biphasic systems are given in the Table 3. Moreover, their corresponding triangular phase diagrams are illustrated in Figure 2. As illustrated in Figure 2, the biphasic system (PEG6000 + sodium citrate + water) has more extended two-phase region in comparison with another biphasic systems. On the basis of the phase diagrams, biphasic systems (PEG6000 + sodium tartrate + water) and (PEG6000 + sodium citrate + water) were chosen as suitable ATPS for cephalexin extraction because of a more extended two-phase region in the phase diagram.

100 1+

PEG6000 + sodium tartrate

(8)

where CTCeph and CBCeph are the concentration of cephalexin in the top phase and the bottom phase, respectively. Moreover, yield recovery from top and bottom phases is determined with the following equations:25 RT% =

PEG6000 + sodium succinate

(9)

(10) C



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⎛ 100 − XB − YB ⎞ ⎛ 100 − X T − YT ⎞ ln⎜ ⎟ = c + d ln⎜ ⎟ XB YT ⎠ ⎝ ⎝ ⎠

Table 3. Adjustable Parameters for Merchuk Equation for Biphasic Systems aqueous two-phase systems

A

B

C

R2 a

PEG6000 + sodium citrate + water PEG6000 + sodium tartrate + water PEG6000 + sodium succinate + water

83.9909

−0.5134

0.0005

0.9983

118.8930

−0.5855

0.0003

0.9982

299.2396

−0.7006

0.00004

0.9979

a

where a, b, c, and d are adjustable parameters obtained by using the regression of the experimental data. The values of the fitted parameters and R2 values are given in Table 6. Effective Excluded Volume and Salting-out Abilities of Salts. Guan et al. developed a simple model for the binodal curve in polymer−polymer aqueous two-phase systems based on statistical geometry.27 The equation predicts the binodal curves using only one parameter. This equation can be written as

For the following equation, n is the number of observations: n

R2 = 1 −

∑1 (Yexp − Ymodel)2 n

∑1 (Yexp − Y ̅ )2

n

,

Y̅ =

∑1 Yexp n

⎛ WP ⎞ W * * S =0 ln⎜v123 ⎟ + v123 MS ⎝ MP ⎠

(13)

where Ws and WP are mass fraction of salt and polymer and MP and Ms are the molecular weight of the polymer and the salt, respectively. The parameter, v123 * represents the effective excluded volume (EEV). In the original application, the Guan equation was used for the correlation of the binodal curve of the (polymer + polymer) system. However, this model has also been used for the correlation of the binodal curves of surfactant/salt,6 ionic liquid/salt,28 and alcohol/salt13 ATPSs. Using the experimental binodal data, the EEV was determined. Furthermore, the EEV can be related with the salting-out strength of the salt, at constant PEG molecular weight.29 The influence of different parameters such as pH and PEG molecular weight on the binodal curve were investigated, and EEV values were reported by Glyk et al. which shows that the EEV has a greater value at higher PEG molecular weight.30 For different salts in the polymer/salt ATPS, the salting-out ability of salts increases with the increase of the EEV and therefore lower concentration of polymer is needed for phase separation. This means that the two-phase region increases with the increase in the value of the EEV. Salts have different anions

The Tie-Line Correlation. As mentioned before the gravimetric method was applied for the determination of tielines. The feed compositions, the composition of each phases, the pH values of the phases and TLL and STL are reported in Tables 4 and 5. Tables 4 and 5 show that TLLs increase with an increase of PEG concentration. The STL is on average constant for all tielines, and tie-lines are almost parallel to each other. Also, Figures 3 and 4 show tie lines for PEG + sodium citrate + water and PEG + sodium tartrate + water, respectively. The tie lines compositions were fitted with the Othmer− Tobias and the Bancroft equations, eqs 9 and 10, respectively. These equations can be used to relate the tie line compositions where the experimental data are not available. They have been widely used in the correlation of polymer−salt, IL−salt, surfactant−salt and hydrophilic alcohol−salt systems.26 ⎛ 100 − XB ⎞ ⎛ 100 − YT ⎞ ln⎜ ⎟ ⎟ = a + b ln⎜ XB YT ⎠ ⎝ ⎝ ⎠

(12)

(11)

Figure 2. Ternary phase diagram for ATPS composed of PEG 6000 + three organic salts + water at 298 K: blue tartrate; green ◆, sodium succinate. D

▲sodium

citrate; red ●, sodium



Article

Table 4. Feed and Phase Equilibrium Compositions, The pH Values of the Phases, Tie-Line Length (TLL), and Slope of TieLine (STL) for the System PEG6000 + Sodium Citrate + Water at 298 Ka feed composition %(w/w)

pH

a

top phase %(w/w)

bottom phase %(w/w)

feed no.

TLL

STL

bottom phase

top phase

PEG

salt

PEG

salt

PEG

salt

1 2 3 4

20.65 25.49 34.29 40.35

−1.92 −1.74 −1.77 −1.59

8.09 8.16 8.14 8.19

8.27 8.32 8.27 8.41

13 15 19 23

10 10 10 10

19.7 22.6 99.97 34.17

6.52 5.67 3.81 2.97

1.36 0.42 0.0997 0.0044

16.03 18.33 20.65 24.45

Standard uncertainties for weight fraction, pH, and temperature are u(W) = 0.79, u(pH) = 0.06 and u(T) = 0.5, respectively.

Table 5. Feed and Phase Equilibrium Compositions, the pH Values of the Phases, Tie-Line Length (TLL) and Slope of Tie-Line (STL) for the PEG6000 + Sodium Tartrate + Water at 298 Ka feed composition %(w/w)

pH

a

top phase %(w/w)

bottom phase %(w/w)

feed no.

TLL

STL

bottom phase

top phase

PEG

salt

PEG

salt

PEG

salt

1 2 3 4

23.62 28.49 32.32 35.84

−1.80 −1.76 −1.66 −1.59

7.67 7.79 7.45 7.44

7.73 7.89 7.47 7.64

15 17 19 21

11 11 11 11

21.84 25.37 27.91 30.44

7.21 6.26 5.64 5.08

1.16 0.57 0.20 0.07

18.64 20.29 22.28 24.10

Standard uncertainties for weight fraction, pH and temperature are u are u(W) = 0.26, u(pH) = 0.06, and u(T) = 0.5.

Table 6. Fitting Parameters for Othmer−Tobias and Bancroft Equation for Biphasic Systems with the Corresponding R2 Values at 298 K Othmer−Tobias aqueous two-phase systems PEG6000 + sodium citrate + water PEG6000 + sodium tartrate + water a

Bancroft

a

b

R2

1.49

2.59

0.961

0.90

0.16

0.9334

R2a

0.76

0.65

0.9558

−0.57

0.42

0.9873

For the following equation, n is the number of observations: n

R2 = 1 − Figure 3. Tie lines for aqueous PEG6000 + sodium citrate system at 298 K.

d

c


∑1 (Yexp − Y ̅ )2

n

,

Y̅ =

∑1 Yexp n

(citrate, tartrate, and succinate) and common sodium cation. The values of the EEV are reported in Table 7. According to Table 7. EEV Values Obtained from eq 13 for Biphasic Systems with the Corresponding R2 Values at 298 K aqueous two-phase systems PEG6000 + sodium citrate + water PEG6000 + sodium tartrate + water PEG6000 + sodium succinate + water a

EEV/g mol−1

R2a

77.55 54.77 40.51

0.9739 0.9713 0.9903

For the following equations, n is the number of observations: n

R2 = 1 −


∑1 (Yexp − Y ̅ )2

n

,

Y̅ =

∑1 Yexp n

the results reported in Table 7, sodium citrate has the highest value of the EEV, while sodium succinate has the smallest ones. These results are in agreement with the fact that higher valence denotes more salting out ability. The mentioned phenomenon can be related to the Gibbs free energy of hydration of the ions;

Figure 4. Tie lines for aqueous PEG6000 + sodium tartrate system at 298 K.

E



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According to these results, GO was chosen as the proper additive to further the investigation. Feed composition partition coefficients of cephalexin in the absence and presence of GO for two aqueous two-phase systems containing PEG6000 and sodium citrate and sodium tartrate are reported in Table 9. Numerous interactions like van der Waals, ionic interaction, isoelectric point (pI), hydrophobic interaction, and hydrogen bonding between biomolecules and surrounding components can affect the partitioning of biomolecules in the ATPS. As can be seen from the results in all studied systems, partition coefficients of cephalexin are larger than unity. This means that cephalexin is mostly partitioned in the top phase. As mentioned above, the partition coefficient of biomolecules depends on its isoelectric point (pI). The pI of the cephalexin is 4.5−5 in aqueous solution.6 As shown in Tables 4 and 5, the pH of phases are above the pI of cephalexin. This means that in all studied systems cephalexin has a negative surface charge. Repulsion between cephalexin and salt anions causes the cephalexin partition to rise to the top phase. According to Table 7, the salting-out ability of citrate is higher than that of tartrate. Thus, it seems that the partition coefficient of cephalexin in the system containing citrate is higher than the other one. This trend is in agreement with the results reported in Table 9. As mentioned above, the partition coefficient can be affected by various parameters (for example, attraction and repulsion between the negatively charged surface of cephalexin and ions). Even though the negatively charged surface of cephalexin causes it to partition to the bottom phase, the salting-out ability of salts (especially anions) leads it to partition to the top phase. In the case of nanoparticles as additive, GO partitioned to the top phase. GO changes the color of the suspension to dark brown. Visual observation showed that the top phase (PEGrich) becomes dark brown while the bottom phase remains very

the ion with higher negative Gibbs free energy had higher salting-out ability.16,31 Partition Behavior of Cephalexin in ATPS Systems. In this work, four different nanoparticles including TiO2, Al2O3, graphene, and graphene oxide (GO) were selected to study their effect on the partition coefficient of cephalexin in the ATPS. These nanoparticles were chosen because they are biocompatible and their biomedical applications were reported in the literature. For example graphene contains a free surface π electron which can interact for drug loading on the surface of graphene, and graphene oxide contains an oxygen functional group which led to hydrogen bonding with the drug.32,33 It should be mentioned here that the graphene oxide was synthesized by the modified Hummers’ method.34 The experimental results for the partition coefficient of cephalexin in the absence and presence of nanoparticles are reported in Table 8. Results show that the addition of Table 8. Partition Coefficient of Cephalexin in Absence and Presence of Nanoparticles for Aqueous PEG6000 + Sodium Citrate System at 298 Ka feed composition (% w/w) nanoparticle TiO2 Al2O3 graphene GO

KCeph

K*Ceph

Aqueous PEG6000 + Sodium Citrate System 13 10 0.01 0.001 2.65 13 10 0.01 0.001 13 10 0.01 0.001 13 10 0.01 0.001

2.2 2.49 2.7 2.89

PEG

salt

cephalexin

nanoparticle

a

Standard uncertainties for partition coefficient with/without nanoparticle are u(KCeph) = 0.1 and u(K*Ceph) = 0.25, respectively.

nanoparticles on the ATPS can affect the partition coefficient. Also, the influence of GO on the partition coefficient of cephalexin is much more than the other nanoparticles.

Table 9. Partition Coefficient of Cephalexin in the Absence and Presence of GO for Two Aqueous Two-Phase Systems Containing PEG6000 and Sodium Citrate and Sodium Tartrate at 298 Ka feed composition (% w/w)

a

feed no.

PEG

salt

cephalexin

1 2 3 4

13 15 19 21

10 10 10 10

0.01 0.01 0.01 0.01

1 2 3 4

15 17 19 21

11 11 11 11

0.01 0.01 0.01 0.01

GO

KCeph

K*Ceph

PEG6000 + Sodium Citrate + Water 0.01 2.65 3.03 0.01 3.11 4.33 0.01 3.61 5.74 0.01 4.17 5.36 PEG6000 + Sodium Tartrate + Water 0.01 1.47 2.168 0.01 1.50 2.167 0.01 1.60 2.08 0.01 1.81 2.60

RTb

RBb

% enhancementb

40.13 39.73 34.38 30.56

17.52 13.49 12.72 11.53

14.6 39.2 59.2 28.7

59.27 57.45 58.23 57.92

24.12 24.76 21.88 18.14

47.5 44.4 30.1 43.8

Standard uncertainties for partition coefficient with/without nanoparticle are u(KCeph) = 0.1, u(K*Ceph) = 0.25. bEquations: RT =

RB =

100% 1+

1 RVK

100% 1 + R VK

%enhancement =

* − K Ceph K Ceph K Ceph F



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Figure 5. FTIR spectra of samples either with GO (blue line) or without GO (red line).

coefficient decreases with increase of GO concentration. This trend suggests that the surface charge of GO becomes more negative at higher concentration. That may be due to the ionization of the surface functional group of GO. The zeta potential of GO in three samples with different concentrations of GO were measured. The zeta potential of GO increases from −716.9 mV at GO concentration 0.005 wt % to −3203.5 mV at 0.01 wt % and −3346.7 at 0.02 wt %. As a result, the surface charge of GO becomes more negative with increasing GO concentration. However, the trend of this increase is much faster at the lower concentrations. It can be concluded that an optimum amount for GO concentration may be determined, while increasing GO concentration has no significant effect on the partition coefficient at higher concentrations. This trend indicates that the optimum point occurs because of two probable affecting factors: hydrogen bonding and surface charge. At low GO concentration, the partition coefficient increases with GO concentration, which can be explained by the fact that the hydrogen bonding between GO and cephalexin is stronger than other affecting factors. In the range of concentration after the optimum point, the surface charge of GO becomes more negative with an increase of GO concentration. This trend was measured by zeta potential analysis and it seems the repulsion between surface charge of GO and cephalexin, which is stronger than hydrogen bonding, decreases the partition coefficient at higher GO concentration.

clear. So it was concluded that GO is portioned to the top phase. Even after 2 weeks, GO was accumulated at the interface of phases. Also, the analysis of the top phase by UV spectrophotometry showed a pick of GO while this pick was not observed at the bottom phase. This occurs because the presence of free surface π electrons in the graphene oxide structure can lead to noncovalent π−π stacking between the PEG and GO sheets. The FTIR spectra was obtained to investigate the possible interactions between GO, PEG, and cephalexin. The samples were scanned in the wavenumber range of 400 to 4000 either with or without GO. As shown in Figure 5, the FTIR spectrum showed the presence of hydroxyl −OH (3403.96 cm−1) and alkoxy C−O (1100 cm−1) which correspond to PEG molecules. The peak at 1963.18 cm−1 is due to the presence of N−H bond in amide III of cephalexin. In the case of sample with GO, the intensity of the N−H bond is weaker compared to that in the sample without GO. It is suggested that the possible interaction between GO and cephalexin occur in the amino functional group in the cephalexin structure (electron acceptor) and various oxygen-containing functional groups in the GO structure (electron donor). In the other word, cephalexin has been attached to GO via the N−H group. Effect of GO Concentration on Partition Coefficient. Figure 6 illustrates the effect of GO concentration on the partition coefficient of cephalexin. The highest partition coefficient of the cephalexin in the ATPS is achieved at 0.01 wt % of GO. After the optimum concentration, the partition

■

CONCLUSIONS In this work the ability of four different nanoparticles (Al2O3, TiO2, graphene, graphene oxide) as an additive to extract cephalexin within the (PEG6000 + sodium citrate + water) and (PEG6000 + sodium tartrate + water) were examined. Liquid−liquid equilibrium data for the (PEG6000 + sodium citrate, sodium tartrate, sodium succinate + water) were measured at 298 K. The binodal curves were fitted to the Merchuk equation, and the effective excluded volume theory was applied to investigate the effect of the type of salt on the binodal. The experimental tie-line compositions were successfully correlated by using the Othmer−Tobias and Bancroft equations. According to the results, it can be concluded that nanoparticles as additive can affect the partition coefficient and results indicated that GO have a higher effect on the partition coefficient of cephalexin. To evaluate the effect of GO concentration, it was varied in the range of 0.005−0.04 wt %.

Figure 6. Effect of GO concentration on the partition coefficient of cephalexin for the systems composed of PEG 19% w/w + sodium citrate 10%w/w + water + cephalexin 0.01%w/w. G



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The highest partition coefficient 5.74 was obtained using PEG 6000 19 wt %, sodium citrate 10 wt %, and graphene oxide 0.01 wt %.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +98-41-33459168. Fax: +9841-33444355. Funding

The authors gratefully acknowledge Iran Nanotechnology Initiative Council for the financial support of this research. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. A. Jafarizad from the department of chemistry of Atatü rk University, Erzurum, Turkey, for providing graphene and graphene oxide.

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REFERENCES

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