CE分析
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Key Words：capillary zone electrophoresis (CZE); micellar electrokinetic capillary chromatography (MECC); migration time; reproducibility; running buffer
CHINESE JOURNAL OF CHROMATOGRAPHY Volume 24, Issue 4, July 2006 Online English edition of the Chinese language journal
Cite this article as: Chin J Chromatogr, 2006, 24(4): 396–401.
Abstract：The effects of running buffer on reproducibility of the migration time in capillary electrophoresis (CE) were investigated in the mode of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC). The parameters include concentration, pH value, and the change of properties of the running buffer. The results showed that the reproducibility of migration times could be improved through three approaches. Firstly, the correlation between running buffer concentration and migration time was established, and the running buffer concentration was determined by conductivities to improve the reproducibility. Secondly, it was found that the residual running buffer on the capillary showed significant influence on the reproducibility of migration times. Thus, appropriate running or washing procedures between separations were necessary. Finally, the inlet reservoir buffer played an extremely important role in affecting the migration behaviors of solutes. By improving the test apparatus, reproducible migration time was achieved as a result of the increase in the frequency of replacement of the inlet reservoir buffer.
RESEARCH PAPER
Study on Factors Affecting Reproducibility of Migration Time in Capillary Electrophoresis
ZHU Jianping1,2, HU Changqin1,*, LIU Wenying3
1National Institute for the Control of Pharmaceut00050, China 2Guangxi Institute for the Control of Pharmaceutical Products, Nanning 530021, China 3China Pharmaceutical University, Nanjing 210009, China
phosphoric acid, hydrochloric acid, and sodium hydroxide were of analytical grade. Sodium laurylsulfate (SDS) and cetyl trimethyl ammonium bromide (CTAB) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Methanol used was HPLC grade. Water was distilled twice before use. The reference standards of sodium ceftriaxone, cefalexin, cefoperazone, cefazolin, ampicillin, and norfloxacin were obtained from the National Institute for the Control of Pharmaceutical and Biological Products, PR China. 1.2 Experimental methods 1.2.1 Running conditions of CE
Capillary electrophoresis (CE) with the characteristics of high-performance, rapid automatism, and requirement of only minute samples [1], is also a kind of liquid separation technique, similar to high-performance liquid chromatography (HPLC). On the basis of different separation mechanisms, various separation modes of CE are developed, among which capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) are the most widely used. Compared with HPLC, poor reproducibility is one of the major shortcomings of CE, which hinders the wide use of CE in pharmaceutical analysis. On account of a number of studies that focused on improving the reproducibility in theoretics, apparatus, and operations in recent years [2–21], that is, dealing with controlling the temperature of columns [3, 4], choosing injection modes [5–9], improving capillary characteristics [6, 10–12], adding inner standards [6, 8, 9, 13–15], optimizing the washing procedure [16–18], optimizing the component of running buffer [5, 19–21] etc., the precision of quantitative analysis of CE has been improved to a large extent. However, good reproducibility of migration time is an objective that
ZHU Jianping et al. / Chinese Journal of Chromatography, 2006, 24(4): 396–401
pH meter and an AKTA prime conductivity meter were used. Acetone, NaH2PO4·2H2O, Na2HPO4·12H2O, boracic acid,
remains to be achieved. In CE separation, solutes are separated through electromo-
tion. Electrical particles and the inner surface of the capillary have double-layer and Zeta potential, respectively, which are sensitive to the intensity of ions and the pH of the running buffer. Using a group of β-lactam antibiotics as model compounds, the relationship between the running buffer and the reproducibility of migration time is investigated in this article. The experimental results show that the reproducibility of migration behaviors can be improved significantly by accurately controlling the composition and pH value of the running buffer, choosing the rational washing procedure, and increasing the renewal frequencies of the running buffer.
1 Experimental
1.1 Instruments and reagents The high-performance capillary electrophoresis system
consisted of an Agilent 3D instrument and a Quickenet fused silica capillary (75-µm i.d., 49.5 cm in length, 41 cm to detector), controlled by Agilent Chemostation. A Beckman φ 660
Received August 23, 2005; revised November 09, 2006 *Corresponding author. Tel: +86-10-67095308; E-mail: hucq@
Copyright © 2006, Chinese Chemical Society and Dalian Institute of Chemical Physics, Chinese Academy of Sciences. Published by Elsevier BV. All rights reserved.
CHINESE JOURNAL OF CHROMATOGRAPHY Volume 24, Issue 4, July 2006 Online English edition of the Chinese language journal
Cite this article as: Chin J Chromatogr, 2006, 24(4): 396–401.
Abstract：The effects of running buffer on reproducibility of the migration time in capillary electrophoresis (CE) were investigated in the mode of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC). The parameters include concentration, pH value, and the change of properties of the running buffer. The results showed that the reproducibility of migration times could be improved through three approaches. Firstly, the correlation between running buffer concentration and migration time was established, and the running buffer concentration was determined by conductivities to improve the reproducibility. Secondly, it was found that the residual running buffer on the capillary showed significant influence on the reproducibility of migration times. Thus, appropriate running or washing procedures between separations were necessary. Finally, the inlet reservoir buffer played an extremely important role in affecting the migration behaviors of solutes. By improving the test apparatus, reproducible migration time was achieved as a result of the increase in the frequency of replacement of the inlet reservoir buffer.
RESEARCH PAPER
Study on Factors Affecting Reproducibility of Migration Time in Capillary Electrophoresis
ZHU Jianping1,2, HU Changqin1,*, LIU Wenying3
1National Institute for the Control of Pharmaceut00050, China 2Guangxi Institute for the Control of Pharmaceutical Products, Nanning 530021, China 3China Pharmaceutical University, Nanjing 210009, China
phosphoric acid, hydrochloric acid, and sodium hydroxide were of analytical grade. Sodium laurylsulfate (SDS) and cetyl trimethyl ammonium bromide (CTAB) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Methanol used was HPLC grade. Water was distilled twice before use. The reference standards of sodium ceftriaxone, cefalexin, cefoperazone, cefazolin, ampicillin, and norfloxacin were obtained from the National Institute for the Control of Pharmaceutical and Biological Products, PR China. 1.2 Experimental methods 1.2.1 Running conditions of CE
Capillary electrophoresis (CE) with the characteristics of high-performance, rapid automatism, and requirement of only minute samples [1], is also a kind of liquid separation technique, similar to high-performance liquid chromatography (HPLC). On the basis of different separation mechanisms, various separation modes of CE are developed, among which capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) are the most widely used. Compared with HPLC, poor reproducibility is one of the major shortcomings of CE, which hinders the wide use of CE in pharmaceutical analysis. On account of a number of studies that focused on improving the reproducibility in theoretics, apparatus, and operations in recent years [2–21], that is, dealing with controlling the temperature of columns [3, 4], choosing injection modes [5–9], improving capillary characteristics [6, 10–12], adding inner standards [6, 8, 9, 13–15], optimizing the washing procedure [16–18], optimizing the component of running buffer [5, 19–21] etc., the precision of quantitative analysis of CE has been improved to a large extent. However, good reproducibility of migration time is an objective that
ZHU Jianping et al. / Chinese Journal of Chromatography, 2006, 24(4): 396–401
pH meter and an AKTA prime conductivity meter were used. Acetone, NaH2PO4·2H2O, Na2HPO4·12H2O, boracic acid,
remains to be achieved. In CE separation, solutes are separated through electromo-
tion. Electrical particles and the inner surface of the capillary have double-layer and Zeta potential, respectively, which are sensitive to the intensity of ions and the pH of the running buffer. Using a group of β-lactam antibiotics as model compounds, the relationship between the running buffer and the reproducibility of migration time is investigated in this article. The experimental results show that the reproducibility of migration behaviors can be improved significantly by accurately controlling the composition and pH value of the running buffer, choosing the rational washing procedure, and increasing the renewal frequencies of the running buffer.
1 Experimental
1.1 Instruments and reagents The high-performance capillary electrophoresis system
consisted of an Agilent 3D instrument and a Quickenet fused silica capillary (75-µm i.d., 49.5 cm in length, 41 cm to detector), controlled by Agilent Chemostation. A Beckman φ 660
Received August 23, 2005; revised November 09, 2006 *Corresponding author. Tel: +86-10-67095308; E-mail: hucq@
Copyright © 2006, Chinese Chemical Society and Dalian Institute of Chemical Physics, Chinese Academy of Sciences. Published by Elsevier BV. All rights reserved.