The Research and Mechanism of Extracting Vitamin B6 Using Aqueous Two-Phase Systems

School of Water Resources and Environment, Hebei Province Key Laboratory of Sustained Utilization and Development of Water Resources, Hebei Province Collaborative Innovation Center for Sustainable Utilization of Water Resources and Optimization of Industrial Structure, Hebei GEO University, Shijiazhuang 050031, China School of Geology and Environment, Xi’an University of Science and Technology, Xi’an 710064, Shaanxi, China College of Chemical and Pharmaceutical Engineering, Hebei University of Science and Technology, Shijiazhuang 050018, China


Introduction
Vitamin B 6 , as an indispensable part of human body, can regulate body fluids, prevents hair loss, and has a central role in amino acid metabolism and modulating the activity of steroid and other hormones [1]. In recent years, events concerning food safety have increased public attention. Vitamin B 6 exists in all kinds of food, and it is important to know how much it is contained in food. erefore, a rapid, accurate, and safe measurement analysis must be established [2].
Since aqueous biphasic systems were proposed by Beijernick in 1896 [3], the aqueous two-phase system (ATPS) as a new technology that is safe and easy to control and has a simple process is now gradually taking the place of traditional organic-water solvent extraction systems. e ATPS is usually composed of two or more polymers, a polymer, and a salt [4,5]. ey have mild separation conditions, rapid balance processes, and high rate of product recovery. As a "green chemistry," ATPS now is widely used in the separation of protein [6,7], DNA [8], drugs [9], antibiotic [10,11], and metal ion [12]. So, we pay more attention to find a new ATPS to extract vitamin B 6 .
With the development of science and technology, a new system is born. Small-molecule organic solvent ATPSs are led to our increased use in searching ATPSs. For example, binodal data for ethanol, 2-propanol, and 1-propanol + MgSO 4 / ZnSO 4 + water systems experimentally determined at 303.15 K and 313.15 K [13,14] and ethanol + salt [(NH 4 ) 2 SO 4 [15], NaOH [16], NaF [17], and K 2 HPO 4 [18] ATPSs have been reported. Comparing traditional ATPSs with the small-molecule organic solvent ATPS, the small-molecule organic solvent ATPS has some advantages. For example, it is easy to control, has a low cost, and is environmentally friendly. So, it has been successfully used in the separation and purification of hesperidin [19] and pigments [20]. However, it also has shortcomings, such as large amount of phase-forming materials used and having a higher volatility of alcohol. In order to reduce these situations, we use quaternary systems to replace ternary systems. So, the ATPS containing polymer and small-molecule, such as polymer/alcohol and binary alcohol to form an ATPS, is studied in the following passage.
In this work, phase diagrams and liquid-liquid equilibrium (LLE) data for the quaternary system polyethylene glycol + ethanol + salt (Na 2 SO 4 ) + water were investigated at T � 308.15 K, 318.15 K, and 328.15 K, and those for the system isopropyl alcohol + ethanol + salt (Na 2 SO 4 /Na 2 CO 3 ) + water were examined at T � 308.15 K and those at atmospheric pressure have been studied. In the above, system polyethylene glycol/isopropyl alcohol and ethanol were mixed in different proportions and formed homogeneous and stabilized solutions. From what we discussed, the bimodal curves were fitted to three nonlinear equations. What is more, we have discussed what the temperature, salts, and different composition of organic solution can do on the phase separation abilities of these systems. In addition, some factors such as salt, pH, and temperature were discussed in the system of vitamin B 6 extraction.

Materials.
Analytical-grade Na 2 CO 3 , Na 2 SO 4 , ethanol, polyethylene glycol, and isopropyl alcohol were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), Tianjin Baishi Chemical Co., Ltd. (Tianjin, China), and Tianjin Fuyu Fine Chemical Co., Ltd. (Tianjin, China), respectively. All the above materials have a minimum mass fraction of 99.5%. Vitamin B 6 was purchased from a local supermarket and was ultrasonically degassed in advance. Double-distilled deionized water was also used in the experiments. e sources and purities of chemicals used in the study are shown in Table 1.

Determination Method of Binodal Curves Data.
Before the experiment, we added polyethylene glycol and isopropyl alcohol into ethanol until the mass fraction of ethanol reached 0.200 and 0.333, respectively. In the following text, "MA" denotes the mass fraction of ethanol in the mixture of polyethylene glycol and ethanol or isopropyl alcohol and ethanol. So, the values of MA 1 and MA 2 were 0.200 and 0.333, respectively. e binodal curves were determined by the cloud point method at 308.15, 318.15, and 328.15 K. First, known mass fractions of polyethylene glycol alcohol and ethanol were added drop-wise using a small-sized pipette to create heterogeneous systems until the mixture became cloudy. en, known amounts of double-distilled deionized water were subsequently added to the tube to create a transparent onephase system. After recording the data, the mass of the tube was measured again until there was some precipitation at the bottom of the vessel for obtaining sufficient data for the construction of a phase diagram. e data of isopropyl alcohol and ethanol at T = 308.15 K was obtained by the same method. roughout this preparation procedure, the tubes were incubated in an external jacket through which water at a constant temperature was circulated using a DC-2008 water thermostat (Tianjin Taisite Instrument Factory, China). e temperature was controlled to within ±0.05 K. e masses of the starting and added solutions were measured using an analytical balance (BC 124S, Beijing Sartorius Instrument Co., Ltd., China) with a precision of ±0.0001 g.

Determination Method of Tie-Line Data.
After the collecting of the binodal data, five tie-lines were determined for each of the studied systems. Systems with a known polyethylene glycol/isopropyl alcohol and ethanol composition (total weight of approximately 10 g) were vigorously shaken in separate tubes and then placed in a thermostat bath for at least 48 h until the solution separated into two clear phases to reach a phase equilibration. en the upper phase was carefully removed by pipette. e mass of the upper phase was determined by an analytical balance, and the mass of the lower phase was obtained by subtraction. Both the top phase and bottom mixture solutions with concentrations in the range of 1%-50% w/w were prepared, and their refractive indexes were determined at the experimental temperatures using an Abbe refract meter (modal WAY [2WAJ], Shanghai Physical Optics Instrument Co., Ltd.). After that, the salt solutions with concentration in the range of 0.1%-25% w/w were added to the above alcohol mixture solutions, and the corresponding reactive indexes were determined at the experimental temperatures. A comparison of the refractive indexes for the two groups of solutions revealed that there was some influence of salt on the refractive index when solved in the alcohol solutions. A comparison of the refractive indexes revealed that the salt content influence on the refractive index is not obvious. Hence, the salt concentrations in the two phases were determined directly by atomic absorption spectrophotometer. Since the refractive index of mixture depends on the mass fractions of alcohol and salt, the alcohol concentrations were determined by Abbe refractometer and in accordance with the following equation: where n 0 is the refractive index of deionized water; a 1 and a 2 are experimental fitting parameters; w 1 and w 2 are mass fractions of alcohol and salt, respectively. e values of parameters were given in Table S1 (Supplementary Materials). For the measurement of alcohol, we used an Abbe refractometer with an uncertainty of 0.0002 to determine the refractive indexes. e refraction indexes were put into the corresponding equation to obtain the concentration of the alcohol mixture in the phases. e mass fraction of water in both the top phase and bottom phase was obtained from the law of mass conservation.
When ion exchanging exists in both the top phase and bottom phase, it may affect the validity of the concentration of both the top phase and bottom phase. Aiming to check it, we used the method above and corrected it with mathematic model. Firstly, the concentration of both the top phase and bottom phase was accurately determined; then MATLAB software was used to deal with these dates and the dates met with the "lever principle" [21]. e method was used in the literature in recent years [22,23]. e tie-line length (TLL) and the slope of the tie-line (S) at different compositions were calculated using equations (2) and (3), respectively [24]: where w t 1 , w b 1 , w t 2 , and w b 2 represent the equilibrium mass fractions of the polyethylene glycol/isopropyl alcohol and salt (Na 2 SO 4 /Na 2 CO 3 ) in the top and bottom phases, respectively.

Vitamin B 6 Analysis.
A certain portion of sample solution containing isopropyl alcohol, ethanol, Na 2 CO 3 , and H 2 O was transferred into a 10 mL centrifuge tube. en the centrifuge tube with ATPS was put on the analytical balance, and the 2 g of the standard working condition B 6 prepared in advance was added drop-wise to the tube using a syringe. e mixture was stirred thoroughly at room temperature (25°C). Still, an 802 centrifuge was used at 2000 rpm for 25 min to ensure that phase separation operated completely. To finish these, the centrifuge tubes were then placed in water thermostat DC-2008 (Tianjin Taisite Instrument Co., China) with an uncertainty of 0.05°C at the setpoint temperature of 35°C for 12 h to reach phase equilibrium. After that, B 6 and alcohols were enriched in top phase and salt was enriched in bottom phase. e volume of top phase was read accurately and part of them was aspirated into a 50 μL microsyringe and directly injected into the HPLC system for analysis. e pH value was adjusted by hydrochloric acid and ammonia water. e extraction efficiency of B 6 was defined as the ratio between the amount of the B 6 in the top phase and the initial amount of the B 6 .
where C t is the concentration of B 6 in the top phase after the phase equilibrium, V t is the volume of the top phase after the phase equilibrium, and m s is the amount of B 6 added initially.

HPLC-UV Analysis.
A 1200 HPLC system (Agilent, USA) essentially comprised of a quaternary pump and a variable wavelength UV-VIS detector (Agilent, USA) was employed for the determination of DBP. All data processing and facility control were conducted via Agilent Chem Station software. Acetonitrile and water (88 : 12 by volume) were used as the mobile phase with gradient elution on the reversed phase chromatography column (Shim-pack VP-ODS column, 150 × 4.6 mm, 5 μm), and the flow rate was 1.0 mL/min. e injected volume was 5 μL. e detection wavelength was 228 nm using the UV-VIS detector.

Phase Behaviors of the IL-Salt ILATPS.
e binodal data of the quaternary system polyethylene glycol + ethanol + Na 2 SO 4 + water determined at T � 308.15, 318.15, and 328.15 K, system isopropyl alcohol + ethanol + Na 2 SO 4 + water at T � 308.15 K, and system isopropyl alcohol + ethanol + Na 2 CO 3 + water at T � 308.15 K are presented in Table 2.
e binodal data were fit to the following system of equations using the empirical nonlinear expression developed by Merchuk et al. [25]: where w 1 is the mass fraction of the mixture of the alcohol, w 2 is the mass fraction of the salt, and a, b, and c are fitting parameters. e fitting parameters obtained from fitting the experimental binodal data to equation (5) are presented in Table 3, along with the correlation coefficients (R 2 ) and the corresponding standard deviations (sd a ).  In order to obtain a more accurate fitting result, we use another two nonlinear empirical equations to correlate the experimental binodal data [26,27]: where w 1 and w 2 are the mass fractions of the alcohol and the salt, respectively. e fitting parameters a, b, c, and d along with the corresponding standard deviations (sd a ) and correlation coefficient (R 2 ) are listed in Tables 4 and 5. Equations (4)- (6) have been successfully used for the correlation of binodal data of IL-based ATPSs [28][29][30], polymer-based ATPSs [31,32], and hydrophilic alcohol-based ATPSs [33,34]. Based on the obtained R 2 and sd a values in Tables 3-5, it can be concluded that equations (5)-(7) are Table 3: Parameters of equation (5) for the mixture of polyethylene glycol/isopropyl alcohol + ethanol + salt (Na 2 CO 3 /Na 2 SO 4 ) + water ATPSs at different temperatures.
is the experimental mass fraction of the alcohol mixture, w cal i is the corresponding data calculated using equation (5), and "N" represents the number of binodal data points.

Journal of Chemistry
perfectly suitable for correlating the binodal data of the investigated systems. Furthermore, equation (6) shows the most satisfactory accuracy among the three expressions due to its R 2 .

Tie-Line Data and Correlation.
e tie-line data of the ATPSs composed of polyethylene glycol + ethanol + Na 2 SO 4 and water determined at three different temperatures and isopropyl alcohol + ethanol + Na 2 SO 4 /Na 2 CO 3 + H 2 O system at T � 308.15 K are listed in Table 6.
e Setschenow-type equation [35] has been successfully used to correlate the tie-line data for polymer + salt ATPSs [36]. is equation has the following form: In this equation, k S represents the salting-out coefficient, c 1 and c 2 are the molarities of the alcohol mixture and salts, respectively, and k MA is a parameter relating the activity coefficient of the alcohol mixture to its concentration. e superscripts "t" and "b" represent the top phase and the bottom phase, standing for the salt-rich phase and alcoholrich phase. e fitting parameters, the corresponding intercepts, the correlation coefficients (R 2 ), and the standard deviations (sd a ) are shown in Table 7.
As we can see from Table 7, the Setschenow-type equation can be satisfactorily applied to the correlation of tie-line data for these systems.
A relatively simple equation was also used to correlate the tie-line data: where k signifies the salting-out coefficient and β represents a constant related to the activity coefficient. e superscripts "t" and "b" represent the top phase and the bottom phase, respectively. e fitting parameters of the equation, the corresponding correlation coefficient values (R 2 ), and the standard deviations (sd a ) are provided in Table 8.
is equation was successfully used to correlate the tieline data for polymer-salt ATPSs [37]. is expression shows strong agreement when correlated with the experiment tieline data. It can be seen from Tables 7 and 8 that equation (9) has a better accuracy for the investigated system. Moreover, the results show good reliability of the experimental tie-line data and calculation methods used.

Effect of Temperature on Binodal Curves. It has been
reported that temperature plays a positive role in forming the ATPS consisting of 2-propanol, NaOH, and water [16]. Figure 1 shows the binodal curves for the systems consisting of polyethylene glycol + ethanol + Na 2 SO 4 + water at T � 308.15, 318.15, and 328.15 K to discuss the influence of temperature on the binodal curves. It shows that temperature has little influence on the phase equilibrium compositions, so there is no significant change in the slope of the binodal curves with increased temperature. is situation mainly occurs because when the salt concentration in the system with the alcohol mixture of (MA 1 � 0.200), Na 2 SO 4 , and the water is low than 25% by mass fraction, the system   Journal of Chemistry will be less sensitive to changes in temperature, and the liquid-liquid equilibrium condition will be maintained.

Effect of the Mass Composition of the Alcohol on
Binodal Curves. Figure 2 shows the binodal curves for the systems consisting of polyethylene glycol + ethanol + Na 2 SO 4 + water at T � 308.15 K and the systems consisting of isopropyl alcohol + ethanol + Na 2 SO 4 + water at T � 308.15 K to discuss the influence of the mass composition of the alcohol on binodal curves. It shows that mass composition of the alcohol can affect the phase equilibrium composition, so there are some changes in the slope of the binodal curves with the change of the mass composition of the alcohol. e change can be described as follows: when the salt concentration is greater than 7.3% by mass fraction, the biphasic region of the polyethylene glycol + ethanol + Na 2 SO 4 + water system is larger than that of    (8) for the mixture of polyethylene glycol/isopropyl alcohol + ethanol + salt (Na 2 CO 3 /Na 2 SO 4 ) + water ATPSs at different temperatures.
where N is the number of tie-lines and j is the number of components in each phase. δ (k s ) b is the standard deviation for the fitting parameters of "k s ." δ (intercept) c is the standard deviation of the fitting parameters of the "intercept." isopropyl alcohol + ethanol + Na 2 SO 4 + water system. Hence, the salting-out ability of the former system is greater than the latter. When the salt composition is lower than 7.3% by mass fraction, the biphasic region of the isopropyl alcohol + ethanol + Na 2 SO 4 + water system is larger than that of polyethylene glycol + ethanol + Na 2 SO 4 + water system. Hence, the salting-out ability of the latter is greater than the former.
Furthermore, the binodal curves of (polyethylene glycol x + ethanol)/polyethylene glycol 1500 [38]/polyethylene glycol 4000/polyethylene glycol 8000 [39] + Na 2 SO 4 + water systems were shown in Figure S1 (in Supplementary Materials). It can be seen from the figure that the ATPSs containing polyethylene glycol 1500, polyethylene glycol 4000, and polyethylene glycol 8000 have larger two-phase ranges, which show that the ATPS containing polyethylene glycol can more easily form two-phase system than the combination of polyethylene glycol and ethanol.

Effect of Salt on Binodal Curves.
System concerning isopropyl alcohol + ethanol + Na 2 SO 4 /Na 2 CO 3 + water ATPSs at T � 308.15 K is also discussed in this paper. As we can see from Figure 3 the salting-out ability of system containing Na 2 CO 3 is better than that containing Na 2 SO 4 . And the salting-out ability increased when salt composition increased.
is may be because Na 2 CO 3 has a smaller molecular weight than that of Na 2 SO 4 in the same salt composition. Also, we can see from Table 6 that the absolute value of entropy (S) of Na 2 SO 4 is larger than that of Na 2 SO 4 . e larger the absolute value of entropy (S), the easier to form a salting-out ability. Figure 4 shows the tie-line for the systems consisting of polyethylene glycol + ethanol + Na 2 SO 4 + water at T � 308.15, 318.15, and 328.15 K to discuss the influence of temperature on the tieline. It can be seen from the figure that the tie-lines at the three temperatures are almost parallel and it demonstrates that temperature has no effect on tie-line. And this result is similar to the effect of temperature on binodal curves.

Effect of Salt on Tie-Line.
System concerning isopropyl alcohol + ethanol + Na 2 SO 4 /Na 2 CO 3 + water ATPSs at T � 318.15 K is also discussed in this paper. As we can see from Figure 5, the salting-out ability of system containing Na 2 CO 3 is better than that containing Na 2 SO 4 . e saltingout ability increased when salt composition increased. Also, the trend is similar to the effect of salt on binodal curves.

Separation Behaviors of B 6 in ILATPS.
According to what has been discussed above, ATPS (isopropyl alcohol + ethanol + Na 2 CO 3 + H 2 O), with a better ability of   Journal of Chemistry salting-out than other systems above, has been chosen to find out whether ATPSs have high-performance extraction when dealing with vitamin B 6 . Vitamin B 6 in real samples was conceivably concentrated in the salt-rich top phases and determined by the proposed HPLC method. e results are shown in Figure 6. From the figure, it is critical that ATPS (isopropyl alcohol + ethanol + Na 2 CO 3 + H 2 O) coupled with HPLC-UV can satisfactorily concentrate and quantitatively determine vitamin B 6 in real samples. Meanwhile, some factors such as salts, pH, and temperature are also discussed in the following passage.

Effect of the Salts on Extraction
Efficiency. e discussions above show that Na 2 CO 3 has a better salting-out ability than Na 2 SO 4 . Experiments have been done to find out what salt can do to affect the extraction efficiency. Different salts (Na 2 CO 3 , Na 2 SO 4 , and NaH 2 PO 3 ) with the same weight were added into three centrifuge tubes containing the same systems (isopropyl alcohol + ethanol + H 2 O) individually. en 2 g of the standard working B 6 was added into the centrifuge tubes. e mixtures were then stirred thoroughly at T � 308.15 K. An 802 centrifuge was used at 2000 rpm for 25 min to ensure phase separation. To finish these, the centrifuge tubes were then placed in a thermostatic water bath at the setpoint temperature of T � 308.15 K for 12 h to reach phase equilibrium. And the HPLC system was used for analysis of these systems. e result is shown in Table 9; we can read from the table that the extraction efficiency of Na 2 CO 3 is 84.24% and it is the largest among the three ATPSs. It is perfectly similar to the study we have discussed earlier.

Effect of Temperature on the Extraction Efficiency.
Temperature is always a factor that affects chemical reactions, so it can affect the extraction efficiency. Effect of temperature on the ATPS (isopropyl alcohol + ethanol + Na 2 CO 3 + H 2 O) was also investigated in our study. In the beginning, several ATPSs (isopropyl alcohol + ethanol + Na 2 CO 3 + H 2 O) of identical component contents were formed by mixing four components in centrifuge tubes; then 2 g of the standard working B 6 was added into the centrifuge tubes. e mixtures were then stirred thoroughly at T � 308.15 K. An 802 centrifuge was used at 2000 rpm for 25 min to ensure phase separation. To finish these, the centrifuge tubes were then placed in a thermostatic water bath at six different setpoint temperatures (303. 15, 308.15, 313.15, 318.15, 323.15, and 328.15) for 12 h to reach phase equilibrium. e top phase was analyzed using the HPLC method finally. e results are shown in Figure 7; we can see that temperature did affect the extraction efficiency: the extraction efficiency initially increased and then decreased during the increase in temperature. e maximum value of extraction efficiency reached 83.01% when temperature is 308.15 K.

Effect of pH on the Extraction
Efficiency. pH can not only affect the phase separation but also be a factor that acts on extraction efficiency. So, the extraction of B 6 was studied  100 w 2 Figure 5: Effect of salt on the tie-line system concerning isopropyl alcohol + ethanol + Na 2 SO 4 /Na 2 CO 3 + water ATPSs at T � 308.15 K; ■ Na 2 SO 4 ; ○ Na 2 CO 3 .
in different pH range from 3.25 to 7.47. As shown in Figure 8, the extraction efficiency initially increased and then decreased during the increase of pH. e maximum value of extraction efficiency reached 89.32% when pH was 5.02.

Conclusions
Liquid-liquid equilibrium data have been determined for polyethylene glycol + ethanol + Na 2 SO 4 + water ATPSs at T � 308.15, 318.15, and 328.15 K and isopropyl alcohol + ethanol + Na 2 SO 4 /Na 2 CO 3 + water ATPSs at T � 308.15 K. e Merchuk equation and other equations were successfully used to correlate binodal data, and the tie-lines were described by the Setschenow-type equation and a two-parameter equation. e effect of temperature on the binodal curves of the investigated systems is that the temperature did not affect the separated two-phase area. Saltingout ability can be ranked as Na 2 SO 4 < Na 2 CO 3 at the same temperature, and the phase separation ability of the alcohol mixture was that when the salt concentration is higher than 7.3% by mass fraction, the salting-out ability of the polyethylene glycol + ethanol + Na 2 SO 4 + water ATPSs system at T � 308.15 K is greater than isopropyl alcohol + ethanol + Na 2 SO 4 + water ATPSs at T � 308.15 K, and when the salt composition is lower than 7.3% by mass fraction, the salting-out ability of isopropyl alcohol + ethanol + Na 2 SO 4 + water ATPSs system at     T � 318.15 K is greater than the other. As a viable pretreatment technique for complicated samples, ATPS, combined with a HPLC method, has been successfully used to quantitatively determine vitamin B 6 . e optimum working conditions were considered for an aqueous twophase system composed of isopropyl alcohol + ethanol + Na 2 CO 3 operating at T � 308.15 K and at a pH of 5.02.

Data Availability
e data supporting the conclusions of this work are included in this manuscript. Other datasets generated and analyzed during the current work are available from the corresponding author on reasonable request.

Conflicts of Interest
e authors declare no conflicts of interest.