阅读说明：本技术 一种高效钠升级液相色谱柱的分压填充方法 (Partial pressure filling method of high-efficiency sodium upgrading liquid chromatography column ) 是由 万建 汪兵 万翠红 李兵 周权 熊国梅 阮君 龚思颖 于 2019-11-18 设计创作，主要内容包括：一种高效钠升级液相色谱柱的分压填充方法,解决了钠升级色谱柱价格昂贵,色谱柱的填充效率较低的问题。其包括如下步骤：a)准备聚酰亚胺石英毛细管一根；b)采用拉针仪将聚酰亚胺石英毛细管一端拉细至5um,拉出的聚酰亚胺石英毛细管的针尖平滑；c)色谱柱填充；d)聚酰亚胺石英毛细管色谱柱针尖处理；e)色谱柱组装,将聚酰亚胺石英毛细管色谱柱与四通阀完成组装；f)填充液置换,利用钠升级液相色谱,将填充色谱柱的甲醇利用流动相进行置换后置于洁净干燥处保存。本发明填充钠升级色谱柱的成本在80元-150元之间,极大程度上降低了成本,且分离效率较高。(A partial pressure filling method of a high-efficiency sodium upgrading liquid chromatographic column solves the problems of high price and low filling efficiency of the sodium upgrading liquid chromatographic column. Which comprises the following steps: a) preparing one polyimide quartz capillary tube; b) one end of the polyimide quartz capillary tube is thinned to 5um by adopting a needle drawing instrument, and the needle point of the drawn polyimide quartz capillary tube is smooth; c) filling a chromatographic column; d) processing a polyimide quartz capillary chromatographic column needle tip; e) assembling a chromatographic column, namely assembling a polyimide quartz capillary chromatographic column and a four-way valve; f) and (3) replacing the filling liquid, namely upgrading the liquid chromatography by using sodium, replacing the methanol filled in the chromatographic column by using a mobile phase, and then placing the methanol in a clean and dry place for storage. The cost of the sodium-filled upgraded chromatographic column is between 80 yuan and 150 yuan, the cost is greatly reduced, and the separation efficiency is higher.)

1. A partial pressure filling method of a high-efficiency sodium upgrading liquid chromatographic column is characterized by comprising the following steps: a) preparing one polyimide quartz capillary tube;

b) one end of the polyimide quartz capillary tube is thinned to 5um by adopting a needle drawing instrument, and the needle point of the drawn polyimide quartz capillary tube is smooth;

c) column packing, 1, mixing column packing with methanol (taking C18 packing as an example), 2, connecting a packing device, fixing a drawn polyimide quartz capillary tube on the packing device, 3, setting partial pressure packing: observing the overflowing state of turbid liquid of the needle point of the polyimide quartz capillary tube to ensure that the needle point is normal and is not damaged, setting a pressure value of 4MPa when filling is started, setting a pressure value of 4.2MPa when filling is 1/3 of the total column length of the polyimide quartz capillary tube, setting a pressure value of 4.5MPa when filling is 2/3 of the total column length, closing a nitrogen valve until filling is finished, and taking down the polyimide capillary tube chromatographic column after the filling device automatically releases pressure;

d) treating the needle point of the polyimide quartz capillary chromatographic column, soaking the needle point of the polyimide quartz capillary chromatographic column in acetonitrile for 30 minutes, taking out the soaked needle point of the polyimide quartz capillary chromatographic column, and scrubbing the needle point with dust-free paper;

e) assembling a chromatographic column, namely assembling a polyimide quartz capillary chromatographic column and a four-way valve;

f) and (3) replacing the filling liquid, namely upgrading the liquid chromatography by using sodium, replacing the methanol filled in the chromatographic column by using a mobile phase, and then placing the methanol in a clean and dry place for storage.

2. The partial pressure packing method of the high efficiency sodium upgrading liquid chromatography column as claimed in claim 1, wherein the pin drawing instrument draws the polyimide quartz capillary tube for a plurality of cycles, cycle 1: heating index is 400 ℃, voltage value is 25V, and delay time is 200S;

circulation 2, heating index is 390 ℃, voltage value is 15V, and delay time is 200S;

circulating 3, heating index is 390 ℃, voltage value is 15V, and delay time is 200S;

cycle 4, heating index 400 ℃, voltage value 20V and delay time 200S;

cycle 5, heating knowledge 410 ℃, voltage value 15V, delay time 110S, and tension value 240N.

Technical Field

The invention relates to the technical field of liquid chromatographic column filling, in particular to a partial pressure filling method of a high-efficiency sodium upgrading liquid chromatographic column.

Background

The combination of sodium upgrading liquid chromatography and high-resolution mass spectrometry is a commonly used technical means in proteomics at present. Wherein the column efficiency of the chromatographic column is a key index for determining the identification quality of the protein. The sodium upgrading chromatographic column technology commercialized at present is mature. However, due to the requirements of different experiments and different protein sampling techniques, new requirements are put on the packing of chromatographic columns and the diversity of packing methods. The proteomics experiment sample population is large, the stability and the continuity of the chromatographic column are important, and therefore, the economy and the packing effect efficiency of the free-ranging column are necessary to be ensured in a controllable range. The price of the commercial sodium upgrading chromatographic column in the market is 8000-12000 yuan mostly.

Disclosure of Invention

Aiming at the situation, in order to overcome the defects of the prior art, the invention provides a partial pressure filling method of a high-efficiency sodium upgrading liquid chromatographic column, which effectively solves the problems of high price and low filling efficiency of the sodium upgrading liquid chromatographic column.

In order to achieve the above object, the present invention comprises the steps of: a) preparing one polyimide quartz capillary tube;

b) one end of the polyimide quartz capillary tube is thinned to 5um by adopting a needle drawing instrument, and the needle point of the drawn polyimide quartz capillary tube is smooth;

c) column packing, 1, using a methanol mixed column packing (taking C18 packing as an example), 2, connecting a packing device, fixing a drawn polyimide quartz capillary column on the packing device, 3, setting partial pressure packing: observing the overflowing state of a turbid liquid of the needle point of the polyimide quartz capillary tube to ensure that the needle point is normal and is not damaged, setting a pressure value of 4MPa when filling is started, setting a pressure value of 4.2MPa when filling is 1/3 of the total column length of the polyimide quartz capillary tube, setting a pressure value of 4.5MPa when filling is 2/3 of the total column length, closing a nitrogen valve until filling is finished, and taking down the polyimide quartz capillary tube chromatographic column after the filling device automatically releases pressure;

d) treating the needle point of the polyimide quartz capillary chromatographic column, soaking the needle point of the polyimide quartz capillary chromatographic column in acetonitrile for 30 minutes, taking out the soaked needle point of the polyimide quartz capillary chromatographic column, and scrubbing the needle point with dust-free paper;

e) assembling a chromatographic column, namely assembling a polyimide quartz capillary chromatographic column and a four-way valve;

f) and (3) replacing the filling liquid, namely upgrading the liquid chromatography by using sodium, replacing the methanol filled in the chromatographic column by using a mobile phase, and then placing the methanol in a clean and dry place for storage.

The cost of the polyimide quartz capillary tube filled with the sodium upgrading chromatographic column is between 80 yuan and 150 yuan, the cost is greatly reduced, the separation efficiency is higher, and the ionization efficiency is high when the polyimide quartz capillary tube is combined with a mass spectrum.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 shows the effect of the needle tip observed under the perspective mirror of the present invention.

FIG. 2 is a schematic diagram of the chromatographic assembly of the present invention.

FIG. 3 is a schematic diagram of column efficiency detection of a chromatographic column according to the present invention

Detailed Description

The following describes in further detail embodiments of the present invention with reference to fig. 1-3.

As can be seen from fig. 1-3, the present invention comprises the following steps: a) preparing one polyimide quartz capillary tube;

the specification of the polyimide quartz capillary tube commonly used at present is 360um of external diameter, and the internal diameter has three specifications of 75um, 100um, 150 um.

b) One end of the polyimide quartz capillary tube is thinned to 5um by adopting a needle drawing instrument, and the needle point of the drawn polyimide quartz capillary tube is smooth;

as shown in FIG. 1, the tip of the drawn polyimide quartz capillary is smooth, the tip of the drawn polyimide quartz capillary is drawn to be about 5um, the ionization efficiency of the drawn polyimide quartz capillary in the liquid chromatography-mass spectrometry combination is determined by the shape of the tip, and the needle drawing instrument adopted in the invention is a P-2000 needle drawing instrument of SUTTER company.

Drawing a polyimide quartz capillary tube by a needle drawing instrument by adopting multiple cycles, wherein the cycle is 1: heating index is 400 ℃, voltage value is 25V, and delay time is 200S;

cycle 2, heating index of 390 ℃, voltage value of 15V and delay time of 200S

Circulating 3, heating index is 390 ℃, voltage value is 15V, and delay time is 200S;

cycle 4, heating index 400 ℃, voltage value 20V and delay time 200S;

cycle 5, heating knowledge 410 ℃, voltage value 15V, delay time 110S, and tension value 240N.

The method of the needle drawing instrument is set to influence the needle drawing effect, the parameter is obtained through multiple tests, the tip of the drawn polyimide quartz capillary is smooth, the filling is easy, and the ionization efficiency is high when the liquid chromatogram and the mass spectrum are combined.

c) Column packing, 1, using a methanol mixed column packing (taking C18 packing as an example), 2, connecting a packing device, fixing a drawn polyimide quartz capillary column on the packing device, 3, setting partial pressure packing: observing the overflowing state of turbid liquid of the needle point of the polyimide quartz capillary tube to ensure that the needle point is normal and is not damaged, setting a pressure value of 4MPa when filling is started, setting a pressure value of 4.2MPa when filling is 1/3 of the total column length of the polyimide quartz capillary tube, setting a pressure value of 4.5MPa when filling is 2/3 of the total column length, closing a nitrogen valve until filling is finished, and taking down the polyimide capillary tube chromatographic column after the filling device automatically releases pressure;

the filling device is the prior art, a plurality of filling devices can use the method at present, the C18 filler is taken as an example in the application for explanation, the filling device utilizes a magnetic stirrer to continuously stir to obtain C18 methanol mixed suspension of the C18 filler, the high-pressure nitrogen presses the uniformly mixed methanol suspension of the filler into a polyimide quartz capillary chromatographic column, and the needle point can ensure that the filler cannot leak.

d) Treating the needle point of the polyimide quartz capillary chromatographic column, soaking the needle point of the polyimide quartz capillary chromatographic column in acetonitrile for 30 minutes, taking out the soaked needle point of the polyimide quartz capillary chromatographic column, and scrubbing the needle point with dust-free paper;

e) assembling a chromatographic column, namely assembling a polyimide quartz capillary chromatographic column and a four-way valve;

the chromatographic column assembly is shown in FIG. 2, which comprises an HPLC pump, an automatic sample injector, a pre-column, a four-way valve, a polyimide quartz capillary tube (as an analytical column), a hole-through steering valve, etc., and is completed by a 360um four-way valve with a charging end, or by a four-way valve with an inner diameter of 1/32 inches and a 1/32 to 360um sleeve,

f) replacing the filling liquid, namely upgrading the liquid chromatogram by using sodium, replacing the methanol filled in the chromatographic column by using a mobile phase, and then storing the methanol in a clean and dry place;

g) chromatographic column efficiency detection

And connecting the filled polyimide quartz capillary column to a Saimerfi Q active Plus liquid chromatography mass spectrometer. Mobile phase: phase A is pure water containing 0.1% formic acid; phase B was acetonitrile containing 10% pure water with 0.1% formic acid added. The flow rate was 300nl/min and the elution gradient is shown in the table below. The chromatography effluent sample was directed through a HESI ion source into the mass spectrometer. The sample size of the liquid chromatography is 1 ul.

Chromatographic elution gradient

And (4) carrying out detection analysis on the polypeptide components subjected to nanoliter liquid chromatography separation by using a mass spectrum. The main mass spectrum parameters are as follows: the ion source voltage is 2 KV; the mass spectrum acquisition mode is a positive ion mode; the primary mass spectrum is used for collecting the molecular weight range of 350-2000 m/z; resolution 70000; the maximum ion implantation time is 20 ms; the secondary mass spectrum acquisition resolution is 17500; the maximum ion implantation time is 50 ms; TopN is set to 20; the fragmentation energy was set to 27 eV.

The protein sequence of human holoprotein (BSA) was looked up on the open protein database website Uniport (http:// www.uniprot.org /), and downloaded in FSATA format [11 ]. The protein was introduced into the protein discover 2.1 SP1 software for protein database search. And (3) search parameter setting: comparing the polypeptide mass range of 350Da-5000 Da; signal-to-noise ratio (S/N) > 1.5; the number of amino acids in the polypeptide fragment ranges from 6 to 144; the parent ion error is less than 10 PPM; the error of the polypeptide fragment is less than 0.2 Da; the comparison of the positive database and the negative database is checked to be 0.05 obvious and 0.01 extremely obvious.

Identification of proteins was accomplished using professional proteomics analysis software, Proteome discover 2.1 SP1 [14 ]. According to the button-up strategy, large protein is firstly enzymolyzed into polypeptide fragments, and the assembly of the protein is completed according to the coverage degree of the polypeptide fragments by identifying the polypeptide fragments, thereby completing the identification of the protein [15 ]. 42625 mass spectrograms with better quality are obtained by screening, 22592 mass spectrograms on the mass spectrograms are matched by comparing with human whole protein amino acid sequences in a Uniport database website, 15368 peptide fragment sequences are identified, and the peptide fragments are matched with 3617 protein sequences in the database. The removal of these protein sequence repeats finally identified 2939 proteins, the number of protein identifications being comparable to the effect of commercial chromatographic columns.

The cost of the sodium-filled upgrading chromatographic column is between 80 yuan and 150 yuan, the cost is greatly reduced, the separation efficiency is higher, and the ionization efficiency is high when the sodium-filled upgrading chromatographic column is combined with mass spectrum.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. 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.