CN115078602A - Capillary high-pressure liquid chromatographic column and ultrasonic preparation method and application thereof - Google Patents
Capillary high-pressure liquid chromatographic column and ultrasonic preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 32
- 239000007788 liquid Substances 0.000 title claims abstract description 27
- 238000012856 packing Methods 0.000 claims abstract description 52
- 238000000034 method Methods 0.000 claims abstract description 32
- 239000002245 particle Substances 0.000 claims abstract description 10
- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 69
- 229910052757 nitrogen Inorganic materials 0.000 claims description 34
- 238000000926 separation method Methods 0.000 claims description 19
- 238000004811 liquid chromatography Methods 0.000 claims description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 238000004850 capillary HPLC Methods 0.000 claims description 11
- 239000000945 filler Substances 0.000 claims description 11
- 239000003960 organic solvent Substances 0.000 claims description 8
- 229920001184 polypeptide Polymers 0.000 claims description 8
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 8
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 7
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 claims description 6
- 239000011261 inert gas Substances 0.000 claims description 6
- RLQWHDODQVOVKU-UHFFFAOYSA-N tetrapotassium;silicate Chemical compound [K+].[K+].[K+].[K+].[O-][Si]([O-])([O-])[O-] RLQWHDODQVOVKU-UHFFFAOYSA-N 0.000 claims description 6
- 238000000265 homogenisation Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 3
- 238000011160 research Methods 0.000 claims description 3
- 238000007664 blowing Methods 0.000 claims description 2
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- 238000002604 ultrasonography Methods 0.000 claims description 2
- 239000007921 spray Substances 0.000 abstract description 5
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- 238000004587 chromatography analysis Methods 0.000 description 9
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- 238000001514 detection method Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 238000007789 sealing Methods 0.000 description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
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- 230000002797 proteolythic effect Effects 0.000 description 2
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- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 238000003776 cleavage reaction Methods 0.000 description 1
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- 229910001873 dinitrogen Inorganic materials 0.000 description 1
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/22—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the construction of the column
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/56—Packing methods or coating methods
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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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Abstract
The invention belongs to the field of capillary chromatographic column preparation, and particularly relates to a capillary high-pressure liquid chromatographic column and an ultrasonic preparation method and application thereof. The invention prepares the chromatographic packing into homogenate and keeps the homogenate in a state of continuous and uniform stirring, so that the capillary with the plunger is communicated with the homogenate, ultrasonic treatment is applied to a capillary column, large-area direct contact type ultrasonic is used, the packing particles in the capillary are dispersed more, and the packing is accumulated and filled in the capillary more uniformly. In addition, longer capillary columns are prepared using multiple cycles of packing. In addition, the method can be used for filling a capillary column with a smaller particle size of 1.9 mu m and a length of 30cm or less, and the filled capillary column is matched with a metal spray needle for use, so that the method has a longer service life and thus has a good value in practical application.
Description
Technical Field
The invention belongs to the field of capillary chromatographic column preparation, and particularly relates to a capillary high-pressure liquid chromatographic column and an ultrasonic preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The rapid development of proteomics urgently requires a high-efficiency nanoflow liquid chromatography separation technology. The capillary chromatographic column is a necessary tool for realizing high-efficiency separation of complex trace polypeptide and is an important part in a nano-flow liquid chromatographic separation technology.
The packing of capillary chromatography columns directly affects their separation performance. A chromatographic column with poor filling is filled, the distribution of the filling materials in the chromatographic column is uneven or not compact, the detection separation degree is low, and the chromatographic peak pattern is poor; or the column efficiency drops significantly after a short period of use. Good packing methods are important to obtain capillary chromatography columns with high column efficiency and high stability.
The complex plunger preparation and column packing aspects limit the efficiency of capillary column preparation. In recent years, a capillary column in which a separation column and a nozzle are integrated has been reported. However, the capillary drawing tip requires expensive special equipment, the success rate is not high, and it is difficult to prepare a long capillary column; in addition, the needle tip is easily damaged, which causes poor spraying effect and even loss of column packing, and is difficult to realize longer column service life.
The homogenization packing method is a commonly used packing method of a liquid chromatography column. The homogenate packing was continuously pressed into the column tube under pressure provided by nitrogen to form a column bed of the chromatographic column. The back pressure generated by the bed is related to the particle size of the packing, the length of packing, and the packing with particle size less than 2 μm is usually packed in a column less than 10 cm. If a filling method is not improved, longer capillary columns are filled, the column filling pressure is often higher, special equipment such as a vacuum pump is required to be configured, and the column filling cost and the potential safety hazard are increased.
Disclosure of Invention
Aiming at the problems of low capillary column preparation success rate, short service life, difficult preparation of long length of smaller filler particle size under medium and low pressure and the like in the existing column packing technical method, the invention provides a capillary high-pressure liquid chromatographic column and an ultrasonic preparation method and application thereof. The invention prepares the chromatographic packing into homogenate and keeps the homogenate in a state of continuous and uniform stirring, so that the capillary with the plunger is communicated with the homogenate, ultrasonic treatment is applied to a capillary column, large-area direct contact type ultrasonic is used, the packing particles in the capillary are dispersed more, and the packing is accumulated and filled in the capillary more uniformly. In addition, longer capillary columns are prepared using multiple cycles of packing. In addition, the method can be used for filling a capillary column with a smaller particle size of 1.9 mu m and a length of 30cm or less, and the filled capillary column is matched with a metal spray needle for use, so that the method has a longer service life and thus has a good value in practical application.
Specifically, the invention relates to the following technical scheme:
the invention provides an ultrasonic preparation method of a capillary high-pressure liquid chromatography column, which comprises the following steps of filling a capillary column by adopting a medium-pressure homogenization ultrasonic filling method:
preparing a chromatographic column filler into homogenate by using an organic solvent, and keeping the uniform dispersion of the chromatographic column filler; inserting one end of a capillary column into the homogenate, simultaneously carrying out ultrasonic treatment on the capillary column, and moving the filler to the capillary plunger and gradually accumulating under the action of inert gas and ultrasonic vibration; when the mixture is filled to a position of 2-3 cm (preferably 3cm), the pressure of the inert gas is adjusted to 4-5 MPa, and the mixture is filled while maintaining a constant pressure.
Wherein the organic solvent is a chromatographic grade organic solvent, and in one embodiment of the invention, the organic solvent is methanol.
The chromatographic column packing can be reverse chromatographic packing, such as C18 reverse chromatographic column packing, the grain size of the packing is 1.9-5 μm, and the preparation method is particularly suitable for the small-grain-size chromatographic column packing.
The inert gas may be nitrogen.
The capillary column is a capillary tube with a sieve plate, and the preparation method of the capillary tube with the sieve plate comprises the following steps:
the preparation ratio is kasil 1624: kasil 1: and (3) inserting the capillary tube into the homogeneous liquid with a ratio of 1-3: 1:1, quickly pulling out the capillary tube when the homogeneous liquid rises to be more than 1cm, and then placing the capillary tube in a high-temperature (such as 80-90 ℃) environment for 3-4 hours to obtain the formamide-containing homogeneous liquid.
Meanwhile, aiming at capillary columns with different column lengths, the invention adopts different filling strategies for filling, specifically:
when the length of the capillary column is within 20cm (including 20cm), filling by using a one-step method directly, namely continuously filling under the constant pressure until the required length is reached, and drying by using nitrogen; then reducing the nitrogen pressure to zero, and unloading the capillary column; connecting the capillary column to a liquid chromatography pump, and compacting the chromatographic packing in the capillary column by utilizing sectional gradual pressure increase to obtain a usable capillary column;
when the length of the capillary column is 20cm-25cm (including 25cm), filling by using a two-step method, specifically, firstly filling to 20cm according to the constant pressure, then adjusting the capillary column to be above the homogenate liquid level, blowing the capillary column by using nitrogen, secondly repeatedly inserting the capillary column below the homogenate liquid level, and filling the capillary column at the constant pressure until the required length is reached; then reducing the nitrogen pressure to zero, and unloading the capillary column; connecting the capillary column to a liquid chromatography pump, and compacting the chromatographic packing in the capillary column by utilizing sectional gradual pressure increase to obtain a usable capillary column;
when the length of the capillary column is 25cm-30cm, filling by using a three-step method, specifically, continuously filling to 20cm, drying by using nitrogen, filling to 25cm in the second step, and filling to the required length in the third step; the third specific method comprises the following steps: reducing the nitrogen pressure to zero, and unloading the capillary column; connecting the capillary column to a liquid chromatography pump, and compacting the chromatographic packing in the capillary column by utilizing sectional gradual pressure increase to obtain the usable capillary column. It should be noted that the high pressure generated by the liquid chromatography pump is used, and the maximum pressure is between 200bar and 500bar according to the length of the column, so as to ensure the packing to be compact and obtain high column efficiency.
In a second aspect of the invention, a capillary high pressure liquid chromatography column obtained by the above ultrasonic preparation method is provided. According to the preparation method, the capillary column with the particle size of 1.9 mu m and the length of 30cm can be prepared; meanwhile, the prepared capillary column has good separation degree reproducibility and high column efficiency; and the service life is long; meanwhile, compared with the capillary column with the integrated separation column and the spray needle, the damage of the needle tip to the whole capillary column is avoided.
In a third aspect of the present invention, there is provided the use of the capillary high pressure liquid chromatography column described above in proteomics research.
Specifically, the application comprises the step of carrying out high-efficiency separation on complex and trace polypeptides by using the capillary high-pressure liquid chromatography column.
The beneficial effects of one or more of the above technical solutions are as follows:
1. the technical scheme can realize the preparation of the capillary column with the filler with smaller particle size of 1.9 mu m and the length of 30 cm;
2. the capillary column prepared by the technical scheme has good separation degree reproducibility and high column efficiency;
3. the success rate of filling the capillary column in the technical scheme is as high as 95 percent;
4. the capillary column prepared by the technical scheme has long service life, and more than 1000 separable polypeptide samples can be obtained;
5. compared with the capillary column with the integrated separation column and the spray needle, the capillary column prepared by the technical scheme can effectively avoid the loss of the whole capillary column due to the damage of the needle point;
6. above-mentioned technical scheme uses miniature dress post equipment, and the in-process does not need special instrument and equipment, and required pressure is less, and operation safety realizes easily.
In conclusion, the technical scheme provides a preparation method for preparing a capillary column, which has the advantages of low cost, high success rate of column filling, high column efficiency, low requirement on high-pressure tightness of filling equipment and long preparation time at medium pressure, and has good practical application value.
Drawings
FIG. 1 is a schematic packing diagram of a capillary chromatography column of the present invention; in the figure, 1, a homogenate tank; 2. homogenizing the chromatographic packing; 3. a magneton; 4. a magnetic stirrer; 5. a capillary column; 6. a capillary column sieve plate; 7. an ultrasonic device; 8. a nitrogen gas cylinder; 9. and (4) controlling the valve.
FIG. 2 is a total ion flow diagram obtained using the capillary chromatography column prepared in example 1.
FIG. 3 is a graph of extracted ion peaks obtained using the capillary chromatography column prepared in example 1.
Fig. 4 is a chromatogram obtained using the capillary chromatography column prepared in example 2.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the following detailed description is given with reference to specific embodiments.
The test materials used in the examples of the present invention were all conventional in the art and commercially available.
In one embodiment of the present invention, a method for preparing a capillary high pressure liquid chromatography column by using ultrasound is provided, which comprises:
1. capillary chromatographic column sieve plate manufacture
The preparation ratio is kasil 1624: kasil 1: and (3) inserting the capillary tube into the homogeneous liquid with the ratio of formamide to 1:1, quickly pulling out the capillary tube when the homogeneous liquid rises to be more than 1cm, and then placing the capillary tube in an environment at 90 ℃ for 4 hours to manufacture the capillary tube with the sieve plate.
2. Capillary column packing
The filling method belongs to a medium-pressure homogenization ultrasonic filling method, is suitable for filling a capillary column with a sieve plate at one end, and has the filling flow shown in figure 1. Preparing a chromatographic packing into homogenate with a certain concentration by using mass spectrum methanol, and carrying out ultrasonic oscillation for 2 min; placing the glass tube containing the homogenate and the stirrer in a homogenate tank, and opening a magnetic stirrer to keep the homogenate uniformly dispersed; inserting one end of a capillary tube into the homogenate liquid through the homogenate tank cover, and sealing the homogenate tank; opening a pressure reducing valve, slowly and gradually opening a control valve and introducing nitrogen; simultaneously, ultrasonic treatment is applied to the capillary column; under the action of nitrogen and ultrasonic vibration, the homogenate slowly and uniformly enters the capillary; when the capillary column is filled with the filler by about 3cm, all control valves are opened, the pressure of nitrogen is 5Mpa, and the filling is carried out by keeping constant pressure.
The filling method of capillary columns with different lengths comprises the following steps:
2.1 the length of the capillary column is 20cm and less, the filling is carried out by using a 1-step method, the filling is continuously carried out under constant pressure until the required length is reached, and the nitrogen is dried; slowly reducing the nitrogen pressure to zero, and unloading the capillary column; connecting the capillary column to a liquid chromatography pump, and compacting the chromatographic packing in the capillary column by utilizing sectional gradual pressure increase to obtain the usable capillary column.
2.2 the capillary column is 20cm-25cm long, filling by using a 2-step method, firstly filling to 20cm under constant pressure, then adjusting the capillary column to be above the homogenate liquid level, drying by using nitrogen, secondly repeatedly inserting the capillary column below the homogenate liquid level, and filling under constant pressure until the required length is reached; slowly reducing the nitrogen pressure to zero, and unloading the capillary column; connecting the capillary column to a liquid chromatography pump, and compacting the chromatographic packing in the capillary column by using sectional type gradual pressure increase to obtain the usable capillary column.
2.3 capillary column length 25cm-30cm, using 3 steps method to fill, first continuously filling to 20cm, nitrogen blow drying, second filling to 25cm, third filling to required length. Slowly reducing the nitrogen pressure to zero, and unloading the capillary column; connecting the capillary column to a liquid chromatography pump, and compacting the chromatographic packing in the capillary column by utilizing sectional gradual pressure increase to obtain the usable capillary column.
The chromatographic column packing is reverse chromatographic packing, and the grain size of the packing is 1.9-5 microns. The high pressure generated by the liquid chromatography pump is used, the maximum pressure is between 200bar and 500bar according to different column lengths, the packing is ensured to be compact, and high column efficiency is obtained. Under the pressure of nitrogen, the homogenate liquid slowly moves towards the sieve plate of the capillary column, when reaching the sieve plate of the capillary column, the solvent of the homogenate liquid flows out, and the chromatographic packing stays down to block the packing at the back from moving forwards. Since the homogenate in the homogenization tank moves upward without stop, the chromatographic packing gradually fills the capillary column.
In another embodiment of the present invention, a capillary high pressure liquid chromatography column obtained by the above-mentioned ultrasonic preparation method is provided. According to the preparation method, the capillary column with the grain diameter of 1.9 mu m and the length of 30cm can be prepared; meanwhile, the prepared capillary column has good separation degree reproducibility and high column efficiency; and has long service life; meanwhile, compared with the capillary column with the integrated separation column and the spray needle, the damage of the needle tip to the whole capillary column is avoided.
In another embodiment of the present invention, there is provided a use of the capillary high pressure liquid chromatography column described above in proteomics research.
Specifically, the application comprises the step of carrying out high-efficiency separation on complex and trace polypeptides by using the capillary high-pressure liquid chromatography column.
Example 1 analysis of the separation Performance of a capillary chromatography column with a column length of 25cm on a 293T cell Trypsin lysate
Preparing 500 mu l of 50mg/ml 1.9 mu m C18 reverse chromatographic filler homogenate by using a 2ml glass sample bottle, wherein a solvent is methanol, a magneton 3 with the diameter of 5mm is placed in the homogenate, and the homogenate is subjected to ultrasonic treatment for 2 min; placing the homogenate tank connected with the nitrogen device on a magnetic stirrer 4, and stirring homogenate liquid at a constant speed; putting the sample bottle into a homogenate tank 1; a capillary column 5 having an inner diameter of 150 μm, an outer diameter of 360 μm and a length of 30cm was inserted into the homogenate 2 as shown in FIG. 1; sealing the homogenate tank by using a sealing screw; adjusting the ultrasonic device 7 to a proper position, and turning on the ultrasonic (1.6W); opening a nitrogen pressure reducing valve, and adjusting the nitrogen pressure to be 5 MPa; controlling a valve 9, and slowly increasing the nitrogen pressure at the beginning, wherein the filler slowly moves to the capillary plunger and gradually accumulates under the nitrogen pressure; when the pressure was 3cm, the pressure was adjusted to 5MPa and kept constant. Under the action of nitrogen pressure and ultrasonic vibration, the homogenate uniformly and slowly enters the capillary; filling by using a 2-step method, namely firstly filling to 20cm at constant pressure, adjusting the capillary column to be above the homogenate liquid level, drying by using nitrogen, then repeatedly inserting the capillary column below the homogenate liquid level, filling to more than 25cm at constant pressure, and drying by using nitrogen; the nitrogen pressure is slowly reduced to zero, and the capillary column is dismounted. Connecting the capillary column to a liquid chromatography pump, and compacting the chromatographic packing in the capillary column by using the sectional pressure rise until 450 bar; and cutting off a tail hollow pipe to obtain a capillary column with the length of 25 cm. Polypeptide separation is carried out by using a capillary column, and then mass spectrum detection is carried out.
The chromatographic conditions were as follows: mobile phase a, 0.1% formic acid, mobile phase B, 0.1% formic acid-80% acetonitrile; mobile phase B increased linearly from 4% to 7% in 1min, to 25% in 94min, to 40% in 16min, to 100% in 5min and then maintained for 4min at a flow rate of 700 nl/min. Mass spectrum conditions: in a full scanning range of 350-2000 m/z, after one full scanning is finished, selecting 20 strongest peaks for secondary scanning; the MS1 scan resolution was 60,000 and the MS2 scan resolution was 15,000. The 3-time repeated detection chromatograms are shown in figure 2 (total ion flow diagram) and figure 3 (extracted ion peak diagram), and the reproducibility and the separation degree of complex samples are good. The apparatus used was: thermo company EASY-nLC 1200 liquid phase system and QE HFX mass spectrometer. Sample preparation: 293T cells 1. mu.g of proteolytic enzyme-cleaved polypeptide.
Example 2 analysis of the separation Performance of a capillary chromatographic column having a column length of 15cm on a cleavage lysate of bovine serum albumin
1.9 μm C18 chromatography packing 15cm reverse capillary chromatography column was packed as in example 1 except that the packing was done using a 1-step method. The chromatographic conditions were as follows: mobile phase a, 0.1% formic acid, mobile phase B, 0.1% formic acid-80% acetonitrile; mobile phase B increased linearly from 6% to 25% in 23min, to 40% in 4min, to 100% in 2min and then maintained for 1min at a flow rate of 1000 nl/min. Mass spectrum conditions: in a full scanning range of 350-2000 m/z, after one full scanning is finished, selecting 20 strongest peaks for secondary scanning; the MS1 scan resolution was 60,000 and the MS2 scan resolution was 15,000. The 3 repeated detections overlap chromatogram map is shown in figure 4, and the reproducibility and the sample separation degree are good. The instrument comprises the following steps: thermo company EASY-nLC 1200 liquid phase system and QE HFX mass spectrometer. Sample preparation: bovine serum albumin BSA 0.03. mu.g of proteolytic polypeptide.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An ultrasonic preparation method of a capillary high-pressure liquid chromatographic column is characterized by comprising the following steps of filling a capillary column by adopting a medium-pressure homogenization ultrasonic filling method, specifically:
preparing a chromatographic column filler into homogenate by using an organic solvent, and keeping the uniform dispersion of the chromatographic column filler; inserting one end of a capillary column into the homogenate, simultaneously carrying out ultrasonic treatment on the capillary column, and moving the filler to the capillary plunger and gradually accumulating under the action of inert gas and ultrasonic vibration; and when the mixture is filled to the position of 2-3 cm, adjusting the pressure of the inert gas to 4-5 MPa and keeping constant pressure for filling.
2. The ultrasonic preparation method of claim 1, wherein the organic solvent is a chromatographic grade organic solvent, preferably the organic solvent is methanol.
3. The ultrasonic preparation method of claim 1, wherein the chromatographic column packing is reverse chromatographic packing comprising C18 reverse chromatographic column packing, and the packing has a particle size of 1.9 μm to 5 μm.
4. The ultrasonic method of claim 1, wherein the inert gas is nitrogen.
5. The method of claim 1, wherein the capillary column is a capillary tube with a frit, preferably wherein the method of preparing the capillary tube with the frit comprises:
the preparation ratio is kasil 1624: kasil 1: and (2) inserting a capillary tube into the homogeneous liquid with a ratio of 1-3: 1:1, quickly pulling out the capillary tube when the homogeneous liquid rises to be more than 1cm, and then placing the capillary tube in a high-temperature (such as 80-90 ℃) environment for 3-4 hours to obtain the formamide-containing homogeneous liquid.
6. The ultrasonic preparation method of claim 1,
when the length of the capillary column is within 20cm (including 20cm), filling by using a one-step method directly, namely continuously filling under the constant pressure until the required length is reached, and drying by using nitrogen; then reducing the nitrogen pressure to zero, and unloading the capillary column; connecting the capillary column to a liquid chromatography pump, and compacting the chromatographic packing in the capillary column by utilizing sectional gradual pressure increase to obtain a usable capillary column;
when the length of the capillary column is 20cm-25cm (including 25cm), filling by using a two-step method, specifically, firstly filling to 20cm according to the constant pressure, then adjusting the capillary column to be above the homogenate liquid level, blowing the capillary column by using nitrogen, secondly repeatedly inserting the capillary column below the homogenate liquid level, and filling the capillary column at the constant pressure until the required length is reached; then reducing the nitrogen pressure to zero, and unloading the capillary column; connecting the capillary column to a liquid chromatography pump, and compacting the chromatographic packing in the capillary column by utilizing sectional type gradual pressure increase to obtain a usable capillary column;
when the length of the capillary column is 25cm-30cm, filling by using a three-step method, specifically, continuously filling to 20cm, drying by using nitrogen, filling to 25cm in the second step, and filling to the required length in the third step; the third specific method comprises the following steps: reducing the nitrogen pressure to zero, and unloading the capillary column; connecting the capillary column to a liquid chromatography pump, and compacting the chromatographic packing in the capillary column by utilizing sectional gradual pressure increase to obtain the usable capillary column.
7. The ultrasound preparation method of claim 6, wherein the high pressure maximum pressure generated by the liquid chromatography pump is controlled between 200bar and 500 bar.
8. Capillary high pressure liquid chromatography column obtained by the ultrasonic preparation method according to any one of claims 1 to 7.
9. Use of a capillary high pressure liquid chromatography column according to claim 8 in proteomics research.
10. The use of claim 9, wherein the use comprises high efficiency separation of complex trace amounts of the polypeptide using a capillary high pressure liquid chromatography column according to claim 8.
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