CN107037169B - A kind of full two-dimensional highly effective liquid phase chromatographic instrument and chromatography separating method with Multifunctional switching valve - Google Patents

Abstract

The invention provides a comprehensive two-dimensional high-performance liquid chromatograph with a multi-functional switching valve. The comprehensive two-dimensional high-performance liquid chromatograph with a multi-functional switching valve consists of a one-dimensional chromatographic mobile phase reservoir M1, a one-dimensional chromatographic high-pressure Pump P1, one-dimensional chromatographic sampling valve V1, one-dimensional chromatographic column C1, one-dimensional chromatographic detector D1, two-dimensional chromatographic mobile phase reservoir M2, two-dimensional chromatographic high-pressure pump P2, multi-functional switching valve V, multiple Composed of two-dimensional separation columns C2-1 to C2-8, two-dimensional chromatographic detector D, fraction collector F. The outstanding effects of the present invention are: 1. Multiple two-dimensional chromatographic columns are used for two-dimensional separation, and there are many types of stationary phases to choose from, high separation efficiency and good separation effect, and the separation and analysis of all components of complex samples can be realized. 2. The system is equipped with multiple detectors, which can ensure the precise control of the separation process and improve the separation effect.

Description

A comprehensive two-dimensional high performance liquid chromatograph with a multifunctional switching valve and its chromatographic separation method

technical field

The invention relates to a comprehensive two-dimensional high-performance liquid chromatograph and a method for comprehensive two-dimensional chromatographic separation, belongs to the technical field of instrument and equipment manufacturing, and is a high-performance liquid chromatography separation and analysis device.

Background technique

High-performance liquid chromatography uses high-pressure delivery of mobile phase, high-efficiency stationary phase and high-sensitivity detector for detection. It has the characteristics of high efficiency, rapidity, and wide application. The key technology is widely used in the fields of pharmacy, life science, medicine, food science, petrochemical industry and environmental protection.

The usual high-performance liquid chromatography is composed of a liquid reservoir, a high-pressure pump, a sampler, a chromatographic column, a detector, a recorder, etc. During the application process, a chromatographic column is often used for separation, that is, one-dimensional chromatographic separation. In the one-dimensional chromatographic separation process, the eluent in the liquid reservoir is transported through the sample injector by a high-pressure pump, and the sample in the sample injector is carried into the chromatographic column for separation, and the separated components flow out from the chromatographic column and enter the detection column in turn. device. For complex systems such as natural products and body fluids, a sample usually contains dozens or even hundreds of components, and the structures of the components are similar, but the differences in quantity are usually large, which is limited by the resolution of one-dimensional liquid chromatography and Limitations such as peak capacity cannot meet the needs of complex separations.

Two-dimensional liquid chromatography is a technology developed on the basis of high-performance liquid chromatography. It uses a one-dimensional chromatographic column and a two-dimensional chromatographic column to separate the one-dimensional chromatographic column through technical and equipment improvements. The fractions are introduced into a second chromatographic column for two-dimensional separation. Compared with the one-dimensional liquid chromatography system, the advantage of two-dimensional chromatography is that it provides a larger peak capacity, so that it can more effectively solve the analysis problems of complex system samples.

As early as 1990, Jorgenson et al. realized the "comprehensive two-dimensional cutting" for the first time on the basis of the "heart cutting" technology. They connected two quantitative tubes to an eight-way valve, and the two quantitative tubes sampled alternately, Injection, to achieve two-dimensional separation. Chinese patent "A preparative multidimensional liquid chromatograph" (application number 201110256302.1), using a one-dimensional separation column, a two-dimensional separation column, two enrichment columns and a two-position ten-way valve, the two-dimensional separation column will The two enrichment columns are connected to the two-position ten-port valve, and the two quantitative tubes are enriched and injected alternately, realizing two-dimensional separation. The Chinese patent "A Method for Separating Natural Products" (Application No. 201210022094.3) uses a one-dimensional chromatography column, a two-dimensional chromatography column, a enrichment column and a two-position six-way valve. The two-dimensional separation is achieved by column enrichment and alternate injection. Chinese patent "a comprehensive two-dimensional capillary liquid chromatography separation system" (application number 200410098443.5), using one one-dimensional separation column, two two-dimensional separation columns, one enrichment column, two six-way valves, one two-position ten Through the valve, two-dimensional separation is realized. The Chinese patent "comprehensive two-dimensional ultra-high pressure high-performance liquid chromatography separation system" (application number 201010256470.6) adopts a conventional high-pressure liquid chromatography column, an ultra-high pressure liquid chromatography column, two quantitative loops, and a two-position ten-way valve. The conventional high-pressure liquid chromatography column is a one-dimensional separation column, and the ultra-high pressure liquid chromatography column is a two-dimensional separation column. Using the rotation of the two-position ten-way valve, the two quantitative loops are alternately sampled and injected to realize two-dimensional separation.

At present, the comprehensive two-dimensional or multi-dimensional chromatography mode is to introduce the fraction separated by one-dimensional chromatographic column into the second chromatographic column for two-dimensional separation through interface modes such as six-way valve, eight-way valve, and ten-way valve. In the separation process, the two-dimensional separation column is required to be short and the analysis speed is fast, which limits the overall separation effect. For large-capacity preparation and separation, the existing technology cannot meet the demand, and cannot be directly applied to the separation and preparation of components. In the prior art, one one-dimensional chromatographic column and one two-dimensional chromatographic column are used for separation. In the separation process, only two kinds of stationary phase chromatographic columns can be used for separation each time, which is difficult to meet the separation needs of complex samples.

Contents of the invention

In order to solve the defects existing in the existing two-dimensional chromatographic technology, the present invention provides a comprehensive two-dimensional high performance liquid chromatography with a multi-functional switching valve. Two-dimensional separation is carried out in a two-dimensional chromatographic column, and the fraction separated by two-dimensional chromatography is collected by a fraction collector.

In order to achieve the above object, the technical scheme adopted in the present invention is:

A comprehensive two-dimensional high-performance liquid chromatograph with a multi-functional switching valve. The comprehensive two-dimensional high-performance liquid chromatograph with a multi-functional switching valve consists of a one-dimensional chromatographic mobile phase reservoir M1, a one-dimensional chromatographic high-pressure pump P1, and a one-dimensional chromatographic high-pressure pump P1. Two-dimensional chromatography sampling valve V1, one-dimensional chromatography column C1, one-dimensional chromatography detector D1, two-dimensional chromatography mobile phase liquid reservoir M2, two-dimensional chromatography high-pressure pump P2, multi-functional switching valve V, multiple two-dimensional separation columns C2-1 to C2-8, two-dimensional chromatographic detector D, fraction collector F.

The connection relationship between the various components is: channel 0 of the multifunctional switching valve V is connected to the pipeline M1→P1→V1→D1, channels 1-8 of the multifunctional switching valve V are respectively connected to the two-dimensional chromatographic column C2-1 - the inlet port of C2-8 is connected, the outlet ports of two-dimensional chromatographic columns C2-1 to C2-8 are respectively connected to a detector D, and the outlet of detector D is connected to fraction collector F again. Channels 9 to 16 of the multi-function switching valve V are connected with a multi-way outlet, and the multi-way inlet is connected with the M2→P2 pipeline. The flow path during use is: M1→P1→V1→D1→multifunctional switching valve V (channel 0)→multifunctional switching valve V (channels 1-8 in sequence), M2→P2→multifunctional switching valve V (channel 9-16) → two-dimensional chromatographic column (respectively C2-1-C2-8) → detector D (respectively) → fraction collector F.

The channel number and channel number of the multi-functional switching valve V are not limited to the above-mentioned fixed channel number, but also include other connection situations in which the design of the multi-functional switching valve V is similar to that of the present invention.

The liquid reservoirs M1 and M2 may be glass containers or organic material containers resistant to organic solvents and corrosion.

The high-pressure pumps P1 and P2 may be constant-flow pumps or constant-pressure pumps that can provide stable pressure and flow rate.

The sampling valve V1 may be a fixed volume sampling valve or a sampling pump or the like.

The chromatographic column C1 and multiple two-dimensional separation columns C2-1, C2-2, C2-3, C2-4, C2-5, C2-6, C2-7, C2-8 can be made of glass, stainless steel, PEEK Wait. The filled fillers can be ion exchange materials, normal phase chromatography fillers, reverse phase chromatography fillers, gel fillers, etc., and the amount of fillers can be analytical type, semi-preparative type, preparative type, etc.

The multifunctional switching valve V is a multi-position multi-way selector valve with one input and multiple outputs.

The detectors D1 and D may be flow-through detectors such as ultraviolet detectors, fluorescence detectors, and differential refractive index detectors. The detection cell of the detector D1 is a high pressure resistant detection cell.

The present invention also proposes a chromatographic separation method for the comprehensive two-dimensional high-performance liquid chromatography: the eluent in the liquid reservoir M1 is transported through the injection valve V1 through the high-pressure pump P1, and the sample in the injection valve V1 is carried into the The chromatographic column C1 performs one-dimensional separation, and the effluent of the chromatographic column C1 enters the No. 0 through hole of the multi-function switching valve V, and is switched in sequence by rotating the multi-function switching valve V into the No. 1-8 through holes connected to the multi-function switching valve V. Chromatographic columns C2-1, C2-2, C2-3, C2-4, C2-5, C2-6, C2-7, C2-8. The eluent in the liquid reservoir M2 is transported through the high-pressure pump P2 and enters the chromatographic columns C2-1, C2-2, C2-3, and C2 through holes 9-16 and 1-8 of the multi-function switching valve V. -4, C2-5, C2-6, C2-7, C2-8, perform two-dimensional separation, the two-dimensional separation effluent enters the detector D for detection, and the effluent of the detector D enters the fraction collector F for collection. The flow route of one-dimensional separation eluent in the separation process is: M1→P1→V1→D1→V(0)→V(1-8)→C2-1 to C2-8 (in sequence), and the flow of two-dimensional separation eluent The route is: M2→P2→V(9-16)→V(1-8)→C2-1 to C2-8 (parallel)→D (parallel)→F (parallel).

The invention discloses a comprehensive two-dimensional high-performance liquid chromatograph with a multifunctional switching valve and a method for the comprehensive two-dimensional chromatographic separation, which have super strong separation ability.

In addition, the outstanding effect of the present invention is:

1. Multiple two-dimensional chromatographic columns are used for two-dimensional separation. There are many types of stationary phases to choose from, high separation efficiency and good separation effect, which can realize the separation and analysis of all components of complex samples.

2. The system is equipped with multiple detectors, which can ensure the precise control of the separation process and improve the separation effect.

3. Using multiple two-dimensional chromatographic columns for two-dimensional separation, there is no limitation on the size of one-dimensional chromatographic columns and the flow rate of mobile phase, which can be used for preparative separation of large sample volumes.

4. Multiple two-dimensional chromatographic columns are used for two-dimensional separation, there is sufficient time for two-dimensional separation, and there is no restriction that the two-dimensional chromatographic column must be a short column.

5. The system design is simple, the separation effect is good, it is suitable for the separation and purification of various complex mixtures, and it is extremely practical.

Description of drawings

Fig. 1 is a schematic structural diagram of a comprehensive two-dimensional high-performance liquid chromatograph according to an embodiment of the present invention.

Fig. 2 is a schematic diagram of the output signal of the one-dimensional detector of the embodiment.

Fig. 3 is a schematic diagram of the output signal of the two-dimensional detector of the embodiment.

Detailed ways

The present invention will be described in detail below in conjunction with the embodiments and drawings, but the scope of protection is not limited thereto.

Embodiment 1 As shown in Figure 1, a comprehensive two-dimensional high-performance liquid chromatograph with a multifunctional switching valve and a method for comprehensive two-dimensional chromatographic separation, the comprehensive two-dimensional high-performance liquid chromatograph consists of a one-dimensional chromatographic mobile phase Liquid reservoir M1, one-dimensional chromatography high-pressure pump P1, one-dimensional chromatography sampling valve V1, one-dimensional chromatography column C1, one-dimensional chromatography detector D1, two-dimensional chromatography mobile phase reservoir M2, two-dimensional chromatography high-pressure pump P2 , a multifunctional switching valve V, two-dimensional chromatographic columns C2-1 to C2-8, a two-dimensional chromatographic detector D, and a fraction collector F.

The eluent in the reservoir M1 is transported by the high-pressure pump P1 through the injection valve V1, and the sample carried in the injection valve V1 enters the chromatographic column C1 for one-dimensional separation, and the effluent of the chromatographic column C1 enters the multi-functional switching valve V Through hole No. 0 of the multi-function switching valve V, the chromatographic columns C2-1, C2-2, C2-3, C2-4, C2-5, C2-6, C2-7, C2-8. The eluent in the liquid reservoir M2 is transported through the high-pressure pump P2 and enters the chromatographic columns C2-1, C2-2, C2-3, and C2 through holes 9-16 and 1-8 of the multi-function switching valve V. -4, C2-5, C2-6, C2-7, C2-8, perform two-dimensional separation, the two-dimensional separation effluent enters the detector D for detection, and the effluent of the detector D enters the fraction collector F for collection. The flow route of the one-dimensional separation eluent in the separation process is: M1→P1→V1→D1→V(0)→V(1-8)→C2-1-8, and the flow route of the two-dimensional separation eluent is: M2→ P2 → V (9-16) → V (1-8) → C2-1 to C2-8 (parallel) → D (parallel) → F (parallel). .

According to the above connection and operation mode, the comprehensive two-dimensional chromatographic separation of Sophora japonicus extract:

The first dimension chromatographic column is a C18 column with a length of 10 cm. The second dimension chromatographic columns C2-1, C2-2, C2-3, C2-4, C2-5, C2-6, C2-7, C2-8 are C4 column, C4 column, benzene column, benzene column, C8 column, C8 column, C18 column, C18 column, the length is 10cm. The one-dimensional mobile phase is methanol-water (volume ratio 40/60), the flow rate is 1ml/min, the two-dimensional mobile phase is acetonitrile-0.1% formic acid gradient elution, 0-30min, acetonitrile changes from 20% to 80%, The flow rate of the two-dimensional mobile phase was 1.0 ml/min.

See accompanying drawings 2 and 3 for the one-dimensional chromatogram and two-dimensional chromatogram when the embodiment of the present invention is separated, which shows that the present invention has high separation efficiency and strong practicability, and is especially suitable for the separation and separation of all components of complex systems such as traditional Chinese medicine and body fluids. Separation of difficult-to-separate substances such as chiral compounds and compounds with similar structures.

The above is only the embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, some improvements and modifications can also be made, and these improvements and modifications should also be regarded as within the protection scope of the present invention.

The completion of this invention benefits from the National Natural Science Foundation of China (project approval number: 21675071).

Claims (3)

1. A comprehensive two-dimensional high-performance liquid chromatograph with a multifunctional switching valve, characterized in that it consists of a one-dimensional chromatographic mobile phase liquid reservoir M1, a one-dimensional chromatographic high-pressure pump P1, a one-dimensional chromatographic sampling valve V1, a Two-dimensional chromatography column C1, one-dimensional chromatography detector D1, two-dimensional chromatography mobile phase reservoir M2, two-dimensional chromatography high-pressure pump P2, multi-function switching valve V, two-dimensional separation columns C2-1 to C2-8, two-dimensional chromatography Chromatographic detector D and fraction collector F; channel 0 of multi-functional switching valve V is connected with pipeline M1→P1→V1→D1, channels 1-8 of multi-functional switching valve V are respectively connected to two-dimensional chromatographic column The inlets of C2-1-C2-8 are connected, and the outlets of two-dimensional chromatographic columns C2-1 to C2-8 are each connected to a detector D, and the outlet of detector D is connected to fraction collector F; multi-functional switching valve Channels 9 to 16 of V are connected to a multi-way outlet, and the multi-way inlet is connected to the M2→P2 pipeline; the liquid reservoirs M1 and M2 are organic solvent-resistant and corrosion-resistant glass containers or organic materials container; the high-pressure pumps P1 and P2 are constant-flow pumps or constant-pressure pumps that can provide stable pressure and flow rate; the injection valve V1 is a fixed-volume injection valve or injection pump; the chromatographic column C1 and two Dimensional separation columns C2-1, C2-2, C2-3, C2-4, C2-5, C2-6, C2-7, C2-8 are made of glass, stainless steel or PEEK, filled with ion exchange material, normal phase chromatography packing, reversed phase chromatography packing or gel packing; the multifunctional switching valve V is a multi-position multi-way selection valve with one input and multiple outputs; the detectors D1 and D are ultraviolet detectors, fluorescence Detector or differential refraction detector; the detection cell of detector D1 is a high pressure resistant detection cell. 2. The comprehensive two-dimensional high-performance liquid chromatograph of band multifunctional switching valve as claimed in claim 1, is characterized in that, the amount of packing is analytical type, semi-preparative type or preparative type. 3. the chromatographic separation method of the comprehensive two-dimensional high-performance liquid chromatograph of band multifunctional switching valve as claimed in claim 1, it is characterized in that: the eluent in the liquid reservoir M1 is transported through the sampling valve through the high pressure pump P1 V1, the sample in the carrier tape injection valve V1 enters the chromatographic column C1 for one-dimensional separation, and the effluent of the chromatographic column C1 enters the No. Chromatographic columns C2-1, C2-2, C2-3, C2-4, C2-5, C2-6, C2-7, C2-8 on through holes 1-8 of the multifunctional switching valve V; The eluent in the device M2 is transported through the high-pressure pump P2 and enters the chromatographic columns C2-1, C2-2, C2-3, and C2-4 through No. 9-16 through holes and No. 1-8 through holes of the multifunctional switching valve V. .