On-line Fast Purification of Three Kinds of Active Proteins from Egg White with Two-dimensional Liquid Chromatography Using a Single Column
- Corresponding author: Bai Quan, baiquan@nwu.edu.cn
Citation: Wang Jianshan, Xia Hongjun, Wan Guangping, Liu Jiawei, Jin Lihua, Bai Quan. On-line Fast Purification of Three Kinds of Active Proteins from Egg White with Two-dimensional Liquid Chromatography Using a Single Column[J]. Acta Chimica Sinica, ;2016, 74(3): 265-270. doi: 10.6023/A15120761
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(a) HIC mode; mobile phase: Solution A, 3.0 mol/L (NH4)2SO4+0.02 mol/L KH2PO4 (pH 6.5) ; Solution B, 0.02 mol/L KH2PO4 (pH 6.5) ; (b) SCX mode; mobile phase: Solution A, 0.02 mol/L KH2PO4 (pH 6.5) , Solution B, 0.02 mol/L KH2PO4+1.0 mol/L NaCl (pH 6.5) . The flow rate was 1.0 mL/min; linear gradient elution: 0→100% mobile phase B, 30 min; detection wavelength: 280 nm. Pressure: HIC mode 2.0~3.1 MPa, IEC mode 2.0~2.4 MPa. Peaks: 1. cytochrome c; 2. RNase A; 3. myoglobin; 4. ovalbumin; 5. lysozyme; 6. α-amylase; 7. insulin; 8. α-chymotrypsin A; 9. RNase B
1, Sample injector; 2, box for maintaining the temperature at 4 ℃; 3 and 4, eight-position valves; 5~8, three-position valves
Mobile phases in the HIC mode: Solution A, 3.0 mol/L (NH4)2SO4+0.02 mol/L KH2PO4 (pH 6.5) ; Solution B, 0.02 mol/L KH2PO4 (pH 6.5) . The flow rate was 1.0 mL/min, linear gradient elution: 30 min, 0~100% mobile phase B; detection wavelength: 280 nm
Mobile phase is the same as that of HIC in Figure 1, Solutions B and A in the SCX mode and Solutions A and B in the HIC mode. Flow rate is 1.0 mL/min except for re-sampling with 5.0 mL/min and the detection wavelength is at 280 nm. The linear gradient elution program is represented by the dashed line in the chromatogram. Column pressure is about 2.0~3.5 MPa except for re-sampling with 15.0~15.8 MPa. Peaks: 1, BSA; 2, ovalbumin; 3, insulin; 4, RNase B; 5, RNase A; 6, α-chymotrypsin A; 7, cytochrome C; 8, lysozyme
(a) Chromatographic condition: Solution 1, 20 mmol/L KH2PO4 (pH 6.5) ; Solution 2, 20 mmol/L KH2PO4+3.0 mol/L (NH4)2SO4 (pH 6.5) , linear gradient elution: 0~30 min, Solution 1%~33% Solution 2; 30~40 min, 100% Solution 2; 40~70 min, Solution 2~100% solution 1. The flow rate is 1.0 mL/min except for re-sampling with 5.0 mL/min and the detection wavelength is at 280 nm, Column pressure is about 2.0~3.5 MPa except for re-sampling with 15.0~15.8 MPa pressure. Sample size: 500 mL. (b) Lane: 1. marker; 2. fraction of peak 1; 3. fraction of peak 3; 4. fraction of peak 4; 5. Ovotransferrin; 6. fraction of peak 2; 7. lysozyme; 8. ovalbumin