CN105572266B - A kind of method for preparing supercritical fluid chromatograph and being purified for preparative separation with multiple-way valve - Google Patents

Abstract

With multiple-way valve supercritical fluid chromatograph is prepared the invention provides a kind of and for preparative separation purification process, switched by the switch of multiple-way valve, so that clastotype of the COMPLEX MIXED component to be separated by the pre-separation of pre-separation post, the trapping of many trapping columns, how main separation post separation is efficiently separated, the separated component opened switches in collector by the switch of valve and collected, and the mobile phase without separated component returns to system circulation and used.Supercritical fluid chromatograph of the present invention design is simple, good separating effect, it is adaptable to which all kinds of complex mixtures are isolated and purified, and practicality is extremely strong.

Description

A preparative supercritical fluid chromatograph with a multi-way valve and it is used for preparative separation and purification Methods

technical field

The invention belongs to the technical field of instrument and equipment manufacturing, in particular to a preparative supercritical fluid chromatograph with a multi-way valve and a method for preparative separation and purification.

Background technique

Supercritical fluid chromatography (SFC) using supercritical fluid as mobile phase is a chromatographic process that relies on the solvating ability of the mobile phase for separation and analysis. It is a new technology developed and perfected in the 1980s.

Supercritical fluid is a state of matter above the critical pressure and critical temperature. It has some properties of gas and liquid, with low viscosity of gas, high density of liquid and high density between gas and liquid. Diffusion coefficient and other characteristics, SFC is a supplement to GC and LC, SFC can solve the problem of gas-liquid chromatography analysis, it can analyze non-volatile samples that are difficult to vaporize in gas chromatography, and has higher efficiency than high-performance liquid chromatography, analysis time Shorter. At present, supercritical fluid chromatography has developed into an important separation technology widely used in chemistry, life science, pharmaceutical and other fields.

The Chinese invention patent document of application publication number CN 103376297 A (application number 201210114350.1) discloses a supercritical fluid chromatograph and an asymmetric six-way sampling valve used therein, which consists of a liquefied fluid tank, a pressure reducing valve, a pressure gauge, Plate heat exchanger, liquid CO2 delivery pump, modifier delivery pump, mixer, autosampler, chromatographic column, detector, automatic back pressure regulator, pressure sensor, component collector. A new type of asymmetrical six-port sampling valve is used, with non-equal rotor and asymmetric design, so that the supercritical CO2 in the quantitative loop can be released in advance during the sample injection switching process. Combined with the check valve, the supercritical fluid in the sample injection quantitative loop can be slowed down. Explanation, to solve the problem of vaporization shock.

A typical supercritical fluid chromatograph consists of a carbon dioxide cylinder, a carbon dioxide pump, an improver pump, a sample injector, a chromatographic column, a detector, a recorder, etc., and the separation mode is one-dimensional separation. However, when performing sample separation, it is difficult to obtain an ideal separation effect for complex samples, and the purity of the product obtained by one-dimensional separation is sometimes not ideal. In addition, when separating samples, a large amount of eluent needs to be used, which also increases the cost of separation.

Contents of the invention

In order to solve the above-mentioned defects in the separation of common supercritical fluid chromatographs, the present invention provides a preparative supercritical fluid chromatograph with a multi-way valve, which allows the complex mixed components to be separated to pass through The separation mode of pre-separation column pre-separation, multi-trapping column trapping, and multi-main separation column separation can be separated efficiently, and the separated components are collected in the collector through the switch of the valve, and the flow without the separated components phase back to the system for recycling.

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

A preparative supercritical fluid chromatograph with a multi-way valve, consisting of a pre-separation system and a main separation system. The pre-separation system consists of a delivery system 1, a sample injection system Cs, a pre-separation column C1, a detector D1, and a multi-way valve V1 , trapping column system C2, multi-way valve V2, detector D2, and back pressure valve B1; the main separation system consists of conveying system 2, multi-way valve V3, trapping column system C2, multi-way valve V4, and multi-way valve V5 , separation column system C3, multi-way valve V6, detector D3, multi-way valve V7, component collector S, and back pressure valve B2. Delivery system 1 is composed of supercritical carbon dioxide delivery pump P1 and improver 1 delivery pump P2, and delivery system 2 is composed of supercritical carbon dioxide delivery pump P3 and improver 2 delivery pump P4.

The connection relationship among the various components is: the outlet of the delivery system 1 is connected to the Cs®C1®D1® pipeline, the inlet of the multi-port valve V1 is connected to the outlet of D1, and the multi-outlets of V1 are respectively connected to the traps in the trapping column system C2. The columns a, b, c, d, e, and f are connected, and the trapping columns a, b, c, d, e, f are respectively connected to multiple inlets of the multi-way valve V2, and the outlet of the multi-way valve V2 is connected to the inlet of D2. Connected, the outlet of D2 is connected with the back pressure valve B1, and B1 is connected with the delivery system 1 to form the delivery system 1 ® Cs ® C1 ® D1 ® V1 ® C2 (a, b, c, d, e, f) ® V2 ® D2 ® B1 ® Connection type of conveyor system 1. The outlet of the delivery system 2 is connected to the inlet of the multi-way valve V3, and the multi-outlets of V3 are respectively connected to the trapping columns a, b, c, d, e, f in the trapping column system C2, and the trapping columns a, b, c, d, e, f are respectively connected with multiple inlets of the multi-way valve V4, the outlet of the multi-way valve V4 is connected with the inlet of the multi-way valve V5, and the multiple outlets of the multi-way valve V5 are respectively connected with the separation column system C3 The inlets of the separation columns I, II, III, and IV are connected, the outlets of the separation columns I, II, III, and IV are respectively connected with the multi-inlets of the multi-way valve V6, the outlet of the multi-way valve V6 is connected with the detector D3, and the detector The outlet of D3 is connected to the inlet of the multi-way valve V7 in the component collection system, the outlet of V7 is connected to the component collector S, the outlet of the component collector S is connected to the inlet of the back pressure valve B2, and the outlet of B2 is connected to the delivery system 2 , forming a delivery system 2 ® V3 ® C2 (a, b, c, d, e, f) ® V4 ® V5 ® C3 (I, II, III, IV) ® V6 ® D3 ® V7 ® Fraction Collector S ® Connection method of B2® Conveyor System 2.

In the above supercritical fluid chromatograph, the supercritical carbon dioxide delivery pumps P1 and P3 can be constant flow pumps that can provide stable pressure and flow rate, or constant pressure pumps, etc. The transported fluid includes not only supercritical carbon dioxide, but also inorganic and organic solvents such as water, methanol, ethanol, acetonitrile, petroleum ether, hexane, methylene chloride, ethyl acetate, and chloroform.

In the above supercritical fluid chromatograph, the improver 1 delivery pump P2 and the improver 2 delivery pump P4 are constant flow pumps that can provide stable pressure and flow rate. The delivered improver includes water, methanol, ethanol, acetonitrile, petroleum ether, hexane, methylene chloride, ethyl acetate, chloroform and other inorganic and organic solvents.

In the above-mentioned supercritical fluid chromatograph, the sampling system Cs may be a fixed-volume sampling valve, a sampling pump, or a sampling column.

In the above-mentioned supercritical fluid chromatograph, the pre-separation column C1, trapping column C2, and separation column C3 can be made of glass, stainless steel, PEEK, etc. The filled filler can be normal phase chromatography filler, reversed phase chromatography filler, gel filler, etc., and the amount of filler can be analytical type, semi-preparative type, preparative type, etc.

In the above supercritical fluid chromatograph, the multi-port valves V1-V7 may be multi-position multi-port valves with one input and multiple outputs, or multi-position multi-port valves with multiple inputs and one output.

In the above-mentioned supercritical fluid chromatograph, the detectors D1, D2, and D3 may be flow-through detectors such as ultraviolet detectors, fluorescence detectors, differential refraction detectors, etc.; the detection cell of the detector is a high-pressure resistant detection cell.

In the above supercritical fluid chromatograph, the back pressure valves B1 and B2 may be back pressure valves that can provide stable system back pressure, or other electronic valve devices that can provide stable back pressure.

The present invention also provides the separation and purification method for preparing the supercritical fluid chromatograph, the steps are as follows:

(1) The mixed components are eluted and separated by the pre-separation system: the carbon dioxide is transported by the supercritical carbon dioxide transport pump P1, and the improver 1 is transported by the improver transport pump P2. After the two are combined and mixed, they pass through the sampling system Cs, and the Cs is carried The sample in the pre-separation column C1 is pre-separated, and the effluent of the pre-separation column C1 enters the detector D1 for detection. After passing through the multi-way valve V1, it is switched to each trapping column in the trapping column system C2, and the corresponding components are separated. The effluent is collected by the trapping column, and the effluent enters the detector D2 through the multi-way valve V2. The switching of each trapping column is jointly controlled according to the signals of the detectors D1 and D2, and the pre-separation and component trapping of the mixed components are completed. At this time, the flow route of the supercritical fluid in the pre-separation system is: delivery system 1 ® Cs ® C1 ® D1 ® V1 ® C2 (a, b, c, d, e, f) ® V2 ® D2 ® B1 ® delivery system 1. The composition change of the supercritical fluid in the pre-separation system is controlled by the flow rate or flow ratio of P1 and P2.

(2) The components trapped in each trapping column are eluted and separated by the main separation system: carbon dioxide is transported by supercritical carbon dioxide transport pump P3, improver 2 is transported by improver transport pump P4, and the two are combined and mixed After the multi-way valve V3 is switched, the trapped components in the trapping columns of the trapping column system C2 are eluted into the separation column system C3 through the multi-port valves V4 and V5 for separation, and the effluent enters the detection column through the multi-port valve V6. After detection by device D3, the corresponding fractions are collected by the component collection system, and the carbon dioxide is recycled. At this time, the flow route of the supercritical fluid in the main separation system is: Formation of the delivery system 2 ® V3 ® C2 (a, b, c, d, e, f) ® V4 ® V5 ® C3 (I, II, III, IV) ® V6 ® D3 ® V7 ® Fraction Collector ® B2 ® Delivery System 2. The change of supercritical fluid composition in the main separation system is controlled by the flow rate or flow ratio of P3 and P4.

The present invention is a preparation supercritical fluid chromatograph with a multi-way valve and a method for preparing separation and purification. Dehydration and recycling, low cost and strong practicability. In addition, the outstanding effect of the present invention is:

1. After the complex sample is separated by the pre-separation column, the capture is switched to capture, and now it is separated by the main separation system, which has super separation ability and is suitable for the separation and purification of complex samples.

2. The system is equipped with 3 detectors, which can ensure the precise control of component capture and collection switching, and improve the separation effect.

3. The trapping column and the separation column of the main separation system can be used in cross-combination, which is suitable for the separation and purification of components in a wide polar range from non-polar to strong polar components, and is suitable for separating more types of samples.

4. The system design is simple, the separation effect is good, it is suitable for the separation and purification of various complex mixtures, and has strong practicability.

Description of drawings

Fig. 1 is a schematic structural diagram of a supercritical fluid chromatograph according to an embodiment of the present invention.

Fig. 2 is a schematic diagram of the output signal of the detector D3 of the embodiment.

detailed description

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 As shown in Figure 1, a preparative supercritical fluid chromatograph with a multi-port valve is composed of a pre-separation system and a main separation system. The pre-separation system consists of delivery system 1, sample injection system Cs, pre-separation column C1, detector D1, multi-way valve V1, trapping column system C2, multi-way valve V2, detector D2, and back pressure valve B1; the main separation The system consists of delivery system 2, multi-way valve V3, trapping column system C2, multi-way valve V4, multi-way valve V5, separation column system C3, multi-way valve V6, detector D3, multi-way valve V7, component collector S, composed of back pressure valve B2. Delivery system 1 is composed of supercritical carbon dioxide delivery pump P1 and improver 1 delivery pump P2, and delivery system 2 is composed of supercritical carbon dioxide delivery pump P3 and improver 2 delivery pump P4.

The separation and purification method of supercritical fluid chromatography is as follows: (1) The mixed components are eluted and separated through the pre-separation system: carbon dioxide is transported by supercritical carbon dioxide delivery pump P1, improver 1 is delivered by improver delivery pump P2, and the two are combined After mixing, it passes through the sample injection system Cs, and the sample in the carrier Cs enters the pre-separation column C1 for pre-separation, and the effluent of the pre-separation column C1 enters the detector D1 for detection, and passes through the multi-port valve V1 to switch to the trapping column system C2 respectively. In each trapping column, the corresponding components are trapped by the trapping column, and the effluent enters the detector D2 through the multi-way valve V2. The switching of each trapping column is jointly controlled according to the signals of the detectors D1 and D2, and the mixed components are completed. pre-separation and component capture. At this time, the flow route of the supercritical fluid in the pre-separation system is: delivery system 1 ® Cs ® C1 ® D1 ® V1 ® C2 (a, b, c, d, e, f) ® V2 ® D2 ® B1 ® delivery system 1. The composition change of the supercritical fluid in the pre-separation system is controlled by the flow rate or flow ratio of P1 and P2.

(2) The components trapped in each trapping column are eluted and separated by the main separation system. The carbon dioxide is transported by the supercritical carbon dioxide transport pump P3, and the improver 2 is transported by the improver transport pump P4. After the two are combined and mixed, they are switched through the multi-way valve V3 to separate the trapped components in each trapping column of the trapping column system C2 through The multi-way valves V4 and V5 are eluted and enter the separation column system C3 for separation. After the effluent passes through the multi-way valve V6 and enters the detector D3 for detection, the corresponding fractions are collected by the component collection system, and the carbon dioxide is recycled. At this time, the flow route of the supercritical fluid in the main separation system is: Formation of the delivery system 2 ® V3 ® C2 (a, b, c, d, e, f) ® V4 ® V5 ® C3 (I, II, III, IV) ® V6 ® D3 ® V7 ® Fraction Collector S ®B2 ® Delivery System 2. The change of supercritical fluid composition in the main separation system is controlled by the flow rate or flow ratio of P3 and P4.

The number of trapping columns in the trapping column system, the number of separation columns in the separation column system, and the number of V channels of the multi-way valve in the preparative supercritical fluid chromatograph with a multi-way valve are not limited to the above-mentioned fixed numbers, but also include the realization of the present invention. Other numbers of situations.

The preparation supercritical fluid chromatograph with a multi-way valve of the present invention has strong separation ability and can complete separation tasks that are difficult for ordinary chromatographs, and at the same time, the separation cost is low because the supercritical fluid is recycled. According to the above connection and operation method, separate and purify the components in Salvia miltiorrhiza: according to the above connection and operation method, the pre-separation column C1 is a stainless steel column with a diameter of 25mm and a length of 200mm, and the column is filled with 10mm amino-bonded silica gel filler, and the six trapping columns are respectively Pack C-18, C-8, C-4, benzene, CN, amino, and diol-bonded silica gel fillers, and the four separation columns are filled with C-18, benzene, CN, and diol-bonded silica gel fillers. The pump 1 of the delivery system delivers carbon dioxide, and the pump 2 delivers methanol. Carbon dioxide and methanol adopt a gradient change. The gradient method is as follows: 0-30min, the flow ratio of carbon dioxide changes from 100% to 70%, and the flow ratio of methanol changes from 0 to 30%. According to the detection signal of detector D1, the eluted fractions are trapped into different trapping columns. In the delivery system, pump 3 delivers carbon dioxide, and pump 4 delivers ethanol. Carbon dioxide and ethanol adopt a gradient change. The gradient method is as follows: 0-60min, the flow ratio of carbon dioxide changes from 100% to 20%, and the flow ratio of ethanol changes from 0 to 20%. The components in the trapping column are eluted to the separation column for separation, and the detector D3 detects the effluent of the separation column, and collects each fraction according to the signal of D3 to obtain each component. Figure 2 shows the separation of the ethyl acetate extract of Salvia miltiorrhiza. During the separation process, the detector D3 records the output signal. According to the signal collection, 12 components can be obtained in one separation, and their purity is higher than 98% as determined by HPLC. The 12 components determined by modern spectrum analysis are: Tanshinone ⅡA, Tanshinone, New Purple Tanshinone A, Tanshinone Ⅰ, Dihydrotanshinone Ⅰ, Dihydroisotanshinone Ⅰ, Cryptotanshinone, Hydroxytanshinone ⅡA, Methylene Tanshinone , Methyl tanshinate, tanshinone B, nortanshinone.

Finally, it should be noted that the embodiments are only the best specific implementation of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it is still The technical solutions described in the foregoing embodiments may be modified, or some technical features thereof may be equivalently replaced. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (8)

1. A preparative supercritical fluid chromatograph with a multi-way valve is composed of a pre-separation system and a main separation system, and is characterized in that the pre-separation system is composed of a delivery system 1, a sampling system Cs, a pre-separation column C1, and a detector D1, multi-way valve V1, trapping column system C2, multi-way valve V2, detector D2, back pressure valve B1; the main separation system consists of delivery system 2, multi-way valve V3, trapping column system C2, multi-way valve V4, multi-way valve V5, separation column system C3, multi-way valve V6, detector D3, multi-way valve V7, component collector S, back pressure valve B2; delivery system 1 consists of supercritical carbon dioxide delivery pump P1 and improved agent 1 delivery pump P2, delivery system 2 is composed of supercritical carbon dioxide delivery pump P3 and improver 2 delivery pump P4; the outlet of delivery system 1 is connected to Cs®C1®D1® pipeline, and the inlet of multi-way valve V1 is connected to D1 The multiple outlets of V1 are respectively connected with the trapping columns a, b, c, d, e, f in the trapping column system C2, and the trapping columns a, b, c, d, e, f are connected with the trapping columns respectively Multiple inlets of the through valve V2 are connected, the outlet of the multi-way valve V2 is connected with the inlet of D2, the outlet of D2 is connected with the back pressure valve B1, and B1 is connected with the delivery system 1 to form the delivery system 1 ® Cs ® C1 ® D1 ® V1 ® C2 (a, b, c, d, e, f) ® V2 ® D2 ® B1 ® connection mode of delivery system 1; the outlet of delivery system 2 is connected to the inlet of multi-way valve V3, and the multi-ports of V3 are respectively connected to the trapping column The trapping columns a, b, c, d, e, and f in the system C2 are connected, and the trapping columns a, b, c, d, e, f are respectively connected with multiple inlets of the multi-way valve V4, and the multi-way valve The outlet of V4 is connected to the inlet of multi-way valve V5, and the multiple outlets of multi-way valve V5 are respectively connected to the inlets of separation columns I, II, III, and IV in the separation column system C3, and the separation columns I, II, III, and IV The outlets of the multi-way valve V6 are respectively connected to the multi-inlets of the multi-way valve V6, the outlet of the multi-way valve V6 is connected to the detector D3, the outlet of the detector D3 is connected to the inlet of the multi-way valve V7 in the component collection system, and the outlet of V7 is connected to the component The collector S is connected, the component collector S outlet is connected to the back pressure valve B2 inlet, and the B2 outlet is connected to the delivery system 2 to form a delivery system 2®V3® C2 (a, b, c, d, e, f)® V4 ® V5 ® C3 (I, II, III, IV) ® V6 ® D3 ® V7 ® Fraction Collector S ® B2 ® Delivery System 2 connection. 2. a kind of preparative supercritical fluid chromatograph with multi-port valve as claimed in claim 1, is characterized in that, described supercritical carbon dioxide delivery pump P1, P3 are constant pressure pumps. 3. a kind of preparative supercritical fluid chromatograph with multi-way valve as claimed in claim 1, is characterized in that, described improver 1 delivery pump P2, improver 2 delivery pump P4 can provide stable pressure and flow rate constant flow pump; the transported improvers include water, methanol, ethanol, acetonitrile, petroleum ether, hexane, methylene chloride, ethyl acetate, and chloroform. 4. a kind of preparative supercritical fluid chromatograph with multiport valve as claimed in claim 1, is characterized in that, described sampling system Cs is the sampling valve of fixed volume, sampling pump or sampling column. 5. a kind of preparative supercritical fluid chromatograph with multiport valve as claimed in claim 1, is characterized in that, described pre-separation column C1, trapping column C2, separation column C3 are glass, stainless steel, PEEK; Packing packing is normal phase chromatography packing, reverse phase chromatography packing, gel packing. 6. a kind of preparative supercritical fluid chromatograph with multi-way valve as claimed in claim 1, is characterized in that, described detector D1, D2, D3 are ultraviolet detector, fluorescence detector or differential refraction detector; The detection cell of the detector is a high pressure resistant detection cell. 7. A kind of preparative supercritical fluid chromatograph with multiport valve as claimed in claim 1, is characterized in that, described back pressure valve B1, B2 is the back pressure valve that can provide stable system back pressure. 8. a kind of separation and purification method of the preparation supercritical fluid chromatograph of band multiport valve as claimed in claim 1, is characterized in that, step is as follows: (1) The mixed components are eluted and separated by the pre-separation system: the carbon dioxide is transported by the supercritical carbon dioxide transport pump P1, and the improver 1 is transported by the improver transport pump P2. After the two are combined and mixed, they pass through the sampling system Cs, and the Cs is carried The sample in the pre-separation column C1 is pre-separated, and the effluent of the pre-separation column C1 enters the detector D1 for detection. After passing through the multi-way valve V1, it is switched to each trapping column in the trapping column system C2, and the corresponding components are separated. The trapping column traps, and the effluent enters the detector D2 through the multi-way valve V2, and the switching of each trapping column is jointly controlled according to the signals of the detectors D1 and D2, and the pre-separation and component capture of the mixed components are completed; at this time The supercritical fluid flow route of the pre-separation system is: delivery system 1 ® Cs ® C1 ® D1 ® V1 ® C2 (a, b, c, d, e, f) ® V2 ® D2 ® B1 ® delivery system 1; pre-separation system The change of supercritical fluid composition is controlled by the flow rate or flow ratio of P1 and P2; (2) The components trapped in each trapping column are eluted and separated by the main separation system: carbon dioxide is transported by supercritical carbon dioxide transport pump P3, improver 2 is transported by improver transport pump P4, and the two are combined and mixed After the multi-way valve V3 is switched, the trapped components in the trapping columns of the trapping column system C2 are eluted into the separation column system C3 through the multi-port valves V4 and V5 for separation, and the effluent enters the detection column through the multi-port valve V6. After the detection by device D3, the corresponding fractions are collected by the component collection system, and the carbon dioxide is recycled; at this time, the flow route of the supercritical fluid in the main separation system is: the formation of the delivery system 2 ® V3 ® C2 (a, b, c, d, e, f ) ® V4 ® V5 ® C3 (I, II, III, IV) ® V6 ® D3 ® V7 ® Fraction Collector S ® B2 ® Conveyor System 2; main separation system supercritical fluid composition changes by P3, P4 flow rate or flow rate proportional control.