Presentation Information
[P04-536]Slug Flow Formation and Protein Separation using Aqueous Two-Phase Systems for protein seperation: from the viewpoints of dimensionless number, hydrophobicity, and internal flow state
○Toshinori Shimanouchi1, Tatsumi Fujii1, Satoko Fujioka2, Koichi Terasaka2, Yukitaka Kimura1 (1. Okayama University (Japan), 2. Keio University (Japan))
Keywords:
Aqueous two-phase partitioning method,microcapirally,protein extraction,slug flow
[Purpose]
Aqueous two-phase systems (ATPS) are used for extraction and separation of biomolecules such as proteins. There are some problems such as the time required for phase separation and the need for improvement of agitation condition due to high viscosity. In order to shorten the phase separation time, a method of forming a parallel two-phase flow in a microchannel has been proposed. However, it seems that this flow limited low contact interfacial area between the two phases, which linking to the low separation performance. formation of slug flow using ATPS was, in this study, investigated to improve the seperation efficiency.
[Method]
Polyethylene glycol 8000 (PEG, 6-24 wt%, continuous phase) and K2HPO4 (KPi, 6-14 wt%, dispersed phase) were prepared, and each was flowed at a flow rate of 0.110 ml/min to make a slug flow through a T-junction. The temperature was kept at room temperature, and the residence time was adjusted by changing the length of the SUS tube. Amino acids, lysozyme (Mw= 15000), BSA (Mw= 65000), and beta-lactoglobulin (Mw=18400) were used as separation objects, and all were mixed in the KPi phase.
[Results]
Slug flow formation was attempted by changing the concentration, flow rate and flow rate ratio of PEG solution (continuous phase; C) and KPi solution (dispersed phase; D). The feasibility of slug flow formation was found to depend on the flow rate and flow rate ratio. The operating conditions of the two phases were expressed as dimensionless functions CaCReC0.5 and CaD0.7ReD0.5, respectively. The results showed that the slug flow formation region of the conventional water/organic solvent two-phase systems was almost consistent with that of the conventional water/organic solvent two-phase system. Hydrophobicity factors HF [mol/kJ] of 20 kinds of ATPS were measured by the ATPS method. The larger the HF value, the more the two phases were separated. The HF could be estimated using PEG phase concentration and KPi concentration by the multiple regression model (R2 = 0.714), and the slug flow was formed stably when the HF value was larger than a certain value.
Next, the extraction behavior of various proteins in PEG (9 wt%)/KPi (10 wt%) was examined. The distribution concentration of all proteins reached a constant value after 100 s of residence time, and especially BSA accumulated at the interface. This was considered to be due to the internal circulation flow, and the flow state was visualized. Unlike the usual water/organic two-phase system, the circulation flow occurred throughout the slag. Therefore, the internal circulation frequency fd of the dispersed phase was determined. The dispersed phase of ATPS generally had a high fd value. The interfacial accumulation of protein was plotted by defining F = fd× (molecular weight)2/3× (hydrophobicity). The interfacial accumulation was suppressed up to a certain F value.
[Conclusion]
It was shown that the possibility of slug flow formation by ATPS could be judged from two aspects: (1) the composition and physical properties of the dispersed and continuous phases; and (2) the HF value. Amino acids and proteins could be separated continuously using this system. It is expected that the operation conditions which can suppress the accumulation of proteins at the interface will be clarified and contribute to the improvement of separation yield.
Aqueous two-phase systems (ATPS) are used for extraction and separation of biomolecules such as proteins. There are some problems such as the time required for phase separation and the need for improvement of agitation condition due to high viscosity. In order to shorten the phase separation time, a method of forming a parallel two-phase flow in a microchannel has been proposed. However, it seems that this flow limited low contact interfacial area between the two phases, which linking to the low separation performance. formation of slug flow using ATPS was, in this study, investigated to improve the seperation efficiency.
[Method]
Polyethylene glycol 8000 (PEG, 6-24 wt%, continuous phase) and K2HPO4 (KPi, 6-14 wt%, dispersed phase) were prepared, and each was flowed at a flow rate of 0.110 ml/min to make a slug flow through a T-junction. The temperature was kept at room temperature, and the residence time was adjusted by changing the length of the SUS tube. Amino acids, lysozyme (Mw= 15000), BSA (Mw= 65000), and beta-lactoglobulin (Mw=18400) were used as separation objects, and all were mixed in the KPi phase.
[Results]
Slug flow formation was attempted by changing the concentration, flow rate and flow rate ratio of PEG solution (continuous phase; C) and KPi solution (dispersed phase; D). The feasibility of slug flow formation was found to depend on the flow rate and flow rate ratio. The operating conditions of the two phases were expressed as dimensionless functions CaCReC0.5 and CaD0.7ReD0.5, respectively. The results showed that the slug flow formation region of the conventional water/organic solvent two-phase systems was almost consistent with that of the conventional water/organic solvent two-phase system. Hydrophobicity factors HF [mol/kJ] of 20 kinds of ATPS were measured by the ATPS method. The larger the HF value, the more the two phases were separated. The HF could be estimated using PEG phase concentration and KPi concentration by the multiple regression model (R2 = 0.714), and the slug flow was formed stably when the HF value was larger than a certain value.
Next, the extraction behavior of various proteins in PEG (9 wt%)/KPi (10 wt%) was examined. The distribution concentration of all proteins reached a constant value after 100 s of residence time, and especially BSA accumulated at the interface. This was considered to be due to the internal circulation flow, and the flow state was visualized. Unlike the usual water/organic two-phase system, the circulation flow occurred throughout the slag. Therefore, the internal circulation frequency fd of the dispersed phase was determined. The dispersed phase of ATPS generally had a high fd value. The interfacial accumulation of protein was plotted by defining F = fd× (molecular weight)2/3× (hydrophobicity). The interfacial accumulation was suppressed up to a certain F value.
[Conclusion]
It was shown that the possibility of slug flow formation by ATPS could be judged from two aspects: (1) the composition and physical properties of the dispersed and continuous phases; and (2) the HF value. Amino acids and proteins could be separated continuously using this system. It is expected that the operation conditions which can suppress the accumulation of proteins at the interface will be clarified and contribute to the improvement of separation yield.
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