Research Article  Open Access
Selvaraj Raja, Vytla Ramachandra Murty, "Optimization of Aqueous TwoPhase Systems for the Recovery of Soluble Proteins from Tannery Wastewater Using Response Surface Methodology", Journal of Engineering, vol. 2013, Article ID 217483, 10 pages, 2013. https://doi.org/10.1155/2013/217483
Optimization of Aqueous TwoPhase Systems for the Recovery of Soluble Proteins from Tannery Wastewater Using Response Surface Methodology
Abstract
Aqueous twophase system (ATPS) composed of polyethylene glycol 6000 (PEG 6000) and sodium citrate (SC) has been proposed to recover the valuable soluble proteins from tannery wastewater. A sequential optimization strategy which included fractional factorial design (fFD) and central composite design (CCD) was employed to enhance the recovery. From this strategy, a secondorder polynomial model was obtained for the protein recovery and it was validated. The optimum recovery was found as 93.46% when pH, NaCl concentration, and temperature were kept at 7.5, 0.1 M, and 33°C, respectively, for a phase system composed of 20% (w/w) PEG 600015% (w/w) SC. Thus the proposed ATPS can serve as an alternative to the conventional precipitation method to recover the soluble proteins from tannery wastewater.
1. Introduction
The leather industry is one of the major foreign exchange earners in India nearly over last thirty years [1]. It is reported that the Indian market has been fragmented with about 2200 tanneries [2]. During the traditional leather processing, the skin and hides are subjected to various operations such as soaking, dehairing, liming, deliming, bating, degreasing, and pickling [3]. When the skin is subjected to the alkali treatment, the soluble proteins present on the surface of the skin are discharged as waste. Yearly, nine million tons of skins/hides are being processed worldwide [4]. Literature reveals that, for every 100 kg of raw hides, 15 kg of solubilized protein is discharged as waste in the early stages of the process of transforming hides into leather [5]. The value of these solubilized proteins is enormous and they find applications in food and pharmaceutical industries [6]. The presence of these proteins in the tannery effluents increases the biological oxygen demand and chemical oxygen demand and leads to pollution. By removing these proteins from the waste, a reduction of the tax on wastewater can be achieved and the recovered proteins can be used in food and pharmaceutical industries.
There are a few reports available in the literature for the recovery of soluble proteins from industry effluents using membrane separation processes, but the major drawback of these processes is membrane fouling [7, 8]. The conventional method of protein recovery from tannery wastewater is “precipitation method” which has been well addressed by Kabdasli et al. [9] and Marsal et al. [10]. However the precipitation method has the limitation of protein denaturation and low recovery. For example, only a recovery of 50–70% and 68–78% of soluble proteins from the effluents of tannery beamhouse operations was possible in the precipitation methods developed by Kabdasli et al. [9] and Marsal et al. [10], respectively.
In the context of clean environment and pollution prevention, nanotechnology could play a key role [11]. However, aqueous twophase system (ATPS) attracts more attention because of simple process, low cost, and easy scaleup procedures. For example, very recently, ATPS composed of PEG and sodium citrate was successfully used for textile effluent dye removal [12]. It has been proven by many researchers that ATPS is an efficient and economical process when compared to other separation processes like precipitation, chromatography, and so forth [13–15].
Therefore, a benign technique for the proteins, ATPS, has been proposed in the present study in order to recover the soluble proteins from the tannery wastewater. ATPS is a downstream processing method which uses the principles of liquidliquid extraction. It can be formulated by mixing two hydrophilic polymers (Poly Ethylene Glycol (PEG)Dextran) with water or one hydrophilic polymer (PEG) and inorganic salts (phosphates, sulfates, and citrates) with water [16]. It has been reported that polymersaltbased ATPS has many advantages over polymerpolymer ATPS and few of them includes easy scaleup, low cost, low interfacial tension, possibility of process integration, and less viscous phases [17].
For the first time, Saravanan et al. [18] research group developed an ATPS made up of PEG/sulfate to recover the soluble proteins from tannery wastewater. Since then, ATPS has received much attention to recover the biomolecules from various industrial effluents such as fish industry [19], dairy industry [20], and prawn industry [21]. Recently, our research group has addressed the partitioning of tannery wastewater proteins in ATPS composed of PEG 10000 plus different citrate salts [22]. The proposed method of ATPS in this paper is environmentbenign since the phase components used are nontoxic and biodegradable.
For the maximization of recovery of proteins from tannery wastewater, a sequential method of optimization using response surface methodology (RSM) has been employed which includes the following steps: (i)screening of significant process variables which affect the protein partitioning in ATPS by a fractional factorial design (fFD),(ii)crude optimization of the most significant variables by a full factorial design (FFD) with center points,(iii)final optimization of the most significant variables by central composite design (CCD) using response surface methodology (RSM),(iv)development and verification of mathematical model and expressing the relationship between the protein partitioning and significant process variables.
2. Materials and Methods
2.1. Materials
PEG 6000 was purchased from Merck and used without further purification. Trisodium citrate, citric acid, and sodium chloride were also purchased from Merck and MilliporeMilliQ water was used in all the experiments.
2.2. Preparation of Tannery Wastewater
The tannery wastewater sample was prepared as discussed elsewhere [23]. In this method, known weight of raw skin/hides was treated with alkali solution. This sectional stream wastewater was used as a protein source in partition experiments to recover the soluble proteins.
2.3. Preparation of TwoPhase Systems
Calculated amounts of trisodium citrate and citric acid were taken, and pH of the system was adjusted. ATPS was prepared by mixing appropriate amounts of PEG and citrate solutions, with tannery waste sample as described in the previous section in 15 mL graduated tubes. By the addition of water, the weight of the system was maintained at 10 g. The systems were well mixed in a vortex mixer and left in a water bath at various temperatures for overnight.
2.4. Quantification of Tannery Wastewater Soluble Proteins
The soluble protein from the tannery wastewater was quantified by Bradford method [24]. For the determination of protein concentration, samples were withdrawn from each phase and diluted if necessary with distilled water, and its absorbance was measured using Shimadzu spectrophotometer at 595 nm.
2.5. Partition Coefficient and Recovery
The partitioning of soluble proteins in ATPS is characterized by two factors, namely, partition coefficient and the percentage bottom phase protein recovery : where and are the volume of PEG rich and sodium citrate phases, respectively.
2.6. Screening of Significant Process Variables
Partitioning of proteins in ATPS is a complex phenomenon. It depends on many factors like type and concentration of phaseforming components, pH, temperature, presence of neutral salts, and so forth. Based on prior experiments (data not shown), the following five factors namely, concentration of PEG 6000, concentration of SC, pH of the system, concentration of NaCl, and temperature were chosen as the factors that affect the protein partitioning.
Consequently, a fraction, 2level factorial design for five factors (2^{5−1} = 16 experiments) was employed to investigate the significant factors. Table 1 gives both coded and uncoded values of these factors in fFD with the percentage recovery. The table shows a wide variation in percentage recovery ranging from 34% to 83% which reflects the importance to attain higher percentage recovery.
 
^{
a}Data in brackets; ^{b}Data without brackets. 
2.7. Crude Optimization
It has been observed from the analysis that the three factors, namely, pH, NaCl, and temperature are the significant factors which enhance the protein recovery. Since PEG and SC do not play a significant role in partitioning, PEG was fixed at 20% (positive effect) and SC was fixed at 15% (negative effect) for all the upcoming experiments. The three significant variables were further optimized using a 2^{3} FFD (Table 2, Experiment nos. 1–8) with three center points (Table 2, Experiment nos. 9–11) to determine the optimum operating conditions. These experiments were done to make sure that the proposed optimization process was in the appropriate region [25].

2.8. Final Optimization
In order to include the curvature, few more experiments were done by adding 6 axial (star) points (Table 2, Experiment nos. 12–17) and 3 more center points (Table 2, Experiment nos. 18–20) to the previous FFD setup. This entire set of 20 experiments is a central composite design (CCD) for three factors, an RSM technique [26].
In this methodology, the effects of the variables on the protein recovery were fit to the secondorder polynomial model according to the following equation: where is the response variable (percentage recovery), and are the independent variables in coded units, is the average response, and , , and are the measures of the , , , and of linear, quadratic, and interaction effects, respectively.
For the statistical calculations, the variables were coded according to the following equation: where is the independent variable in the coded unit, is the real value of independent variable, is center point the real value of the independent variable, and is the step change value. By analyzing the contour plots, the optimum values of the significant variables were obtained. The statistical analysis of the model was represented in the form of analysis of variance (ANOVA).
3. Results and Discussion
3.1. Screening of Significant Process Variables by fFD and Crude Optimization
The fFD showing the recovery of protein from each experiment combination shown in Table 1 was used for statistical analysis. The results were analysed by MINITAB15.0 (CA, USA). Figure 1 represents the normal probability plot of the effect estimates. This plot is used to analyze the significant factors based on the (=0.05) value. The significant factors do not conform to the normal plot and lie away from the normal line. From the figure, it is clear that the factors pH, NaCl, temperature, and the interaction between PEG concentration and temperature are significant.
The plot of the mean percentage recovery and experiment levels (Figure 2) illustrates the main effects of the operating conditions on the recovery. PEG, pH, NaCl, and temperature increased the protein recovery at high level. A decrease in mean percentage recovery was observed for SC.
Table 3 summarizes the analysis of variance for fFD. The model sum of squares is , and this accounts for 95.82% (high ) of the total variability in recovery. Moreover, the variables are statistically significant when the value (defined as the smallest level of significance which leads to the rejection of null hypothesis) is less than 0.05 (95% confidence level). Based on this, pH, NaCl, temperature, and the interaction between PEG and temperature are considered as the significant factors.
 
; Sq = 95.82%; Sq(adj) = 93.73%. *Significant at 95% confidence level. 
The positive effect of PEG at high concentration may be because of the volume occupied by the PEG molecules with the increase in concentration decreases the free space available for the molecules in the top phase. Therefore, because of “volume exclusion effect” all the biomolecules tend to partition towards the bottom phase and thus percentage recovery in the bottom phase increases [27].
In contrast to this, SC had a negative effect on percentage recovery which can be explained based on the “saltingout effect”. At high salt concentrations of salt, the ions decrease the solubility of biomolecules which makes them to move to the PEG rich top phase and therefore the percentage recovery in the bottom phase decreases [28].
The pH presented a statistically significant positive effect for the partitioning of proteins to the bottom phase. It can be explicated with respect to the isoelectric point of the proteins. The wastewater proteins present in tannery wastewater are soluble and globular proteins [29] and therefore they have net negative charge at pH > 5. Hence, the negatively charged protein molecules partition to the bottom phase at high pH. Similar results were obtained by many researchers [30–32].
NaCl presence in the ATPS showed a significant positive effect which may be due to the alterations of hydrophobic interactions or changes in the electrostatic potential difference. For the NaCl concentrations studied in this study (0.1 M to 0.3 M), the interaction of biomolecules with the salt rich bottom phase increases because of the changes in the electrostatic potential difference [33].
The temperature also indicated a significant positive effect on the percentage recovery. The increase in temperature not only alters the structure of biomolecules but also changes the phase composition of the ATPS. Therefore the increase in temperature increases the protein recovery in the bottom phase [34].
As a conclusion from fFD, the factors pH, NaCl, and temperature are confirmed as significant factors and therefore selected for further optimization to maximize the percentage recovery. From the Table 2, it is evident that the average recovery in the center of the experimental region is 90.27%, while the average recovery at the corners is 82.88%. Since this difference is significant, the recovery will be a curved function of all three factors. Moreover, because of the presence of curvature, the response could not be explained by a linear model and there is a need for a quadratic model which is discussed in the following section.
3.2. Final Optimization
After crude optimization of pH, NaCl, and temperature by 2^{3} FFD with three center points and ascertaining of optimal region, additional experiments were performed with 6 axial points and 3 more center points to frame a complete CCD (Table 2). The CCD of 20 experimental runs was used to analyze and optimize the significant factors. Table 4 lists the Minitab output of estimated regression coefficients, standard errors, values, and values.
 
= 84.86%; (adj) = 71.22%. *Significant at 95% confidence level. 
As discussed earlier, at 95% confidence level, the terms having values <0.05 are considered to be statistically significant. By substituting these statistically significant parameters’ regression coefficients in (2), the following model was obtained in coded units: In addition to the linear effects, RSM helps to evaluate the interaction and quadratic effects. It is clear from the table that interaction effects are also significant in this process. The regression coefficients and values were determined as 84.86% and 71.22%, respectively.
Table 5 represents the ANOVA for the quadratic model developed. Higher values indicate that the term is statistically significant. Another convenient measure to test the significance of the terms is value. It is evident that linear, interaction, and quadratic effects were statistically significant () for the developed model. Nevertheless, a low value for lack of fit indicated that it is also statistically significant, and therefore it is necessary to identify possible outliers. It is done by examination of residual plot as shown in Figure 3 which suggested that a data point corresponding to experimental run 16 could be a possible outlier.
 
*Significant at 95% confidence level. 
Consequently, this data point was omitted and the regression was repeated for the remaining data. The regression coefficients and the ANOVA values after the omission of outlier are given in Tables 6 and 7, respectively.
 
= 97.27%; (adj) = 94.54%. *Significant at 95% confidence level. 
 
*Significant at 95% confidence level. 
Substituting the new regression coefficients into (2) gives the following new modified model: The new regression coefficients and values were determined as 97.27% and 94.54%, respectively, which were higher than the previous values. Thus, 97.27% of variation in yield was explained while only 2.73% was left to the residuals. Apart from this, the value for lack of fit increased from 0.001 to 0.053 (Table 7) after the omission of the outlier. From Table 7, it is obvious that the new model is valid and linear, and quadratic and interaction terms should be included in the model. A good normal distribution of the model (Figure 4) with a linear line for the percentage recovery confirmed that the model was well fitted with the experimental results and all the major assumptions of the model [35] have been validated.
The threedimensional response surface plots (Figures 5(a) and 5(c)) show the effects of operating parameters on the percentage recovery while the contour plots (Figures 5(b) and 5(d)) reflect the nature and degree of these effects. As seen from the figures, the response surface plots are concave, indicating that it is possible to obtain a maximum value within the range of the levels investigated. The curved lines in the contour plots confirmed that interaction between the factors () was present and these interaction terms were included in the new model (5). The new regression model was solved for the maximum recovery using the response optimizer tool in MINTAB 15.0, and the optimum values of pH, 7.45; NaCl, 0.1 M; and temperature, 32.72°C were obtained with a maximum predicted response of 94.40% recovery.
(a)
(b)
(c)
(d)
3.3. Verification of the Model by Experiment
In order to validate these results, experiments were done in triplicates (Table 8) by using the modified optimized values (pH: 7.5; NaCl: 0.1 M; Temperature: 33°C). A minimum partition coefficient of 0.056 was obtained with a recovery of 93.46 ± 7%. The partition coefficient obtained is in good agreement with the literature for the soluble protein BSA [36]. The good correlation between the observed and predicted recoveries confirmed that the validity of the new model was adequate. This recovery is comparatively high when compared to the precipitation method as discussed earlier [9, 10]. Therefore, this ATPS can be an alternative to the conventional method of protein recovery from tannery wastewater. Moreover, in the current system studied, most of the protein is partitioned to the salt rich bottom phase and it is possible to recycle the PEG from the top phase by ultrafiltration [37, 38]. Therefore recycling of the phase components decreases the overall cost of the process.

4. Conclusions
A sequential optimization method which consisted of fFD and CCD was used to obtain the optimum values of significant factors for the recovery of soluble proteins from tannery wastewater in PEG 6000SC ATPS. The fFD revealed that only pH, concentration of NaCl, and temperature were the significant factors. From the CCD studies, the optimized values of these significant factors were determined: pH 7.5, NaCl 0.1 M, and temperature 33°C for a phase system composed of 20% (w/w) PEG 600015% (w/w) SC. The predicted and observed recoveries were 94.40% and 93.46%, respectively, which confirmed that the proposed quadratic model was valid. Thus, it is concluded that ATPS can be used as an alternative method to recover the valuable soluble proteins from tannery wastewater.
Conflict of Interests
The authors declare that they have no conflict of interests.
Acknowledgment
The authors gratefully acknowledge the Department of Biotechnology, MIT, Manipal University, for providing the facilities to carry out the research work.
References
 S. Sanyal, S. Banerjee, and S. Majumder, “India’s leather in the world market: exploration of recent trends,” Trade and Development Review, vol. 3, no. 1, pp. 22–58, 2010. View at: Google Scholar
 http://sinetinfo.com/pdf/chapters/leather1.pdf.
 N. R. Kamini, C. Hemachander, J. G. S. Mala, and R. Puvanakrishnan, “Microbial enzyme technology as an alternative to conventional chemicals in leather industry,” Current Science, vol. 77, no. 1, pp. 80–96, 1999. View at: Google Scholar
 P. Thanikaivelan, J. R. Rao, B. U. Nair, and T. Ramasami, “Recent trends in leather making: processes, problems, and pathways,” Critical Reviews in Environmental Science and Technology, vol. 35, no. 1, pp. 37–79, 2005. View at: Publisher Site  Google Scholar
 A. Marsal, E. Bautista1, S. Cuadros, F. Maldonado, A. M. Manich, and J. Font, “Diminution of toxicity of beamhouse operations by precipitation of solubilized proteins,” in Proceedings of the 30th Congreso IULTCS, Beijing, China, 2009. View at: Google Scholar
 J. Benavides and M. RitoPalomares, “Practical experiences from the development of aqueous twophase processes for the recovery of high value biological products,” Journal of Chemical Technology and Biotechnology, vol. 83, no. 2, pp. 133–142, 2008. View at: Publisher Site  Google Scholar
 H. Paredes and R. B. Bórquez, “Development of an alternative treatment system for fishing industry waste waters using ultrafiltration,” Latin American Applied Research, vol. 31, pp. 359–365, 2001. View at: Google Scholar
 M. D. Afonso and R. B. Bórquez, “Nanofiltration of wastewaters from the fish meal industry,” Desalination, vol. 151, pp. 131–138, 2002. View at: Google Scholar
 I. Kabdasli, T. Olmez, and O. Tunay, “Nitrogen removal from tannery wastewater by protein recovery,” Water Science Technology, vol. 48, pp. 215–223, 2003. View at: Google Scholar
 A. Marsal, E. Hernández, S. Cuadros, R. Puig, E. Bautista, and J. Font, “Recovery of proteins from wastewater of tannery beamhouse operations: influence on the main pollution parameters,” Water Science Technology, vol. 62, no. 3, pp. 658–666, 2010. View at: Google Scholar
 C. Darnault, K. Rockne, A. Stevens, G. A. Mansoori, and N. Sturchio, “Fate of environmental pollutants,” Water Environmental Research, vol. l77, pp. 2576–2658, 2005. View at: Google Scholar
 D. Z. Ivetic, M. B. Sciban, V. M. Vasic, D. V. Kukic, J. M. Prodanovic, and M. G. Antov, “Evaluation of possibility of textile dye removal from wastewater by aqueous twophase extraction,” Desalination and Water Treatment, vol. 51, pp. 1–6, 2012. View at: Google Scholar
 O. Aguilar, V. Albiter, L. SerranoCarreón, and M. RitoPalomares, “Direct comparison between ionexchange chromatography and aqueous twophase processes for the partial purification of penicillin acylase produced by E. coli,” Journal of Chromatography B, vol. 835, no. 12, pp. 77–83, 2006. View at: Publisher Site  Google Scholar
 K. Naganagouda and V. H. Mulimani, “Aqueous twophase extraction (ATPE): an attractive and economically viable technology for downstream processing of Aspergillus oryzaeαgalactosidase,” Process Biochemistry, vol. 43, no. 11, pp. 1293–1299, 2008. View at: Publisher Site  Google Scholar
 V. Yazbik and M. AnsorgeSchumacher, “Fast and efficient purification of chloroperoxidase from C. fumago,” Process Biochemistry, vol. 45, no. 2, pp. 279–283, 2010. View at: Publisher Site  Google Scholar
 P. A. Albertsson, Partitioning of Cell Particles and Macromolecules, John Wiley and Sons, New York, NY, USA, 3rd edition, 1987.
 R. HattiKaul, Aqueous Two Phase Systems: Methods and Protocols, Humana Press, Totowa, NJ, USA, 2000.
 S. Saravanan, J. R. Rao, T. Murugesan, B. U. Nair, and T. Ramasami, “Recovery of valueadded globular proteins from tannery wastewaters using PEGsalt aqueous twophase systems,” Journal of Chemical Technology and Biotechnology, vol. 81, no. 11, pp. 1814–1819, 2006. View at: Publisher Site  Google Scholar
 R. Iyyaswami, P. D. Belur, B. Girish, and V. H. Nagaraj, “Development and evaluation of PEGlithium citrate salt based aqueous two phase system and its application in partitioning of proteins from fish industry Effluent,” Separation Science and Technology, vol. 47, pp. 591–598, 2012. View at: Google Scholar
 M. Perumalsamy and T. Murugesan, “Extraction of Cheese whey proteins (alphalactalbumin and betalactoglobulin) from dairy effluents using environmentally benign aqueous biphasic system,” International Journal of Chemical and Environmental Engineering, vol. 3, pp. 55–59, 2012. View at: Google Scholar
 D. Ramyadevi, A. Subathira, and S. Saravanan, “Central composite design application for optimization of aqueous twophase extraction of protein from shrimp waste,” Journal of Chemical and Pharmaceutical Research, vol. 4, pp. 2087–2095, 2012. View at: Google Scholar
 S. Raja and V. R. Murty, “Development and evaluation of environmentally benign aqueous two phase systems for the recovery of proteins from tannery waste water,” ISRN Chemical Engineering, vol. 2012, Article ID 290471, 9 pages, 2012. View at: Publisher Site  Google Scholar
 S. Saravanan, J. R. Rao, T. Murugesan, B. U. Nair, and T. Ramasami, “Recovery of valueadded globular proteins from tannery wastewaters using PEGsalt aqueous twophase systems,” Journal of Chemical Technology and Biotechnology, vol. 81, no. 11, pp. 1814–1819, 2006. View at: Publisher Site  Google Scholar
 M. M. Bradford, “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding,” Analytical Biochemistry, vol. 72, no. 12, pp. 248–254, 1976. View at: Google Scholar
 J. Lawson and J. Erjavec, Modern Statistics for Engineering and Quality Improvement, Thomson Publihsers, Australlia, 1st edition, 2002.
 D. C. Montgomery, Design and Analysis of Experiments, John Wiley and Sons, Hoboken, NJ, USA, 1st edition, 2005.
 T. S. Porto, G. M. Medeiros e Silva, C. S. Porto et al., “Liquidliquid extraction of proteases from fermented broth by PEG/citrate aqueous twophase system,” Chemical Engineering and Processing: Process Intensification, vol. 47, no. 4, pp. 716–721, 2008. View at: Publisher Site  Google Scholar
 F. Luechau, T. C. Ling, and A. Lyddiatt, “Selective partition of plasmid DNA and RNA in aqueous twophase systems by the addition of neutral salt,” Separation and Purification Technology, vol. 68, no. 1, pp. 114–118, 2009. View at: Publisher Site  Google Scholar
 S. Saravanan, J. R. Rao, T. Murugesan, B. U. Nair, and T. Ramasami, “Partition of tannery wastewater proteins in aqueous twophase poly (ethylene glycol)magnesium sulfate systems: effects of molecular weights and pH,” Chemical Engineering Science, vol. 62, no. 4, pp. 969–978, 2007. View at: Publisher Site  Google Scholar
 L. P. Malpiedi, G. Picó, and B. Nerli, “Features of partitioning pattern of two pancreatic enzymatic precursors: trypsinogen and chymotrypsinogen in polyethyleneglycolsodium citrate aqueous biphasic systems,” Journal of Chromatography B, vol. 870, no. 1, pp. 1–7, 2008. View at: Publisher Site  Google Scholar
 A. Boaglio, G. Bassani, G. Picó, and B. Nerli, “Features of the milk whey protein partitioning in polyethyleneglycolsodium citrate aqueous twophase systems with the goal of isolating human alpha1 antitrypsin expressed in bovine milk,” Journal of Chromatography B, vol. 837, no. 12, pp. 18–23, 2006. View at: Publisher Site  Google Scholar
 G. Tubío, L. Pellegrini, B. B. Nerli, and G. A. Picó, “Liquidliquid equilibria of aqueous twophase systems containing poly(ethylene glycols) of different molecular weight and sodium citrate,” Journal of Chemical and Engineering Data, vol. 51, no. 1, pp. 209–212, 2006. View at: Publisher Site  Google Scholar
 M. Perumalsamy and T. Murugesan, “Partition behavior of bovine serum albumin in peg2000sodium citratewater based aqueous twophase system,” Separation Science and Technology, vol. 42, no. 9, pp. 2049–2065, 2007. View at: Publisher Site  Google Scholar
 C. A. S. da Silva, J. S. R. Coimbra, E. E. G. Rojas, L. A. Minim, and L. H. M. da Silva, “Partitioning of caseinomacropeptide in aqueous twophase systems,” Journal of Chromatography B, vol. 858, no. 12, pp. 205–210, 2007. View at: Publisher Site  Google Scholar
 D.C. Montomery and G.C. Runger, Applied Statistics and Probability For Engineers, John Wiley and Sons, New York, NY, USA, 5th edition, 2011.
 M. Perumalsamy and T. Murugesan, “Partition behavior of bovine serum albumin in peg2000sodium citratewater based aqueous twophase system,” Separation Science and Technology, vol. 42, no. 9, pp. 2049–2065, 2007. View at: Publisher Site  Google Scholar
 Y. M. Lu, Y. Z. Yang, X. D. Zhao, and C. B. Xia, “Bovine serum albumin partitioning in polyethylene glycol (PEG)/potassium citrate aqueous twophase systems,” Food and Bioproducts Processing, vol. 88, no. 1, pp. 40–46, 2010. View at: Publisher Site  Google Scholar
 G. Johansson, Methods in Enzymology, Aqueous TwoPhase Systems, vol. 228, Academic Press, San Diego, Calif, USA, 1994.
Copyright
Copyright © 2013 Selvaraj Raja and Vytla Ramachandra Murty. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.