Preparation of Open Tubular Capillary Electrochromatography Column with Nano-chitosan Coating and Its Application for Basic Proteins Analyzing†

doi:10.7503/cjcu20140978

Abstract

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

CLC Number:

O657.8

TrendMD:

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

Figures/Tables 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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