纳米壳聚糖修饰的开管毛细管电色谱柱的制备及对碱性蛋白质的分离

doi:10.7503/cjcu20140978

摘要/Abstract

摘要：

采用离子交联法制得纳米壳聚糖粒子溶液, 通过静电吸附作用将其修饰于石英毛细管内表面形成涂层毛细管柱. 通过场发射扫描电子显微镜观察到毛细管内壁有小丘状突起, 表明纳米壳聚糖粒子已吸附于柱内层. 检测了不同pH 值下的电渗流变化趋势并与裸柱比较, 结果显示修饰后的柱电渗流受到明显抑制, 并在pH<4.7的环境中能产生反向电渗流, 该柱在不同pH值下电渗流的相对标准偏差(RSD)<6%, 日内、日间和柱间的RSD分别为1.46%, 4.64%和14.43%, 表明柱稳定性较好. 通过分离3种中性物质甲苯、苯酚和硫脲考察了该柱的色谱行为, 出峰顺序与其极性大小顺序一致, 表明纳米涂层起到极性固定相的作用. 用该柱成功分离了3种碱性蛋白质溶菌酶、细胞色素c和核糖核酸酶A, 柱效分别为 39481, 42610和245373 Plate/m, 重现性良好, 表明纳米涂层可有效抑制碱性蛋白质的吸附作用.

关键词: 纳米壳聚糖, 开管毛细管电色谱, 碱性蛋白质分离

Abstract:

Nano-chitosan particles(N-CS) prepared by ionic crosslinking technology were coated onto the inner wall of fused silica capillary via electrostatic adsorption to get a nano-chitosan coating capillary column. N-CS particles were successfully coated on the inner wall of the capillary as hillocks observed by field emission scanning electron microscopy. The electroosmotic flow(EOF) was tested in buffer at different pH values and compared to an uncoated capillary. The result showed that it was definitely inhibited after modification and reversed when pH<4.7. It was also found that EOF RSD of the prepared column was less than 6% in different acidity buffer and RSD of intra-day, inter-day and column to column were 1.46%, 4.64% and 14.4%, respectively. Those results indicated that the stability of N-CS coated column was good. The chromatographic behavior of the coated column was tested by separating three neutral analytes namely toluene, phenol and thiourea, the eluotropic sequence was consistent with their polarity, which indicated the nano-chitosan coatings played the role of hydrophilic stationary phase. Three basic proteins including lysosomes, cytochrome C and ribonuclease A were successfully separated with the resulting column, and their column efficiency were 39481, 42610 and 245373 Plate/m, respectively. The results showed that the adsorption between basic proteins and fused silica capillary is prevented effectively in the N-CS coated capillary.

Key words: Nano-chitosan particle, Open tubular capillary electrochromatography, Separation of basic proteins

中图分类号:

O657.8

TrendMD:

张欢欢, 陈继涢, 周孙英. 纳米壳聚糖修饰的开管毛细管电色谱柱的制备及对碱性蛋白质的分离. 高等学校化学学报, 2015, 36(4): 631.

ZHANG Huanhuan, CHEN Jiyun, ZHOU Sunying. Preparation of Open Tubular Capillary Electrochromatography Column with Nano-chitosan Coating and Its Application for Basic Proteins Analyzing^†. Chem. J. Chinese Universities, 2015, 36(4): 631.

图/表 9

Fig.1 Inner surface morphologies of uncoated fused silica capillary(A) and N-CS-coated capillary(B)

Fig.2 Chromatograms of three neutral compounds in capillaries with varying coating thickness of N-CS a. Uncoated; b. coated 1 time; c. coated 2 times; d. coated 3 times. Peak 1: toluene; peak 2: phenol; peak 3: thiourea. Electrophoretic conditions: buffer: 50 mmol/L phosphate solution of pH=8.0; injection: 10 s, 1 kPa; voltage: 10 kV; wavelength: 214 nm.

Fig.3 Effect of buffer pH on the electroosmotic flow of N-CS-coated(a) and uncoated(b) capillaries Electrophoretic conditions: buffer: pH=3.0—5.0, 50 mmol/L citrate solution; pH=6.0—9.0, 50 mmol/L phosphate solution; injection: 10 s with 1 kPa; voltage: 10 kV; wavelength: 214 nm.

Table 1 Stability of the N-CS-coated capillary at different pH values*

pH	2	3	4	5	6	7	8	9	10
RSD(%)(n=6)	5.79	3.67	4.88	0.28	0.37	0.34	1.65	1.51	1.83

Fig.4 Separation of three basic proteins at different pH values pH: a. 6.0; b. 6.2; c. 6.4; d. 6.5; e. 7.0. Peak 1: lysozyme; peak 2: ribonuclease A; peak 3: cytochrome c; peak 4: solvent. Electrophoretic conditions: buffer: 100 mmol/L phosphate solution; injection: 10 s, 1 kPa; voltage: 10 kV; wavelength: 280 nm.

Fig.5 Separation of three basic proteins in diffe-rent concentrations of phosphate buffer Concentration/(mmol·L-1): a. 120; b. 100; c. 90; d. 75; e. 50. Peak 1: lysozyme; peak 2: ribonuclease A; peak 3: cytochrome c; peak 4: solvent. Electrophoretic conditions: buffer: pH=6.2 phosphate solution; injection: 10 s, 1 kPa; voltage: 10 kV; wavelength: 280 nm.

Fig.6 Separation of three basic proteins at diffe-rent detection voltages Voltage/kV: a. 12; b. 10; c. 9; d. 6. Peak 1: lysozyme; peak 2: ribonuclease A; peak 3: cytochrome c; peak 4: solvent. Electrophoretic conditions: buffer: 100 mmol/L phosphate solution of pH=6.2; injection: 10 s, 1 kPa; wavelength: 280 nm.

Table 2 Repeatability of protein separation*

Analyte	RSD(%) of retention time		RSD(%) of peak area
Analyte	Intra-day(n=6)	Inter-day(n=3)	Intra-day(n=6)	Inter-day(n=3)
Cytochrome c	1.55	3.55	3.21	11.52
Lysosomes	1.57	3.52	2.12	7.19
Ribonuclease A	1.56	4.13	1.45	12.36

Fig.7 Separation of three basic proteins on N-CS-coated capillary Peak 1: lysozyme; peak 2: ribonuclease A; peak 3: cytochrome c; peak 4: solvent. Electrophoretic conditions: buffer: 100 mmol/L phosphate solution of pH=6.2; injection: 10 s, 1 kPa; voltage: 10 kV; wavelength: 280 nm.

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