WO2023155629A1 - 一种多模式抗基质的萃取柱 - Google Patents

一种多模式抗基质的萃取柱 Download PDF

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WO2023155629A1
WO2023155629A1 PCT/CN2023/070265 CN2023070265W WO2023155629A1 WO 2023155629 A1 WO2023155629 A1 WO 2023155629A1 CN 2023070265 W CN2023070265 W CN 2023070265W WO 2023155629 A1 WO2023155629 A1 WO 2023155629A1
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column
extraction column
filler
inner diameter
mode anti
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PCT/CN2023/070265
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English (en)
French (fr)
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王峰
郁凯
王志明
吴开电
张国余
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湖南德米特仪器有限公司
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Priority to CN202380009521.0A priority Critical patent/CN116997793A/zh
Publication of WO2023155629A1 publication Critical patent/WO2023155629A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column

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  • the invention relates to the field of liquid chromatography columns, in particular to a multi-mode anti-matrix extraction column.
  • Chromatographic columns are often used for sample pretreatment in pure extraction systems, and the chromatographic columns used for sample processing are called extraction columns.
  • the extraction column is mainly used for the primary separation, purification and concentration of the sample. During this process, the extraction column is subjected to pollution from various impurities in the sample, and is easy to accumulate at the inlet end, resulting in high pressure, which seriously affects the service life of the extraction column.
  • the structure of the extraction column usually includes a column tube, a sieve plate and a column cap.
  • the sieve plate in the column cap is generally a stainless steel sintered mesh plate with a pore size of 0.1 ⁇ m-3 ⁇ m.
  • the extraction column usually bears a larger injection volume than ordinary chromatographic columns, and the extraction column often has to withstand a sample injection volume of 100 microliters or more, so the extraction column is more prone to clogging. Generally, the lifespan of the extraction column for sewage treatment or biomass samples is difficult to exceed 200 needles.
  • the existing solution is generally to ultrasonically clean the extraction column or replace the column head packing to repair the column head, but this requires a high degree of specialization, and the ability to solve problems is limited.
  • CN02106928.X discloses a conical high performance liquid chromatography preparative column. It is mainly composed of a column head, a column body and a column tail, and is a tapered cylinder whose inner diameter (2R) of the inlet of the column head is larger than the inner diameter (2r) of the outlet of the column tail.
  • the column head consists of a conical liquid flow guide groove, a distribution plate and a sieve plate to form a liquid flow distribution system.
  • the distribution plate is composed of a plurality of radial liquid flow channels, concentric liquid flow collection channels and seepage holes.
  • the column length L 5cm ⁇ 100cm.
  • the cone angle is in the range of 1° ⁇ 20°, and the cone can be filled Various types of chromatographic separation media.
  • This tapered preparative column can significantly increase column efficiency and sample loading capacity, and the dilution effect on the separated components is lower than that of cylindrical chromatographic columns.
  • the processing of this chromatographic column is complicated, and it is not a conventional cylindrical chromatographic column.
  • CN201110071563.6 discloses a parabolic high performance liquid chromatography preparative column, the capillary liquid chromatography preparative column comprises: a column head located at the upper part of the column, a parabolic tubular column and a column tail located at the lower part of the column; wherein, the parabolic The inlet end and the outlet end of the tubular column have a special seal extension respectively, and the column is designed in a parabolic shape, and the parabolic equation corresponds to the flow rate, so that a perfect plug-shaped chromatographic band can be obtained, and the column efficiency of the chromatographic column is greatly improved;
  • the inner diameter of the column gradually decreases, which increases the sample loading capacity of the chromatographic column and has a certain amount of enrichment effect on the sample. This kind of chromatographic column should only stay in the theoretical stage, and the processing cost is high.
  • the purpose of the first invention is to provide an extraction column, which can solve the problem that the extraction column is easily blocked, is easy to clean, and improves the service life of the chromatographic column.
  • the second object of the invention is to provide an extraction column, which can be adapted to large-volume sample injection, and still has good column efficiency when large-volume sample injection is performed. And as the injection volume increases, the peak area increases linearly.
  • the large-volume injection refers to an injection volume of 200 ⁇ L or more.
  • the third object of the invention is to provide an extraction column, which rectifies the manifold and flow velocity of the fluid entering the extraction column, so that the target objects in the sample are rearranged after rectification, and move forward in unison. After entering the detector, the obtained A relatively normal peak shape maintains high column efficiency.
  • the fourth object of the invention is to provide an extraction column, which can be adapted to the extraction and separation of complex samples, and when separating complex samples, the separation effect is good and the column efficiency is still good.
  • a multi-mode anti-matrix extraction column with the inflow end of the mobile phase as the upper end, from top to bottom, the extraction column includes a column head, a front end column cap, a column tube, an end column cap and a column head connected in sequence, and the inside of the column tube
  • the cavity is cylindrical, and the column tube is divided into an upper section and a lower section, the inner diameter of the upper section is greater than the inner diameter of the lower section; the upper end of the front end column cap is provided with a pressure variable pore structure, and the pressure variable pore structure includes upper, middle and lower layers, from From top to bottom are elastic soft plate, polymer filter membrane and supporting orifice plate.
  • the elastic soft plate includes a fixed ring and an elastic sieve plate located in the fixed ring.
  • the material of the fixing ring is preferably a PEEK ring, the material is polyether ether ketone, and the material of the PE sieve plate is preferably polyethylene.
  • the material has the characteristics of high strength and low dissolution, and the thickness of the PE sieve plate is between 0.5mm-8mm.
  • the elastic soft board has a thickness of 0.1mm-10mm, preferably 0.5mm-8mm, and a pore size of 5 microns-10 microns.
  • the polymer filter membrane has a thickness of 0.05mm-3mm, preferably 0.1-2mm, more preferably 0.1mm-0.5mm, and can retain substances with a molecular weight > 3000Da, more preferably substances with a molecular weight > 3000Da.
  • the polymer filter membrane is made of a material capable of withstanding organic and acid-base reagents, such as polyethersulfone resin or polypropylene.
  • the inlet end of the column tube is provided with a front-end column cap
  • the outlet end of the column tube is provided with a terminal column cap
  • the inlet end and the outlet end of the column tube are arranged in the column head
  • the column head has internal threads
  • the column tube and the column head are connected by threads .
  • the structure of the supporting orifice plate can be exactly the same as that of the elastic soft plate.
  • the length of the upper section accounts for 1/5-3/10 of the entire length of the column tube extraction column.
  • the length of the upper section accounts for 1/5-3/10 of the entire length of the column tube extraction column.
  • the ratio of the inner diameter of the upper section to the lower section is 1.2-1.5. After a lot of research by the inventor, it is more suitable within this range, which can be adapted to large-volume sample injection, while maintaining overall good column efficiency, without greatly increasing the cost and analysis time.
  • the outer circumference of the column tube is provided with a first connecting section and a first connecting section for connecting with the column head, and the outer diameter of the first connecting section and the first connecting section is greater than the outer diameter of the column tube.
  • the column head is provided with an inflow hole for the mobile phase to flow in or an outflow hole for the mobile phase to flow out.
  • a non-equal diameter extraction column, the column tube and the column head are made of stainless steel.
  • the upper section and the lower section are filled with different fillers respectively.
  • the upper section can be filled with reverse and ionic fillers
  • the lower section can also be filled with reverse and ionic fillers
  • the particle size of the filler in the upper section is larger than that of the lower section, and the adsorption effect of the upper section is better than that of the lower section. filler.
  • the two fillers are separated by a metal sieve plate.
  • the second technical solution of the present invention is:
  • the column tube is filled with a stationary phase, and a polymer liquid-impermeable membrane is provided in the stationary phase near the inflow end of the mobile phase, and the outer diameter of the polymer liquid-impermeable membrane is smaller than that of the column.
  • the inner diameter of the tube, and the central axis of the polymer liquid-impermeable membrane coincides with the central axis of the column tube, and the polymer liquid-impermeable membrane is arranged parallel to the cross-section of the column tube.
  • the material of the polymer liquid-impermeable membrane can be any liquid-impermeable membrane as long as it is a fluorine-containing polymer material, preferably a polyester film or a nylon film.
  • the gap between the outer periphery of the polymer liquid-impermeable membrane and the inner wall of the column tube is less than 1/4 of the inner diameter of the column tube, and it is reasonable to control it within this range.
  • the gap between the outer periphery of the polymer liquid-impermeable membrane and the inner wall of the column tube is 0.6-3. According to the inventor's extensive research, the rectification effect is better when the gap is 0.6-3.
  • a metal sieve plate is arranged above the polymer liquid-impermeable membrane for supporting the polymer liquid-impermeable membrane and preventing its deformation.
  • the structure of the supporting orifice plate can be exactly the same as that of the elastic soft plate.
  • the third technical solution of the present invention is:
  • the packing in the stationary phase is at least three layers, and different packings are filled respectively, and the different packing layers are separated by metal sieve plates.
  • the stationary phase includes an adsorption layer, a focusing layer and a separation layer.
  • the focusing layer is selected from phenyl bonded silica gel packing or octadecylsilane bonded silica gel packing;
  • the separation layer is selected from cyano bonded silica gel packing, sulfonic acid cationic polymer packing or octadecyl Silane bonded silica filler.
  • the depth of the adsorption layer is between 1-30 mm
  • the depth of the focusing layer is between 1-20 mm
  • the depth of the separation layer is between 5-50 mm.
  • the particle size of the filler in the adsorption layer is 5 ⁇ m-20 ⁇ m
  • the particle size of the filler in the focusing layer is 2 ⁇ m-5 ⁇ m
  • the particle size of the filler in the separation layer is 2 ⁇ m-10 ⁇ m.
  • a pressure-variable pore soft plate and a polymer membrane are provided in the column head at the inlet end.
  • the background pressure corresponding to the one-dimensional mobile phase of the extraction column is generally about 2MPa, which is not enough to form a high pressure.
  • High pressure is formed, generally reaching above 10MPa, the elastic soft plate shrinks downward, the pore size becomes smaller, intercepts and filters particles, finer impurities are intercepted and filtered by the polymer membrane, and the supporting orifice supports the elastic soft plate and the polymer membrane;
  • the pressure decreases, the elastic soft plate deforms and expands, and the impurities intercepted by the organic polymer filter membrane and the elastic soft plate are reversely eluted and discharged.
  • An extraction column with pressure-variable pores can more easily reversely wash out impurities at the inlet end of the extraction column, thereby improving the service life of the extraction column.
  • the stationary phase loses its adsorption capacity, making the target in the sample do various irregular movements, and the closest to the chromatographic column
  • the flow velocity in the center is the largest, and the flow velocity near the chromatographic column is small and uneven.
  • the present invention shapes the fluid by adding a polymer membrane with deformation characteristics. The fluid first contacts the polymer impermeable membrane.
  • H is the theoretical plate height
  • A, B, and C are constants
  • u represents the linear velocity of the mobile phase in the chromatographic column.
  • the linear velocity of the mobile phase in the chromatographic column is inversely proportional to the square of the inner diameter of the chromatographic column, that is to say, the smaller the inner diameter of the chromatographic column, the greater the linear velocity of the mobile phase in the chromatographic column, and the column efficiency worse.
  • the inventor After a lot of research by the inventor, the inventor has developed a multi-section non-equal-diameter chromatographic column, which can not only meet the requirements of large-volume sample injection, but also maintain good column efficiency, and can also save costs and the analysis time is appropriate.
  • the inner diameter of the upper section of the multi-section non-equal-diameter chromatographic column is larger than the inner diameter of the lower section.
  • the linear velocity of the mobile phase in the upper section of the chromatographic column is small, which is conducive to separation and aggregation, and can be well adapted to large-volume sample injection, while maintaining overall good column efficiency.
  • the inner diameter of the upper section is relatively large and the pressure is low, which can reduce the damage to the chromatographic column during large-volume sample injection and improve the service life of the extraction column.
  • the inner diameter of the lower section of the chromatographic column does not need to be as large as that of the upper section, and the inner diameter can be reduced, and the flow rate of the mobile phase will be increased compared with the upper section, which can reduce the analysis time, reduce the amount of packing, and save costs.
  • the upper section and the lower section are filled with different packing materials to meet the separation or enrichment requirements at different flow rates.
  • the upper section can be filled with SCX ion packing with strong adsorption.
  • the lower section can be filled with fillers such as C18, C8, phenyl and naphthyl. After optimization of the internal packing, the enrichment effect is better.
  • the particle size of the upper filler is larger than that of the lower filler, the upper particle size is generally selected between 5-50 ⁇ m, and the lower particle size is generally selected between 2 ⁇ m-10 ⁇ m: the enrichment ability of the upper filler for the target It is greater than the enrichment ability of the lower filler for the target substance.
  • the sample of the present invention After the sample of the present invention enters the chromatographic column through the mobile phase, some ionic compounds are initially adsorbed in the adsorption layer by the force of ions, and the target is eluted from the adsorption layer through the mobile phase, and after being focused by an auxiliary mobile phase with a certain flow rate, The flow state of the sample is rearranged. After reaching the focusing layer, some other impurities are effectively adsorbed by the non-polar force between functional groups, surface electric field force, etc., and the target objects are rearranged and separated effectively through the separation layer.
  • the advantage of the present invention is:
  • the extraction column of the present invention has a pressure change pore structure, which can more easily reversely clean the impurities at the inlet end of the extraction column, thereby improving the service life of the extraction column.
  • the extraction column of the present invention has a rectification structure, which rectifies the manifold and flow velocity of the fluid entering the chromatographic column, so that the sample is dispersed more evenly after rectification, which is more conducive to the separation of samples, the column efficiency is improved, and the peak shape of the extraction column is improved. better.
  • the extraction column of the present invention has a non-equal diameter structure.
  • the inner diameter of the upper section of the column tube is larger than the inner diameter of the lower section.
  • the extraction column of the present invention has a packing structure with functional partitions, and the functional division of the extraction column is performed by filling different packings in sections, which has a better separation effect for complex samples. At the same time, it has good retention effect for some difficult-to-analyze drugs.
  • Fig. 1 is the schematic diagram of the sectional structure of the extraction column of Example 1 of the present invention.
  • Example 2 is a schematic diagram of the split structure of the extraction column of Example 1 of the present invention.
  • Fig. 3 is a schematic top view structure diagram of the elastic flexible board of the present invention.
  • Example 4 is a schematic structural view of the column tube of the extraction column in Example 1 of the present invention.
  • Fig. 5 is the schematic diagram of the sectional structure of the extraction column of Example 3 of the present invention.
  • Fig. 6 is a schematic diagram of the enlarged structure at A in Fig. 5;
  • Fig. 7 is that embodiment 1 extraction column tests peak figure
  • Fig. 8 is a peak figure of comparative example 1 conventional extraction column test
  • Fig. 9 is that embodiment 2 extraction column tests peak figure
  • Fig. 10 is a peak figure of comparative example 2 conventional extraction column test
  • Fig. 11 is the peak figure of different volumes of sample injection of the extraction column in Example 2.
  • Fig. 12 is a peak figure of different volumes of sample injection of the extraction column of Comparative Example 2;
  • Fig. 13 is embodiment 2 and comparative example 2 the relation contrast diagram of extraction column peak area and different volume injection;
  • Fig. 14 is embodiment 3 and comparative example 3 extraction column test clozapine separation and impurity removal effect comparative figure;
  • Fig. 15 is a comparison diagram of the focusing effect of phenobarbital tested by the extraction column of Example 3 and Comparative Example 3;
  • Fig. 16 is a schematic structural view of the extraction column column tube of embodiment 2;
  • Fig. 17 is the structural diagram of the conventional extraction column described in comparative example 1 and comparative example 2;
  • 1 is the column head
  • 2 is the polymer filter membrane
  • 3 is the elastic soft board
  • 4 is the front column cap
  • 5 is the column tube
  • 5-1 is the first connecting section
  • 5-2 is the second connecting section
  • 5- 3 is the upper section
  • 5-4 is the lower section
  • 6 is the end column cap
  • 7 is the stationary phase
  • 7-1 is the adsorption layer
  • 7-2 is the focusing layer
  • 7-3 is the separation layer
  • 8 is the support plate
  • 9 is Fixed ring
  • 10 is a sieve plate
  • 11 is a polymer liquid-impermeable membrane
  • 12 is a metal sieve plate.
  • Embodiment 1 As shown in Figure 1-2: an extraction column with a pressure-stable pore structure, with the inflow end of the mobile phase as the upper end, the extraction column includes a column head 1 and a front-end column cap connected in sequence from top to bottom 4.
  • Column tube 5, end column cap 6 and column head, the front end of the front end column cap 4 is sequentially provided with elastic soft plate 3, polymer filter membrane 2 and supporting orifice plate 8 (elastic soft plate 3, high
  • the molecular filter membrane 2 and the supporting orifice plate 8 form a pressure change pore structure).
  • the column head 1 is provided with holes for flowing in or out of the mobile phase.
  • the inside of the column tube 5 is filled with a stationary phase 7 .
  • the column tube 5 is divided into an upper section 5-3 and a lower section 5-4, and the inner diameter of the upper section 5-3 is greater than the inner diameter of the lower section 5-4.
  • the length of the upper section 5-3 accounts for 1/4 of the entire length of the column tube 5 extraction column.
  • the ratio of the inner diameter of the upper section 5-3 to the inner diameter of the upper section 5-3 is 1.3.
  • the inlet end of the column tube 5 is provided with a front end column cap 4, the outlet end of the column tube 5 is provided with a terminal column cap 6, the inlet end and the outlet end of the column tube are arranged in the column head 1, and there is an internal thread inside the column tube, and the column tube and the column head pass through threaded connection.
  • the support orifice 8 may be a support mesh, or the support orifice 8 is composed of a PEEK ring and a PE sieve, the PEEK ring is made of polyether ether ketone, and the PE sieve is made of polyethylene.
  • the material has the characteristics of high strength and easy dissolution.
  • the column tube 5 and column head 1 are made of stainless steel.
  • the thickness of the elastic soft plate 3 is 0.5 mm, and the pore size is 5 microns.
  • the thickness of the polymer filter membrane 2 is 0.1 mm, which can hold off substances with a molecular weight greater than 3000 Da.
  • the stationary phase 7 is an octadecyl bonded silica gel filler.
  • the blood sample of complex matrix After the blood sample of complex matrix is processed by protein precipitation, it enters the extraction column with the mobile phase of liquid chromatography.
  • the impurities with large molecular weight in the sample are intercepted when they pass through the elastic soft plate, and the substances with small and medium molecular weight pass through the elastic filter plate.
  • the organic polymer filter membrane When passing through the organic polymer filter membrane, it is intercepted and filtered for the second time, and the small molecular weight analyte and some impurities in the final sample enter the stationary phase of the extraction column through the second-stage elastic soft plate and anti-blocking filter cap, and the chromatographic effect of the extraction column is fixed.
  • the analyte is separated from the sample under force, flows into the pipeline through the outlet filter cap, and enters other separation systems or detection systems.
  • the extraction column with elastic blocking After the extraction column with elastic blocking has completed the complex sample processing multiple times or once, off-line or on-line, it can be reverse-phase cleaned and purified by using a mobile phase extraction column with a certain elution strength.
  • the organic polymer filter membrane and The impurities intercepted by the elastic soft plate can be deformed in the expansion and contraction of the elastic soft plate to promote the discharge of impurities and complete the self-purification of the extraction column.
  • the inner diameter of the upper section of the multi-section non-equal diameter chromatographic column is larger than the inner diameter of the lower section, and the linear velocity of the mobile phase in the upper section of the chromatographic column is small, which is conducive to separation and aggregation, and can be well adapted to large-volume sample injection, while maintaining overall good column efficiency.
  • the inner diameter of the upper section is relatively large and the pressure is low, which can reduce the damage to the chromatographic column during large-volume sample injection and improve the service life of the extraction column.
  • the extraction column of embodiment 1, with the conventional extraction column of comparative example 1 (not possessing pressure change pores and non-equal diameter structure) uses two-dimensional liquid chromatography system to carry out comparative test, the content of valproic acid in the test human blood, blood Take 5% perchloric acid for treatment.
  • the test instrument detector is Shimadzu SPD-20A, the wavelength is 215nm, it is 30% acetonitrile: 70% water, the flow rate is 1.0mL/min, the auxiliary mobile phase is water, the auxiliary flow rate is 2.0mL/min, and the auxiliary time is 0.6min , the column temperature was 40°C, and the injection volume was 500 ⁇ L.
  • Example 1 has pressure change pores and non-equal-diameter extraction columns.
  • the peak shape still has no diffusion, still has a good peak shape, and the pressure remains stable, and can withstand the impact of 1000 needles. See Figure 7.
  • the peak shape becomes wider as shown in Figure 8, and the pressure gradually increases at the same time.
  • the specific pressure performance indicators are shown in Table 1.
  • Comparative Example 1 is a conventional extraction column, and other structures are the same as in Example 1, the difference 1 is that a conventional existing filter is added to the front end to filter impurities.
  • the difference 2 is that its inner diameter is 4.6mm, which is an equal-diameter structure.
  • the two-dimensional liquid chromatographic systems all have a reverse cleaning function. Both the conventional chromatographic column and the extraction column of the example are tested in a chromatographic system with a reverse cleaning function, one needle analysis is performed and then the reverse cleaning is performed once.
  • Table 1 is the pressure comparison table of the extraction column of embodiment 1 and comparative example 1
  • the extraction column of Example 1 is compared with the conventional extraction column (without pressure change pores and non-isodiametric structure) in a two-dimensional liquid chromatography system, and the test valproic acid debugging solution has a concentration of 500 ⁇ g/mL, and the test instrument
  • the detector is Shimadzu SPD-20A, the wavelength is 215nm, the mobile phase is 30% acetonitrile: 70% water, the flow rate is 1.0mL/min, the auxiliary mobile phase is water, the auxiliary flow rate is 2.0mL/min, and the auxiliary time is 0.6min , the column temperature was 40°C, and the injection volume was set to 10 ⁇ L, 25 ⁇ L, 50 ⁇ L, 100 ⁇ L, and 200 ⁇ L.
  • the other structures are the same as in Embodiment 1, and a polymer liquid-impermeable membrane 11 is provided in the stationary phase 7 near the inflow end of the mobile phase, and the outer diameter of the polymer liquid-impermeable membrane 11 is smaller than the inner diameter of the column tube 5 .
  • the central axis of the polymer liquid-impermeable membrane 11 coincides with the central axis of the column tube 5 and the polymer liquid-impermeable membrane 11 is arranged parallel to the cross-section of the column tube 5 .
  • the gap between the outer periphery of the polymer liquid-impermeable membrane and the inner wall of the column tube is 0.8mm.
  • the polymer liquid-impermeable membrane 11 is made of fluorine-containing polymer material, which has no permeability to liquid.
  • the column tube 5 is divided into an upper section and a lower section bounded by a polymer liquid-impermeable membrane 11 , and the upper section and the lower section can be filled with the same stationary phase 7 .
  • the packed stationary phase 7 may be C8.
  • the present invention shapes the fluid by adding a polymer impermeable membrane.
  • the fluid first contacts the deformable polymer membrane. It does not have selective permeability, so the manifold of the fluid changes from vertical to horizontal, and then flows out evenly from around the polymer impermeable membrane. Cloth, moving forward in unison, the peak shape of the extraction column is significantly improved.
  • test sample was levetiracetam biological sample
  • the injection volume was 100 ⁇ L
  • the detector was Shimadzu SPD-20A
  • the wavelength was 232nm
  • the flow rate is 1.0mL/min
  • the auxiliary mobile phase is water
  • the auxiliary flow rate is 0.5mL/min
  • the auxiliary time is 0.5min.
  • the temperature is 40°C.
  • Example 2 On the basis of Example 2, take processing a 3.5 ⁇ 25 mm, 5 ⁇ m extraction column as an example.
  • the extraction column of Example 3 also includes a stationary phase packed in the column tube 5, the stationary phase is divided into three sections, and a metal sieve is arranged between any two sections of the stationary phase.
  • the adsorption layer 7-1 is an SCX cationic filler
  • the focusing layer 7-2 is a phenyl bonded silica gel filler
  • the separation layer 7-3 is an octadecyl bonded silica gel filler.
  • the depth of the adsorption layer 7-1 section is 5mm
  • the depth of the focusing layer 7-2 section is 5mm
  • the separation layer 7-3 section has a depth of 15mm.
  • the particle size of the filler in section 7-1 of the adsorption layer is 15 ⁇ m
  • the particle size of the filler in section 7-2 of the focusing layer is 3.5 ⁇ m
  • the particle size of the filler in section 7-3 of the separation layer is 5 ⁇ m.
  • the other structures are the same as those in Example 3, except that no functional division is carried out, and the filler is octadecyl bonded silica gel filler.
  • clozapine After clozapine is metabolized, it will produce the metabolite norclozapine, which will affect the determination of clozapine in the determination of human blood samples by liquid chromatography. At the same time, phenobarbital has a relatively low content in human blood samples and is easy to diffuse, so it is difficult to determine with conventional liquid chromatography columns.
  • Adopt two-dimensional liquid chromatography system (with reverse cleaning function) to carry out the biological sample determination of clozapine
  • chromatographic condition is: detector is SPD-20A, and wavelength is 290nm, and mobile phase is 26% acetonitrile: 74% water (pH 7.0), the flow rate is 0.7mL/min, the column temperature is 40°C, the auxiliary solvent is aqueous solution, the auxiliary flow rate is 2.0mL/min, and the auxiliary time is 1.0min.
  • the injection volume was 200 ⁇ L.
  • the test results are shown in Figure 14.
  • the biological sample determination of phenobarbital is carried out by a two-dimensional liquid chromatography system.
  • the chromatographic conditions are: the detector is SPD-20A, the wavelength is 235nm, the mobile phase is 30% acetonitrile: 70% water, and the flow rate is 1.0mL/min.
  • the column temperature is 40°C, the auxiliary solvent is 0.5% formic acid solution, the auxiliary flow rate is 2.0 mL/min, and the auxiliary time is 0.6 min.
  • the test results are shown in Figure 15.

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Abstract

一种多模式抗基质的萃取柱，以流动相的流入端作为上端，从上至下所述萃取柱包括依次连接的柱头（1）、前端柱帽（4）、柱管（5）、末端柱帽（6）和柱头，所述柱管（5）内填充有固定相（7），所述柱管（5）内腔为圆柱形，且柱管（5）分为上段（5-3）和下段（5-4），上段（5-3）的内径＞下段（5-4）的内径；所述前端柱帽（4）的上端设有压变孔隙结构，所述压变孔隙结构包括上中下三层，从上至下依次为弹性软板（3）、高分子滤膜（2）以及支撑孔板（8）。该萃取柱可以解决萃取柱容易堵塞的问题，容易清洗，提高色谱柱使用寿命和提高柱效、以及可以大体积进样等多种功能。

Description

一种多模式抗基质的萃取柱 技术领域
本发明涉及液相色谱柱领域，具体涉及多模式抗基质的萃取柱。
背景技术
在纯萃取系统中经常使用色谱柱进行样品的前处理，用于样品处理的色谱柱称为萃取柱。在二维液相色谱分析检测中，萃取柱主要用于样品的初级分离、纯化和浓缩。在此过程中，萃取柱承受了来自样品中各种杂质的污染，容易在入口端聚集，造成压力高，使得萃取柱的使用寿命受到严重影响。
萃取柱的结构通常包括柱管、筛板和柱帽，柱帽中筛板的一般为不锈钢烧结网板，孔径为0.1μm-3μm微米。流动相从色谱柱中流过，一般先会被筛板拦截，但是由于样品中有大量杂质，特别是生物样品中的蛋白被筛板拦截之后会发生质聚现象，因此，随着色谱柱进样次数越多，时间越长，筛板堵塞的程度就越厉害，这也是现代液相色谱柱最为常见的问题。萃取柱通常要承受比普通色谱柱更大的进样量，萃取柱经常要承受100微升甚至更大的进样量，所以萃取柱更加容易发生堵塞。一般处理污水处理或者生物质样品的萃取柱一般使用寿命很难超过200针。
现有的解决方法一般是对萃取柱进行超声清洗或者更换柱头填料，对柱头进行修补，但是这需要高度的专业化，解决问题的能力有限。
申请人曾获得一种具有在线清洗功能的液相色谱仪的专利(申请号201410705067.5)，主要针对二维液相色谱的第一色谱柱和中间色谱柱的在线清洗，具有反向清洗功能。当然，这只是其中的一种具体的色谱系统。清洗色谱柱可以在线清洗也可以离线反向清洗。
色相色谱分析时，某些样品成分比较复杂，目标物含量低，需要在线萃取时大体积进样，从而实现富集纯化目标物的功能。但是针对大体积进样，常规萃取柱难以承受(一般常规的进样量为20-50微升左右)。同时常规萃取柱随着进样体积的增大，峰面积呈非线性增长。常规萃取柱大多为等径萃取柱，柱体内流动相的线速度维持一致，对于一些不容易聚焦的药物，就会出现聚焦能力差等问题。同时，流动相会有颗粒物等杂质产生，当柱 头的半径越小，柱压越容易上升，影响到萃取柱的使用寿命。
经检索发现，现有技术中也有非等径的色谱柱，例如CN02106928.X公开了一种锥形高效液相色谱制备柱。主要由柱头、柱体及柱尾构成，是柱头入口内径(2R)大于柱尾出口内径(2r)的锥形柱体。柱头由锥形液流导向槽、分配盘及筛板构成液流分配系统。分配盘由多个辐射状液流通道、同心圆液流收集通道及渗液孔构成。柱体入口内径为2R＝10mm～2000mm、出口内径2r＝3mm～1800mm、柱长L＝5cm～100cm的均匀过渡的锥体，锥体锥角在1°～20°范围内，锥体内可填充各种类型的色谱分离介质。这种锥形制备柱可以显著地提高柱效及样品负载量，对分离组分的稀释效应低于圆柱形色谱柱。但是该色谱柱加工复杂，且并不是常规圆柱形色谱柱。
CN201110071563.6公开了一种抛物线形高效液相色谱制备柱，该毛细液相色谱制备柱包括：位于柱体上部的柱头、抛物线形管状柱体和位于柱体下部的柱尾；其中，抛物线形管状柱体的入口端和出口端分别具有一个特殊的密封伸入件，以及柱体为抛物线形设计，且抛物线方程与流速对应，能得到完美的塞子状色谱带，大大提高色谱柱柱效；柱体内径逐渐减小，增加了色谱柱载样量，且对样品有一定量的富集效应。这种色谱柱应该还只停留在理论阶段，加工成本高。
另外，常规的在线萃取柱，样品随着流动相，经过固定相后，由于传质阻力的存在，会做各种不规则运动，使得色谱柱的理论塔板高度升高，导致萃取柱的柱效下降。
再有，常规的萃取柱，一般用C18做为固定相，对于较为复杂的样品提取和分离效果不是很理想，通过一种填料进行固相萃取后，依然有较多的杂质干扰目标化合物的分析。
因此，研究一种多模式抗基质的萃取柱解决上述现有技术中存在的技术问题，具有重要意义。
发明内容
(一)针对现有技术的不足，本发明的发明目是：
第一个发明目的是提供一种萃取柱，该萃取柱可以解决萃取柱容易堵塞的问题，容易清洗，提高色谱柱使用寿命。
第二个发明目的是提供一种萃取柱，该萃取柱可以适应大体积进样，在大体积进样时仍然有较好的柱效。且随着进样体积的增大，峰面积呈线性增长。所述大体积进样是指进样量在200μL及以上的进样量。
第三个发明目的是提供一种萃取柱，对进入萃取柱的流体的流形和流速进行整流，使得样品中目标物经过整流之后重新排布，齐步往前运动，进入检测器后，得到一个比较正 态的峰型，保持高柱效。
第四个发明目的是提供一种萃取柱，该萃取柱可以适应复杂样品的萃取和分离，在分离复杂样品时，分离效果好，依然有较好的柱效。
(二)为了实现发明目的，本发明的技术方案是：
针对发明目的一和发明目的二，本发明的技术方案一是：
一种多模式抗基质的萃取柱，以流动相的流入端作为上端，从上至下所述萃取柱包括依次连接的柱头、前端柱帽、柱管、末端柱帽和柱头，所述柱管内腔为圆柱形，且柱管分为上段和下段，上段的内径＞下段的内径；所述前端柱帽的上端设有压变孔隙结构，所述压变孔隙结构包括上中下三层，从上至下依次为弹性软板、高分子滤膜以及支撑孔板。
优选的，所述弹性软板包括固定环和位于固定环内的弹性筛板。
优选的，固定环材质优选为PEEK圈，材质为聚醚醚酮，PE筛板材质优选为聚乙烯。该材质具有高强度且低溶出等特性，PE筛板的厚度在0.5mm-8mm之间。
优选的，所述弹性软板的厚度为0.1mm-10mm，优选的0.5mm-8mm，孔径为5微米-10微米。
优选的，所述高分子滤膜的厚度为0.05mm-3mm，优选的0.1-2mm，进一步优选的0.1mm-0.5mm，可截留分子量＞3000Da物质，进一步优选的可截留分子量＞3000Da物质。
优选的，所述高分子滤膜的材质为能够耐受有机、酸碱试剂的材料，可以是聚醚砜树脂或者聚丙烯。
优选的，所述柱管入口端设有前端柱帽，柱管出口端设有末端柱帽、柱管入口端和出口端设置在柱头内，柱头内部有内螺纹，柱管和柱头通过螺纹连接。
优选的，支撑孔板的结构可以和弹性软板的结构完全相同。
优选的，所述上段的长度占柱管萃取柱整长度的1/5-3/10。经过发明人的大量研究，在这个范围内较为合适，可以适应于大体积进样，而同样保持整体良好的柱效，且不会大量增加成本和分析时间。
优选的，所述上段的内径与下段之比为1.2-1.5。经过发明人的大量研究，在这个范围内较为合适，可以适应于大体积进样，而同样保持整体良好的柱效，且不会大量增加成本和分析时间。
优选的，所述柱管外周设有用于与柱头连接的第一连接段和第一连接段，所述第一连接段和第一连接段的外径＞柱管的外径。
优选的，所述柱头内设有用于流动相流入的流入孔或者用于流动相流出的流出孔。
优选的，一种非等径萃取柱，所述柱管和柱头的材质均为不锈钢。
优选的，上段与下段分别填装不同的填料。
优选的，上段可以装填反向类和离子类等填料，下段也可装填反向类和离子类等填料，且上段的填料粒径大于下段填料，对于目标物的吸附效果上段填料要优于下段填料。两种填料之间通过金属筛板隔开。
再进一步的针对发明目的三，本发明的技术方案二是：
在技术方案一的基础上，所述柱管内填充有固定相，在所述固定相内靠近流动相流入端设有高分子不透液膜，所述高分子不透液膜的外径小于柱管的内径，且所述高分子不透液膜的中心轴与柱管的中心轴重合且高分子不透液膜平行于柱管的横截面设置。
优选的，所述高分子不透液膜的材质只要为不透液的膜均可，可以为含氟高分子材料，优选为聚脂薄膜或者尼龙薄膜。
优选的，所述高分子不透液膜的外周与柱管的内壁之间的间隙小于柱管内径的1/4，控制在此范围内比较合理。进一步优选的，所述高分子不透液膜的外周与柱管的内壁之间的间隙为0.6-3，根据发明人的大量研究，间隙为0.6-3时整流效果较好。
优选的，所述高分子不透液膜的上方设有金属筛板，用于对高分子不透液膜进行支撑，防止其发生形变。
优选的，支撑孔板的结构可以和弹性软板的结构完全相同。
再进一步的针对发明目的四，本发明的技术方案三是：
在技术方案一或技术方案二的基础上，进一步的所述固定相内的填料至少为三层，且分别填装不同的填料，不同填料层之间通过金属筛板隔开。
从流动相流入端到流出端，所述固定相包括吸附层、聚焦层以及分离层，所述吸附层选自十八烷基硅烷键合硅胶填料、磺酸基阳离子聚合物填料或者萘基键合硅胶填料；所述聚焦层选自苯基键合硅胶填料或者十八烷基硅烷键合硅胶填料；分离层选自氰基键合硅胶填料、磺酸基阳离子聚合物填料或者十八烷基硅烷键合硅胶填料。
优选的，所述吸附层的深度在1～30mm之间，所述聚焦层的深度在1～20mm之间，分离层的深度在5～50mm之间。
优选的，所述吸附层填料粒度为5μm-20μm，所述聚焦层填料粒度为2μm-5μm，所述分离层填料粒度为2μm-10μm。
(三)本发明的原理说明：
1、关于具有压变孔隙的萃取柱便于清洗可以提高萃取柱的使用寿命的原理说明：
本发明通过在入口端的柱头内设有压变孔隙软板和高分子膜，萃取柱在一维流动相所对应的背景压力一般2MPa左右，不足以形成高压，在大流速辅助流动相进入时，形成高压，一般到达10MPa以上，弹性软板向下收缩，孔径变小，拦截过滤颗粒，更细一点的杂质被高分子膜拦截过滤，支撑孔板对弹性软板和高分子膜进行支撑；在在线和离线反向清洗时，压力降低，弹性软板发生形变扩张，有机高分子滤膜及弹性软板拦截的杂质被反向洗脱排出。一种具有压变孔隙的萃取柱，能够更容易的将萃取柱入口端杂质反向清洗出去，从而提升萃取柱的使用寿命。
2、关于高分子不透液膜可以对流动相进行整形从而提高柱效的原理说明：
正常的色谱柱，样品随着流动相流经固定相后，随着样本中蛋白等杂质的积累，固定相失去了吸附能力，使得样本中目标物做各种不规则的运动，最靠近色谱柱中心的流速最大，靠近色谱柱四周的流速小，流速不均匀，本发明通过增加一张具有形变特性的高分子膜对流体进行整形，流体先接触高分子不透液膜，由于膜不具备选择透过性，因此流体的流形从横向变纵向，再从具有形变特性的高分子膜的四周均匀流出，流速变均匀，流向变规则，使得样品中目标物经过整流之后重新排布，齐步往前运动，萃取柱的峰形得到明显改善。
3、关于非等径萃取柱可以适应大体积进样的原理说明：
范德姆特方程最常用的形式如下式所示，
Figure PCTCN2023070265-appb-000001
式中H为理论塔板高度，A,B,C为常数，u表示色谱柱内流动相的线速度。该式直观地反映了色谱柱内流动相的线速度对于分离的影响。色谱柱内流动相的线速度越大柱效越差。
另外，根据色谱柱数理常数理论，色谱柱内流动相的线速度跟色谱柱的内径的平方成反比，也就是说色谱柱的内径越小，色谱柱内流动相的线速度越大，柱效越差。
那么，针对大体积进样，为了得到较好的柱效，可以考虑从改变柱管内径的角度来解决大体积进样的问题，但是，如果纯粹只是扩大萃取柱的内径也会有其他问题产生，例如：1、需要填装更多的填料，增加萃取柱成本；2、填料越多，分析时间越长；3、色谱柱内径越大、内含的溶剂就越多，内含的溶剂在转移到下一级色谱柱的时候会产生溶剂的扩散，对后续的分析产生影响等。
经过发明人的大量研究，发明人研发了多段式非等径色谱柱，既可以满足大体积进样的要求，且保持良好的柱效，还可以节约成本且分析时间合适。
多段式非等径色谱柱的上段内径＞下段内径，上段色谱柱内流动相的线速度小，利于分离和聚集，可以很好的适应于大体积进样，而同样保持整体良好的柱效，同时上段内径相对较大，压力低，可以减少大体积进样时对色谱柱的伤害，提高萃取柱的使用寿命。通过色谱柱上段的缓冲，色谱柱下段的内径不用与上段内径一样大，可以缩小内径，流动相的流速相对上段会有所提高，这样可以减少分析时间，同时减少填料的用量，节约成本。
进一步的，因为上段和下段的流速不同，我们进一步研究了填料的填装，上段和下段分别填装不同的填料，满足不同流速下分离或者富集所需要求。上段可以装填吸附性较强的SCX离子类填料。下段可以填补C18、C8、苯基和萘基等填料。经过对内部填料的优化，使得富集效果更好。
填料的选择原则：上段填料的粒径大于下段填料的粒径，上段粒径一般在5-50μm之间选择，下段粒径一般在2μm-10μm之间选择：上段填料对目标物的富集能力大于下段填料对目标物的富集能力。
4、关于分段填充不同填料(功能分区)可以提高分离效果的原理说明：
本发明的样品通过流动相进入色谱柱后，部分离子化合物先在吸附层中通过离子作用力进行初步吸附，目标物经过流动相从吸附层洗脱下来，经过一定流速的辅助流动相聚焦后，样品流动状态进行重新排布，到了聚焦层后，其他一些杂质通过官能团之间的非极性作用力、表面电场力等得到有效吸附，目标物又重新进行排布，通过分离层进行有效分离。
(四)与现有技术相比，本发明的优势是：
1、本发明的萃取柱，具有压变空隙结构能够更容易的将萃取柱入口端杂质反向清洗出去，从而提升萃取柱的使用寿命。
2、本发明的萃取柱，具有整流结构，对进入色谱柱的流体的流形和流速进行整流，使得样品经过整流之后分散更均匀，更利于样品的分离，柱效提高，萃取柱的峰形更好。
3、本发明的萃取柱，具有非等径结构，柱管上段的管径内径大于下段的内径，大体积进样时，上段的流速低，利于分离和聚集，可以很好的适应于大体积进样，而同样保持整体良好的柱效，提高萃取柱的使用寿命，且不会大量增加成本和分析时间。
4、本发明的萃取柱，具有功能分区的填料结构，通过分段填补不同的填料进行萃取柱的功能区分，针对复杂样品有更好的分离效果。同时对于部分难分析药物具有良好的保留效果。
以下结合附图和具体实施方式对本发明的详细结构作进一步描述。
附图说明
图1为本发明实施例1的萃取柱剖面结构示意图；
图2是本发明实施例1的萃取柱拆分结构示意图；
图3是本发明所述弹性软板俯视结构示意图；
图4是本发明实施例1的萃取柱柱管结构示意图；
图5是本发明实施例3的萃取柱剖面结构示意图；
图6是图5的A处放大结构示意图；
图7是实施例1萃取柱测试出峰图；
图8是对比例1常规萃取柱测试出峰图；
图9是实施例2萃取柱测试出峰图；
图10是对比例2常规萃取柱测试出峰图；
图11是实施例2萃取柱进样不同体积出峰图；
图12是对比例2萃取柱进样不同体积出峰图；
图13是实施例2与对比例2萃取柱峰面积与不同体积进样的关系对比图；
图14是实施例3与对比例3萃取柱测试氯氮平分离及除杂效果对比图；
图15是实施例3与对比例3萃取柱测试苯巴比妥聚焦效果对比图；
图16是实施例2的萃取柱柱管结构示意图；
图17是对比例1和对比例2所述的常规萃取柱的结构图；
其中，1是柱头、2是高分子滤膜、3是弹性软板、4是前端柱帽、5是柱管、5-1是第一连接段、5-2是第二连接段、5-3是上段、5-4是下段、6是末端柱帽、7是固定相、7-1是吸附层、7-2是聚焦层、7-3是分离层、8是支撑孔板、9是固定环、10是筛板、11是高分子不透液膜，12是金属筛板。
具体实施方式
实施例1：如图1-2所示：一种具有压变孔隙结构的萃取柱，以流动相的流入端作为上端，从上至下所述萃取柱包括依次连接的柱头1、前端柱帽4、柱管5、末端柱帽6和柱头，所述前端柱帽4的前端从上至下依次设有弹性软板3、高分子滤膜2以及支撑孔板8(弹性软板3、高分子滤膜2以及支撑孔板8组成压变孔隙结构)。所述柱头1内设有用于流动相流入或者流出的孔。所述柱管5内部填充有固定相7。所述柱管5分为上段5-3和下段5-4，上段5-3的内径＞下段5-4的内径。所述上段5-3的长度占柱管5萃取柱 整长度的1/4。上段5-3的内径与上段5-3的内径之比为1.3。
所述柱管5入口端设有前端柱帽4，柱管5出口端设有末端柱帽6、柱管入口端和出口端设置在柱头1内，柱头内部有内螺纹，柱管和柱头通过螺纹连接。
所述支撑孔板8可以为支撑网板，或者支撑孔板8由PEEK圈和PE筛板组成，PEEK圈材质为聚醚醚酮，PE筛板材质为聚乙烯。该材质具有高强度且易溶出等特性。
所述柱管5和柱头1的材质均为不锈钢。
所述弹性软板3的厚度为0.5mm，孔径5微米，所述高分子滤膜2的厚度为0.1mm，可截留分子量大于3000Da物质。
所述固定相7为十八烷基键合硅胶填料。
工作原理说明：复杂基质的血样经蛋白沉淀处理后，随液相色谱流动相进入萃取柱，样品中大分子量的杂质在经过弹性软板时被拦截，中小分子量的物质穿过弹性过滤板后在经过有机高分子滤膜时被第二次拦截过滤，最终样品中小分子量被测物及部分杂质经过第二级弹性软板及防堵滤帽进入萃取柱固定相，在萃取柱固定性的色谱作用力下将被测物从样品中初级分离出来，通过出口滤帽流入管路，进入其他的分离系统中或检测系统中。此具有弹性阻拦的萃取柱在多次或单次，离线或在线完成复杂样品处理后，可反相使用一定洗脱强度的流动相对萃取柱进行反相清洗净化，此时有机高分子滤膜及弹性软板拦截的杂质可以在弹性软板的扩张及收缩弹性内发生形变，促进杂质的排出，完成萃取柱的自我净化。同时多段式非等径色谱柱的上段内径＞下段内径，上段色谱柱内流动相的线速度小，利于分离和聚集，可以很好的适应于大体积进样，而同样保持整体良好的柱效，同时上段内径相对较大，压力低，可以减少大体积进样时对色谱柱的伤害，提高萃取柱的使用寿命。
实施例1的萃取柱，与对比例1的常规萃取柱(不具备压变孔隙和非等径结构)使用二维液相色谱系统中进行对比测试，测试人血液中丙戊酸的含量，血液采取5％高氯酸进行处理。测试仪器检测器为岛津SPD-20A，波长为215nm，为30％乙腈：70％水，流速为1.0mL/min，辅助流动相为水，辅助流速为2.0mL/min，辅助时长为0.6min，柱温为40℃，进样量为500μL。实施例1具有压变孔隙及非等径的萃取柱，在测试200针后，峰型仍然没有扩散，仍然具有较好的峰形，且压力保持稳定，并且能耐受1000针的冲击，见图7。常规萃取柱在测试200针后(无压变孔隙及非等径结构，其他结构一致)，峰型变宽具体见图8，同时压力逐渐升高，具体压力性能指标见表1。
对比例1为常规萃取柱，其他结构同实施例1，不同点1在于其前端另外加了一个常规现有过滤器用于过滤杂质。不同点2在于其内径为4.6mm，为等径结构。所述用于二 维液相色谱系统均带有反向清洗功能。常规色谱柱与实施例萃取柱都是在带反向清洗功能的色谱系统内进行测试，做一针分析然后反向清洗一次。
表1为实施例1与对比例1萃取柱压力对比表
Figure PCTCN2023070265-appb-000002
实施例1的萃取柱，与常规萃取柱(不具备压变孔隙及非等径结构)使用二维液相色谱系统中进行对比测试，测试丙戊酸调试溶液，浓度为500μg/mL，测试仪器检测器为岛津SPD-20A，波长为215nm，流动相为30％乙腈：70％水，流速为1.0mL/min，辅助流动相为水，辅助流速为2.0mL/min，辅助时长为0.6min，柱温为40℃，进样量设定为10μL、25μL、50μL、100μL、200μL。查看进样体积与峰面积是否呈线性，具体结果见图9、图10和图11，可见本申请的萃取柱可以承受大体积进样，而常规萃取柱在大体积进样后出现平头峰及峰面积与进样体积不呈线性。
实施例2
其他结构同实施例1，在所述固定相7内靠近流动相流入端设有高分子不透液膜11，所述高分子不透液膜11的外径小于柱管5的内径。所述高分子不透液膜11的中心轴与柱管5的中心轴重合且高分子不透液膜11平行于柱管5的横截面设置。所述高分子不透液膜的外周与柱管的内壁之间的间隙为0.8mm。
所述高分子不透液膜11的材质为含氟高分子材料，对液体没有透过性。
所述柱管5以高分子不透液膜11为界限分为上段和下段，上段和下段可以装填相同的固定相7。所装填的固定相7可以为C8。
工作原理说明：正常的色谱柱，样品随着流动相流经固定相后，随着样本中蛋白等杂质的积累，固定相失去了吸附能力，使得样本中目标物做各种不规则的运动，最靠近色谱柱中心的流速最大，靠近色谱柱四周的流速小，流速不均匀，本发明通过增加一张具有高分子不透液膜对流体进行整形，流体先接触形变特性高分子膜，由于膜不具备选择透过性，因此流体的流形从纵向变横线，再从具有高分子不透液膜的四周均匀流出，流速变均匀，流向变规则，使得样品中目标物经过整流之后重新排布，齐步往前运动，萃取柱的峰形得到明显改善。
与常规萃取柱(不具备整流结构)使用二维液相色谱系统中进行对比测试，测试样品为左乙拉西坦生物样本，进样体积为100μL，检测器为岛津SPD-20A，波长为232nm，流动相为4％甲醇：96％水(1mmol磷酸盐，pH＝3.2)，流速为1.0mL/min，辅助流动相为水，辅助流速为0.5mL/min，辅助时长为0.5min，柱温为40℃。由测试结果可知，实施例2增加了分子截留膜，随着进样针数的积累，峰会渐渐变宽，但色谱峰未见明显拖尾情况。对比例2常规萃取柱(无高分子不透液膜)可明显感受到色谱峰变宽和拖尾情况。具体测试结果见图12和图13。
实施例3
在实施例2的基础上，以加工3.5×25mm，5μm萃取柱为例。
实施例3的萃取柱还包括填装在柱管5内的固定相，所述固定相分为三段，任意两段固定相之间设有金属筛片。所述吸附层7-1为SCX阳离子填料、聚焦层7-2为苯基键合硅胶填料以及分离层7-3为十八烷基键合硅胶填料。
所述吸附层7-1段的深度为5mm，所述聚焦层7-2段的深度为5mm，分离层7-3段的深度为15mm。所述吸附层7-1段填料粒度为15μm，聚焦层7-2段填料粒度为3.5μm，分离层7-3段填料粒度为5μm。
对比例3
其他结构同实施例3，只是不进行功能分区，所填填料为十八烷基键合硅胶填料。
氯氮平经代谢后，会产生代谢产物去甲氯氮平，在液相色谱法测定人体血样时，会影响到氯氮平的测定。同时苯巴比妥在人体血样内，相对含量较低，同时容易扩散，常规液相色谱柱难以测定。
采用二维液相色谱系统(带反向清洗功能)进行氯氮平的生物样本测定，色谱条件为：检测器为SPD-20A，波长为290nm，移动相为26％乙腈：74％水(pH为7.0)，流速为0.7mL/min，柱温为40℃，辅助溶剂为水溶液，辅助流速为2.0mL/min，辅助时间为1.0min。进样体积为200μL。测试结果见图14。
采用二维液相色谱系统进行苯巴比妥的生物样本测定，色谱条件为：检测器为SPD-20A，波长为235nm，移动相为30％乙腈：70％水，流速为1.0mL/min，柱温为40℃，辅助溶剂为0.5％甲酸溶液，辅助流速为2.0mL/min，辅助时间为0.6min。测试结果见图15。
由图14和图15，可见实施例3的萃取柱对含氯氮平等复杂生物样品的分离能力优于对比例3的萃取柱。同时实施例3对于较难检测含苯巴比妥的生物样品能够实现有效截取 窗口。
以上所述为本发明的具体实施方式，但本发明的保护范围不局限于此，任何熟悉技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其构思加以等同替换或改变，都应涵盖在本发明权利要求的保护范围之内。

Claims (13)

  1. 一种多模式抗基质的萃取柱，以流动相的流入端作为上端，从上至下所述萃取柱包括依次连接的柱头(1)、前端柱帽(4)、柱管(5)、末端柱帽(6)和柱头，其特征是，所述柱管(5)内腔为圆柱形，且柱管(5)分为上段(5-3)和下段(5-4)，上段(5-3)的内径＞下段(5-4)的内径；所述前端柱帽(4)的上端设有压变孔隙结构，所述压变孔隙结构包括上中下三层，从上至下依次为弹性软板(3)、高分子滤膜(2)以及支撑孔板(8)。
  2. 根据权利要求1所述多模式抗基质的萃取柱，其特征是，所述柱管(5)内填充有固定相(7)，在所述固定相(7)内靠近流动相流入端设有高分子不透液膜(11)，所述高分子不透液膜(11)的外径小于柱管(5)的内径，且所述高分子不透液膜(11)的中心轴与柱管(5)的中心轴重合且高分子不透液膜(11)平行于柱管(5)的横截面设置。
  3. 根据权利要求1或2所述多模式抗基质的萃取柱，其特征是，所述固定相(7)内的填料至少为两层，且分别填装不同的填料，不同填料层之间通过金属筛板(12)隔开。
  4. 根据权利要求1或2所述多模式抗基质的萃取柱，其特征是，所述弹性软板(3)包括固定环(9)和位于固定环(9)内的弹性筛板(10)。
  5. 根据权利要求1或2所述多模式抗基质的萃取柱，其特征是，所述弹性软板(3)的厚度为0.1mm-10mm，孔径为5微米10微米。
  6. 根据权利要求1或2所述多模式抗基质的萃取柱，其特征是，所述高分子滤膜(2)的厚度为0.05mm-3mm，可截留分子量＞3000Da物质。
  7. 根据权利要求2所述多模式抗基质的萃取柱，其特征是，所述高分子不透液膜(11)的外周与柱管(5)的内壁之间的间隙小于柱管(5)内径的1/4。
  8. 根据权利要求2所述多模式抗基质的萃取柱，其特征是，所述高分子不透液膜(11)的外周与柱管(5)的内壁之间的间隙为0.6-3mm。
  9. 根据权利要求1或2所述多模式抗基质的萃取柱，其特征是，所述上段(5-3)的长度占柱管(5)萃取柱整长度的1/5-3/10。
  10. 根据权利要求1或2所述多模式抗基质的萃取柱，其特征是，所述上段(5-3)的内径与下段(5-4)的内径之比为1.2-1.5。
  11. 根据权利要求3所述多模式抗基质的萃取柱，其特征是，从流动相流入端到流出端，所述固定相包括吸附层(7-1)、聚焦层(7-2)以及分离层(7-3)，所述吸附层(7-1) 选自十八烷基硅烷键合硅胶填料、磺酸基阳离子聚合物填料或者萘基键合硅胶填料；所述聚焦层(7-2)选自苯基键合硅胶填料或者十八烷基硅烷键合硅胶填料；所述分离层(7-3)选自氰基键合硅胶填料、磺酸基阳离子聚合物填料或者十八烷基硅烷键合硅胶填料。
  12. 根据权利要求3所述多模式抗基质的萃取柱，其特征是，所述吸附层(7-1)的深度在1～30mm之间，所述聚焦层(7-2)的深度在1～20mm之间，所述分离层(7-3)的深度在5～50mm之间。
  13. 根据权利要求3所述多模式抗基质的萃取柱，其特征是，所述吸附层(7-1)填料粒度为5μm-20μm，所述聚焦层(7-2)填料粒度为2μm-5μm，所述分离层(7-3)填料粒度为2μm-10μm。
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CN215894510U (zh) * 2021-07-16 2022-02-22 上海通微分析技术有限公司 一种液相色谱柱
CN113759053A (zh) * 2021-08-16 2021-12-07 江苏同济分析仪器有限公司 一种防堵塞的高压液相色谱柱筛板
CN216259551U (zh) * 2021-09-15 2022-04-12 依利特(苏州)分析仪器有限公司 一种可更换过滤膜的双重在线过滤器
CN115078615A (zh) * 2022-08-22 2022-09-20 湖南德米特仪器有限公司 一种用于液相色谱的萃取柱
CN115253382A (zh) * 2022-08-22 2022-11-01 湖南德米特仪器有限公司 一种用于萃取柱的压变孔隙结构以及一种萃取柱
CN115337673A (zh) * 2022-08-22 2022-11-15 湖南德米特仪器有限公司 一种非等径萃取柱
CN217909071U (zh) * 2022-08-22 2022-11-29 湖南德米特仪器有限公司 一种功能分区的萃取柱
CN217909065U (zh) * 2022-08-22 2022-11-29 湖南德米特仪器有限公司 一种具有整流特性的萃取柱
CN218157741U (zh) * 2022-08-22 2022-12-27 湖南德米特仪器有限公司 一种具有自膨胀特性的液相色谱柱

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