基于捕集柱阵列的制备型二维液相色谱纯化烟草中的4个组分

doi:10.3724/SP.J.1123.2022.08021

摘要/Abstract

摘要：

二维液相色谱(2D-LC)因具有较高的峰容量,在复杂样品的分离分析中获得了广泛的关注。然而,制备型2D-LC以纯化高纯单体为目标,在方法开发和设备构成等方面与分析型2D-LC有较大的不同,目前尚未得到充分的开发,在大规模的制备纯化中应用较少。本文以一套制备液相色谱模块为分离系统,以稀释泵、切换阀和捕集柱阵列为接口,构建了新型的制备型2D-LC系统,旨在规模化纯化多个活性成分。以烟叶中可以用作医药原料的烟碱、绿原酸、芦丁和茄尼醇等组分为目标物,考察了不同类型填料对样品的捕集效率、过载条件下的色谱保留行为等,优化了制备色谱条件。进而利用在线2D-LC系统实现了烟叶提取物的纯化,通过一次运行获得了4个高纯化合物。该系统具有中压色谱纯化成本低、系统在线运行自动化程度高、稳定性好及容易放大等优点。烟叶中活性化学成分的回收利用对促进烟草行业的发展及带动地方农业经济开发具有重大的意义。

关键词: 制备型二维液相色谱, 高纯化合物, 烟草, 捕集柱阵列, 中压色谱

Abstract:

Two-dimensional liquid chromatography (2D-LC) has gained increased attention because of its high peak capacity for separating complex samples. However, preparative 2D-LC aimed at isolating compounds is significantly different compared with one-dimensional liquid chromatography (1D-LC) in terms of method development and system configuration; thus, it is less developed than its analytical counterpart. The use of 2D-LC in large-scale product preparation has rarely been reported. Hence, a preparative 2D-LC system was developed in this study. The system was composed of one set of preparative LC modules as a separation system, with a dilution pump, switch valves, and trap column array as the interface, to enable the simultaneous isolation of several compounds. Tobacco was used as a sample, and the developed system was applied to isolate nicotine, chlorogenic acid, rutin, and solanesol. The chromatographic conditions were developed by investigating the trapping efficiency of different types of trap column packings, and chromatographic behaviors under different overload conditions. The four compounds were isolated in one 2D-LC run with high purity. The developed system features low cost because it employs medium-pressure isolation, excellent automation owing to its use of an online column switch, high stability, and capability for large-scale production. The isolation of chemicals from tobacco leaves as pharmaceutical raw materials could aid in the development of the tobacco industry and promote the local agricultural economy.

Key words: preparative two-dimensional liquid chromatography, high-purity compounds, tobacco, trapping column array, medium-pressure chromatography

中图分类号:

O658

沙云菲, 熊骏威, 翟钰霖, 王宝雷, 钟志华, 费婷, 李笃信, 吴达. 基于捕集柱阵列的制备型二维液相色谱纯化烟草中的4个组分[J]. 色谱, 2023, 41(3): 281-286.

SHA Yunfei, XIONG Junwei, ZHAI Yulin, WANG Baolei, ZHONG Zhihua, FEI Ting, LI Duxin, WU Da. Trapping array-based preparative two-dimensional liquid chromatography for the purification of four components in tobacco leaves[J]. Chinese Journal of Chromatography, 2023, 41(3): 281-286.

图/表 6

图1 2D-LC系统的结构图

Fig. 1 Schematic of the two-dimensional liquid chromatography system a. first-dimensional liquid chromatography (1D-LC) operation setup; b. second-dimensional liquid chromatography (2D-LC) operation setup; c. photograph of the system.

图2 不同载样量及流动相梯度条件下烟草提取物的一维分离色谱图

Fig. 2 Chromatograms of 1D-LC separation of tobacco extracts obtained under different sample loads and gradient elution programs Chromatographic conditions: C18 column (460 mm×15 mm i.d., 50 μm); mobile phase A: 50 mmol/L ammonium phosphate (pH 6.5); mobile phase B: ethanol; detection wavelength: 210 nm; gradient elution programs: 0-5 min, 5%B; 5-25 min, 5%B-50%B; 25-35 min, 50%B-100%; 35-50 min, 100%B; c, d. 0-40 min, 5%B-60%B; 40-50 min, 60%B-100%B.

图3 一维液相色谱纯化烟叶提取物及馏分分析色谱图

Fig. 3 Chromatograms of the 1D-LC separation of tobacco extract and its fraction analysis 1D-LC conditions: C18 (460 mm×15 mm i.d., 50 μm); mobile phase A: 50 mmol/L ammonium phosphate (pH 6.5); mobile phase B: ethanol; gradient elution program: 0-5 min, 5%B; 5-40 min, 5%B-55%B; 40-41 min, 55%B; 41-50 min, 55%B-100%B; 50-70 min, 100%; detection wavelength: 210 nm; sample load: 10 g.Fraction analysis conditions: C18 column (250 mm×4.6 mm i.d., 5 μm); mobile phase A: 50 mmol/L ammonium phosphate (pH 6.5); mobile phase B: ethanol; gradient elution program: 0-5 min,5%B; 5-25 min,5%B-50%B;25-35 min,50%B-100%;35-45 min,100%B; detection wavelength: 210 nm.

表1 2D-LC接口中捕集柱的捕集效率

Table 1 Trapping efficiency of different trap columns in the 2D-LC interface%

Compound	C₁₈			WAX (undiluted)	LH-20 (undiluted)
Compound	Undiluted	Diluted with water	Diluted with 0.1% TFA	WAX (undiluted)	LH-20 (undiluted)
Nicotine	65.41	54.02	47.03	10.68	43.97
Chlorogenic acid	32.70	56.37	36.14	100	76.10
Rutin	38.30	88.30	95.67	-	-
Solanesol	61.77	96.13	94.30	-	-

图4 在线制备2D-LC系统纯化烟叶中的活性成分

Fig. 4 Online preparative 2D-LC isolation of bioactive compounds in tobacco extract 1D conditions: C18 column (460 mm×15 mm i.d., 50 μm); mobile phase A: 50 mmol/L ammonium phosphate (pH 6.5); mobile phase B: ethanol; gradient elution program: 0-5 min, 5%B; 5-40 min, 5%B-55%B; 40-41 min, 55%B; 41-50 min, 55%B-100%B; 50-70 min, 100%B; detection wavelength: 210 nm; sample load: 10 g.2D conditions: C18 column (460 mm×15 mm i.d., 50 μm); mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: ethanol; gradient elution program: 0~5 min, 5%B; 5~25 min, 5%B~50%B; 25~35 min, 50%B~100%; 35~45 min, 100%B; detection wavelength: 210 nm.Purity analysis: C18 column (250 mm×4.6 mm, i.d., 5 μm); mobile phase A: 0.1%TFA aqueous solution; mobile phase B: ethanol; gradient elution program for compound 4: 0-5 min, 90%B-100%B; 5-15 min, 100%B; gradient program for other compounds: 0-5 min, 5%B; 5-25 min, 5%B-55%B; 25-35 min, 55%B-100%; 35-45 min, 100%B.For peaks, see Table 2.

表2 烟草分离组分的色谱保留时间和纯度

Table 2 Chromatographic retention times and purities of tobacco fractions

Peak No. in Fig.4	Compound	t_R/min	Purity/%
1	nicotine	3.23	96.96
2	chlorogenic acid	12.89	98.23
3	rutin	18.12	95.71
4	solanesol	8.59	81.01
5	unknown	9.91	86.83
6	unknown	11.94	41.82
7	unknown	13.24	45.24

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