Comprehensive two-dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography for analysis of toad skin

doi:10.1016/j.aca.2017.01.038

Analytica Chimica Acta

Volume 962, 15 April 2017, Pages 114-120

https://doi.org/10.1016/j.aca.2017.01.038 Get rights and content

Highlights

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A novel thermal evaporation assisted adsorption (TEAA) interface was designed for 2D NPLC × RPLC system.
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Rapid on-line solvent exchange between NP and RP dimensions was achieved within a short modulation time of 190 s.
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The first time to realize on-line comprehensive analysis of a moderate polar natural product by coupling of NPLC and UHPLC.
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A high orthogonality of 75.2% was achieved within 300 min' analysis.

Abstract

An analytical two–dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography (2D NPLC × RPLC) system was constructed with a newly developed thermal evaporation assisted adsorption (TEAA) interface. This novel TEAA interface with heating temperature above solvent boiling point allowed fast removal of organic NPLC solvent and successfully solved the solvent incompatibility problem between NPLC and RPLC. The system achieved rapid on-line solvent exchange between the two dimensions within a short modulation time of 190 s and was applied in the analysis of an extract from the skin of Bufo bufo gargarizans. This is the first time to realize the on-line comprehensive analysis of a moderate polar natural product by coupling NPLC with reversed phase ultra-high performance liquid chromatography (UHPLC). To be highlighted, with the TEAA interface, the 2D NPLC × RPLC system provided excellent resolution and orthogonality (75.2%), when compared with that of 2D RPLC × RPLC.

Graphical abstract

Introduction

In recent years, two-dimensional liquid chromatography (2D-LC) has outperformed conventional one-dimensional (1D-LC) techniques in term of complex sample separation with higher peak capacity. In 2D-LC, many separation mechanisms, such as normal phase liquid chromatography (NPLC), hydrophilic interaction (HILIC), reversed phase liquid chromatography (RPLC), size exclusion (SEC), ion exchange (IEX), and affinity chromatography (AC), usually apply in pairs [1]. Of all the combination, NPLC × RPLC is probably the most orthogonal [2], though, difficult to achieve because of the problem of mobile phase immiscibility in each dimension.

Recently, Yun Zhang [3] developed an off-line 2D NPLC × RPLC method for the isolation and identification of bufadienolides in toad skin, where orthogonality of 49.5% was achieved. However, the off-line mode suffered from the disadvantages of difficult automation, poor reproducibility and time-consuming. In contrasted to that, the on-line comprehensive NPLC × RPLC method was preferred. As a typical example, Murphy et al. [4] applied aqueous solvents in both NPLC and RPLC systems to isolate alcohol ethoxylates, which successfully avoided the solvent immiscibility. However, the NPLC selectivity and the orthogonality of the 2D-LC system were affected. Wei et al. [5] developed a NPLC × RPLC system based on the modification of mobile phases, without considering its versatility. Dugo et al. [6] constructed a 2D-LC system with the use of a microbore silica column operated in first dimensional NP mode and a monolithic type C18 column operated in second dimensional RP mode. The small NPLC column inner diameter and the low NPLC flow rate permitted the direct injection of a small volume of NPLC solvents onto the second dimension. However, the small sized column reduced sample capacity and detection sensitivity. Tian et al. [7], [8] and Ding et al. [9] designed a new vacuum assisted dynamic evaporation loop-type valve interface for 2D-LC system. In this design, desired fractions from NPLC were eluted into the loop-interface and underwent solvent evaporation, before RP column separation. To be mentioned, due to the limitation of second dimension's long separation time, the system could only be operated in a stop-flow mode.

In previous reports, we have showed the construction of airflow assisted adsorption (AAA) interface [10] and vacuum evaporation assisted adsorption (VEAA) interface [11] for on-line preparative 2D NPLC × RPLC systems. In details, the NPLC solvent was evaporated when it flowed into the NP enrichment column, under the blowing of airflow (for AAA) or a vacuum condition (for VEAA). To be highlighted, both AAA and VEAA interfaces could achieved the NPLC solvent removal, adsorption of the solutes on the NP enrichment column, and further solute transition from the first dimension to the second one, which completely solved the problem of mobile phase incompatibility.

In this report, we design a novel thermal evaporation assisted adsorption (TEAA) interface, which allows complete NPLC solvent removal without vacuum condition at a relative low flow rate., TEAA technique overcomes the defect of VEAA interface and is more suitable for establishing on-line analytical 2D NPLC × RPLC system. Furthermore, the performance of the on-line NPLC × RPLC system with new TEAA interface has also been evaluated by using toad skin as a complex model.

Section snippets

Chemicals and materials

Acetonitrile, n-hexane and anhydrous alcohol (HPLC grade) were obtained from TEDIA (Fairfield, USA). A Milli-Q system (Billerica, MA, USA) was utilized for water supply. Analytical grade solvents applied for the toad skin pretreatment were purchased from Xilong Chemical Company, Shantou, China. The dried toad skin of Bufo bufo gargarizans were supplied by Luyan Pharmaceuticals (Xiamen, China) and identified by Professor Quan-Cheng Chen (Xiamen University, Xiamen, China).

Instrumentation

Two Agilent 1100 series

Coupling of NPLC and RPLC

Although NPLC × RPLC has been mentioned in many reports, the connotations of NPLC and RPLC have been defined from various different perspectives. According to the polarity of analytes, as well as the stationary and mobile phases, combination of NPLC × RPLC includes three types. For the separation of high polar components, HILIC or amino columns [12] were used in the NP dimension, with aqueous mobile phase systems. On the other hand, for the analysis of fat-soluble samples, such as lipids [13],

Conclusion

A new on-line NPLC × RPLC system with a thermal evaporation assisted adsorption (TEAA) interface was developed and successfully applied in the 2D analysis of the crude toad skin extract. Based on the fast evaporation of NP solvent under elevated temperature, the interface realized on-line solvent exchange from the first dimension to the second and solved the problem of mobile phase incompatibility completely. When compared with our previously reported VEAA interface, the novel TEAA approach

Acknowledgment

The project was supported by the National Natural Science Foundation of China (No. 81302652, 21303145). The authors would also like to acknowledge the financial supports from Fujian Key Science and Technology Program (No.2013N0019), Xiamen Science and Technology Program (No. 3502Z20143001), and Xiamen South Marine Center project (13GZP002NF08).

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¹: These authors contribute equally to this paper.

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Comprehensive two-dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography for analysis of toad skin

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Chemicals and materials

Instrumentation

Coupling of NPLC and RPLC

Conclusion

Acknowledgment

J. Chromatogr. A

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