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Two-dimensional highly effective liquid phase chromatographic system and its uses

A high-performance liquid chromatography and analytical column technology, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of difficult to fully utilize peak capacity, difficult to achieve column head focusing, complicated flow path design, etc., to simplify the pretreatment process. , The effect of reducing accidental error and good separation effect

Active Publication Date: 2008-04-30
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

However, the construction of LC×LC involves the change of mobile phase, and it is necessary to comprehensively consider the matching of multiple factors such as the analysis object, column system, flow mutual solubility, flow rate, valve switching frequency, etc., and the design of the flow path is relatively complicated.
In addition, because there is no independent modulator between the two dimensions, it is difficult to achieve column head focusing, and the range of applicable samples is narrow
Coupled with the existence of problems such as long analysis time and difficulty in fully utilizing the peak capacity, so far, most of the research is still limited to the separation and identification of proteins and peptides in the field of proteomics (document 3.Opiteck G J, Jorgenson J W. Anal. Chem. 1997, 69: 2283-2291. Document 4. Opiteck G J, Ramirez S M, Jorgenson J W, M. Moseley III A. Anal. Biochem. 1998, 258: 349-361. Document 5 . Wagner K, Miliotis T, Marko-Varga G, Bischoff R, Unger K K. Anal. Chem. 2002, 74: 809-820. Literature 6. Machtejevas E, John H, Wagner K, L, Marko-Varga G, Forssmann W, Bischoff R, Unger K K.J.Chromatog.B.2004, 803 (1): 121-130.), but progress is slow in the research field of other complex systems, only a few literature reports for Analysis of polymer composition (document 7.Horst A, Schoenmakers PJ.J.Chromatog.A.2003,1000:693-709.), separation of structural isomers (document 8.GrayM J, Sweeney A P, Dennis G R, Slonecker P J, Shalliker R.A.Analyst.2003, 128:598-604.), analysis of surfactants (document 9.Haefliger O P., Anal.Chem.2003, 75:371-378.), determination of polysaccharides in environmental samples Ring aromatic hydrocarbons (document 10.TsuyoshiMurahashi.Analyst.2003,128:611-615.) and separation of certain lipid compounds (document 11.Paola Dugo et al.Anal.Chem., 2004,76,2525-2530. Document 12 .P.Dugo et al., J.Chromatogr.A, 2006, 1112, 269-275.) etc.
However, for functionally important (molecular weight less than 1000) small molecular substances, especially hydrophilic small molecular substances, in complex samples (especially biological samples), no corresponding separation method has been proposed, resulting in the Loss of information prevents full understanding of sample properties

Method used

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  • Two-dimensional highly effective liquid phase chromatographic system and its uses
  • Two-dimensional highly effective liquid phase chromatographic system and its uses
  • Two-dimensional highly effective liquid phase chromatographic system and its uses

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Embodiment 1: Single column separation condition optimization

[0039] Prepare a mixed solution of standard samples containing 25 components, including 12 kinds of hydrophilic samples (uracil, uridine, pseudouridine, palmatine hydrochloride, nicotine, proparolol, atenolol, p-chlorobenzoic acid, p-hydroxybenzene Formic acid, hippuric acid, isonicotinic acid, Trp-Phe) and 13 kinds of hydrophobic samples (benzene, phenol, 2,6-dimethylphenol, toluene, o-chlorotoluene, 1,2,4-trimethylbenzene, biphenyl , 4-chlorobiphenyl, naphthalene, 2,3-dimethylnaphthalene, acenaphthene, pyrene, fluoranthene).

[0040] The mixture of standards was separated on a single column.

[0041] The HILIC column separation conditions are as follows: Atlantis TM HILIC Silica column (3μm, 2.1×150mm), phase A 100mM NH 4 Aqueous COOH, phase B acetonitrile. Gradient elution (93% B phase maintained for 8 min, then decreased to 85% B within 12 min, maintained for 5 min). The flow rate is 0.25mL / min, a...

Embodiment 2

[0044] The trapping capacity investigation of embodiment 2 component transfer column

[0045] Using the HILIC column as the first-dimensional analysis column, according to the single-column separation spectrum, it is determined that the switching time of the ten-way valve is 2.7 minutes. A C18 chromatographic column (column length 50mm) with the same inner diameter and packing material is used for the transfer of hydrophobic samples between two dimensions. In order to investigate the ability of the component transfer column to capture this part of the component, a detector is connected to the ⑤ position of the six-way valve to observe whether there is component outflow. The volume of the mixer connected to P5 in the flow path is 2.6mL, and the flow rate of P5 is set to 0.75mL / min. Therefore, starting from the switching of the ten-port valve, after 3.5min, all the residual components in the mixer will be flushed to the group. onto the transfer column. If the column cannot cap...

Embodiment 3

[0046] Example 3 The mixed standard sample was separated by a simultaneous separation flow platform.

[0047] According to the separation optimization results of a single column, the analysis conditions were set as follows. The flow path is shown in Figure 1. The first dimension uses a HILIC column, and the mobile phase conditions are the same as in Example 1. The second dimension uses an RP column, the mobile phase A is water, and other chromatographic conditions are the same as in Example 1, but the gradient is delayed by 6.2 minutes compared with Example 1. The flow rate of the pump 5 was set at 0.75 mL / min, and the mobile phase was an aqueous solution containing 5% acetonitrile. The switching time of the ten-way valve is 2.7 minutes, and the switching time of the six-way valve is 6.2 minutes. The first-dimension detector adopts a semi-micro detection cell, the second-dimension detector adopts a conventional detection cell, and the detection wavelength is 254nm. as atta...

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Abstract

The present invention relates to high-performance liquid chromatography, specifically a two-dimensional high-performance liquid chromatography system and its application, consisting of five high-pressure liquid chromatography solvent delivery pumps, a solvent mixer, a ten-way valve, and a six-way valve 1. Two liquid chromatography analysis columns suitable for the separation of hydrophobic components and hydrophilic components and a component transfer column. The system of the present invention is suitable for the separation and detection of a variety of different hydrophobic small molecular components in complex system samples. It is easy to operate, good in flexibility, strong in universality, short in time consumption, and can be automatically separated on two chromatographic columns at the same time. The hydrophilic and hydrophobic components in complex system samples can significantly improve the resolution and peak capacity of the analysis on the basis of meeting the qualitative and quantitative analysis requirements of the sample components.

Description

technical field [0001] The invention relates to high-performance liquid chromatography, in particular to a two-dimensional high-performance liquid chromatography system and its application. Background technique [0002] With the rapid development of research in the fields of proteomics, modernization of traditional Chinese medicine, and environmental protection, multidimensional liquid chromatography has quickly become one of the hot spots in the field of chromatographic science research in the face of the requirements of high-efficiency separation and high-sensitivity detection of non-volatile extremely complex system samples. one. The core idea of ​​this technology is to couple two chromatographic columns with different separation mechanisms that are independent of each other, and to improve peak capacity and separation selectivity by designing the valve / column interface and optimizing process parameters. [0003] There are currently two main ways to construct 2D-LC. The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/24
Inventor 许国旺王媛
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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