Method for improving detection efficiency of liquid chromatography

Abstract

The invention relates to a method for improving liquid chromatogram detection efficiency, which is characterized in that two same trap columns are arranged in a ten-way valve of a liquid chromatogram system valve module, the trap column 2 is connected with a chromatographic column, and the trap column 1 is connected with a loading pipeline; after the sample injection and load of the first needle are completed, the trap column 1 is connected with the chromatographic column through the ten-way valve cutting valve, the sample on the trap column 1 is subjected to gradient elution, and meanwhile, the sample injection and load of the second needle sample are performed through the six-way valve cutting valve in the sample injection module, so that the second needle sample is combined on the trap column 2 to complete load; the trap column 2 and the chromatographic column are connected through the ten-way valve cutting valve to start gradient elution of a second needle sample, and meanwhile, the six-way valve cutting valve carries out sample injection and load of a next needle sample. The two trap columns alternately carry out elution and sample introduction, sample gradient elution and next sample introduction can be carried out at the same time under the condition that a gradient elution program is not changed, the two trap columns run independently, the two trap columns cannot appear in the same flow path at the same time during sample analysis, the retention time of each sample is not affected, and the two trap columns are suitable for detection of various types of samples.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and more specifically to a method for improving the continuous detection efficiency of liquid chromatography. Background Art

[0002] Liquid chromatography (LC) is an analytical technique used to separate, identify and quantitatively analyze components in a mixture. The LC-MS instrument runs a sample including three processes: injection, pre-concentration (load), and run. Injection and load are invalid waiting times, usually called blank time, which is the time from the start of sample injection to the completion of sample pre-concentration. The time from the first substance in the sample eluting from the trap column to the end of the chromatographic peak of the last substance is the peak time, and the detection time of a sample includes blank time and peak time. The longer the blank time, the longer the invalid waiting time for each analysis and detection, resulting in lower efficiency of liquid chromatography detection and smaller detection flux. Although injection and load are invalid times, they are essential processes in LC-MS detection.

[0003] In order to improve the detection efficiency of liquid chromatography, conventional operation mainly improves the efficiency of continuous detection of liquid chromatography by increasing column temperature or optimizing injection volume to shorten blank time, but shortening blank time may affect the efficiency and peak shape of chromatographic separation. In the prior art, in order to reasonably utilize blank time, advance injection is adopted to improve detection efficiency, such as patent CN111289640B discloses a liquid chromatography detection method for improving detection efficiency by advance injection, which shortens the detection time of the sample and improves the use efficiency of HPLC by arranging the peak area time of the previous sample in the blank time area of ​​the latter sample, but this method requires that the sample peak time is less than the blank time. Moreover, this method is complicated to operate, and the most important retention time of the sample is inaccurate, and the substance corresponding to each chromatographic peak can only be judged according to the characteristics of the chromatographic peak and the time sequence, which is not applicable in liquid chromatography-mass spectrometer. In proteomics, sample retention time is a very important dimension of the sample itself, so it is of great significance to study a method that can improve the detection efficiency of liquid chromatography and is applicable to various types of sample detection. Summary of the invention

[0004] In view of the above defects or improvement needs of the prior art, the present invention provides a method for improving the detection efficiency of liquid chromatography, which is to set two identical trap columns in the ten-way valve of the valve module of the liquid chromatography system to alternately perform elution and advance injection, so that the sample gradient elution and the injection and loading of the next needle sample can be performed simultaneously without changing the gradient elution program, which significantly shortens the invalid time required for waiting for the injection and loading of the next needle sample, and even after the gradient elution of the current sample is completed, the gradient elution of the next needle sample can be started without waiting, which greatly shortens the blank time required for continuous detection, thereby improving the detection efficiency of liquid chromatography. This solves the technical problem that the invalid time required for sample injection and loading is long and the detection efficiency is low during conventional detection of existing liquid chromatography.

[0005] To achieve the above object, according to one aspect of the present invention, a method for improving the detection efficiency of liquid chromatography is provided, which can simultaneously perform sample gradient elution and injection of the next sample without changing the gradient elution program, and specifically comprises the following steps:

[0006] Two identical trap columns are arranged in the ten-way valve of the valve module of the liquid chromatography system, wherein the trap column 2 is connected to the chromatography column, and the trap column 1 is connected to the loading pipeline;

[0007] The first needle sample is sucked into the loop to complete the injection, and the sample is combined with the trap column 1 to complete the loading. The ten-way valve is cut to connect the trap column 1 with the chromatographic column, and the sample on the trap column 1 is gradient eluted. At the same time, the six-way valve in the injection module is cut to perform the injection and loading of the second needle sample, so that the second needle sample is combined with the trap column 2 to complete the loading;

[0008] When the gradient elution of the first sample is completed and the injection and loading of the second sample are completed, the ten-way valve is turned off to connect the trap column 2 with the chromatographic column to start the gradient elution of the second sample, and at the same time, the six-way valve is turned off to inject and load the next sample, so that the sample is combined with the trap column 1; the above operations are repeated in sequence.

[0009] Preferably, the method for improving the efficiency of liquid chromatography detection is for conventional detection using a liquid chromatography-mass spectrometry analyzer, and the peak time in the sample spectrum is ≥ the blank time, according to the following steps:

[0010] Without changing the sample gradient elution program, the six-way valve is cut to inject the second sample during the gradient elution time of the first sample, and the sample is combined on trap column 2 to complete the loading before the gradient elution of the first sample is completed;

[0011] When the gradient elution of the first sample is completed, the ten-way valve is turned off to connect trap column 2 to the chromatographic column, and the gradient elution of the second sample is started. At the same time, the six-way valve is turned off to inject and load the next sample; and the process is repeated in sequence.

[0012] Preferably, the method for improving the efficiency of liquid chromatography detection is for conventional detection using a liquid chromatography-mass spectrometry analyzer, and the peak time in the sample spectrum is less than the blank time, according to the following steps:

[0013] Without changing the sample gradient elution program, when the gradient elution of the first sample begins, the six-way valve is cut to inject the second sample. After the sample is combined with the trap column 2 and loaded, the ten-way valve is cut to connect the trap column 2 with the chromatographic column, and the gradient elution of the second sample begins. At the same time, the six-way valve is cut to inject and load the next sample. Repeat this process.

[0014] Preferably, the method for improving the detection efficiency of liquid chromatography increases the flow rate of the loading pump to shorten the time for waiting for the completion of loading of the N+1th needle sample after the gradient elution of the Nth needle sample.

[0015] Preferably, the method for improving the detection efficiency of liquid chromatography controls the flow rate of the loading pump so that the N+1th needle sample is combined on another trap column to complete the loading when or before the gradient elution of the Nth needle sample is completed.

[0016] Preferably, in the method for improving the efficiency of liquid chromatography detection, in the ten-way valve, the trap column 1 is connected to the holes 3 and 6, and the trap column 2 is connected to the holes 1 and 8.

[0017] Preferably, in the method for improving the efficiency of liquid chromatography detection, the ten-way valve 1-2 is connected to connect the trap column 2 with the chromatographic column, the trap column 1 is connected with the loading pipeline, and the injection and loading of the first needle sample are started. After completion, the ten-way valve 1-10 is connected to perform gradient elution on the sample on the trap column 1. During the gradient elution time of the sample on the trap column 1, the six-way valve is cut to start the injection and loading of the next needle sample, so that the next needle sample is combined with the trap column 2 to complete the loading.

[0018] Preferably, in the method for improving the efficiency of liquid chromatography detection, the second needle sample completes loading, the ten-way valve cuts valve 1-2 to connect the sample on the trap column 2 for gradient elution, and the six-way valve cuts valve during the gradient elution time of the sample on the trap column 2 to start the injection and loading of the next needle sample, so that the next needle sample is combined with the trap column 1 to complete the loading.

[0019] Preferably, in the method for improving the efficiency of liquid chromatography detection, the loops in the six-way valve are connected according to hole positions 1-4.

[0020] Preferably, the method for improving the efficiency of liquid chromatography detection is that when the six-way valve 1-6 is connected, the sample injection starts. After the injection is completed, the six-way valve cut valve 1-2 is connected to the loading pump to combine the sample on the loop ring to the trap column to complete the sample loading.

[0021] In general, compared with the prior art, the above technical solution conceived by the present invention can achieve the following beneficial effects by setting two identical trap columns in the ten-way valve of the valve module of the liquid chromatography system to alternately perform elution and pre-load samples:

[0022] The method is to connect two identical trap columns in the ten-way valve of the valve module of the liquid chromatography system, wherein the trap column 2 is connected to the chromatographic column, the trap column 1 is connected to the loading pipeline, the first needle sample is injected and loaded, the ten-way valve is cut to connect the trap column 1 to the chromatographic column, the sample on the trap column 1 is gradient eluted, and the six-way valve in the injection module is cut to inject and load the second needle sample; the ten-way valve is cut to connect the trap column 2 to the chromatographic column, the sample on the trap column 1 is gradient eluted, and the six-way valve in the injection module is cut to inject and load the third needle sample; and the method is repeated in sequence. Since the two trap columns operate independently of each other, the injection and loading of the next needle sample can be realized while the sample is gradient eluted by coordinating with the valve switching, which can shorten the invalid time required for waiting for sample injection and loading during continuous detection, thereby improving the detection efficiency and detection throughput, and there is no need to change the sample gradient elution program, and it will not affect the retention time of each sample, and it is suitable for the detection of various types of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the TIC diagram of the sample injected according to this method.

[0024] Figure 2 This is a schematic diagram of the connection between the ten-way valve and the six-way valve corresponding to the N-th needle sample injection; Figure 2 1 is the NC pump module including A and B pumps and pipeline group; 2 is the loading pump module including loading pump and pipeline; 3 is the valve module including ten-way valve, chromatographic column, trap column and pipeline; 4 is the injection module including syringe, six-way valve, injection tray and pipeline.

[0025] Figure 3 This is a connection diagram of the ten-way valve and the six-way valve corresponding to the load of the N-th needle sample.

[0026] Figure 4It is a schematic diagram of the connection of the ten-way valve and the six-way valve corresponding to the simultaneous injection of the N+1th needle sample within the gradient elution time zone of the Nth needle sample.

[0027] Figure 5 It is a schematic diagram of the connection of the ten-way valve and the six-way valve corresponding to the N+1th needle sample loading during the Nth needle sample gradient elution time zone.

[0028] Figure 6 It is a TIC diagram of samples injected according to the conventional method using liquid chromatography-mass spectrometry. DETAILED DESCRIPTION

[0029] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0030] The liquid chromatography detection system includes an injection module, an NC pump module, a loading pump module and a valve module. The injection module contains an automatic sampler, a three-way or six-way valve and an injection tray; the NC pump module is composed of A and B pumps, which can control the ratio and flow rate of phase A and phase B; the loading pump module contains a loading pump and connecting pipes, which are used to transport the sample from the loop to the trap column, and the trap column is used to adsorb the target to be detected in the sample; the valve module contains a ten-way valve, a trap column (only one trap column is currently available) and a chromatographic column, wherein the target on the trap column is eluted by the mobile phase, and the components of the target to be detected in the sample are separated by the chromatographic column, and the detection of each component is achieved by connecting with a detector such as a mass spectrometer.

[0031] When using LC-MS to detect samples, the entire process of conventional detection methods from injection to completion of one-shot sample detection is as follows:

[0032] (1) Injection: The sample is sucked from the injection bottle into the loop by the injector. After the injection, the automatic injector will automatically clean the injection pipeline to reduce the residue of the injector.

[0033] (2) Trap column enrichment: The loading pump transports the sample from the loop to the trap column (capturing column) for pre-concentration. The sample is adsorbed on the trap column according to the different distribution coefficients of the filler in the trap column, completing the enrichment and purification of the sample.

[0034] (3) Gradient elution and mass spectrometry detection: According to the sample gradient elution procedure, the target analyte on the trap column is eluted by mobile phase gradient elution and transported to the chromatographic column (analytical column); the chromatographic column operates continuously under the set conditions, and the mobile phase passes through the chromatographic column, carrying the sample components through the stationary phase (the filler in the chromatographic column) to achieve separation. The separated components enter the detector such as a mass spectrometer in turn for detection and analysis.

[0035] The total time for detecting one injection of sample includes injection time, loading time, and sample gradient elution time, wherein the sample gradient elution time is the time period for the sample to peak.

[0036] This application is the first to set two trap columns for alternate elution in the ten-way valve of the liquid chromatography system valve template, which can achieve the gradient elution of the sample and the injection of the next needle sample at the same time without changing the original gradient elution detection procedure. Specifically:

[0037] Two trap columns are connected in the ten-way valve of the valve module of the liquid chromatography system to form a new liquid chromatography pipeline connection method, in which trap column 2 is connected to the chromatography column, and trap column 1 is connected to the loading pipeline. By switching the valve, the injection and loading of the next sample can be started during the gradient elution detection of the current sample.

[0038] If the next sample has been loaded before the last substance in the current sample has finished eluting, the gradient elution of the next sample can be started after the gradient elution of the current sample is completed without waiting for the injection and loading of the sample.

[0039] If the last substance in the current sample has finished eluting, but the next sample has not finished loading, the gradient elution of the sample can start after the next sample has finished loading.

[0040] In order to shorten the time of waiting for the next sample to be loaded after the gradient elution of the current sample is completed, the flow rate of the loading pump can be increased to promote the loading of the sample as soon as possible and reduce the waiting time. In particular, the flow rate of the loading pump is controlled so that the next sample is loaded when or before the gradient elution of the current sample is completed.

[0041] Based on this, the present invention proposes a method for improving the detection efficiency of liquid chromatography, which can simultaneously perform sample gradient elution and injection of the next needle sample without changing the gradient elution program, and specifically includes the following steps:

[0042] A ten-way valve in a valve module of a liquid chromatography system is connected to two identical trap columns, wherein trap column 2 is connected to a chromatography column, and trap column 1 is connected to a loading pipeline;

[0043] The first needle sample is sucked into the loop to complete the injection, and the sample is combined with the trap column 1 to complete the loading. The ten-way valve is cut to connect the trap column 1 with the chromatographic column, and the sample on the trap column 1 is gradient eluted. At the same time, the six-way valve in the injection module is cut to allow the second needle sample to be sucked into the loop to complete the injection of the second needle sample;

[0044] When the gradient elution of the first sample is completed and the second sample is combined with the trap column 2 to complete the loading, the ten-way valve is cut to connect the trap column 2 with the chromatographic column to start the gradient elution of the second sample, and the six-way valve is cut to inject and load the next sample.

[0045] After the gradient elution and detection of the second sample is completed and the loading of the third sample is completed, the ten-way valve is switched again to connect trap column 1 to the chromatographic column to start the gradient elution and detection of the third sample. At the same time, the six-way valve is switched to inject and load the next sample. The valves are switched alternately in sequence and the cycle is repeated.

[0046] In some embodiments, the method for improving the detection efficiency of liquid chromatography is shown in the schematic diagram of the detection. Figure 1 As shown, the specific steps include:

[0047] Two identical trap columns are connected to the ten-way valve in the valve module of the liquid chromatography system, wherein trap column 1 is connected to holes 3 and 6, and trap column 2 is connected to holes 1 and 8, wherein trap column 2 is connected to the chromatographic column, and trap column 1 is connected to the loading pipeline. The injection module is a six-way valve, and loops 1-4 are connected.

[0048] like Figure 2 The six-way valves 1-6 shown are connected to start the injection of the first needle sample, and the autosampler draws the first needle sample into the loop.

[0049] After the injection is completed, the six-way valve 1-6 and the valve 1-2 are connected as follows Figure 3 As shown, the loading pump will push the sample out of the loop and flow through the trap column 1 at a fixed flow rate or a variable speed, so that the target in the first needle sample is bound to the trap column 1, completing the loading.

[0050] Then Figure 4 As shown, the ten-way valve 1-10 is connected to connect the trap column 1 with the chromatographic column. During the time period of the gradient elution of the first needle sample, the six-way valve 1-6 is switched to start the injection of the second needle sample. The automatic sampler sucks the second needle sample into the loop ring. After the preset injection volume is completed, the following steps are performed. Figure 5The six-way valve shown is switched to connection 1-2, and the loading pump will push the sample out of the loop and flow through trap column 2 at the same flow rate as the first needle sample, so that the target in the second needle sample is bound to trap column 2, completing the loading.

[0051] When the first sample is eluted (the last substance is eluted) and the second sample is loaded, Figure 2 The ten-way valve shown is switched from 1-10 connection to 1-2 connection, so that trap column 2 is connected to the chromatographic column. At the same time, the six-way valve is switched to 1-6 connection, and the automatic sampler starts the injection of the third needle sample and draws the sample into the loop.

[0052] Then as Figure 3 The six-way valve shown is cut to connect valve 1-2, and the loading pump loads the third sample onto trap column 1. When the gradient elution of the second sample is completed and the loading of the third sample is completed, the ten-way valve and the six-way valve are cut to start the gradient elution of the third sample and the injection of the next sample, and the process is repeated in sequence.

[0053] It should be noted that the ten-way valve upper door in the figure has 10 connection holes, and the six-way valve upper door has 6 connection holes. The numbering in the present invention is only for the convenience of indicating which hole it is, and it is not a limitation. Other hole connections that can realize this method should also fall within the scope of protection of the present invention.

[0054] In some embodiments, for liquid chromatography detection of samples that do not require pre-concentration, the sample is sucked into the loop in advance according to the above steps. After the previous sample is analyzed, the six-way valve is cut, the loop is connected to the analysis flow path, and the sample in the loop is loaded onto the chromatographic column, which can also reduce the injection time during liquid chromatography detection.

[0055] In this method, there is no special limitation on the size relationship between the sample peak time and the blank time, because the peak time is generally set manually in liquid chromatography detection. The injection time generally depends on the liquid phase model, and the continuous detection of liquid chromatography is carried out under the condition of the same liquid phase model and injection method, and the injection time is relatively fixed. Although the load time is related to the maximum flow rate allowed by the trap column, the sample load time is relatively fixed for the same trap column, and the analysis time can be controlled manually. For example, a mass spectrometer with a high acquisition speed can shorten the analysis time appropriately without affecting the amount of protein detected, while a mass spectrometer with a low acquisition speed can increase the analysis time appropriately to increase the amount of protein detected.

[0056] For example, liquid phase injection takes 2 minutes and loading takes 5-10 minutes. This is generally related to trap. The mass spectrometry acquisition speed used is relatively low. We set a 30-minute liquid-mass method, that is, the actual total time: 30min+2min (injection time), peak time: 30min-(load sample time), blank time: injection time+load time. At this time, peak time>blank time, then the elution of the next sample can start after the current sample gradient elution.

[0057] The mass spectrometry acquisition speed is high. We set a 10-min LC-MS method. The actual total time is: 10min+2min (injection time), peak time: 10min-5min (sample loading time), blank time: injection time + loading time. At this time, the peak time is less than the blank time. After the current sample is gradient eluted, wait for the next sample to complete loading before starting elution.

[0058] Since this method uses two trap columns, it can start the injection of another sample while the sample is being detected, that is, inject and load are performed simultaneously during the liquid phase operation, which can significantly shorten the ineffective waiting time required for the injection and loading of each sample during the continuous detection process, thereby significantly improving the efficiency of liquid chromatography continuous detection without affecting the retention time of the target object.

[0059] For samples with many impurities after liquid chromatography detection, such as plasma samples, the overall operation is the same, but there are differences in the gradient setting. This method can reserve 5-10 minutes of high organic phase to flush the trap column and the chromatographic column after the sample peaks out, so as to activate the column and remove impurities and residues. When using this method for continuous detection, the details are as follows:

[0060] like Figure 2 The six-way valves 1-6 shown are connected to start the injection of the first needle sample, and the automatic sampler draws the first needle sample into the loop to complete the injection.

[0061] After injection Figure 3 As shown, the six-way valve 1-6 is connected to the cut valve 1-2, and the loading pump will push the sample out of the loop and flow through the trap column 1 at a fixed flow rate or a variable speed, so that the target in the first needle sample is bound to the trap column 1, completing the loading.

[0062] like Figure 4 The ten-way valve shown cuts off the valve 1-10 connection to connect the trap column 1 with the chromatographic column. When the gradient elution of the first needle sample begins, the six-way valve switches the 1-6 connection to start the injection of the second needle sample until the preset injection volume is completed.

[0063] Then Figure 5The six-way valve shown in the figure switches to connection 1-2, and the loading pump will push the sample out of the loop and flow through trap column 2 at a fixed flow rate or variable speed, so that the target in the sample binds to trap column 2 to complete the loading.

[0064] like Figure 2 The ten-way valve shown is switched from 1-10 connection to 1-2 connection, so that the trap column 2 is connected to the chromatographic column, and the elution and detection of the second sample can be started; at the same time, the six-way valve is switched to 1-6 connection, and the blank sample or organic phase is first flushed into the tap column 1 and then the third sample is introduced. After the loading is completed, the ten-way valve is switched from 1-2 connection to 1-10 connection, so that the trap column 1 is connected to the chromatographic column, and the elution of the third sample is started, and the process is repeated in sequence.

[0065] The following are examples

[0066] The following embodiment detection schematic diagram is as follows Figure 1 As shown, two trap columns are set to start the injection and loading of the next sample during the sample gradient elution. Starting from the second sample, the gradient elution of the sample can be started without waiting for a long time for injection and loading, which can greatly shorten the blank time of invalid waiting for each sample during continuous detection of liquid chromatography, and the gradient elution program in this method does not change, and has no effect on the detection of the target. The specific instructions are as follows:

[0067] Example 1 Continuous detection of Class A samples using LC-MS

[0068] The pre-concentrated LC-MS analysis was performed and the A-type samples were detected according to the conventional method to obtain their spectra. The blank time and peak time of the A-type samples were compared. If the peak time was ≥ the blank time, the method was used for continuous detection, that is, the next sample injection and loading were performed simultaneously in the sample gradient elution time zone, which can significantly save the time for subsequent sample injection and sample pre-concentration. After the gradient elution of the current sample is completed, the gradient elution of the next sample can be started without waiting.

[0069] The details are as follows:

[0070] When using pre-concentrated LC-MS analysis, two identical trap columns are connected to the ten-way valve of the valve module of the liquid chromatography system. Trap column 1 ( Figure 2 The trap column on the right side of the middle) is connected to holes 3 and 6, and trap column 2 ( Figure 2 The trap column on the left is connected to holes 1 and 8, where trap column 2 is connected to the chromatographic column, and trap column 1 is connected to the loading pipeline. Figure 2 The ten-way valve has 10 connection holes on the upper door, and the six-way valve has 6 connection holes on the upper door. The numbers in the present invention are only for the convenience of explaining which hole is which, and are not limiting.

[0071] The specific operation process of its continuous detection is as follows:

[0072] (1) Sample injection: Figure 2 As shown, the ten-way valve 1-2 is connected to connect the trap column 2 to the chromatographic column, the trap column 1 to the loading pipeline, and the six-way valve 1-6 is connected to start the injection of the first needle sample. The automatic sampler sucks the first needle sample into the loading ring. After the injection, the automatic sampler will automatically clean the injection pipeline to reduce the residue of the sampler.

[0073] (2) Trap column enrichment (load): The first injection of sample is completed, such as Figure 3 The six-way valve is switched to the 1-2 connection, so that the loading pump pushes the sample from the loop to flow through the trap column 1 at a fixed flow rate or a variable speed, wherein the composition and concentration of the mobile phase of the loading pump are not limited in the present invention and can be adjusted according to the characteristics of the target object.

[0074] For example, when testing proteomics in samples, the loading mobile phase is 2% acetonitrile / 0.1% formic acid water, and the trap column contains C18 beads. According to different distribution coefficients, the target in the sample will bind to the trap column 1. The loading time is generally 5 to 10 minutes.

[0075] (3) Gradient elution: After the first injection of sample is loaded, Figure 4 As shown, the ten-way valve cut valves 1-10 are connected, and according to the gradient elution program, the AB pump controls the proportional gradient elution of the mobile phase A and the mobile phase B on the sample on the trap column 1, and at the same time, the six-way valve cut valves 1-6 are connected to start the injection of the second needle sample.

[0076] The eluted target flows are separated again through the chromatographic column and then enter the mass spectrometer for detection and analysis. That is, within the time zone of the sample on the gradient elution trap column 1 (running the first sample), the injection of the next sample can be started, that is, the six-way valve cuts the valves 1-6 to connect, and the automatic sampler or manual injection sucks the second sample into the loop.

[0077] After the injection is completed Figure 5 As shown, the six-way valve cuts the valve 1-2 connection, and the loading pump loads the second needle sample on the loop ring onto the trap column 2. After the gradient elution of the first needle sample on the trap column 1 is completed, the ten-way valve cuts the valve to start the gradient elution of the next needle sample. At the same time, the six-way valve cuts the valve to start the injection of the next needle sample, and the process is repeated in sequence. Specifically:

[0078] When the first needle sample is eluted and tested, the ten-way valve cuts valve 1-2 to connect as follows Figure 2As shown, the trap column 2 is connected to the chromatographic column, and the gradient elution and detection of the second sample are started; within the gradient elution time zone of the second sample, the six-way valve cuts the valves 1-6 and connects, and the automatic sampler or manual injection sucks the third sample onto the loop.

[0079] After the third injection of the sample, Figure 3 The six-way valve shown in the figure switches the 1-2 connection, and the loading pump loads the third needle sample on the loop ring onto the trap column 1. After the loading is completed, wait until the gradient elution of the second needle sample is completed and switch the ten-way valve from the 1-2 connection to the 1-10 connection to connect the trap column 1 to the chromatographic column, and start the gradient elution and detection of the third needle sample. At the same time, the six-way valve switches the valve to perform the injection and loading of the next needle sample, alternating in sequence and repeating.

[0080] When the target is a peptide, mobile phase A is 0.1% formic acid water, and mobile phase B is 0.1% formic acid acetonitrile. Gradient elution can be performed according to the existing elution conditions of the target peptide. The two trap columns are operated independently and will not affect the retention time of the sample on the trap column.

[0081] In this embodiment, the composition, concentration and elution conditions of the mobile phase are not limited in the present invention and can be adjusted or selected according to the characteristics of the target object. Any mobile phase and elution conditions that can elute the target object are acceptable; preferably, the mobile phase and elution conditions whose gradient elution time of the target object is equivalent to the total time required for injection and loading are used.

[0082] In addition, the separation conditions of the chromatographic column are not limited, and any chromatographic conditions that can achieve separation of the components of the target substance may be used, but chromatographic conditions with a shorter separation time are preferred.

[0083] Example 2 Continuous detection of Class B samples using LC-MS

[0084] A conventional LC / MS analyzer is used to detect Class B samples to obtain a complete detection spectrum of a needle sample. Compare the blank time and peak time of Class B samples. If the peak time is less than the blank time, this method is used for continuous detection, that is, the next needle is injected and loaded at the same time as the sample gradient elution time. After the gradient elution of the current sample is completed, wait for the next needle sample to complete loading, cut the valve to start the gradient elution of the needle sample, and inject and load the subsequent samples at the same time, as follows:

[0085] The first injection of sample and loading are the same as in Example 1.

[0086] The ten-way valves 1-10 are connected. When the gradient elution of the first sample starts, the six-way valve cuts valves 1-6 and connects, and the syringe draws the second sample onto the loop; after the injection is completed, the six-way valve cuts valves 1-2 and connects, and the loading pump loads the second sample on the loop onto trap column 2. After the loading is completed, the ten-way valve cuts valves 1-2 and connects to start the gradient elution of the second sample. At the same time, the six-way valve cuts valves 1-6 and connects, and the syringe draws the third sample onto the loop. After the injection is completed, the six-way valve cuts valves 1-2 and connects, and the loading pump loads the third sample on the loop onto trap column 1. After the loading is completed, the ten-way valve cuts valves 1-10 and connects to start the gradient elution of the third sample. At the same time, the six-way valve cuts valves to start the injection and loading of the next sample, and the cycle is repeated in sequence.

[0087] The flow rate of the loading pump can be increased to shorten the waiting time for loading the sample after the gradient elution of the sample is completed. Preferably, the flow rate of the loading pump is controlled so that the N+1th sample is adsorbed on the trap column before the Nth sample completes the gradient elution. When the Nth sample completes the gradient elution detection, the ten-way valve is cut to start the gradient elution and detection of the N+1th sample, and the next sample is injected and loaded at the same time.

[0088] Example 3 Continuous detection of Class C samples using LC-MS

[0089] A conventional LC-MS analyzer is used to detect Class C samples to obtain a complete detection spectrum of a needle sample. Among them, Class C samples have a lot of impurities after the test, and it is necessary to use a blank sample to elute the residue before injecting the next needle sample. This detection method is used, as follows:

[0090] The first injection of sample and loading are the same as in Example 1.

[0091] The ten-way valves 1-10 are connected, and while the gradient elution of the first sample begins, the six-way valve cuts valves 1-6 and connects. The syringe draws the second sample onto the loop. After the injection is completed, the six-way valve cuts valves 1-2 and connects. The loading pump delivers the second sample on the loop to trap column 2. After loading is completed, the ten-way valve cuts valves 1-2 and connects trap column 2 to the chromatographic column. After the gradient elution of the first sample is completed, the gradient elution of the second sample begins. At the same time, the six-way valve cuts valves 1-6 and connects. The syringe draws the blank sample onto the loop. After the injection is completed, the six-way valve cuts valves 1-2 and connects. The loading pump delivers the blank sample to trap column 1 to rinse trap column 1.

[0092] After the gradient elution of the second sample is completed, the ten-way valve cuts valves 1-10 and connects, and the mobile phase is used to flush the chromatographic column. At the same time, the six-way valve cuts valves 1-6 and connects. The syringe draws the blank sample onto the loop. After injection, the six-way valve cuts valves 1-2 and connects. The loading pump transports the blank sample on the loop to trap column 2 to flush trap column 2.

[0093] After the trap column 2 is flushed, the six-way valve cuts valves 1-6 and connects, and the syringe draws the third needle sample onto the loop. After injection, the six-way valve cuts valves 1-2 and connects, and the loading pump delivers the third needle sample on the loop to trap column 1. After loading is completed, the gradient elution and detection of the third needle sample begins.

[0094] In the present invention, according to experimental requirements, when the Nth sample starts gradient elution, the six-way valve cuts valves 1-6 to start injecting blank samples and flushing another trap column; after the Nth sample is detected, the ten-way valve cuts valves to start flushing the chromatographic column, and at the same time, the six-way valve cuts valves 1-6 to start injecting blank samples and flushing the trap column that adsorbs the Nth sample, and then injects, loads and elutes for detection.

[0095] When detecting a sample with residual impurities, the method can start to introduce a blank sample during the sample gradient elution to flush another trap column, which can reduce the time required to wait for the blank sample to flush the impurities and improve the liquid chromatography detection efficiency.

[0096] Comparative Document 1 Conventional LC-MS Detection Method

[0097] The pre-concentrated LC-MS analysis was performed and the samples were tested according to the conventional method to obtain a complete test spectrum of the sample. Figure 6 The detection time of one sample includes the injection time, the loading time, and the sample gradient elution time, among which the injection time and the loading time are invalid time, i.e., blank time. The invalid waiting time required for each sample during detection is long, resulting in low efficiency of continuous detection of liquid chromatography.

[0098] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for improving liquid chromatography detection efficiency, characterized in that: Without changing the gradient elution program, the sample gradient elution and the next sample injection can be performed simultaneously, which specifically includes the following steps: Two identical trap columns are arranged in the ten-way valve of the valve module of the liquid chromatography system, wherein the trap column 2 is connected to the chromatography column, and the trap column 1 is connected to the loading pipeline; The first needle sample is sucked into the loop to complete the injection, and the sample is combined with the trap column 1 to complete the loading. The ten-way valve is cut to connect the trap column 1 with the chromatographic column, and the sample on the trap column 1 is gradient eluted. At the same time, the six-way valve in the injection module is cut to perform the injection and loading of the second needle sample, so that the second needle sample is combined with the trap column 2 to complete the loading; When the gradient elution of the first sample is completed and the injection and loading of the second sample are completed, the ten-way valve is turned off to connect the trap column 2 with the chromatographic column to start the gradient elution of the second sample, and at the same time, the six-way valve is turned off to inject and load the next sample, so that the sample is combined with the trap column 1; the above operations are repeated in sequence.

2. The method for improving liquid chromatography detection efficiency according to claim 1, characterized in that: For routine testing using LC / MS analyzer, if the peak time in the sample spectrum is ≥ blank time, follow the steps below: Without changing the sample gradient elution program, the six-way valve is cut to inject the second sample during the gradient elution time of the first sample, and the sample is combined on trap column 2 to complete the loading before the gradient elution of the first sample is completed; When the gradient elution of the first sample is completed, the ten-way valve is turned off to connect trap column 2 to the chromatographic column, and the gradient elution of the second sample is started. At the same time, the six-way valve is turned off to inject and load the next sample; and the process is repeated in sequence.

3. The method for improving liquid chromatography detection efficiency according to claim 1, characterized in that: For routine testing using LC / MS analyzer, if the peak time in the sample spectrum is less than the blank time, follow the steps below: Without changing the sample gradient elution program, when the gradient elution of the first sample begins, the six-way valve is cut to inject the second sample. After the sample is combined with the trap column 2 and loaded, the ten-way valve is cut to connect the trap column 2 with the chromatographic column, and the gradient elution of the second sample begins. At the same time, the six-way valve is cut to inject and load the next sample. Repeat this process.

4. The method for improving the detection efficiency of liquid chromatography according to claim 3, characterized in that: By increasing the flow rate of the loading pump, the time required to wait for the N+1th needle sample to complete loading after the gradient elution of the Nth needle sample can be shortened.

5. The method for improving liquid chromatography detection efficiency as claimed in claim 4, characterized in that: The flow rate of the loading pump is controlled so that the N+1th needle sample is combined with another trap column to complete the loading when or before the gradient elution of the Nth needle sample is completed.

6. The method for improving the detection efficiency of liquid chromatography according to any one of claims 1 to 5, characterized in that: In the ten-way valve, the trap column 1 is connected to the hole positions 3 and 6, and the trap column 2 is connected to the hole positions 1 and 8.

7. The method for improving liquid chromatography detection efficiency according to claim 6, characterized in that: The ten-way valve 1-2 is connected to connect the trap column 2 to the chromatographic column, and the trap column 1 is connected to the loading pipeline to start the injection and loading of the first sample. After completion, the ten-way valve 1-10 is connected to perform gradient elution on the sample on trap column 1. During the gradient elution time of the sample on trap column 1, the six-way valve is cut to start the injection and loading of the next sample, so that the next sample is combined with trap column 2 to complete the loading.

8. The method for improving liquid chromatography detection efficiency according to claim 7, characterized in that: After the second needle sample is loaded, the ten-way valve cuts valve 1-2 to connect and gradient elute the sample on trap column 2. During the gradient elution time of the sample on trap column 2, the six-way valve cuts valve to start the injection and loading of the next needle sample, so that the next needle sample is combined with trap column 1 to complete the loading.

9. The method for improving liquid chromatography detection efficiency according to claim 1, characterized in that: In the six-way valve, the loops are connected according to hole positions 1-4.

10. The method for improving liquid chromatography detection efficiency according to claim 9, characterized in that: When the six-way valve 1-6 is connected, the sample injection begins. After the injection is completed, the six-way valve cuts off valve 1-2 and connects the loading pump to combine the sample on the loop ring to the trap column to complete the sample loading.

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