A chromatographic analysis method based on low pressure gradient elution and high pressure gradient reconstruction

Abstract

The application discloses a chromatographic analysis method based on low-pressure gradient elution and high-pressure gradient reconstruction. The method comprises three steps of low-pressure gradient elution, temporary storage decoupling of a trapping column and high-pressure gradient reconstruction, and forms a gradient decoupling chromatographic analysis process. The application introduces gradient elution in the low-pressure sample processing stage, realizes gradual release of analytes, temporarily stores the eluent through the trapping column, and sets a fluid control valve between the trapping column and a high-pressure chromatographic analysis passage in this state, so that the two are isolated from each other in fluid, the transmission of pressure changes and fluid disturbance in the high-pressure chromatographic analysis passage to the eluent is blocked, and fluid disturbance and analyte diffusion in the system switching process are reduced.

Description

Technical Field

[0001] This invention relates to the field of liquid chromatography analysis technology, specifically to a chromatographic analysis method based on low-pressure gradient elution and high-pressure gradient reconstruction. Background Technology

[0002] Liquid chromatography (LC) is widely used for the separation and detection of small molecule compounds as well as complex samples such as proteins, peptides, metabolites, and lipids. In many chromatographic systems, to improve analytical throughput and system utilization efficiency, an online sample preparation step is usually set up before chromatographic analysis. For example, analytes in the sample are captured and enriched by using a capture column or adsorption medium before being introduced into the analytical column for separation.

[0003] In existing technologies, capture and elution are typically performed under low-pressure conditions, while chromatographic separation is usually performed under high-pressure conditions. Therefore, a switch in the fluid pathway is usually required between the low-pressure elution stage and the high-pressure chromatographic analysis stage.

[0004] The inventors discovered that during the switching process described above, if the eluent formed by low-pressure elution is directly introduced into the high-pressure chromatography analysis pathway, the pressure changes and fluid disturbances in the high-pressure chromatography system are easily transmitted to the eluent at the moment of switching, thereby causing changes in the composition of the eluent, diffusion of analytes, and deterioration of chromatographic peak shape, which in turn leads to retention time drift and decreased quantitative repeatability.

[0005] Existing technologies typically mitigate these problems by reducing flow rate, increasing pipeline length, or employing complex valve structures, but these methods often increase system complexity or reduce analysis efficiency.

[0006] Therefore, it is necessary to provide a new chromatographic analysis method to achieve stable and reproducible fluid switching between low-pressure elution and high-pressure chromatographic analysis. Summary of the Invention

[0007] The purpose of this invention is to solve the problems of the prior art and provide a chromatographic analysis method based on low-pressure gradient elution and high-pressure gradient reconstruction. By introducing a controlled temporary storage and switching process between the low-pressure elution stage and the high-pressure chromatographic analysis stage, the low-pressure elution process and the high-pressure chromatographic analysis process are made independent of each other in terms of pressure and gradient control, thereby reducing analyte diffusion and fluid disturbance during system switching.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] S1 capture

[0010] A liquid sample is introduced into a capture pathway containing an adsorption medium, so that the analytes in the sample are captured on the adsorption medium.

[0011] parameter Preferred range Capture column inner diameter 0.05 -2 mm Capture column length 5 -30 mm Adsorption medium C18, C8, polymer reverse materials, etc. Particle size 1.5 - 10 µm Flow rate 1 -200 µL / min Operating pressure 1 -100 bar

[0012] S2 low-pressure gradient elution

[0013] While maintaining the capture path at a low pressure,

[0014] An eluent with a time-varying composition is delivered to the adsorption medium via a low-pressure fluid drive unit, thereby eluting the analyte from the adsorption medium in a gradient manner and forming an eluent containing the analyte.

[0015] parameter Preferred range Initial organic phase 2–10 % endpoint organic phase 40–80 % Gradient time 10–120 s Flow rate 5–200 µL / min pressure 1–100 bar

[0016] In this invention, the gradient elution is performed as part of the sample injection stage, rather than as a separation gradient on the chromatographic column.

[0017] S3 temporary storage

[0018] The eluent is temporarily stored in a trapping column. In this state, a fluid control valve between the trapping column and the high-pressure chromatography analysis path is used to fluidly isolate them, thus preventing pressure changes and fluid disturbances in the high-pressure chromatography analysis path from being transmitted to the eluent. Before and after switching the fluid control valve, the solvent composition of the eluent in the trapping column is controlled to change by less than ±5%.

[0019] In this invention, the solvent composition variation within the trapping column is kept less than ±5% by controlling the following parameters:

[0020] (1) Control the volume of the collection column to be close to or the same as the volume of the eluent;

[0021] (2) Keep the temporary storage time within the range of 0.1–10 s;

[0022] (3) Close the high-pressure passage during the temporary storage stage to keep the collection column in a low-pressure isolation state.

[0023] Under the above conditions, the eluent undergoes only limited diffusion within the collection column, thus maintaining the original gradient composition.

[0024] parameter Preferred range Capture column volume 1–200 µL Preferred volume 1–50 µL Temporary storage time 0.1–10 s

[0025] The volume of the trapping column is preferably close to the volume of the eluent to reduce diffusion. Experimental results show that, under conditions where the storage time is less than 10 s, the diffusion of the solution within the trapping column has a negligible effect on chromatographic separation, and therefore does not reduce separation efficiency.

[0026] S4 State Switching

[0027] By changing the connectivity of the fluid pathway using a fluid control valve, the trapping column can be switched from being connected to the capture pathway to being connected to the high-pressure chromatography analysis pathway.

[0028] Optimal switching time: 10–500 ms.

[0029] S5 High Pressure Gradient Reconstruction and Chromatographic Analysis

[0030] After the trapping column is connected to the high-pressure chromatography analysis pathway, a gradient mobile phase for separation is generated by at least two independently controlled high-pressure fluid drive units under pressure conditions higher than those of the low-pressure gradient elution in step S2, so as to perform chromatographic separation and detection of the analytes.

[0031] parameter Preferred range Operating pressure 100–1200 bar Flow rate 10 nL / min–200 µL / min gradient 5–95 % B time 1–180 min

[0032] Chromatographic analysis columns can be:

[0033] type Preferred range inner diameter 50 µm–4.6 mm length 30–150 mm filler C18 / C8 / HILIC

[0034] Through the above steps, the low-pressure elution process and the high-pressure chromatography analysis process are made independent of each other in terms of pressure and gradient generation, thereby achieving stable chromatographic analysis.

[0035] In the method of the present invention, the low-pressure gradient elution stage and the high-pressure chromatography analysis stage employ independent gradient generation mechanisms.

[0036] In step S2, a gradient eluent is generated under low pressure by the first fluid drive unit, so that the analyte is gradually released from the first adsorption medium and forms an eluent.

[0037] Subsequently, in step S3, the eluent is temporarily stored in a trapping column, in which the low-pressure elution system and the high-pressure chromatography system are fluidly isolated from each other, thereby keeping the composition of the eluent formed by low-pressure elution stable before entering the high-pressure system.

[0038] In step S5, a gradient mobile phase for chromatographic separation is generated by at least two independently controlled high-pressure fluid drive units. This high-pressure gradient is generated by the high-pressure fluid drive units according to a preset program, and its gradient change pattern is independent of the gradient formed in the low-pressure elution stage in step S2.

[0039] Therefore, in this invention:

[0040] The gradient formed in step S2 is mainly used to analyze the release process of the analyte from the adsorption medium;

[0041] The gradient formed in step S5 is mainly used for the separation process on the chromatographic column.

[0042] By utilizing the temporary storage function of the trapping column, the two gradient generation processes mentioned above are decoupled from each other in terms of time and pressure conditions, thereby enabling the low-pressure sample processing process and the high-pressure chromatographic separation process to be optimized independently.

[0043] The beneficial effects of this invention are:

[0044] In this invention, a gradient-decoupled chromatographic analysis process is formed through three steps: low-pressure gradient elution, temporary storage and decoupling of the trap column, and high-pressure gradient reconstruction. This invention introduces gradient elution during the low-pressure sample preparation stage to achieve gradual release of the analyte, and by temporarily storing the eluent in the trap column, the low-pressure elution and high-pressure chromatographic analysis are made independent of each other in terms of pressure and gradient control, thereby reducing fluid disturbance and analyte diffusion during system switching. Attached Figure Description

[0045] Figure 1 This is a time-series diagram of the chromatographic analysis method of the present invention;

[0046] Figure 2 This is a schematic diagram illustrating the changes in the gradient mobile phase during the low-pressure gradient elution step and the high-pressure chromatography analysis step in this invention.

[0047] Figure 3 This is a simplified fluid pathway diagram of the present invention;

[0048] Figure 4 This is a comparison of the peak shapes of caffeine and p-hydroxybenzoic acid in Example 1 and Comparative Example 1.

[0049] Figure 5 This is a comparison of the peak shapes of BSA trypsin peptides in Example 2 and Comparative Example 2.

[0050] Figure 6 This is a comparison chart of the baseline stability of caffeine during the system switching process of Example 4 and Comparative Example 4. Detailed Implementation

[0051] To enable those skilled in the art to better understand the present invention, the following detailed description of the embodiments will further illustrate the invention.

[0052] like Figures 1-3 As shown, the method of the present invention executes the capture step, the low-pressure gradient elution step, the eluent temporary storage and decoupling step, the state switching step, and the high-pressure gradient reconstruction and chromatographic analysis step in chronological order.

[0053] In the S1 capture step, a liquid sample is introduced into a capture pathway containing an adsorption medium, so that the analytes in the sample are captured on the adsorption medium.

[0054] In the S2 low-pressure gradient elution step, an eluent with a time-varying composition is delivered to the capture pathway via a low-pressure fluid drive unit, allowing analytes with different retention capacities to elute sequentially from the adsorption medium. The low-pressure fluid drive unit can be a plunger pump, a binary pump, or a quaternary pump, capable of stably outputting the mobile phase within a pressure range of 1–100 bar.

[0055] In the S3 temporary storage and decoupling step, the eluent is temporarily stored in the trapping column after it is formed. In this state, the trapping column is fluidly isolated from the high-pressure chromatography analysis path, so that pressure fluctuations and fluid disturbances in the high-pressure chromatography analysis path are not transmitted to the eluent.

[0056] The trapping column is a short column structure filled with an adsorption medium. Its inner diameter is preferably 0.05–2 mm and its length is 5–30 mm. The packing material can be C18, C8 or polymer reverse phase material, or a trap column can be used.

[0057] In the S3 stage, the eluent is retained in the collection column with near-zero displacement, forming a solution with a spatial concentration gradient. The concentration distribution of this solution retains the linear relationship of low-pressure elution.

[0058] Subsequently, in the S4 state switching step, the trapping column is switched from being connected to the capture pathway to being connected to the high-pressure chromatography analysis pathway by switching the fluid control valve. During the switching process, it is preferable to disconnect the connection with the capture pathway first, and then establish the connection with the high-pressure chromatography analysis pathway. This switching process is usually completed within 10–500 ms.

[0059] In the S5 high-pressure gradient reconstruction and chromatographic analysis step, the mobile phase is delivered to the chromatographic column through at least two independently controlled high-pressure fluid drive units. Under high pressure conditions, a gradient mobile phase for chromatographic separation is generated, completing the chromatographic separation and detection of the analytes. The high-pressure fluid drive unit can be a high-pressure plunger pump, a binary high-pressure pump, or a quaternary high-pressure pump, capable of stably outputting the mobile phase within a pressure range of 100–1200 bar.

[0060] Because there is an order-of-magnitude difference in pressure between low-pressure elution (step S2) and high-pressure separation (step S5) (e.g., 20 bar vs 600 bar), the compressibility of the solvent will cause changes in apparent volume. This invention corrects for concentration disturbances caused by pressure switching by reconstructing the gradient using an independent high-pressure fluid-driven unit in stage S5.

[0061] The starting point for high-pressure gradient reconstruction does not necessarily start from 0% organic phase. Instead, it is achieved by calculating the organic phase concentration at the end of S2, thus eliminating baseline fluctuations.

[0062] Specifically, the concentration distribution of the eluent within the trapping column can be approximated as a one-dimensional axial distribution, and its concentration gradient can be expressed as:

[0063] C(x) = C0 + kx

[0064] Where x is the axial position of the trap column, k is the gradient slope, C(x) is the concentration at position x, and C0 is the starting concentration.

[0065] Under conditions where the storage time is less than 30 s, the concentration deviation caused by diffusion can be ignored, thus maintaining the concentration distribution formed by low-pressure gradient elution.

[0066] Example 1 (Small molecule sample)

[0067] Sample: A mixture of caffeine and p-hydroxybenzoic acid.

[0068] The capture column is a 1 mm × 10 mm C18 column.

[0069] S1 capture

[0070] Capture column: 1 mm × 10 mm C18;

[0071] Flow rate: 50 µL / min;

[0072] Mobile phase: water + 0.1% formic acid;

[0073] Pressure: 5 bar.

[0074] S2 low-pressure gradient elution

[0075] Gradient: 0% acetonitrile → 60% acetonitrile;

[0076] Gradient time: 30 s;

[0077] Flow rate: 30 µL / min;

[0078] Pressure: Approximately 25 bar.

[0079] S3 temporary storage

[0080] Eluent was introduced into a 20 µL trapping column;

[0081] Storage time: 5 seconds.

[0082] S4 State Switching

[0083] Valve switching time: 100 ms;

[0084] S5 High Pressure Gradient Reconstruction and Chromatographic Analysis

[0085] Chromatographic column: 50 mm × 2.1 mm C18;

[0086] Pressure: 600 bar;

[0087] Gradient: 5% → 90% acetonitrile;

[0088] Time: 10 min.

[0089] Comparative Example 1

[0090] Under the same conditions described above, step S3 is omitted, allowing the eluent to directly enter the chromatographic column.

[0091] Experimental results:

[0092] index Example 1 Comparative Example 1 Peak width (FWHM) 0.12 min 0.21 min Retention time RSD 1.3 % 4.6 % Peak symmetry 1.05 1.32

[0093] Combination Figure 3 As can be seen, the scheme in Example 1 significantly reduces peak broadening and improves retention time repeatability.

[0094] Example 2 (peptide sample)

[0095] Sample: BSA trypsin digest.

[0096] Capture column: 75µm×20mm C18.

[0097] S1 capture

[0098] Capture column: 75 µm × 20 mm C18;

[0099] Flow rate: 10 µL / min;

[0100] Mobile phase: water + 0.1% formic acid;

[0101] Pressure: 5 bar.

[0102] S2 low-pressure gradient elution

[0103] Gradient: 3% → 50% acetonitrile;

[0104] Time: 60 s;

[0105] Flow rate: 30 µL / min;

[0106] Pressure: Approximately 25 bar.

[0107] S3 temporary storage

[0108] Collection column: 10 µL;

[0109] Storage time: 5 seconds.

[0110] S4 State Switching

[0111] Valve switching time: 100 ms;

[0112] S5 High Pressure Gradient Reconstruction and Chromatographic Analysis

[0113] Chromatographic column: 75 µm × 50 mm C18;

[0114] Pressure: 600 bar;

[0115] Gradient: 3% → 70% acetonitrile;

[0116] Time: 10 min.

[0117] Comparative Example 2

[0118] Under the same conditions described above, step S3 is omitted, allowing the eluent to directly enter the chromatographic column.

[0119] Experimental results:

[0120] index Example 2 Comparative Example 2 Peak width 0.18 min 0.30 min Peptide resolution 1.8 1.1 Retention time RSD 2.0 % 5.5 %

[0121] Combination Figure 4 As can be seen, the peptide separation scheme in Example 2 is significantly improved, and the repeatability is enhanced.

[0122] Example 3 (Metabolite Sample)

[0123] Sample: Plasma metabolite extract.

[0124] S1 capture

[0125] Collection column: 75 µm × 10 mm C18;

[0126] Flow rate: 5 µL / min;

[0127] Mobile phase: water + 0.1% formic acid;

[0128] Pressure: 10 bar.

[0129] S2 gradient elution

[0130] Gradient: 10% → 70% acetonitrile;

[0131] Time: 45 s;

[0132] Flow rate: 50 µL / min;

[0133] Pressure: Approximately 35 bar.

[0134] S3 temporary storage

[0135] Collection column: 15 µL;

[0136] Storage time: 5 seconds.

[0137] S4 State Switching

[0138] Valve switching time: 100 ms;

[0139] S5 High Pressure Gradient Reconstruction and Chromatographic Analysis

[0140] Chromatographic column: 1.7 mm × 100 mm C18;

[0141] Pressure: 800 bar.

[0142] Gradient: 5% → 90% acetonitrile;

[0143] Time: 15 min.

[0144] Comparative Example 3

[0145] Under the same conditions described above, steps S2 and S3 are omitted, and the plasma metabolite sample from S1 is directly introduced into the chromatographic analysis column.

[0146] Experimental results:

[0147] index Example 3 Comparative Example 3 Peak width 0.15 min 0.35 min Retention time drift 0.8 % 3.9 %

[0148] The above results indicate that the metabolite separation stability of the scheme in Example 3 is significantly improved compared with that of Comparative Example 3.

[0149] Example 4 (Gradient Decoupling Verification)

[0150] This embodiment is used to verify the gradient decoupling effect.

[0151] Experimental Design

[0152] The samples and experimental procedures are the same as in Example 1.

[0153] Comparative Example 4

[0154] The sample and experimental procedure are the same as in Example 1, but the gradient coupling of S2 and S5 is not optimized.

[0155] In the experiments of Example 4 and Comparative Example 4, the baseline changes of caffeine before and after gradient coupling optimization were monitored using an ultraviolet detector.

[0156] Experimental results:

[0157] index Example 4 Example 5 Gradient offset < 2 % > 10 % Baseline perturbation No significant fluctuations Significant fluctuations

[0158] Combination Figure 6 Therefore, the solution in Example 4 effectively blocks pressure disturbances and achieves gradient decoupling.

[0159] In this invention, decoupling is manifested as follows:

[0160] 1. Solvent composition variation within the trapping column is <±5%; 2. Pressure variation within the trapping column does not fluctuate with the high-pressure system; 3. High-pressure gradient is generated independently.

[0161] Example 5 (Optimization of Capture Column Volume)

[0162] The sample and experimental procedure were the same as in Example 1, except that the volume of the trapping column was changed.

[0163] Test capture column volume:

[0164] volume Peak width 5 µL 0.15 min 10 µL 0.12 min 20 µL 0.18 min

[0165] Experimental results: Under the same conditions, the performance is best when the trapping column volume is about 10 µL.

[0166] The above embodiments demonstrate that by employing gradient elution in the low-pressure stage and temporarily storing and decoupling the sample using a trapping column after elution, the low-pressure sample processing and high-pressure chromatographic analysis processes can be made independent of each other in terms of pressure and gradient control, thereby achieving stable chromatographic separation and analysis.

[0167] Traditional chromatographic systems typically minimize dead volume in the fluid pathway, therefore those skilled in the art generally do not actively add an additional trap column structure between the capture column and the analytical column. Existing techniques usually employ direct sample loading or isocratic elution, introducing the sample or eluent directly into the analytical column. This invention introduces gradient elution during the low-pressure sample preparation stage and temporarily stores the eluent in the trap column, making low-pressure elution and high-pressure chromatographic analysis independent of each other in terms of pressure and gradient control, thereby reducing fluid disturbances during system switching.

[0168] Therefore, in this invention, a gradient-decoupled chromatographic analysis process is formed through three steps: low-pressure gradient elution, temporary decoupling of the trapping column, and high-pressure gradient reconstruction. This process makes the low-pressure sample processing stage and the high-pressure chromatographic separation stage independent of each other in terms of pressure conditions and gradient generation methods, thereby reducing fluid disturbances and analyte diffusion during system switching.

[0169] The method of the present invention is applicable to capillary liquid chromatography, microfluidic liquid chromatography or nanofluidic liquid chromatography analysis, and the control program can be executed by the chromatography system controller.

[0170] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A chromatographic analysis method based on low-pressure gradient elution and high-pressure gradient reconstruction, characterized in that: Includes the following steps, S1 capture: A liquid sample is introduced into a capture pathway containing an adsorption medium, so that the analyte is captured on the adsorption medium; S2 Low-pressure gradient elution: While maintaining the capture pathway at a low pressure, eluent is delivered to the adsorption medium through a low-pressure fluid drive unit to elute the analyte from the adsorption medium in a gradient manner, forming an eluent containing the analyte. S3 Temporary Storage: The eluent is temporarily stored in the trapping column. In this state, the trapping column is fluidly isolated from the high-pressure chromatography analysis path to block the transmission of pressure changes and fluid disturbances in the high-pressure chromatography analysis path to the eluent. S4 State Switching: By changing the connectivity of the fluid passage through the fluid control valve, the state of the trapping column is switched from being connected to the capture passage to being connected to the high-pressure chromatography analysis passage; S5 High-Pressure Gradient Reconstruction and Chromatographic Analysis: After the trapping column is connected to the high-pressure chromatographic analysis pathway, a mobile phase is delivered to the chromatographic analysis column through at least two independently controlled high-pressure fluid drive units. A gradient mobile phase for chromatographic separation is generated under pressure conditions higher than those of the low-pressure gradient elution in step S2, so as to perform chromatographic separation and detection of the analytes.

2. The chromatographic analysis method according to claim 1, characterized in that, The gradient elution in step S2 is a time-varying solvent composition gradient, used to develop and elute analytes with different retention capacities under low pressure conditions, while simultaneously feeding them into a chromatographic analyzer.

3. The chromatographic analysis method according to claim 1 or 2, characterized in that, In step S2, the pressure in the low-pressure state is between 1 bar and 100 bar.

4. The chromatographic analysis method according to claim 1, characterized in that, In step S5, the operating pressure of the chromatographic column is 100 bar to 1200 bar.

5. The chromatographic analysis method according to claim 1, characterized in that, The switching time of the fluid control valve in step S4 is from 10 ms to 500 ms.

6. The chromatographic analysis method according to claim 1, characterized in that, In step S2, the gradient for gradient elution can be a linear gradient, a step gradient, or a piecewise gradient, and the gradient elution time is 10–60 s.

7. The chromatographic analysis method according to claim 1, characterized in that, The volume of the trapping column is from 1 µL to 200 µL.

8. The chromatographic analysis method according to claim 1, characterized in that, In step S2, the flow rate of the eluent is from 5 µL / min to 200 µL / min.

9. The chromatographic analysis method according to claim 1, characterized in that, The analytes include proteins, peptides, metabolites, lipids, or combinations thereof.

10. The chromatographic analysis method according to claim 1, characterized in that, This method is applicable to capillary liquid chromatography, microfluidic liquid chromatography, or nanofluidic liquid chromatography analysis.

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