Liquid chromatography analysis device, liquid chromatography analysis method and use thereof

Abstract

The present invention provides a liquid chromatography analysis device and a liquid chromatography analysis method and use, wherein the liquid chromatography analysis device comprises a liquid chromatograph and a solvent effect suppression chromatographic column (1); the liquid chromatograph comprises a liquid reservoir (2), a detector (3) and an infusion pipeline (4); the head end of the infusion pipeline (4) is connected to the liquid reservoir (2), and the tail end of the infusion pipeline (4) is connected to the detector (3); along the liquid injection direction, the infusion pipeline (4) is sequentially provided with a solvent effect suppression chromatographic column (1) and an analytical chromatographic column (5). The liquid chromatography analysis device can better suppress the phenomena of ghost peaks, peak front extension or peak tailing caused by solvent effects, thereby improving the accuracy of chromatographic analysis.

Description

Technical Field

[0001] The invention relates to the field of high performance liquid chromatography technical analysis, and in particular to a liquid chromatography analysis device and a liquid chromatography analysis method and application. Background Art

[0002] In chromatographic analysis, when the solvent strength of the sample diluent is greater than that of the mobile phase, it will cause abnormal sample peaks after injection, resulting in poor peak shape and affecting the analysis results. This phenomenon is common and is called solvent effect. Solvent effect is common in drug analysis and affects the accuracy of the analysis results.

[0003] Chinese patent CN104535699B proposes a peak-front delay suppressor, which increases the dead volume before the column by adding a peak-front delay suppressor with a cavity structure as the core on the infusion pipeline between the analytical chromatographic column and the injector, so as to further dilute the sample solvent in the cavity by the mobile phase, completing the online dilution process. This method of directly increasing the dead volume before the column will directly affect the broadening of the spectral band, resulting in a reduction in the separation column efficiency, and is also prone to solvent stratification and ghost peaks.

[0004] Therefore, it is necessary to develop a suppressor column that can suppress the solvent effect without affecting the chromatographic peak. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a liquid chromatography analysis device and a liquid chromatography analysis method and use, so as to solve the problems in the prior art.

[0006] To achieve the above objectives and other related objectives, the present invention is achieved through the following technical solutions.

[0007] The invention discloses a liquid chromatography analysis device, comprising a liquid chromatograph and a solvent effect suppression chromatographic column; the liquid chromatograph comprises a liquid reservoir, a detector and an infusion pipeline; the head end of the infusion pipeline is connected to the liquid reservoir, and the tail end of the infusion pipeline is connected to the detector; along the liquid injection direction, the solvent effect suppression chromatographic column and the analysis chromatographic column are sequentially arranged on the infusion pipeline; a branch of the infusion pipeline is arranged upstream of the solvent effect suppression chromatographic column, and an injector is connected to the branch of the infusion pipeline; the solvent effect suppression chromatographic column is filled with an inert filler.

[0008] Preferably, the material of the inert filler is selected from one or more of glass, silicon or silicon dioxide, ceramic and C18. The inert filler will not react or be corroded when in contact with the organic solvent, thus avoiding the generation of impurities that affect the analysis effect.

[0009] Preferably, the inert filler of ceramic material is selected from one or more of alumina ceramics, zirconia ceramics, silicon carbide ceramics, boron nitride ceramics and glass ceramics.

[0010] Preferably, the inert filler of glass material is selected from one or more of silicate glass, soda-lime glass, borosilicate glass and silicon carbide glass.

[0011] If the particle size of the chromatographic filler is too small, the back pressure will increase greatly, which may cause delay or advance, and is not suitable for the transplantation of the method; if it is too large, the adsorption effect will be weakened and the mixing effect will be limited. Preferably, the particle size of the inert filler is 0.03-3.5 mm. For example, it can be 0.03-0.5 mm, 0.5-1.5 mm, 1.5-2.5 mm, 2.5-3.5 mm.

[0012] Preferably, the shape of the inert filler is selected from one or both of spherical and elliptical shapes.

[0013] In an optional embodiment, the inert filler is a porous structure and / or a dense structure, and the average pore size of the porous structure is 10 to 200 Å, and the porosity is 10 to 90%.

[0014] The surface of the inert filler has an adsorption effect, which can avoid solvent stratification to a certain extent, and produce the same effect as the pre-column through two-phase distribution, so that the peak time of the sample is slightly advanced, which can save time and cost.

[0015] Preferably, the inert filler is glass wool, and the diameter of the glass wool is 0.03-3.5 mm and the length is 1-100 mm.

[0016] Preferably, the inner diameter of the chromatographic column for suppressing solvent effect is 0.03-10 mm, and the length is 5-300 mm. For example, the inner diameter of the chromatographic column for suppressing solvent effect can be 0.03-1 mm, 1-1.5 mm, 1.5-1.8 mm, 1.8-2.5 mm, 2.5-3.5 mm, 3.5-4.5 mm, 4.5-5.5 mm, 5.5-6.5 mm, 6.5-7.5 mm, 7.5-8.5 mm, 8.5-9.5 mm, 9.5-10 mm, and the inner diameter of the chromatographic column for suppressing solvent effect can be 5-25 mm, 25-30 mm, 30-50 mm, 50-100 mm, 100-200 mm, 200-300 mm.

[0017] Preferably, the inner wall of the chromatographic column for suppressing solvent effect is provided with spiral patterns, which on one hand produce mixing phenomenon, and on the other hand increase the specific surface area in the chromatographic column for suppressing solvent effect, avoid solvent stratification to a certain extent, and help suppress solvent effect.

[0018] Preferably, the solvent effect suppression chromatographic column comprises a column tube, a filter cap and a column head; the filter cap is sleeved on both ends of the column tube; the column head is sleeved on the filter cap and connected to the column tube. Specifically, the connection mode is a fixed connection or a detachable connection, and the detachable connection is a threaded connection.

[0019] The present invention also discloses a liquid chromatography analysis method, which uses the liquid chromatography analysis device as described above to perform quantitative analysis and / or qualitative analysis on a sample solution, wherein the elution capacity of the sample diluent in the sample solution is different from that of the mobile phase.

[0020] Preferably, the mixing of the sample solution and the mobile phase is promoted by the back-mixing effect brought about by the chromatographic column for suppressing solvent effects.

[0021] The present invention also discloses a use of the liquid chromatography analysis device as described above for improving the analysis result defects caused by solvent effect during liquid chromatography analysis.

[0022] Preferably, the analysis result defects include one or more of ghost peaks, peak front extensions and peak tailings.

[0023] Preferably, improving the defects of the analysis results also includes increasing the theoretical plate number and / or half peak width of the chromatographic peak.

[0024] Preferably, the solvent effect refers to that during liquid chromatography analysis, the elution capacity of the sample diluent in the sample solution is greater than that of the mobile phase.

[0025] Preferably, the sample diluent is selected from one or more of water, acetonitrile, tetrahydrofuran, alcohols, alkanes, aromatics and esters; and the mobile phase is selected from an aqueous phase system or an organic phase system.

[0026] More preferably, the alcohol is methanol.

[0027] More preferably, the aqueous phase system is a methanol-water system or an acetonitrile-water system.

[0028] Preferably, the solute of the sample solution is one of tranexamic acid tablets, caffeine and schisandra chinensis alcohol A.

[0029] The invention discloses a liquid chromatography analysis device and a liquid chromatography analysis method and use, which have the following beneficial effects: in a liquid reservoir, a mobile phase and a sample solution enter an infusion pipeline respectively via an injector and a branch, and are mixed in the infusion pipeline. When the mobile phase and the sample solution flow to a chromatographic column for suppressing solvent effect, a back-mixing phenomenon occurs due to the blocking effect of an inert filler therein. The back-mixing phenomenon can achieve the following three effects: on the one hand, accelerated mixing of a sample diluent and a mobile phase is achieved, so that the mixing is more uniform and the mixing efficiency is improved; on the other hand, the introduction of the inert filler increases the specific surface area in the chromatographic column for suppressing solvent effect compared with the hollow column in the prior art, which is beneficial to the redistribution of solutes between the two phases, thereby avoiding the occurrence of solvent stratification caused by too strong a dilution effect; and on the third hand, the dead volume in front of the analytical chromatographic column can be reduced, avoiding the disadvantages caused by a large dead volume in front of the analytical chromatographic column, and improving the column efficiency.

[0030] When the liquid chromatography analysis device disclosed in the present application is used for qualitative and quantitative analysis, ghost peaks, peak front extensions or peak tailing caused by solvent effects can be better suppressed, thereby improving the accuracy of chromatographic analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Shown is a schematic structural diagram of the solvent effect suppression chromatographic column of the present invention.

[0032] Figure 2 Shown is a cross-sectional view of a chromatography column for suppressing solvent effects according to the present invention.

[0033] Figure 3 Shown is a schematic diagram of a liquid chromatography analysis device of the present invention.

[0034] Figure 4 Shown is the liquid chromatogram of Example 1 of the present invention.

[0035] Figure 5 Shown is the liquid chromatogram of Comparative Example 1 of the present invention.

[0036] Figure 6 Shown is the liquid chromatogram of Example 2 of the present invention.

[0037] Figure 7 Shown is the liquid chromatogram of Comparative Example 2 of the present invention.

[0038] Figure 8 Shown is the liquid chromatogram of Example 3 of the present invention.

[0039] Fig. 9 Shown is the liquid chromatogram of Comparative Example 3 of the present invention.

[0040] Description of reference numerals:

[0041] 1. Chromatographic columns to suppress solvent effects

[0042] 11 column tube

[0043] 12 filter caps

[0044] 13. Stigma

[0045] 2 Reservoirs

[0046] 3 detectors

[0047] 4 Infusion lines

[0048] 5Analytical Column

[0049] 6 Injectors. DETAILED DESCRIPTION

[0050] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0051] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention. The test methods in the following examples without specifying specific conditions are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

[0052] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0053] In the present application, a specific Figure 3 The liquid chromatography analysis device shown comprises a liquid chromatograph and a solvent effect suppression chromatographic column 1; the liquid chromatograph comprises a liquid reservoir 2, a detector 3 and an infusion pipeline 4; the head end of the infusion pipeline 4 is connected to the liquid reservoir 2, and the tail end of the infusion pipeline 4 is connected to the detector 3; along the liquid injection direction, the solvent effect suppression chromatographic column 1 and the analysis chromatographic column 5 are sequentially arranged on the infusion pipeline 4; a branch of the infusion pipeline 4 is arranged upstream of the solvent effect suppression chromatographic column 1, and an injector 6 is connected to the branch of the infusion pipeline 4; the solvent effect suppression chromatographic column 1 is filled with an inert filler.

[0054] The structure of the chromatographic column for suppressing solvent effect is as follows: Figure 2 and Figure 3 As shown: the solvent effect suppression chromatographic column 1 comprises a column tube 11, a filter cap 12 and a column head 13; the filter cap 12 is sleeved on both ends of the column tube 11; the column head 13 is sleeved on the filter cap 12 and connected to the column tube 11. More specifically, the inner wall of the column tube 11 is provided with spiral patterns.

[0055] The filling method of the chromatographic column for inhibiting solvent effect is as follows: first, insert the filter cap 12 at one end of the column tube 11, tighten the column head 13, then install the inert filler, insert the filter cap 12 at the other end, and tighten the column head 13. After testing the pressure resistance, fill the cavity with methanol and tighten the two ends with PEEK plugs.

[0056] In the prior art, when a chromatographic instrument is used for qualitative and quantitative analysis, for a determined analyte, the stationary phase chromatographic column is generally screened to avoid problems such as ghost peaks, peak front extensions or peak tailing, thereby improving the accuracy of qualitative and quantitative analysis, and thereby screening out a chromatographic column with relatively no obvious defects in peak shape. However, even so, it is still impossible to further or completely eliminate these problems caused by the solvent effect. With the aid of the chromatographic analysis device containing a chromatographic column that suppresses the solvent effect in the present application, the problems of abnormal peaks such as ghost peaks, peak front extensions, and peak tailing can be further improved through the mixing back effect. Combined with the chemical action of C18, the problems of abnormal peaks such as ghost peaks, peak front extensions, and peak tailing can be further improved. The following are specific examples and explanations, and the technical concept of the present invention is not limited to the following specific chromatographic columns and test systems.

[0057] Example 1

[0058] This embodiment provides a liquid chromatography analysis device, including a liquid chromatograph and a chromatographic column for suppressing solvent effects; the chromatographic column for suppressing solvent effects is arranged between the analytical chromatographic column and the injector of the liquid chromatograph.

[0059] Chromatographic column: ODS-C18, 4.6*250mm / 5μm.

[0060] Columns to suppress solvent effects:

[0061] Inert filler: ceramic balls, particle size 0.5-1.3 mm;

[0062] The inner diameter of the column tube is 1.5 mm and the length of the column tube is 30 mm.

[0063] This embodiment also provides a liquid chromatography analysis method for tranexamic acid tablets, and the analysis method is as follows:

[0064] Testing conditions:

[0065] Mobile phase A: 0.23% sodium dodecyl sulfate solution, mobile phase B: methanol, isocratic elution, mobile phase A: mobile phase B = 60:40;

[0066] Detection wavelength: 220nm; column temperature: 30℃; flow rate: 1.2ml / min; injection volume: 20μl.

[0067] Sample solution preparation: Add sample tranexamic acid tablets into sample diluent water to dissolve and prepare sample solution.

[0068] The prepared sample solution was detected and analyzed using the above-mentioned liquid chromatography analysis device, and the analysis spectrum is shown in Figure 4 .

[0069] Comparative Example 1

[0070] This comparative example is the comparative example of Example 1, except that when the sample solution is detected, the solvent effect suppression chromatographic column is not used, and only the liquid chromatograph is used for analysis. Figure 5 .

[0071] By comparison Figure 4 and Figure 5 It can be seen that after the solvent effect suppression chromatographic column was added to the detection of tranexamic acid tablets, the components in the sample were effectively separated; when it was not installed, the chromatogram had ghost peaks and abnormal peaks.

[0072] Example 2

[0073] This embodiment provides a liquid chromatography analysis device, including a liquid chromatograph and a chromatographic column for suppressing solvent effects; the chromatographic column for suppressing solvent effects is arranged between the analytical chromatographic column and the injector of the liquid chromatograph.

[0074] Chromatographic column: SB-C18, 4.6*150mm / 3.5μm.

[0075] Columns to suppress solvent effects:

[0076] Inert filler: glass balls, particle size 0.5 to 1.3 mm.

[0077] The inner diameter of the column tube is 1.5 mm and the length of the column tube is 29 mm.

[0078] This embodiment also provides a liquid chromatography analysis method for caffeine, and the analysis method is as follows:

[0079] Testing conditions:

[0080] Mobile phase A: methanol, mobile phase B: water;

[0081] Isocratic elution, mobile phase A: mobile phase B = 30:70; elution time: 5 minutes;

[0082] Detection wavelength: 205nm; column temperature: 30℃; flow rate: 1.0ml / min; injection volume: 20μl.

[0083] Sample solution preparation: add sample caffeine into sample diluent methanol to dissolve and prepare sample solution.

[0084] The prepared sample solution was detected and analyzed using the above-mentioned liquid chromatography analysis device, and the analysis spectrum is shown in Figure 6 .

[0085] Comparative Example 2

[0086] This comparative example is the comparative example of Example 2, except that when the sample solution is detected, the solvent effect suppression chromatographic column is not used, and only the liquid chromatograph is used for analysis. Figure 7 .

[0087] By comparison Figure 6 and Figure 7 It can be seen that after the installation of the chromatographic column with suppressed solvent effect in the caffeine detection, the front extension of the chromatographic peak was significantly improved, the peak width (5%) was improved from 0.672 to 0.550, and the peak height was changed from 404.2 to 421.37.

[0088] Example 3

[0089] This embodiment provides a liquid chromatography analysis device, including a liquid chromatograph and a chromatographic column for suppressing solvent effects; the chromatographic column for suppressing solvent effects is arranged between the analytical chromatographic column and the injector of the liquid chromatograph.

[0090] Chromatographic column: 5TC-C18, 4.6*150mm / 5μm.

[0091] Columns to suppress solvent effects:

[0092] Inert filler: silicon balls, particle size 0.5 to 1.5 mm.

[0093] The inner diameter of the column tube is 1.8 mm and the length of the column tube is 30 mm.

[0094] This embodiment also provides a liquid chromatography analysis method for Schisandrin A, and the analysis method is as follows:

[0095] Testing conditions:

[0096] Mobile phase A: methanol, mobile phase B: water;

[0097] Isocratic elution, mobile phase A: mobile phase B = 62:38; elution time: 30 minutes;

[0098] Detection wavelength: 250nm; column temperature: 26℃; flow rate: 1.0ml / min; injection volume: 20μl.

[0099] Preparation of sample solution: add the sample schisandrae alcohol A into the sample diluent methanol to dissolve it and prepare the sample solution.

[0100] The prepared sample solution was detected and analyzed using the above-mentioned liquid chromatography analysis device, and the analysis spectrum is shown in Figure 8 .

[0101] Comparative Example 3

[0102] This comparative example is the comparative example of Example 2, except that when the sample solution is detected, the solvent effect suppression chromatographic column is not used, and only the liquid chromatograph is used for analysis. Fig. 9 .

[0103] By comparison Figure 8 and Fig. 9 It can be seen that after adding a chromatographic column that suppresses solvent effects in the detection of schisandra chinensis alcohol A, the tailing factor of the chromatographic peak is improved to a certain extent, from 0.82 to 1.05, the peak front extension is improved, the symmetry of the peak shape is improved, and the number of plates is improved from 10202 to 11346, which is also a certain improvement.

[0104] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A liquid chromatography analysis device, characterized in that: The invention comprises a liquid chromatograph and a solvent effect suppression chromatographic column (1); the liquid chromatograph comprises a liquid reservoir (2), a detector (3) and an infusion pipeline (4); the head end of the infusion pipeline (4) is connected to the liquid reservoir (2), and the tail end of the infusion pipeline (4) is connected to the detector (3); along the liquid injection direction, the solvent effect suppression chromatographic column (1) and the analytical chromatographic column (5) are sequentially arranged on the infusion pipeline (4); a branch of the infusion pipeline (4) is arranged upstream of the solvent effect suppression chromatographic column (1), and the branch of the infusion pipeline (4) is connected to an injector (6); the solvent effect suppression chromatographic column (1) is filled with an inert filler; the particle size of the inert filler is 0.5-1.5 mm.

2. The liquid chromatography analysis device according to claim 1, characterized in that: The material of the inert filler is selected from one or more of glass, silicon or silicon dioxide, ceramic and C18.

3. The liquid chromatography analysis device according to claim 1, characterized in that: The shape of the inert filler is selected from one or both of spherical and elliptical shapes.

4. The liquid chromatography analysis device according to claim 1, characterized in that: The inner diameter of the solvent effect suppression chromatographic column (1) is 1.5-2.5 mm and the length is 29-300 mm; And / or, the inner wall of the chromatographic column (1) for suppressing solvent effect is provided with spiral patterns.

5. A liquid chromatography analysis method, characterized in that: The liquid chromatography analysis device according to any one of claims 1 to 4 is used to perform quantitative analysis and / or qualitative analysis on a sample solution, wherein the elution capacity of the sample diluent in the sample solution is different from that of the mobile phase.

6. Use of the liquid chromatography analysis method according to claim 5 for improving the analysis result defects caused by solvent effects during liquid chromatography analysis.

7. The use according to claim 6, characterized in that The analysis result defects include one or more of ghost peaks, peak front extensions and peak tailings.

8. The use according to claim 6, characterized in that Improving the analytical results also includes increasing the number of theoretical plates of the chromatographic peak and / or reducing the half-peak width.

9. The use according to claim 8, characterized in that The sample diluent is selected from one or more of water, acetonitrile, tetrahydrofuran, alcohols, alkanes, aromatics and esters; and the mobile phase is selected from an aqueous phase system or an organic phase system.

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