Method for improving ion pair reversed-phase liquid chromatography separation by using inert gas atmosphere protection and chromatographic instrument mobile phase management system

Abstract

The invention discloses a method for improving ion pair reversed-phase liquid chromatography separation by using inert gas atmosphere protection and a chromatographic instrument mobile phase management system, and aims to prevent oxidation denaturation of an ion pair reagent in a mobile phase by using inert gas and prolong the service life of the mobile phase so as to improve the stability and reproducibility of the method. The method comprises the following steps: carrying out headspace replacement on a container filled with a mobile phase by using inert gas, pre-filling the inert gas protection box by using the inert gas, placing the container filled with the mobile phase in the inert gas protection box, carrying out sealed storage, and carrying out small-amount timed supplement on the inert gas protection box by using the inert gas. The method disclosed by the invention can be applied to reversed-phase liquid chromatography separation of a mobile phase added with an ion pair reagent and qualitative and quantitative analysis of combination of the reversed-phase liquid chromatography separation and an optical detector or a mass spectrum; after the method provided by the invention is used, the reproducibility and the stability of each analysis result are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of ion-pair reversed-phase liquid chromatography, and more specifically to a method for improving ion-pair reversed-phase liquid chromatography separation using an inert gas atmosphere and a mobile phase management system for the chromatograph. Background Technology

[0002] Reversed-phase liquid chromatography (RP-LC), as a highly efficient separation technique, is widely used in major fields such as biomanufacturing, pharmaceutical industry, and environmental safety to achieve accurate qualitative and quantitative analysis of analytes in complex samples. Its working principle mainly relies on the differential partitioning and migration of compounds in the nonpolar stationary phase (chromatographic column bed) and the polar mobile phase (elution solution). To further improve the separation efficiency of RPL, since the 1970s, ion-pairing RPL methods have received considerable attention. These methods involve adding ion-pairing reagents to the mobile phase to enhance the retention of polar compounds and improve chromatographic resolution. Ion-pairing reagents typically contain ionizable and nonpolar ends and exert their effects primarily through the following two pathways:

[0003] (1) When it is added to the mobile phase and comes into contact with the chromatographic column bed, its non-polar end will bind with the non-polar groups in the stationary phase through hydrophobic forces, while its ionizable end is exposed and ionized in the mobile phase, and undergoes electrostatic interaction with the polar compound to be tested to enhance retention.

[0004] (2) The ionizable end in the ion-pairing reagent will also first pair with the polar compound to be tested in the mobile phase, thereby improving the hydrophobicity of the compound to be tested and enhancing retention.

[0005] In recent years, RNA drugs based on gene silencing technology, represented by antisense oligonucleotides (ASO) and small interfering RNA (siRNA), have gained widespread popularity. ASO and siRNA drugs typically contain RNA fragments of 12-30 bases. After administration, the RNA drugs inhibit the translation of specific mRNAs in target organs through base pairing, thereby regulating protein expression levels for therapeutic purposes. Liquid chromatography-fluorescence detection (LC-FLD) and liquid chromatography-tandem mass spectrometry (LC-MS / MS), as efficient, accurate, and specific analytical and quantitative methods, play important roles in various stages of RNA drug development, including active ingredient screening, production quality control, and preclinical and clinical bioanalysis. Due to the cyclopentose and base components in the structure of RNA drugs, the compounds are often highly polar, and their high phosphate content makes them easily ionized and negatively charged in aqueous solutions. These chemical characteristics make ion-pair reversed-phase chromatography the preferred method for the separation and analysis of RNA drugs.

[0006] Currently, alkylamines are the primary ion-pairing reagents used for the chromatographic separation of RNA drugs. In aqueous solution, the alkyl portion readily binds to hydrophobic groups on the stationary phase surface, while the nitrogen atom, due to the presence of lone pairs of electrons, combines with the protons in the water to form positively charged ammonium ions. These ions then interact with the negatively charged phosphate backbone of the RNA drug, forming ion pairs. However, precisely because of this characteristic of the nitrogen atom in alkylamines, these reagents are highly susceptible to oxidation. In the presence of dissolved oxygen in water, they are oxidized to generate secondary amines, oximes, or further undergo polymerization reactions, thereby losing their ion-pairing function, reducing chromatographic resolution, and causing significant shifts in retention time. Furthermore, the oxidation products can further affect the ionization efficiency of the RNA drug itself, impacting the quantitative accuracy of LC-MS / MS. Therefore, preventing the oxidative denaturation of ion-pairing reagents is a crucial factor in ensuring the stability and reproducibility of ion-pair reversed-phase chromatography methods.

[0007] To address this issue, current research and production practices often employ a fresh-preparation approach for the mobile phase in ion-pair reversed-phase chromatography. However, this method is extremely wasteful of reagents and human resources and cannot support unsupervised automated analysis of large sample volumes over extended periods. Furthermore, recent literature reports a method using a ternary pump to deliver the mobile phase in an attempt to solve this problem. This method adds a high-pressure pump to mix and deliver a solution containing a high concentration of ion-pair reagents in a pre-set ratio, mitigating reagent loss due to oxidation. However, this significantly increases hardware acquisition and method development costs and introduces errors in mobile phase composition due to uneven mixing. These technical bottlenecks severely restrict the widespread application of ion-pair reversed-phase chromatography in RNA drug analysis. Summary of the Invention

[0008] The technical problem to be solved by the present invention is as follows: In response to the challenges described in the background art, the present invention proposes a low-cost, high-efficiency, simple and feasible method for preventing oxidation of the mobile phase in ion-pair reversed-phase chromatography. By using an inert gas atmosphere to reduce the oxygen exposure content in the local environment of the mobile phase, the usage time of the mobile phase containing ion-pair reagents is significantly extended, and the stability and reproducibility of ion-pair reversed-phase chromatography separation are significantly improved, thereby overcoming the defects caused by the prior art.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0010] In a first aspect, a method for improving ion-pair reversed-phase liquid chromatography separation using an inert gas atmosphere, comprising the following steps:

[0011] The mobile phase management system of the chromatograph is equipped with an inert gas protection box that is pre-filled with inert gas;

[0012] The headspace of the mobile phase container used in the chromatograph is replaced with an inert gas before use.

[0013] The container holding the mobile phase is placed inside the inert gas protection box and sealed.

[0014] The mobile phase pipeline extends through the pre-drilled hole at the top of the inert gas protection box and connects to the inlet of the chromatography pump;

[0015] The inert gas source is connected through an inlet valve built into the side of the inert gas protection box for initial filling and replenishment during use. The inert gas protection box is equipped with an outlet valve on the top.

[0016] The application field of this invention is ion-pair reversed-phase liquid chromatography. The principle of this invention is to replace and protect the local air of the mobile phase with a gas that does not react with the ion-pair reagent, thereby reducing the oxygen content in the local environment of the mobile phase and mitigating the impact of ion-pair reagent oxidation failure on chromatographic resolution and retention time reproducibility. When mass spectrometry is used as the detector, it prevents the oxidation products of the ion-pair reagent from affecting the ionization efficiency of the analyte, thereby improving quantitative accuracy and stability.

[0017] The above-mentioned method for improving ion-pair reversed-phase liquid chromatography separation by using an inert gas atmosphere for protection includes a mobile phase consisting of methanol / water or acetonitrile / water solution with added ion-pairing reagents.

[0018] The above-mentioned method for improving ion-pair reversed-phase liquid chromatography separation by using an inert gas atmosphere for protection includes, wherein the detector used in the ion-pair reversed-phase liquid chromatography method is one or more of the following: ultraviolet-visible spectrometer (UVD), fluorescence spectrometer (FLD), mass spectrometer (MS), tandem mass spectrometer (MS / MS), and high-resolution mass spectrometer (HRMS).

[0019] The above-mentioned method for improving ion-pair reversed-phase liquid chromatography separation using an inert gas atmosphere includes, but is not limited to: triethylamine (TEA), n-butylamine (BA), dibutylamine (DBA), N,N-diisopropylethylamine (DIEPA), n-hexylamine (HA), triethylamine acetate (TEAA), acetic acid (AA), formic acid (FA), and trifluoroacetic acid (TFA). The ion-pairing reagents are reagents that can form electrostatic interactions with the separated compounds or the chromatographic column bed.

[0020] The above-described method for improving ion-pair reversed-phase liquid chromatography separation using an inert gas atmosphere includes an inert gas selected from helium, neon, argon, krypton, xenon, nitrogen, and sulfur hexafluoride. Inert gases are either monatomic or polyatomic gases, meaning gases that do not chemically react with any component in the mobile phase solution.

[0021] The above-mentioned method for improving ion-pair reversed-phase liquid chromatography separation by using an inert gas atmosphere for protection includes an inert gas protection box that is gas-tight.

[0022] The above-mentioned method for improving ion-pair reversed-phase liquid chromatography separation by using an inert gas atmosphere protection includes an inert gas protection box with an oxygen sensor and a humidity sensor built in.

[0023] In the above-mentioned method for improving ion-pair reversed-phase liquid chromatography separation using an inert gas atmosphere, if the mobile phase container does not have a headspace, i.e., the mobile phase container is filled with liquid, then the inert gas replacement step is omitted.

[0024] The above-mentioned method for improving ion-pair reversed-phase liquid chromatography separation by using an inert gas atmosphere for protection includes a sealing ring installed at the reserved hole.

[0025] The above-mentioned method for improving ion-pair reversed-phase liquid chromatography separation using an inert gas atmosphere is described, wherein the inlet valve is a one-way check valve and the outlet valve is a one-way check valve.

[0026] In a second aspect, a chromatograph mobile phase management system includes an inert gas protection box pre-filled with inert gas, a mobile phase container, a mobile phase pipeline, a chromatographic pump, and an inert gas source.

[0027] The mobile phase container is placed inside the inert gas protection box and sealed. One end of the mobile phase pipeline is connected to the mobile phase container, and the other end extends through a pre-drilled hole at the top of the inert gas protection box and is connected to the inlet of the chromatography pump. The inert gas source is connected through an inlet valve on the side of the inert gas protection box, and an outlet valve is installed at the top of the inert gas protection box.

[0028] In the aforementioned mobile phase management system for a chromatograph, a sealing ring is installed at the reserved hole.

[0029] In the above-mentioned mobile phase management system for a chromatograph, the inlet valve is a one-way check valve, and the outlet valve is a one-way check valve.

[0030] The technical solution provided by the present invention, which utilizes an inert gas atmosphere to improve ion-pair reversed-phase liquid chromatography separation, has the following technical advantages:

[0031] When ion-pair reversed-phase chromatography is coupled with optical detectors such as UV or fluorescence, the chromatographic resolution and retention time are stably reproducible over a long period (not less than 120 hours) after using this method; when ion-pair reversed-phase chromatography is coupled with mass spectrometry, the chromatographic peak area is stably reproducible over a long period (not less than 120 hours) after using this method. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the inert gas protection box used in the method described in this invention;

[0033] Figure 2 A comparison of the first injection and the 120-hour injection chromatograms collected using the method described in this invention and without using the method described in this invention in Example 1;

[0034] Figure 3 The scatter plots of the internal standard peak areas collected from the first injection to the 120th hour injection (a total of 223 injections) are compared between Example 2 using the method described in this invention and not using the method described in this invention.

[0035] Figure 4 This is a schematic diagram of the structure of a chromatograph mobile phase management system according to the present invention.

[0036] The reference numerals in the attached figures are as follows:

[0037] Inert gas protection box 100, mobile phase pipeline 200, chromatography pump 300, inert gas source 400, reserved hole 101, inlet valve 102, outlet valve 103, sealing ring 104. Detailed Implementation

[0038] In order to make the technical means, inventive features, objectives and effects of the invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to specific illustrations. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0039] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] It should be noted that the structures, proportions, sizes, etc., illustrated in the accompanying drawings of this specification are only used to complement the content disclosed in the specification for those skilled in the art to understand and read, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0041] Furthermore, the terms such as "upper," "lower," "left," "right," "middle," and "one" used in this specification are merely for clarity of description and are not intended to limit the scope of the invention. Any changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

[0042] Example 1, such as Figure 1-2 As shown:

[0043] The quantification of an antisense oligonucleotide (ASO) drug and its two metabolites, 3'-1 and 3'-2, in a biological matrix using ion-pair reversed-phase chromatography-fluorescence spectroscopy (IP-RPLC-FLD) with external standard under an inert gas atmosphere is performed. The biological matrix in this embodiment includes, but is not limited to, plasma, tissues, and excrement from various preclinical experimental animals, as well as human plasma and excrement. Taking rat plasma as an example, the implementation steps are as follows:

[0044] A. Preparation of standard curves and quality control samples for ASO standards in rat plasma: Accurately weigh 2 mg of ASO and its 3'-1,3'-2 metabolite standards, dissolve in 1 mL of deionized water to obtain a 2 mg / mL mixed standard stock solution. Dilute the stock solution with 30% methanol to prepare a series of standard curve working solutions, add them to rat blank plasma at a volume ratio of 1:19 and mix well to obtain standard curve samples with concentrations of 10, 20, 100, 1000, 2000, 4000, 8000, and 10000 ng / mL, and quality control samples with concentrations of 10, 30, 200, 5000, and 7500 ng / mL, respectively.

[0045] B. Processing of rat plasma samples: Take 30 mL each of blank matrix, standard curve sample and quality control sample, and extract and enrich ASO and two metabolites using TRIzol reagent (Thermo Fisher Scientific, USA) and chloroform liquid-liquid extraction method. Transfer the supernatant to a 96-well plate and concentrate by nitrogen blowing. Add pre-synthesized PNA Atto fluorescent tagging reagent (PNA Bio, USA) and heat to 95°C, then cool to 4°C for later use.

[0046] C. Preparation of mobile phase for ion-pair reversed-phase chromatography with inert gas atmosphere protection: Prepare mobile phase A / B solutions in clean glass containers according to the following proportions: Mobile phase A: aqueous solution containing 0.05% (v / v) dibutylamine and 0.1% (v / v) hexafluoroisopropanol; Mobile phase B: methanol containing 0.05% (v / v) dibutylamine and 0.1% (v / v) hexafluoroisopropanol. Insert an inert tube connected to an argon gas source into the bottom of the bottle and continuously purge for 15 minutes before sealing and storing for later use.

[0047] D. Inert gas protection box 100 pre-charge: Press Figure 1 As shown, connect the inlet valve 102 to the argon source and open the outlet valve 103. Continue to purge for 20 minutes until the oxygen sensor reading is less than 0.5%. Place the mobile phase prepared in step C inside the box and extend the mobile phase pipeline 200 connected to the pump head into the mobile phase bottle through the reserved hole 101 in the box cover. Tighten the cap of the mobile phase bottle to seal the box. The sealing ring 104 can play a sealing role.

[0048] E. Quantitative analysis using IP-RPLC-FLD with inert gas protection: The instrument system used in Example 1 was an LC-40 liquid chromatograph (Shimadzu Corporation, Japan) equipped with an RF-20AXS fluorescence detector. The inert gas protection box 100 containing the mobile phase solution from step D was placed in the solvent management module of the system. The system operating software was opened and the parameters were set as follows:

[0049] Gradient: The content of mobile phase B increases from 20% by volume to 30% within 0 to 8 minutes;

[0050] Column type: Thermo DNAPac™ RP 4mm 2.1×100mm (Thermo Fisher Scientific, USA);

[0051] Flow rate: 0.6 mL / min;

[0052] Fluorescence excitation wavelength: 436 nm;

[0053] Fluorescence detection wavelength: 484nm;

[0054] Injection volume: 10 mL;

[0055] Column oven temperature: 70℃;

[0056] F. Data Processing: Quantification was performed using the external standard method. The theoretical concentrations of the three compounds in the standard curve sample were used as the x-axis, and the peak areas after chromatographic peak integration were used as the y-axis. The least squares method with weighted w = 1 / x2 was used for regression calculation to obtain the linear regression equation, which is the standard curve. The concentrations in each sample were then calculated back from the peak areas of the drug and its metabolites in the sample and the standard curve.

[0057] Example 2, as Figure 3 As shown:

[0058] The sense (AS) and antisense (SS) strands of a siRNA in a biological matrix are quantified using ion-pair reversed-phase chromatography-tandem mass spectrometry (IP-RPLC-MS / MS) with an internal standard method under an inert gas atmosphere. In this embodiment, the biological matrix includes, but is not limited to, plasma, tissues, and excrement from various preclinical experimental animals, as well as human plasma and excrement. Taking rabbit plasma as an example, the implementation steps are as follows:

[0059] A. Preparation of standard curves and quality control samples for ASO standards in rabbit plasma: Accurately weigh 2 mg of siRNA standard and dissolve it in 1 mL of deionized water to obtain a 2 mg / mL standard stock solution. Weigh 1 mg of internal standard nusinersen and dissolve it in 1 mL of deionized water to obtain a 1 mg / mL internal standard stock solution, and dilute it with 30% methanol solution to a 1 μg / mL internal standard working solution. Dilute the stock solution with 30% methanol solution to prepare a series of standard curve working solutions, add them to rabbit blank plasma at a volume ratio of 1:19 and mix well to obtain standard curve samples with concentrations of 10, 20, 100, 1000, 2000, 4000, 8000, and 10000 ng / mL and quality control samples with concentrations of 10, 30, 200, 5000, and 7500 ng / mL, respectively.

[0060] B. Processing of rabbit plasma samples: Take 30 mL each of blank matrix, standard curve sample, and quality control sample. Except for the blank matrix sample, which is added to 30 mL of 30% methanol solution, the other samples are added to 30 mL of internal standard working solution. Extract and enrich the samples using TRIzol reagent (Thermo Fisher Scientific, USA) and chloroform liquid-liquid extraction method. Transfer the supernatant to a 96-well plate and concentrate it by nitrogen blowing. Finally, reconstitute the solution with 100 mL of 10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride solution for later use.

[0061] C. Preparation of mobile phases for ion-pair reversed-phase chromatography with an inert gas atmosphere: Prepare mobile phase A and mobile phase B solutions in clean glass containers according to the following proportions: Mobile phase A: an aqueous solution containing 0.2% (v / v) N,N-diisopropylethylamine and 0.1% (v / v) hexafluoroisopropanol; Mobile phase B: methanol containing 0.2% (v / v) N,N-diisopropylethylamine and 0.1% (v / v) hexafluoroisopropanol. Insert an inert gas tube connected to an argon gas source into the bottom of the container and continuously purge for 15 minutes, then seal the container for later use.

[0062] D. Inert gas protection box 100 pre-charge: Press Figure 1 As shown, connect the inlet valve 102 to the argon source and open the outlet valve 103. Continue to purge for 20 minutes until the oxygen sensor reading is less than 0.5%. Place the mobile phase prepared in step C inside the box and extend the mobile phase pipeline 200 connected to the pump head into the mobile phase bottle through the reserved hole 101 in the box cover. Tighten the cap of the mobile phase bottle to seal the box. The sealing ring 104 can play a sealing role.

[0063] E. Quantitative analysis using IP-RPLC-MS / MS with inert gas protection: The instrument system used in Example 2 was an ACQUITY I-Class UPLC chromatograph (Waters Corporation, USA) equipped with a Triple Quad 6500 mass spectrometer (AB Sciex Corporation, USA). The inert gas protection box 100 containing the mobile phase solution from step D was placed in the solvent management module of the system. The system operating software was opened and the parameters were set as follows:

[0064] Gradient: The content of mobile phase B increases from 10% by volume to 25% within 0 to 2.4 minutes;

[0065] Column type: Thermo DNAPac™ RP 4mm 2.1×100mm (Thermo Fisher Scientific, USA);

[0066] Flow rate: 0.4 mL / min;

[0067] Injection volume: 5 mL;

[0068] Column oven temperature: 80℃;

[0069] Ion source temperature: 550℃;

[0070] Spray voltage: -4500V;

[0071] Purge air flow rate: 30 au;

[0072] Declustering voltage: -60V;

[0073] Collision pool inlet voltage: -10V;

[0074] Collision cell outlet voltage: -11V;

[0075] Collision energy: -130eV;

[0076] F. Data Processing: The internal standard method was used for quantification. The theoretical concentrations of AS and SS in the standard curve samples were used as the abscissa, and the ratio of the peak area after chromatographic peak integration to the internal standard peak area was used as the ordinate. The weighted least squares method with w = 1 / x2 was used for regression calculation to obtain the linear regression equation, which is the standard curve. The concentrations of AS and SS in each sample were then calculated based on this curve.

[0077] Example 3, as Figure 4 As shown, a chromatograph mobile phase management system includes an inert gas protection box 100 pre-filled with inert gas, a mobile phase container, a mobile phase pipeline 200, a chromatographic pump 300, and an inert gas source 400.

[0078] The mobile phase container is placed inside and sealed in the inert gas protection box 100. One end of the mobile phase pipeline 200 is connected to the mobile phase container, and the other end extends through the pre-drilled hole 101 at the top of the inert gas protection box and is connected to the inlet of the chromatography pump 300. The inert gas source 400 is connected through the inlet valve 102 on the side of the inert gas protection box 100 for initial filling and replenishment during use. The inert gas protection box 100 is equipped with an outlet valve 103 at the top for venting.

[0079] References:

[0080] 【1】JHKnox and GRLaird,J.Chromatogr.122(1976):17-34.

[0081] 【2】GJ Guimaraes, JGSaad, V.Annavarapu, and MGBartlett, J.Am.Soc.MassSpectrom.34(2023):2691-2699.

[0082] 【3】P.Li,Y.Gong,J.Kim,X.Liu,J.Gilbert,HMKerns,R.Groth,M.Rooney,Anal.Chem.92(2020):10548–10559

[0083] In summary, the present invention provides a method for improving ion-pair reversed-phase liquid chromatography separation using an inert gas atmosphere. When ion-pair reversed-phase chromatography is coupled with optical detectors such as ultraviolet or fluorescence, the chromatographic resolution and retention time are stably reproducible over a long period (not less than 120 hours) after using this method. When ion-pair reversed-phase chromatography is coupled with mass spectrometry, the chromatographic peak area is stably reproducible over a long period (not less than 120 hours) after using this method.

[0084] The specific embodiments of the invention have been described above. It should be understood that the invention is not limited to the specific embodiments described above, and the devices and structures not described in detail should be understood to be implemented in a manner common to the art; those skilled in the art can make various modifications or alterations within the scope of the claims, and make several simple deductions, variations or substitutions, which do not affect the substantive content of the invention.

Claims

1. A method for improving ion-pair reversed-phase liquid chromatography separation using an inert gas atmosphere, characterized in that, Includes the following steps: The mobile phase management system of the chromatograph is equipped with an inert gas protection box that is pre-filled with inert gas; The headspace of the mobile phase container used in the chromatograph is replaced with an inert gas before use. The container holding the mobile phase is placed inside the inert gas protection box and sealed. The mobile phase pipeline extends through the pre-drilled hole at the top of the inert gas protection box and connects to the inlet of the chromatography pump; The inert gas source is connected through an inlet valve built into the side of the inert gas protection box for initial filling and replenishment during use. The inert gas protection box is equipped with an outlet valve on the top.

2. The method for improving ion-pair reversed-phase liquid chromatography separation using an inert gas atmosphere as described in claim 1, characterized in that, The mobile phase is a methanol / water or acetonitrile / water solution with added ion-pairing reagents.

3. The method for improving ion-pair reversed-phase liquid chromatography separation using an inert gas atmosphere as described in claim 2, characterized in that, The detector used in this ion-pair reversed-phase liquid chromatography is one or more of the following: UV-Vis spectrometer, FLD fluorescence spectrometer, MS mass spectrometer, MS / MS containing tandem mass spectrometry, and HRMS high-resolution mass spectrometry.

4. The method for improving ion-pair reversed-phase liquid chromatography separation using an inert gas atmosphere as described in claim 2, characterized in that, The added ion-pairing reagents include, but are not limited to: triethylamine (TEA), n-butylamine (BA), dibutylamine (DBA), N,N-diisopropylethylamine (DIEPA), n-hexylamine (HA), triethylamine acetate (TEAA), acetic acid (AA), formic acid (FA), and trifluoroacetic acid (TFA).

5. The method for improving ion-pair reversed-phase liquid chromatography separation using an inert gas atmosphere as described in claim 1, characterized in that, The inert gas is one of helium, neon, argon, krypton, xenon, nitrogen, and sulfur hexafluoride.

6. The method for improving ion-pair reversed-phase liquid chromatography separation using an inert gas atmosphere as described in claim 1, characterized in that, The inert gas protection box has gas-tight properties.

7. The method for improving ion-pair reversed-phase liquid chromatography separation using an inert gas atmosphere as described in claim 1, characterized in that, The inert gas protection box has a built-in oxygen sensor and a humidity sensor.

8. The method for improving ion-pair reversed-phase liquid chromatography separation using an inert gas atmosphere as described in claim 1, characterized in that, If the container holding the mobile phase does not have a headspace, that is, the container holding the mobile phase is filled with liquid, then the inert gas replacement step is omitted.

9. The method for improving ion-pair reversed-phase liquid chromatography separation using an inert gas atmosphere as described in claim 1, characterized in that, A sealing ring is installed at the reserved hole.

10. The method for improving ion-pair reversed-phase liquid chromatography separation using an inert gas atmosphere as described in claim 1, characterized in that, The intake valve is a one-way check valve, and the outlet valve is a one-way check valve.

11. A mobile phase management system for a chromatograph, characterized in that, It includes an inert gas protection box pre-filled with inert gas, a mobile phase container, a mobile phase pipeline, a chromatography pump, and an inert gas source; The mobile phase container is placed inside the inert gas protection box and sealed. One end of the mobile phase pipeline is connected to the mobile phase container, and the other end extends through a pre-drilled hole at the top of the inert gas protection box and is connected to the inlet of the chromatography pump. The inert gas source is connected through an inlet valve on the side of the inert gas protection box, and an outlet valve is installed at the top of the inert gas protection box.

12. A chromatograph mobile phase management system according to claim 11, characterized in that, A sealing ring is installed at the reserved hole.

13. A chromatograph mobile phase management system according to claim 11, characterized in that, The intake valve is a one-way check valve, and the outlet valve is a one-way check valve.

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