CN112824895B

CN112824895B - Method for preparing capillary packed column mass spectrum spray needle by photonic crystal fiber and application

Info

Publication number: CN112824895B
Application number: CN201911148893.3A
Authority: CN
Inventors: 马淑娟; 欧俊杰; 叶明亮
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2019-11-21
Filing date: 2019-11-21
Publication date: 2022-03-15
Anticipated expiration: 2039-11-21
Also published as: CN112824895A

Abstract

The invention relates to a method for preparing a capillary packed column mass spectrum electrospray nozzle needle by using a photonic crystal fiber with an axially set porous microstructure. The method specifically comprises the steps of removing an outer protective layer from a photonic crystal fiber with a required length, using a pouring sealant as an adhesive, fixing one part of the fiber with the protective layer removed in the inner wall of a capillary tube (plunger function), extending the other part of the fiber out of the capillary tube (spray needle function), and curing at a high temperature to be used as an integrated mass spectrum electrospray spray needle. The method for manufacturing the electrospray ionization mass spectrometry spray needle has the advantages of few steps, simplicity in operation and good repeatability, and the preparation process can be completed within hours. The prepared mass spectrum spray needle matrix is fused silica, and the mass spectrum spray needle matrix contains hundreds of micro-channels, so that the defects that the traditional spray needle is easy to block and break are overcome, and the mass spectrum spray needle is not easy to swell in an organic reagent, and has very good mechanical stability and thermal stability and long service life.

Description

Method for preparing capillary packed column mass spectrum spray needle by photonic crystal fiber and application

Technical Field

The invention relates to a method for preparing an electrospray mass spectrometry spray needle of a capillary packed column by using a photonic crystal fiber (MSF) with a porous microstructure as a raw material, which is characterized in that a photonic crystal fiber with a required length is firstly subjected to outer skin protection layer removal, then a part of the fiber with the protection layer removed is fixed in the inner wall of a capillary tube by using an adhesive (the function of a plunger), and the other part of the fiber directly extends out of the capillary tube (the function of the spray needle), and the fiber can be used after being solidified.

Background

Mass Spectrometry (MS), also known as Mass spectrometry, provides abundant structural information of a sample in a single analysis, and is therefore widely used in various fields. The combination technique of mass spectrometry and liquid chromatography, especially capillary micro-column liquid chromatography, fully combines the advantages of high sensitivity and high specificity of mass spectrometry, and the advantages of low sample consumption and high separation efficiency of capillary liquid chromatography, and gradually becomes an important analysis means for complex samples, especially rare or precious samples, and plays an increasingly important role in the fields of environment and biological medicine (document 1Simpson. D.C., ect. Electrophoresis,2005,26(7-8): 1291-305.). The mass spectrum electrospray nozzle needle is used as a key component for liquid chromatography-mass spectrometry, and the spray effect of the mass spectrum electrospray nozzle needle can directly influence the reproducibility and sensitivity of mass spectrum identification, so that the accuracy and accuracy of analysis are influenced. Therefore, the development of a spray needle with good spray stability, high efficiency and long service life is very important for LC-MS analysis. At present, the methods for preparing Mass spectrum electrospray nozzles mainly include laser ablation drawing, chemical etching, physical grinding and monolithic column on column (document 2Koerner T., ect., Rapid communication in Mass Spectrometry,2005,19(22): 3279-. The laser ablation drawing method is to melt a quartz capillary tube by laser and then prepare a mass spectrum spray needle with gradually reduced inner diameter and outer diameter under the action of axial traction force. Although the method is the most widely used method at present, the tip of the prepared spray needle is extremely easy to block, and the service life is short. The chemical etching method is to etch the top end of the capillary tube by using a chemical agent (such as hydrofluoric acid), and to control the specification of the needle by controlling the concentration of the chemical agent and the etching time. Although the spray needle prepared by the method has small outer diameter and uniform inner diameter and is not easy to block, in order to avoid hydrofluoric acid from entering the capillary, water, gas or oil needs to be introduced into the capillary in the operation process, the etching time needs to be strictly controlled, and the operation is complex. The physical grinding method is to fix the capillary tube and then polish the capillary tube with sand paper to obtain the spray needle with controllable conical surface and uniform inner diameter. The spray needle obtained by the method has larger outer diameter of the tip of the needle, and is difficult to obtain stable spray at low flow rate. The column monolithic column method is that a monolithic column with a certain length is prepared in situ at the front end of a capillary tube and used as a spray needle, before preparation, the interior of the capillary tube needs to be activated and derived, and the monolithic column spray needles with different apertures are obtained by controlling the polymerization process of the monolithic column. The method has the disadvantages of complicated operation steps, long preparation time and poor repeatability.

Photonic Crystal Fibers (PCFs), also known as microstructured Fibers, are a new type of fiber material, which generally consist of air holes made of silica as a background material, with a complex refractive index distribution in the cross-section, the dimensions of these air holes being approximately on the same scale as the wavelength of the light waves and running through the entire length of the device, and the light waves can be confined to propagate in the core region of the fiber with low refractive index. By varying the arrangement and size of the air guide holes, the dispersion ratio of the fiber will vary. The concept of PCF was first proposed by p.s.j.russell et al in 1992, but was not successfully prepared until 1996 by the photoelectric research center of the university of south ampere and prawns, uk and the university of denmark technology. Compared with the common optical fiber, the photonic crystal optical fiber has the characteristics of super-strong bending resistance, flexible dispersion cutting, good nonlinearity, radiation resistance and the like, thereby showing wide development prospect and being gradually used in the fields of optical fiber communication, optical devices and the like (document 5 Chenwei. special report of communication world: 2017.47-49; document 6 quiet. physical report. 2017.10.112-113).

The patent takes a novel photonic crystal fiber (MSF) with a plurality of air holes as a raw material to prepare a mass spectrum electrospray nozzle needle which is applicable to a capillary filling column with the inner diameter of about 150 mu m. Compared with other methods for manufacturing the capillary electrospray nozzle needle, the method does not need complex equipment or complicated preparation process, is simple to operate, has few steps, low technical difficulty and good repeatability, and the prepared plunger substrate is fused silica, has stable performance, does not swell in an organic solvent like an integral column nozzle needle, and has good mechanical stability and thermal stability.

Disclosure of Invention

The invention aims to rapidly prepare a mass spectrum electrospray nozzle needle suitable for a capillary packed column with the inner diameter of about 150 mu m, and the nozzle needle has better stability and the function of a chromatographic column plug.

In order to achieve the purpose, the invention adopts the technical scheme that: the outer skin protective layer of the photonic crystal optical fiber with the required length is removed, then the pouring sealant is used as an adhesive, the optical fiber part with the protective layer removed is fixed in the inner wall of the capillary, and the optical fiber part extends out of the capillary, and the optical fiber can be used after being solidified.

The specific process is as follows:

1) immersing the optical fiber cut into a length of 2-5 cm into 5-10 mL of toluene solution, soaking for 10-30 min to swell the coating layer on the outer wall of the optical fiber, carefully taking out the optical fiber by using a forceps, and removing the coating layer;

2) uniformly coating the pouring sealant on the outer wall of the optical fiber, wherein the coating length is 1-2 cm, then inserting the part of the optical fiber coated with the pouring sealant into the hollow capillary, and the length extending out of the capillary is 1-3 cm;

3) and (3) putting the capillary tube with the optical fiber into a drying oven at the temperature of 60-100 ℃ for 1-2 hours, and completely curing the pouring sealant for use.

The optical fiber used in the step 1) is a photonic crystal optical fiber, the specification is 148-152 μm of outer diameter (including a coating layer), the thickness of the coating layer is 5-15 μm, 80-130 air holes are contained in the coating layer, and the aperture range is 2-5 μm (preferably 2-3 μm).

The inner diameter of the capillary packed column used in the step 2) is 140-160 μm (preferably 150 μm), and the outer diameter is 360 μm.

Compared with other methods for manufacturing the capillary electrospray nozzle needle, the method does not need complex equipment or complex preparation process, has simple operation steps, controllable process and low technical difficulty, and the obtained nozzle needle has single hole structure, good repeatability, does not swell in organic solvent like an integral column nozzle needle, and has good mechanical stability and thermal stability. And because the optical fiber contains numerous air hole passages, the defect that the integral drawing spray needle is easy to block can be avoided, more importantly, the inner diameter of the conventional spray needle is reduced to about 3um from dozens to hundreds of micrometers in the manufacturing process, the inner wall of the conventional spray needle becomes very thin and is easy to break, and the optical fiber spray needle is not easy to break because the processes of drawing, polishing or corrosion and the like are not carried out in the preparation process and a quartz protective layer with the thickness of more than 20um is arranged outside the hole region, so that the service life of the optical fiber spray needle is prolonged.

Description of the figures/tables

FIG. 1 is a schematic diagram of the preparation of an electrospray mass spectrometry spray needle using a photonic crystal fiber as a substrate.

FIG. 2 is a cross-sectional scanning electron micrograph of a photonic crystal fiber used in example 1.

FIG. 3 shows a capillary packed column with an integral photonic crystal fiber needle (example 1) and b with an integral drawing needle (example 2).

FIG. 4 is a graph comparing the electrospray effect of a photonic crystal fiber needle (example 1) and b drawn capillary needle (example 2).

FIG. 5 is a chromatogram of the BSA enzymatic solution on a 3 μm C18 silica gel capillary packed column with a photonic crystal fiber as the electrospray nozzle needle (example 1) and b as the integrated drawing nozzle needle (example 2).

Table 1 shows the results of the identification of Bovine Serum Albumin (BSA) enzymatic hydrolysate on 3 μm C18 silica gel capillary packed columns with photonic crystal fiber needles (example 1) and integrally drawn needles (example 2), respectively.

Detailed Description

Example 1 preparation of capillary packed column by electrospray nozzle using photonic crystal fiber as mass spectrometer

1) Cutting a photonic crystal fiber (the outer diameter is 125 mu m, the coating layer is 10um and contains 126 air holes) into a length of 2cm, soaking the photonic crystal fiber in 3mL of toluene solution for 10min to swell the coating layer on the outer wall of the optical fiber, carefully taking out the optical fiber by using forceps, and removing the coating layer;

2) uniformly coating the outer wall of the optical fiber with a pouring sealant (Daokning PDMS-184 glue, USA) with the length of 1cm, and then inserting the optical fiber with the pouring sealant into a 30cm hollow capillary tube (with the outer diameter of 360 μm and the inner diameter of 150 μm), wherein the inner length is 1cm, and the outer length is 1 cm;

3) placing the capillary tube obtained in the step 2) into an oven at 80 ℃ for 2 hours to completely cure the pouring sealant;

4) filling a capillary column by using 3 mu m C18 silica gel as a filler by adopting a homogenization method, wherein the length of the filler is 25 cm;

5) and performing a cLC-MS/MS analysis experiment by taking the enzymolysis liquid of the standard protein Bovine Serum Albumin (BSA) as a sample.

Preparation of BSA enzymatic samples: dissolving 1mg BSA in 100mM ammonium bicarbonate solution (pH 8.2) containing 8M urea, adding 80. mu. mol dithiothreitol, keeping the temperature at 60 ℃ for 1h, adding 40. mu. mol iodoacetamide, keeping the temperature away from light for 40min, diluting the urea concentration to 1M with 100mM ammonium bicarbonate solution, adding trypsin according to the mass ratio of the urea to the trypsin of 1:40, reacting in a water bath at 37 ℃ for 16h, desalting the obtained enzymolysis liquid by using a C18 silica gel SPE column, freeze-drying, and storing in a refrigerator at-20 ℃ for later use.

cLC-MS/MS analysis System: the system consisted of a Surveyor HPLC system (Thermo Fisher Scientific, San Jose, USA), a six-way valve, a pre-column, an analytical column, and LTQ Orbitrap Velos) ion trap mass spectrometry (Thermo Fisher Scientific, San Jose, USA). The pre-column was a reverse phase column (4 cm. times.75 μm i.d.. times.360 μm o.d.) packed with C18-silica AQ particles (particle size 5 μm, pore size 12 nm). The analytical column was a capillary packed column with a fiber optic spray needle (26.0 cm. times.150 μm i.d.. times.360 μm o.d.) prepared in example 1.

Chromatographic analysis conditions: mobile phase a was an aqueous solution containing 0.1% (v%) FA, and mobile phase B was an acetonitrile solution containing 0.1% FA (v%). During analysis, a sample to be analyzed enters the pre-column under the drive of the 100% mobile phase A and is captured by the pre-column. Then the mixed solution of the mobile phases A and B is carried into an analytical column by valve switching for gradient elution, and finally the mixture enters a mass spectrum for identification. Elution gradient: the volume of mobile phase B increased from 0 to 5% in 2min, from 5% to 35% in 30min, from 35% to 80% in 3min, and finally maintained for 10 min.

Mass spectrometry conditions: the data acquisition software is Xcalibur 2.1software (thermo Fisher scientific); the data acquisition mode is a data dependence mode; the full scanning range of the primary mass spectrum is 400-2000 (m/z); extracting the first 20 strongest ion peaks, crushing and dissociating to perform secondary mass spectrum scanning, wherein the normalized collision energy is 35%; the electrospray voltage was 2.0 kV; the ion transport capillary temperature was 250 ℃.

Mass spectrometry data analysis process: the ". raw" file from the Xcalibur 2.1 workstation was converted to ". mgf" format by the software of the protome distributor (v1.2.0.208, Thermo, San Jose, CA), and then subjected to search and identification using the Mascot Daemon (version 2.5.1) protein identification software platform (Matrix Science, London, UK). The BSA database used was a bovine library downloaded from the website http:// www.uniprot.org/. The parent ion mass deviation tolerance was set to 20ppm and the fragment ion mass deviation was set to 0.8 Da. The enzyme cutting is carried out by trypsin full enzyme cutting, and at most two missed cutting sites are adopted. The iodoacetylation modification of cysteine is a fixed modification, and the oxidation modification of methionine is a variable modification; when the retrieval result is derived, the score value is set to be higher than 20, and the false positive rate (FDRs) is lower than 1%.

Example 2 capillary packed column was prepared using an integral laser drawn needle as a mass spectrometric electrospray needle

1) Drawing an empty capillary tube (with the inner diameter of 150 mu m) by using a laser to prepare an integrated drawn spray needle capillary tube, wherein the inner diameter of a spray needle is 5 mu m;

2) filling the capillary column obtained in the step by using a homogenization method and 3 mu m C18 silica gel as a filler by using the same method as the embodiment 1, wherein the length of the filler is 25 cm;

3) the sample was digested with Bovine Serum Albumin (BSA), and the cLC-MS/MS analysis was performed in the same manner as in example 1.

FIG. 1 is a schematic diagram of the preparation of an electrospray mass spectrometry spray needle using a photonic crystal fiber as a substrate. The preparation process is very simple, and the photonic crystal fiber part with the coating removed can be used only by fixing the photonic crystal fiber part into the inner wall of the capillary.

FIG. 2 is a cross-sectional scanning electron micrograph (after removal of the cladding layer) of the photonic crystal fiber used in example 1. The fiber contained 126 air channels with a diameter of 2.6 μm inside. Since the diameter of these microchannels is only 2.6 μm, liquid solvents can freely pass through while solid particles are blocked, thus functioning as a column plug. And the 126 channels effectively avoid the problem of blockage of the needle head of the spray needle.

FIG. 3 shows a capillary packed column with an integral photonic crystal fiber needle (example 1) and b with an integral drawn capillary needle (example 2).

FIG. 4 is a graph comparing the electrospray effect of a photonic crystal fiber needle (example 1) and b drawn capillary needle (example 2). In the experimental process, it was found that under the gradient elution condition, the fiber optic needle was similar to the draw needle and a stable electrospray flow was obtained.

During the experiment it was found that the chromatographic separation patterns obtained with these two needles were relatively similar.

TABLE 1

The library searching result shows that the maximum number of polypeptide segments which can be identified by a single experiment of BSA enzymatic hydrolysate on a C18 silica gel capillary packed column with an optical fiber spray needle is 69, and the protein coverage rate is 79.2-83.5%; under the same condition, the protein coverage rate on a capillary packed column with an integrated drawing spray needle is 78.9-80.8%, and the maximum number of BSA peptide fragments identified in a single time is 65. The data results show that the photonic crystal fiber needle has a mass spectrum spraying effect which is not inferior to that of the integral drawing needle, the defect that the integral drawing needle is easy to block can be avoided because the optical fiber contains hundreds of micro-channels, more importantly, the inner diameter of the integral drawing needle is reduced from 150 mu m to about 5 mu m in the drawing process, the inner wall of the integral drawing needle becomes very thin and is easy to break, and the fiber needle is not drawn, and the quartz protective layer with the diameter of more than 20 mu m is arranged outside the hole region, so that the integral drawing needle is not easy to break, and the service life of the needle is greatly prolonged.

Claims

1. A preparation method of a mass spectrum electrospray nozzle needle suitable for a capillary packed column is characterized by comprising the following steps: using a photonic crystal fiber (MSF) with an outer diameter of 125 μm and an inner diameter of which contains 126 axially-set air through holes as a mass spectrum spray needle of a capillary filling column, removing a sheath protection layer from the photonic crystal fiber with a certain length, coating Polydimethylsiloxane (PDMS) pouring sealant on the outer wall surface of the optical fiber with a part of sheath removed, inserting the optical fiber coated with the PDMS pouring sealant into a capillary, and curing the potting sealant to fix a part of the optical fiber in the capillary and extend a part of the optical fiber out of the capillary; the spray needle has double functions, the part fixed in the capillary tube has the function of a packed column plunger, the part extending out of the capillary tube has the function of a mass spectrum electrospray spray needle, and the inner diameter of the capillary tube is 150 mu m; the process is as follows,

1) immersing the photonic crystal fiber cut into 2-5 cm into a toluene solution, soaking for 10-30 min, taking out with a forceps, and removing the outer skin of the coating layer;

2) uniformly coating the PDMS potting adhesive on the outer wall of the optical fiber, wherein the coating length is 1-2 cm, carefully inserting the PDMS potting adhesive into a tube body with one end of a capillary tube with the inner diameter of 150 mu m for 1-2 cm, and leaving the optical fiber with the length of 1-3 cm outside;

3) and (3) placing the capillary tube inserted with the optical fiber into a drying oven at the temperature of 60-100 ℃ for curing for 1-2 h, and then using.

2. An integral mass spectrometric electrospray needle prepared by the method of preparation of claim 1.

3. Use of an integral mass spectrometry electrospray needle according to claim 2.