基于窄内径多孔层毛细管开管柱的纳流高效液相色谱用于N-衍生化氨基酸对映体的分离

doi:10.3724/SP.J.1123.2019.09002

色谱 ›› 2020, Vol. 38 ›› Issue (3): 350-355.DOI: 10.3724/SP.J.1123.2019.09002

基于窄内径多孔层毛细管开管柱的纳流高效液相色谱用于N-衍生化氨基酸对映体的分离

李若男, 王利娟, 张东堂, 王亚楠, 郭广生, 汪夏燕()

环境安全与生物效应卓越中心, 绿色催化与分离北京市重点实验室, 北京工业大学化学化工系, 北京 100124

收稿日期:2019-11-05 出版日期:2020-03-08 发布日期:2020-12-10
通讯作者: 汪夏燕
作者简介:汪夏燕, Tel:(010)67396493, E-mail:xiayanwang@bjut.edu.cn
基金资助:
国家自然科学基金资助项目(21804005);国家自然科学基金资助项目(21625501);北京高校卓越青-科学家计划项目(BJJWZYJH01201910005017)

Application of porous layer open tubular capillary columns with narrow inner diameter in nanoflow-high performance liquid chromatography for N-derivatized amino acid enantiomers

LI Ruonan, WANG Lijuan, ZHANG Dongtang, WANG Yanan, GUO Guangsheng, WANG Xiayan()

Center of Excellence for Environmental Safety and Biological Effects, Beijing Key Laboratory for Green Catalysis and Separation, Department of Chemistry and Chemistry Engineering, Beijing University of Technology, Beijing 100124, China

Received:2019-11-05 Online:2020-03-08 Published:2020-12-10
Contact: WANG Xiayan
Supported by:
National Natural Science Foundation of China(21804005);National Natural Science Foundation of China(21625501);Beijing Outstanding Young Scientist Program(BJJWZYJH01201910005017)

摘要/Abstract

摘要：

窄内径多孔层毛细管开管柱（NPLOT柱）在生命科学领域，特别是单细胞分析领域具有较好的应用前景。本研究采用原位热引发聚合法来制备窄内径奎尼丁类手性固定相多孔层开管柱，在6 μ m i.d.的毛细管中制备有机聚合物多孔层，考察了不同热聚合时间（3、6和9 h）对NPLOT柱形貌的影响，热聚3 h和6 h制备的NPLOT柱形貌均一，多孔层厚度分别为103±51 nm和210±51 nm。将热聚合3 h制备的NPLOT柱用于纳流高效液相色谱分离N-衍生化氨基酸对映体，在2 min内即可实现基本分离，消耗的样品量仅为皮升级别。该研究将为单细胞分析提供研究手段。

关键词: 窄内径多孔层开管柱, 原位热引发聚合法, 纳流高效液相色谱法, 手性分离

Abstract:

Porous layer open tubular capillary columns with a narrow inner diameter (NPLOT) have potential in life science, especially in single-cell analysis. In this work, quinidine-based chiral NPLOT columns were prepared by in-situ thermal-initiated polymerization. The porous layer of the organic polymer was prepared with a 6 μm i. d capillary by in-situ thermal-initiated polymerization, following which the thickness and morphology of the NPLOT column were observed for different durations (3 h, 6 h, and 9 h) of polymerization. The thicknesses of the porous layer were 103±51 nm and 210±51 nm when polymerization was conducted for 3 h and 6 h, respectively. Subsequently, the columns prepared under 3 h of polymerization were applied in nanoflow-high performance liquid chromatography for the enantioseparation of N-derivatized amino acid enantiomers. Baseline separation was achieved in 2 min. The sample volume consumed was of the picoliter level, which would provide a means of research for single-cell analysis.

Key words: porous layer open tubular columns with narrow inner diameter (NPLOT), in-situ thermal-initiated polymerization, nanoflow-high performance liquid chromatography (nano-HPLC), enantioseparation

李若男, 王利娟, 张东堂, 王亚楠, 郭广生, 汪夏燕. 基于窄内径多孔层毛细管开管柱的纳流高效液相色谱用于N-衍生化氨基酸对映体的分离[J]. 色谱, 2020, 38(3): 350-355.

LI Ruonan, WANG Lijuan, ZHANG Dongtang, WANG Yanan, GUO Guangsheng, WANG Xiayan. Application of porous layer open tubular capillary columns with narrow inner diameter in nanoflow-high performance liquid chromatography for N-derivatized amino acid enantiomers[J]. Chinese Journal of Chromatography, 2020, 38(3): 350-355.

图/表 6

图1 纳流高效液相色谱-紫外检测系统的示意图

Fig. 1 Scheme of nano-HPLC-UV detection system PLOT: porous layer open tubular; DAQ: data acquisition.

图2 热聚合不同时间制备得到的poly(MQD-co-HEMA-co-EDMA) PLOT柱的SEM图

Fig. 2 SEM images of poly(MQD-co-HEMA-co-EDMA) PLOT column prepared by in-situ thermal-initiated polymerization for different durations a. bare capillary; b. 3 h; c. 6 h; d. 9 h.

图3 热聚合3 h的窄内径poly(MQD-co-HEMA-co-EDMA) PLOT柱的多孔层

Fig. 3 SEM image of porous layer in poly(MQD-co-HEMA-co-EDMA) PLOT columns with narrow inner diameter by heating for 3 h

图4 N-衍生化氨基酸对映体的色谱图

Fig. 4 Chromatograms of N-derivatized amino acid enantiomers Chromatographic column: poly(MQD-co-HEMA-co-EDMA) PLOT column, 6 μm i.d., total length 44.60 cm, effective length 40.70 cm; polymerization time: 3 h; sampling condition: 1.38 MPa, 20 s; sample concentration: 1.0 g/L; separation condition: 6.89 MPa; mobile phase flow rate: 32.24 nL/min.

图5 N-衍生化氨基酸对映体混合物的色谱图

Fig. 5 Chromatograms of N-derivatized amino acid enantiomers Sample: a. 10.0 g/L 3, 5-DClB-DL-Val; b. 10.0 g/L 3, 5-DClB-DL-Trp; c. complex of 5.0 g/L 3, 5-DClB-DL-Trp and 5.0 g/L 3, 5-DClB-DL-Val. Chromatographic column: poly(MQD-co-HEMA-co-EDMA) PLOT column, 6 μm i.d., total length 59.40 cm, effective length 54.20 cm; polymerization time: 3 h; sampling condition: 1.38 MPa, 20 s; separation condition: 1.38 MPa; mobile phase flow rate: 5.00 nL/min.1. methanol; 2.3, 5-DClB-L-Val; 3. 3, 5-DClB-L-Trp; 4. 3, 5-DClB-D-Val; 5. 3, 5-DClB-D-Trp.

表1 N-衍生化氨基酸对映体的分离度、理论塔板数、保留因子和手性选择性

Table 1 Resolutions, plate numbers, retention factors, and enantioselectivity of N-derivatized amino acid enantiomers

Sample	R	N_L/m	N_D/m	k_L	k_D	α
m-ClB-DL-Met	1.55	601	404	1.17	1.89	1.61
m-ClB-DL-Ala	1.52	925	1185	1.26	1.73	1.38
3, 5-DMB-DL-Leu	1.25	618	302	0.77	1.27	1.65

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基于窄内径多孔层毛细管开管柱的纳流高效液相色谱用于N-衍生化氨基酸对映体的分离

Application of porous layer open tubular capillary columns with narrow inner diameter in nanoflow-high performance liquid chromatography for N-derivatized amino acid enantiomers

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