Trapping array-based preparative two-dimensional liquid chromatography for the purification of four components in tobacco leaves

doi:10.3724/SP.J.1123.2022.08021

Abstract

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

CLC Number:

O658

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.

Figures/Tables 6

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.

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.

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.

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	-	-

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.

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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