Liquid chromatograph suitable for step gradients
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- TOSOH CORP
- Filing Date
- 2022-01-13
- Publication Date
- 2026-06-30
Smart Images
Figure 0007881910000002 
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Figure 0007881910000004
Abstract
Description
Technical Field
[0001] The present invention relates to a liquid chromatograph apparatus suitable for the step gradient elution method.
Background Art
[0002] In clinical test measurements using a liquid chromatograph apparatus, in addition to the requirement of shortening the analysis measurement time per measurement, there is a requirement for precise analysis to separate more peaks and obtain detailed information. Precise analysis usually requires means such as extending the measurement time and adding the number of eluents. However, increasing the number of eluents leads to a decrease in user-friendliness due to an increase in the number of consumables. In addition, the apparatus also becomes larger due to the addition and complication of mechanisms associated with adding eluents.
[0003] To avoid these problems, a gradient system can be used to prepare a minimum amount of eluents and mix them to obtain a desired eluent composition. In order to suppress the increase in the size and cost of the apparatus, a low-pressure gradient method using one liquid delivery pump is often adopted. In the low-pressure gradient method, a plurality of eluent tanks are provided on the suction side of the liquid delivery pump via valve means such as solenoid valves, and the operation of the valve means is controlled according to a gradient program describing the concentration ratio of each eluent.
[0004] Examples of gradients include a linear gradient performed by specifying the eluent mixing concentration at the start point and the eluent mixing concentration at the end point in the time region where the gradient is implemented, and causing a constant concentration change linearly over time, and a step gradient performed by dividing the time region where the gradient is implemented in advance into arbitrary sections, determining a unique eluent mixing concentration in each divided time region, and causing a stepwise concentration change.
[0005] For linear gradients, the gradient start time and the eluent mixing ratio at that point, as well as the gradient end time and the eluent mixing ratio at that point, must be specified in the gradient program. For step gradients, the start time of each step and the eluent mixing ratio for each step must be specified in the gradient program.
[0006] Patent Document 1 discloses a method for measuring hemoglobin using a low-pressure linear gradient. However, to shorten the measurement time and separate more peaks, it is preferable to use a low-pressure step gradient rather than a low-pressure linear gradient. In step gradient measurements, where the measurement time per sample is short, the time interval between each step is shortened, and the time required to switch eluents in each step is also shortened to the absolute minimum. However, under measurement conditions where the number of steps is increased and the opening and closing interval of the valve means is shortened, it was difficult to control the gradient concentration with good reproducibility between multiple measurements. [Prior art documents] [Patent Documents]
[0007] [Patent Document 1] Japanese Patent Publication No. 2019-82437 [Overview of the project] [Problems that the invention aims to solve]
[0008] The object of the present invention is to improve inter-measurement reproducibility by improving the reproducibility of the low-pressure step gradient in a liquid chromatograph apparatus. [Means for solving the problem]
[0009] When performing low-pressure step gradient liquid chromatography measurements sequentially and rapidly on multiple samples, the pulsation of the liquid delivery pump is considered a factor that impairs the reproducibility between measurements. In a liquid delivery pump with a single reciprocating plunger, the liquid delivery driving force acts only during the discharge phase of the plunger's suction and discharge phases, so the eluent is divided at regular intervals and moved intermittently. When switching eluent compositions while delivering the eluent, the way the boundary between the eluents created by the switch is divided by the liquid delivery pump results in differences in the profile of the delivered eluent composition, which manifests as differences in reproducibility between measurements. Therefore, by aligning the timing of activating the eluent valve mechanism with the operating phase of the liquid delivery pump's plunger, the reproducibility between measurements is improved, and the present invention is complete.
[0010] The liquid chromatograph apparatus according to the present invention comprises an analytical column that provides a time difference in elution for components in a sample, a detector that detects components eluted from the analytical column, a sample injection unit that injects the sample into the analytical column, suction channels connected to two or more eluents, a valve means for selecting the open / closed state of the suction channels, which can switch one suction channel to an open state and the remaining suction channels to a closed state, a liquid delivery pump that sequentially draws in eluents from the open suction channels and discharges them to the analytical column, the liquid delivery pump having a plunger that reciprocates at a predetermined period, a phase sensor that detects a predetermined operating phase of the plunger, a step gradient program which arranges step sections, where the mixing ratio of the eluents is a specified time interval, in a time series, and n parameters (n is a natural number) that indicate which suction channel connected to which eluent should be opened, corresponding to each of the step sections, in the order of execution. A valve control table arranged in such a way that the proportion of the n parameters that open an intake passage connected to a specific eluent is equivalent to the mixing ratio; and a control unit that performs a control operation that reads the first parameter of the valve control table corresponding to the subsequent step section and outputs an operating signal corresponding to that parameter to the valve means at one of two consecutive timings spanning before and after the end time of each step section in the step gradient program when the phase sensor detects a predetermined phase of the plunger operation, and reads the second parameter and outputs an operating signal corresponding to that parameter to the valve means at the next timing when the phase sensor detects a predetermined phase, and repeats the control operation in a manner that similarly returns to the first parameter after passing through the nth parameter.
[0011] As described above, the valve control table corresponding to each step section does not contain time information; it simply contains information (parameters) for selecting the eluent to be aspirated, arranged in execution order. The control unit sequentially reads the parameters from the valve control table at predetermined timings when the plunger of the liquid delivery pump operates periodically, and outputs an operating signal to the valve means corresponding to those parameters. The present invention is characterized in that the operation of the valve means is controlled based on the periodic operation of the plunger, rather than on a time scale.
[0012] Because plungers exhibit changes in their mechanical capacity over time and variations between individual machines, valve control based on a time scale results in valve switching occurring under conditions where the phases of plunger operation differ, making it impossible to ensure inter-measurement reproducibility in chromatographic analysis. This phenomenon is particularly pronounced in step gradient elution methods where the measurement time is approximately 1 minute and the step interval includes a step interval of approximately 10 times or less the plunger's operating cycle.
[0013] In contrast, if the valve mechanism is operated under the condition that the operating phase of the plunger is constant, using the plunger's operating cycle as the unit, then even if there are fluctuations, quantitative differences, or intentional changes in the operating cycle itself, uniformity or similarity of the eluent composition profile can be expected, and good reproducibility of the measurement results can be ensured.
[0014] In low-pressure linear gradient systems, it is common to place a mixer upstream or downstream of the liquid delivery pump, but in this invention, this is optional. In step gradients, for step sections where mixing of the eluent is required, the flow path from the valve to the liquid delivery pump, through the sample injection section to the column acts as a static mixer, forming a time-averaged mixing concentration.
[0015] One example of the control unit's configuration is that it repeats a series of control operations, starting with reading the first parameter of the valve control table corresponding to the subsequent step section at the timing when the phase sensor detects a predetermined phase after the end time of each step section of the step gradient program has elapsed. Alternatively, the control unit can also repeat a series of control operations, starting with reading the first parameter of the valve control table corresponding to the subsequent step section at the timing when the phase sensor detects a predetermined phase after a time equal to the plunger's operating cycle has elapsed before the end time of each step section.
[0016] Furthermore, the step gradient program can also include processing information for each step section, specifying whether the control unit repeats a series of control operations starting with reading the first parameter of the valve control table corresponding to the subsequent step section when the phase sensor detects a predetermined phase after the end time of the step section has elapsed, or whether it repeats a series of control operations when the phase sensor detects a predetermined phase after a time equal to the plunger's operating cycle has elapsed before the end time of the step section.
[0017] When performing measurements on multiple samples sequentially and continuously, synchronizing the timing of sample injection with the pump operation is important to improve inter-measurement reproducibility. Therefore, as an embodiment of the present invention, in a step gradient program for a single measurement of a sample, the control unit outputs an operating signal to the sample injection unit at the timing when the phase sensor detects a predetermined phase, after a time equal to the plunger's operating cycle has elapsed before the end of the final step section included in the step gradient program, and simultaneously starts the next measurement from the first step section of the step gradient program. The reason for setting the timing of the next sample injection to the timing when the phase sensor detects a predetermined phase after a time equal to the plunger's operating cycle has elapsed before the end of the final step section is to shorten the measurement time for a single sample.
[0018] Now, the valve control table corresponding to each step section of the step gradient program is an arrangement of n parameters (n is a natural number) in execution order, each representing which suction channel connected to which eluent should be opened. For step sections with a mixing ratio of 100%, where mixing of eluents is not necessary, only one parameter is required (n=1). For step sections where mixing of eluents is necessary, the mixing ratio is expressed by the proportion of the n parameters that open the suction channel connected to a specific eluent. Since the parameter readout period performed by the control unit matches the plunger's operating period, one parameter corresponds to a valve state maintained throughout the plunger's operating period.
[0019] For example, the parameter array can be divided into two groups: one consisting of the first to fifth parameters and the other consisting of the sixth to tenth parameters, with n=10 parameters. One or both of these groups can then be further divided into two subgroups, and parameters corresponding to different eluents can be assigned to these subgroups. This is because the mixing ratio used in step gradient elution is often specified in 10% increments, and it is convenient to assign the mixing ratio to 10 times the number of parameters, using the plunger's operating cycle as the unit.
[0020] When the analysis time is short and the number of steps is large, a single step interval may be smaller than 10 plunger cycles (10 parameters). In such step intervals, it is preferable to create a valve control table keeping in mind that the parameters defined in the preceding group / subgroup will be dominant. Also, when considering the eluent composition of adjacent step intervals, the arrangement of subgroups in two groups may be in the same order or in reverse order.
[0021] While the present invention has a problem of supplying an eluent having a mixing ratio according to a step gradient program for a step section shorter than 10 times the operation cycle of the plunger, it provides technical means that prioritize maximizing the result reproducibility under the same conditions.
[0022] For example, when the mixing ratio of the eluent is 80% of solution A: 20% of solution B (assuming that the component concentration of solution B is higher), one parameter for setting solution B to the open state (liquid passing state) is placed, then four parameters for setting solution A to the open state are placed, then one parameter for setting solution B to the open state is placed again, and four parameters for setting solution A to the open state are placed, creating an array consisting of a total of 10 parameters. That is, the number ratio of each group obtained by dividing the parameter array in the valve control table into two is 1:4 and 1:4 for B:A.
[0023] Next, when it is 70% of solution A: 30% of solution B, two parameters for setting solution B to the open state are placed, three parameters for setting solution A to the open state are placed, and after one parameter for setting solution B to the open state is placed, four parameters for setting solution A to the open state can be placed. That is, the number ratio of each group obtained by dividing the parameter array in the valve control table into two is 2:3 followed by 1:4 for B:A, becoming asymmetric.
[0024] Also, when it is 90% of solution A: 10% of solution B, after one parameter for setting solution B to the open state is placed, four parameters for setting solution A to the open state are placed, and furthermore, five parameters for setting solution A to the open state can be placed. That is, the number ratio of each group obtained by dividing the parameter array in the valve control table into two is 1:4 followed by 0:5 for B:A. Thus, the final mixing ratio can set a functional valve control table by dividing the 10 parameters of the plunger operation cycle into groups of 5 each and allocating the mixing ratio to each group.
[0025] When the opening and closing conditions are different between two groups, that is, "B:A is 2:3" and "B:A is 1:4", if it is set so that the opening frequency ratio of the valve means to which the B liquid with a high component concentration is connected in the first group increases, the effect of step switching from the eluent with a high component concentration will be strongly reflected. Also, depending on the purpose of component separation, there may be cases where the eluent (liquid A) with a low component concentration is set to the open state first within each group.
[0026] The device according to the present invention is suitable for application in the field of continuous measurement with a short measurement time and a large throughput, such as when the main measurement target is hemoglobin in blood and at least one peak of hemoglobin D, hemoglobin S, and hemoglobin C is eluted, and a high retention time reproducibility of the elution peak is required.
Effects of the Invention
[0027] According to the liquid chromatograph device of the low-pressure step gradient method of the present invention configured as described above, the retention time of each peak of the chromatogram is stable, and the repeatability is greatly improved. Even when a plurality of different peaks are adjacent to each other in the vicinity, the possibility of false detection of the peaks can be reduced by the stability of the retention time.
Brief Description of the Drawings
[0028] [Figure 1] It is a schematic configuration diagram of the liquid chromatograph device of the present invention, showing an example of the structure of the liquid delivery pump and the phase sensor. [Figure 2] It is an explanatory diagram of an example related to step gradient control. [Figure 3] It is an example of a chromatogram of general whole blood by the liquid chromatograph device of the present invention. [Figure 4] It is an explanatory diagram of a comparative example related to step gradient control. [Figure 5]This figure shows superimposed chromatograms of various samples containing abnormal hemoglobin, specifically hemoglobin D, hemoglobin S, and hemoglobin C, as measured by the liquid chromatograph apparatus of the present invention. [Modes for carrying out the invention]
[0029] Figure 1 shows a schematic configuration of a liquid chromatograph apparatus that performs liquid delivery using a low-pressure step gradient method. In this apparatus, three different types of eluents 101, 102, and 103 are connected to a single plunger pump (liquid delivery pump) 104. Each eluent is drawn into the plunger pump 104 via a flow path that passes through a degassing unit 105, solenoid valves (valve means) 106, 107, and 108 corresponding to each eluent, and a confluence section 109. A priming mechanism (not shown) can be provided at the start of measurement for removing air bubbles in the flow path related to the valve means and confluence section or for replacing the eluent.
[0030] The valve mechanism may use one valve for each eluent, but it is also possible to switch between multiple eluent suction channels with a single selective switching valve. Examples of solenoid valves include two-way diaphragm valves, pinch valves, and slider valves, and those with high responsiveness and fast operating speed are desirable. If the orifice diameter of the solenoid valve is too large, the opening and closing response will be poor, and if it is too small, the suction side of the plunger pump may tend to become negatively pressurized, so a diameter of about 0.4 mm to 4.0 mm is desirable.
[0031] Furthermore, when using solenoid valves, they are placed upstream of the pump, but because the switching between opening and closing is performed in a short time, back pressure tends to increase, so it is desirable that the maximum fluid pressure of the solenoid valve be 100kPa or higher.
[0032] The sample injection unit 120 has an injection valve and injects the sample to be measured into the flow path between the plunger pump 104 and the column 111. It may also have the function of an autosampler that collects a sample from a blood collection tube or the like before sample injection and performs pretreatment such as dilution or hemolysis as necessary.
[0033] In column 111, the components to be measured are adsorbed and desorbed from the injected sample, and eluted at different times depending on the difference in elution power between the eluent alone or a gradient mixture. Each eluted component is detected by detector 112. Examples of detectors include ultraviolet-visible detectors and fluorescence detectors. Depending on the sample to be measured, the reaction reagents can also be mixed before and after passing through the column.
[0034] The control unit 121 has one or more microcontrollers or CPUs and controls each unit. It may also be integrated with a storage unit for saving measurement results and various parameter values. The storage unit also stores the gradient program and the valve control table read out based on it. A single-plunger pump is preferable for the plunger pump 104 in order to reduce the size of the device and manufacturing costs. The rotation or reciprocating motion of the cam 202, which is linked to the motor 201, causes the plunger 206 to reciprocate, and the eluent in the cylinder separated by the suction-side check valve 208 and the discharge-side check valve 207 is discharged into the analysis channel.
[0035] The reciprocating motion of the plunger can be monitored by a phase sensor consisting of a slit disc 204 integrally connected to the camshaft 203 and a pair of light-emitting and receiving elements 205. The plunger 206 can be designed so that one reciprocating motion, or one stroke, corresponds to one rotation of the cam 202, i.e., the camshaft 203, and the phase sensor can be adjusted to detect one specific phase within one stroke of the plunger 206. For example, it can be designed to detect the plunger's most retracted position. [Examples]
[0036] The details of step gradient control using the present invention will be explained using the time chart shown in Figure 2. The step gradient program shown in the upper part of Figure 2 consists of a sequence of time intervals (step intervals) of mixing ratios of three types of eluents (liquid 1, liquid 2, and liquid 3) with at least one component concentration different. The flow rate during measurement was set to a constant 2.1 mL / min, the measurement time to 40 seconds per sample, and the plunger volume to 8.4 μL (plunger stroke 0.24 seconds). In the step gradient program, 0 to 4 seconds is 100% liquid 1, 4 to 14 seconds is 100% liquid 2, 14 to 15.8 seconds is 80% liquid 2: 20% liquid 3, 15.8 to 18 seconds is 50% liquid 2: 50% liquid 3, 18 to 21 seconds is 100% liquid 3, and 21 to 40 seconds is 100% liquid 1.
[0037] The valve control table shown in the middle of Figure 2 is an arrangement of parameters in execution order that indicate which suction channel connected to which eluent should be opened, corresponding to each step section of the step gradient program. Here, the parameters are represented by unit vectors having components corresponding to each eluent. Specifically,
[0100] indicates opening the suction channel connected to the first eluent,
[0010] indicates opening the suction channel connected to the second eluent, and
[0001] indicates opening the suction channel connected to the third eluent. For convenience, the unit vectors are shown vertically in the middle of Figure 2.
[0038] First, in the step interval from 0 to 4 seconds, the first liquid is 100%, so the corresponding valve control table consists of one parameter,
[0100] . In the step interval from 4 to 14 seconds, the second liquid is 100%, so the corresponding valve control table consists of one parameter,
[0010] . In the step interval from 14 to 15.8 seconds, the mixing ratio is 80% second liquid: 20% third liquid, so it is necessary to associate multiple (n) parameters within this interval.
[0039] In this embodiment, n=10, and the parameters were divided into two groups: one consisting of the first to fifth parameters and the other consisting of the sixth to tenth parameters. Each of these groups was further divided into two subgroups, and parameters corresponding to different eluents were assigned to each subgroup. Specifically, in the first subgroup of the first to fifth parameters, one
[0001] corresponding to the third liquid was assigned, four consecutive
[0010] corresponding to the second liquid were assigned to the second subgroup, and the same parameters as those assigned to the first to fifth parameters were assigned to the sixth to tenth parameters.
[0040] In step interval 15.8 seconds to 18 seconds, the mixing ratio is 50% second liquid: 50% third liquid, so with n=10, from the group consisting of the first to fifth parameters, one
[0001] corresponding to the third liquid was assigned to the first subgroup, four consecutive
[0010] corresponding to the second liquid was assigned to the second subgroup, and the same parameters as the group consisting of the first to fifth parameters were assigned to the group consisting of the sixth to tenth parameters. In step interval 18 seconds to 21 seconds, the third liquid is 100%, so the corresponding valve control table consists of one parameter
[0001] . Then, in step interval 21 seconds to 40 seconds, the first liquid is 100%, so the corresponding valve control table consists of one parameter
[0100] .
[0041] An example of measurement using the liquid chromatograph apparatus of the present invention, based on the above step gradient program and valve control table, is shown below. An automated glycated hemoglobin analyzer HLC-723G11 (variant mode) (manufactured by Tosoh Corporation) was used as the measuring device.
[0042] This device is a liquid chromatograph based on the principle of cation exchange chromatography. It separates hemoglobin in blood into the usual six fractions: hemoglobin A1a, hemoglobin A1b, hemoglobin F, unstable hemoglobin A1c, stable hemoglobin A1c (HbA1c), and A0. In addition, for some samples from hemoglobin disorders (abnormal hemoglobin disorders), it can also separate some abnormal hemoglobin.
[0043] Although this invention does not specify the object to be measured, its effectiveness is particularly evident when the measurement time is shorter than about 2 minutes and there are about 5 or more components to be separated.
[0044] In this example, the analytical column used was a 2.5 mm x 10 mm column repacked with TSKgel G11 (standard mode) (manufactured by Tosoh Corporation). The eluents used were G11 Variant Elution Buffer No. 1 (S) as the first eluent, G11 Variant Elution Buffer No. 2 (S) as the second eluent, and G11 Variant Elution Buffer No. 3 (S) as the third eluent (all manufactured by Tosoh Corporation). The detector was a visible light detector that detects absorbance at a primary wavelength of 415 nm and a secondary wavelength of 500 nm.
[0045] (Example 1) Sample 1, which is a typical whole blood sample, was measured 30 times consecutively. The step gradient program was performed as shown in the upper part of Figure 2. The valve control table shown in the middle part of Figure 2 was also used. Here, the step gradient program and valve control table were pre-programmed and stored in the device storage unit. In this embodiment, control was performed based on these step gradient program, valve control table, and detection by the phase sensor.
[0046] The opening and closing operations of the valve mechanism performed by the device are shown in the lower part of Figure 2. The time periods during which a valve mechanism was in an open state for any of the eluents are represented by time bars. The white circle at the left end of the time bar indicates the opening operation of the valve mechanism, and the black circle at the right end indicates the closing operation of the same mechanism. In this embodiment, since the reciprocating motion of the plunger is synchronized with the rotation of the cam and the detection by the phase sensor, one reciprocating motion of the plunger is also referred to as one rotation of the pump.
[0047] Starting with the initial sample injection, the valve control table is opened according to the parameter
[0100] , and the plunger's movement is detected by a phase sensor. This open state is repeatedly maintained, effectively resulting in continuous fluid delivery. The dotted line to the right of the single unit vector in the middle of Figure 2 indicates repeated execution.
[0048] At the 17th rotation of the plunger, immediately after reaching the end time of the step section, which is 4 seconds, the phase sensor detected the phase of the plunger operation (PD) at the timing of 4.08 seconds. At the same time, the parameter
[0010] described in the valve control table corresponding to the next step section [4 seconds to 14 seconds] was read, the solenoid valve for the first liquid was closed, and the solenoid valve for the second liquid was opened.
[0049] Next, at the 42nd rotation of the plunger, immediately after reaching the end time of the step section of 14 seconds, at the timing of the phase detection of the plunger operation by the phase sensor (14.16 seconds), the first parameter
[0001] described in the valve control table corresponding to the next step section [14 seconds to 15.8 seconds] was read, and one rotation's worth of the third liquid was delivered. Four rotations' worth of the second liquid corresponding to four
[0010] (2nd to 5th) were delivered, then one rotation's worth of the third liquid corresponding to
[0001] (6th) was delivered, and then one rotation's worth of the second liquid corresponding to
[0010] (7th) was delivered.
[0050] While this parameter
[0010] (7th) is being executed, the end time of the step interval, 15.8 seconds, is reached, and at the timing of the next phase detection (15.84 seconds), the valve control table corresponding to the next step interval [15.8 seconds to 18 seconds] is read. Parameters 8 to 10 of the valve control table corresponding to the step interval [14 seconds to 15.8 seconds] are discarded and not used (truncation section).
[0051] Next, in the step section [15.8 seconds to 18 seconds], the third liquid was dispensed for three rotations corresponding to the 1st to 3rd parameters
[0001] , the second liquid was dispensed for two rotations corresponding to two
[0010] (4th to 5th), then the third liquid was dispensed for two rotations corresponding to
[0001] (6th to 7th), and then the second liquid was dispensed for two rotations corresponding to
[0010] (8th to 9th). While this 9th parameter was being executed, the end time of the step section, 18 seconds, was reached, and immediately after (simultaneously) at the phase detection timing (18 seconds), the valve control table corresponding to the next step section was read. The 10th parameter of the valve control table corresponding to the step section [15.8 seconds to 18 seconds] was discarded (truncation section).
[0052] Next, the parameter
[0001] described in the step section [18 seconds to 21 seconds] was read, the solenoid valve for the second liquid was closed, and the solenoid valve for the third liquid was opened. At the 13th rotation, the end time of this step section, 21 seconds, was reached, and at the subsequent phase detection timing (21.12 seconds), the parameter
[0100] described in the valve control table corresponding to the next step section was read, the solenoid valve for the third liquid was closed, and the solenoid valve for the first liquid was opened.
[0053] The next sample was injected via the injection valve at 39.84 seconds, after the plunger's operating cycle (0.24 seconds) had elapsed (corresponding to the 78th rotation at 39.76 seconds), which was the timing when the phase sensor detected a predetermined phase (39.84 seconds), and this marked the start time of the next measurement (0 seconds).
[0054] Under the above conditions, when measurements were performed using a typical whole blood sample, hemoglobin A1a, hemoglobin A1b, and hemoglobin F were sequentially eluted primarily in the step interval [0 seconds to 4 seconds], and unstable hemoglobin A1c (LA1c) and stable hemoglobin A1c (HbA1c) were sequentially eluted primarily in the step interval [4 seconds to 14 seconds]. Hemoglobin A0 was primarily eluted in the step interval [14 seconds to 15.8 seconds]. No peak was eluted in the interval from 15.8 seconds to 21 seconds.
[0055] Figure 3 shows the chromatogram of a typical whole blood sample measured using the apparatus of the present invention, along with the peaks for LA1c, HbA1c, and hemoglobin A0. Sample 1, a typical whole blood sample, was measured 30 times consecutively, yielding an average HbA1c elution time of 0.329 minutes and a coefficient of variation of 0.31%, which is an indicator of reproducibility. This indicates good reproducibility.
[0056] (Example 2) Under the same conditions as in Example 1, Sample 2, which was whole blood containing hemoglobin S (HbS), a representative abnormal hemoglobin, was measured 30 times consecutively. In the step gradient program, hemoglobin A1a, hemoglobin A1b, and hemoglobin F were eluted sequentially, mainly in the step interval [0 seconds to 4 seconds], and unstable hemoglobin A1c (LA1c) and stable hemoglobin A1c (HbA1c) were eluted sequentially, mainly in the step interval [4 seconds to 14 seconds]. Hemoglobin A0 was mainly eluted in the step interval [14 seconds to 15.8 seconds]. Hemoglobin S was eluten in the step interval [15.8 seconds to 21 seconds]. An average eluten time of 0.495 minutes for hemoglobin S and a coefficient of variation of 0.00%, an indicator of reproducibility, were obtained. This showed extremely good reproducibility.
[0057] (Comparative Example 1) Comparative Example 1, as shown in Figure 4, uses the same step gradient program and valve control table as Example 1, but controls the valve means by reading the valve control table according to the time that defines each step interval. That is, starting with the initial sample injection, the first liquid (eluent) is opened according to the parameter
[0100] described in the valve control table, and the same open state is repeatedly maintained while detecting the operation of the plunger with a phase sensor, thereby substantially continuously delivering the liquid.
[0058] At the end of the step interval [0 seconds to 4 seconds], which is 4 seconds (equivalent to 16.7 rotations), the parameter
[0010] described in the valve control table corresponding to the next step interval was read, the solenoid valve for the first liquid was closed, and the solenoid valve for the second liquid was opened.
[0059] Next, at 14 seconds (equivalent to 41.7 rotations), which is the end time of the step interval [4 seconds to 14 seconds], the parameter
[0001] described in the valve control table corresponding to the next step interval [14 seconds to 15.8 seconds] is read, and one rotation's worth of the third liquid is dispensed. Four rotations' worth of the second liquid corresponding to four
[0010] (2nd to 5th) are dispensed, then one rotation's worth of the third liquid corresponding to
[0001] (6th) is dispensed, then one rotation's worth of the second liquid corresponding to
[0010] (7th) is dispensed, and while the second liquid corresponding to
[0010] (8th) is being dispensed, at 15.8 seconds, which is the end time of the step interval, the valve control table corresponding to the next step interval [15.8 seconds to 18 seconds] is read. The 7th and 8th parameters
[0010] of the valve control table corresponding to the step interval [14 seconds to 15.8 seconds] correspond to the second liquid flowing to 1.5 plunger rotations. The 9th and 10th parameters are not used and are discarded (truncation section).
[0060] Next, in step interval [15.8 seconds to 18 seconds], the valve control table corresponding to the next step interval [18 seconds to 21 seconds] is read out when the end time of step interval, 18 seconds, is reached, and the valve control table corresponding to the next step interval [18 seconds to 21 seconds] is read out when the end time of step interval, 18 seconds, is reached, and the valve control table corresponding to the 8th to 10th plunger of the valve control table corresponding to step interval [15.8 seconds to 18 seconds] corresponds to 2.2 plunger rotations of the second liquid.
[0061] Next, corresponding to the parameter
[0001] described in step interval [18 seconds to 21 seconds], the solenoid valve for the second liquid was closed and the solenoid valve for the third liquid was opened. At 21 seconds (equivalent to 12.5 rotations), which is the end time of this step interval, the parameter
[0100] described in the valve control table corresponding to the next step interval was read, the solenoid valve for the third liquid was closed, and the solenoid valve for the first liquid was opened. At 40 seconds, which is the end time of the last step interval described in the step gradient program, the next sample was injected using the injection valve, and the measurement start time for the next measurement (0 seconds) was set.
[0062] In Comparative Example 1, under the above conditions, Sample 1, which is a typical whole blood sample, was measured 30 times consecutively. An average HbA1c elution time of 0.331 minutes and a coefficient of variation of 0.49%, which is an indicator of reproducibility, were obtained. Compared to the results of Example 1, the average elution time remained unchanged, but the coefficient of variation was significantly higher.
[0063] (Comparative Example 2) Under the same conditions as in Comparative Example 1, Sample 2, a hemoglobin S-containing sample, was measured 30 times consecutively, yielding an average hemoglobin S elution time of 0.494 minutes and a coefficient of variation of 0.13%, which is an indicator of its reproducibility. While this coefficient of variation itself is not particularly high, it is significantly higher compared to Example 2.
[0064] (Example 3) In Example 3, the set flow rate from Example 1 was increased by 5% to 2.2 mL / min. All other conditions were the same as in Example 1. When Sample 1, which is typical whole blood, was measured 30 times consecutively, the average elution time for HbA1c was 0.313 minutes, and the coefficient of variation, an indicator of reproducibility, was 0.28%. Compared to Example 1, both the average elution time and the coefficient of variation were almost identical.
[0065] (Example 4) Under the same conditions as in Example 3, Sample 2, a hemoglobin S-containing sample, was measured 30 times consecutively, yielding an average hemoglobin S elution time of 0.486 minutes and a coefficient of variation of 0.11%, which is an indicator of reproducibility. This indicates good reproducibility.
[0066] (Comparative Example 3) In Comparative Example 3, the set flow rate from Comparative Example 1 was increased by 5% to 2.2 mL / min. All other conditions were the same as in Comparative Example 1. In this comparative example, Sample 1, which is a typical whole blood sample, was measured 30 times consecutively, and an average HbA1c elution time of 0.296 minutes and a coefficient of variation of 1.87%, which is an indicator of reproducibility, were obtained. It can be seen that the coefficient of variation is significantly higher compared to Example 3.
[0067] (Comparative Example 4) Under the same conditions as in Comparative Example 3, Sample 2, a hemoglobin S-containing sample, was measured 30 times consecutively, yielding an average hemoglobin S elution time of 0.482 minutes and a coefficient of variation of 0.32%, which is an indicator of its reproducibility.
[0068] (Summary of Examples and Comparative Examples) Table 1 summarizes the results of the above-described examples and comparative examples. In the table, "Pump Synchronization+" refers to a feature of the present invention in which the control operation of the valve means is synchronized with a predetermined phase of the operation of the plunger of the liquid transfer pump at the boundary of each step section (end time) of the step gradient program. "Pump Synchronization-" refers to a method in which the valve means is controlled by reading the valve control table according to the time that defines each step section.
[0069] Table 1 displays the detected species of sample 1, stable hemoglobin A1c (HbA1c), and the detected species of sample 2, hemoglobin S (HbS), in upper and lower rows. The top two rows and bottom two rows of each row correspond to the eluent flow rate conditions of 2.1 mL / min and 2.2 mL / min.
[0070] Example 1 and Comparative Example 1, Example 2 and Comparative Example 2, Example 3 and Comparative Example 3, and Example 4 and Comparative Example 4 differ only in the presence or absence of "pump synchronization." The measurement results for each example (pump synchronization +) showed smaller coefficients of variation and higher reproducibility compared to the measurement results for the corresponding comparative examples (pump synchronization -). Furthermore, Comparative Examples 3 and 4, with a flow rate of 2.2 mL / min, showed larger corresponding coefficients of variation and worse reproducibility compared to Comparative Examples 1 and 2, respectively, with a flow rate of 2.1 mL / min. In contrast, Examples 3 and 4, with a flow rate of 2.2 mL / min, showed almost no difference in corresponding coefficients of variation compared to Examples 1 and 2, respectively, with a flow rate of 2.1 mL / min.
[0071] This suggests that increasing the flow rate in Comparative Examples 3 and 4 compared to Comparative Examples 1 and 2 led to a shift in the plunger position at the timing of valve switching, resulting in variations in the eluent composition and a deterioration in reproducibility. On the other hand, in Examples 3 and 4, where the liquid delivery flow rate was increased compared to Examples 1 and 2, good reproducibility was obtained regardless of the increase in flow rate. Therefore, by aligning the timing of activating the eluent switching valve with the operating phase of the plunger of the liquid delivery pump, it was possible to improve reproducibility between measurements.
[0072] In Table 1, the coefficients of variation for the detected species HbA1c (top four rows) are generally larger than those for the detected species HbS (bottom four rows). This is because the chromatographic peak of HbA1c is smaller than that of HbS. While the coefficient of variation for HbS remained at 0.32% in Comparative Example 4 compared to 0.11% in Example 4, HbA1c showed a significant deterioration in reproducibility, with the coefficient of variation for Comparative Example 3 being 1.87% compared to 0.28% in Example 3 under the same elution conditions. This indicates that the present invention is particularly effective in improving the measurement reproducibility of minute peaks.
[0073] The measurement conditions for liquid chromatography, including step gradient elution, are optimized for the instrument configuration and flow rate. Therefore, changes in conditions, such as column replacement or flow rate adjustment, can significantly impact the measurement results. However, inter-instrument differences within the manufacturing control range and degradation due to use are unavoidable changes in conditions. Even in such situations, the present invention maintains high reproducibility between measurements and stabilizes the elution time.
[0074] Furthermore, under conditions where the measurement time is short and the number of separated peaks is large, the tolerance range for identifying each peak inevitably becomes smaller. While it is possible to report hemoglobin D, hemoglobin S, and hemoglobin C collectively with representative peak names such as variant peaks, it is considered preferable to report them as individual peaks.
[0075] However, if there is a large variation in the elution time of hemoglobin S, hemoglobin S may be mistakenly detected as hemoglobin D or hemoglobin C. Similarly, if there is a large variation in the elution time of hemoglobin D, it may be mistakenly detected as hemoglobin A0 or hemoglobin S. In the present invention, as described above, the elution time can be stabilized, thus avoiding false detections.
[0076] Furthermore, since hemoglobin D, hemoglobin S, and hemoglobin C have different affinities to the column, they are eluted sequentially even within the same salt concentration step interval. Figure 5 shows an example of superimposed chromatograms of hemoglobin D-containing samples, hemoglobin S-containing samples, hemoglobin C-containing samples, and a typical whole blood sample.
[0077] [Table 1] [Explanation of symbols]
[0078] 101 Eluent Solution 1 102 Eluent Solution 2 103 Eluent Solution No. 3 104 Plunger pump 105 Degassing device 106 Solenoid valve (for eluent solution 1) 107 Solenoid valve (for eluent solution #2) 108 Solenoid valve (for eluent solution #3) 109 Assembly area 111 columns 112 detectors 120 Sample injection section 121 Control Unit 201 Motor 202 Cam 203 Camshaft 204 Slit disk 205 Sensor 206 Plunger 207 Discharge side check valve 208 Suction-side check valve
Claims
1. A liquid chromatograph apparatus in which the object to be measured is hemoglobin in the blood, An analytical column that provides a difference in elution time for components in the sample, A detector for detecting the components eluted from the aforementioned analytical column, A sample injection unit for injecting the sample into the analytical column, A suction channel connected to each of the two or more eluents, A valve means for selecting the open / closed state of the intake passage, the valve means capable of switching one intake passage to an open state and the remaining intake passage to a closed state, A liquid transfer pump that sequentially draws in the eluent from an open intake channel and discharges it to the analysis column, and the liquid transfer pump is equipped with a single plunger that reciprocates at a predetermined period, A phase sensor for detecting a predetermined operating phase of the plunger, A step gradient program consisting of step intervals arranged in chronological order, where the mixing ratio of the eluent is specified for each time interval, A valve control table comprising n parameters (n is a natural number) arranged in execution order, corresponding to each of the aforementioned step intervals, which represent which suction channel connected to which eluent is opened, wherein the ratio of the number of parameters among the n parameters that open a specific suction channel connected to a particular eluent corresponds to the mixing ratio, A control unit performs a control operation that, at one of two consecutive timings spanning before and after the end time of each step section in the step gradient program, when the phase sensor detects a predetermined phase of the plunger operation, reads the first parameter of the valve control table corresponding to the subsequent step section and outputs an operating signal corresponding to that parameter to the valve means, and at the timing when the phase sensor next detects a predetermined phase, reads the second parameter and outputs an operating signal corresponding to that parameter to the valve means, and repeats the control operation in a manner that similarly returns to the first parameter after passing through the nth parameter, A liquid chromatograph apparatus equipped with, Liquid chromatograph apparatus, wherein the step gradient program is provided with processing information for each step section, indicating whether the control unit repeats a series of control operations starting with reading the first parameter of the valve control table corresponding to the subsequent step section at the timing when the phase sensor detects the predetermined phase after the end time of the step section has elapsed, or whether the control unit repeats the series of control operations at the timing when the phase sensor detects the predetermined phase after a time equal to the operation cycle of the plunger has elapsed before the end time of the step section.
2. The liquid chromatograph apparatus according to claim 1, wherein the control unit repeats a series of control operations, starting with reading the first parameter of the valve control table corresponding to the subsequent step section at the timing when the phase sensor detects the predetermined phase after the termination time of each step section has elapsed.
3. The liquid chromatograph apparatus according to claim 1, wherein the control unit repeats a series of control operations, starting with reading the first parameter of the valve control table corresponding to the subsequent step section at the timing when the phase sensor detects the predetermined phase after a time equal to the operation cycle of the plunger has elapsed before the end time of each step section.
4. The liquid chromatograph apparatus according to any one of claims 1 to 3, wherein the control unit synchronizes the operation of the sample injection unit with the timing at which the phase sensor detects the predetermined phase.
5. The liquid chromatograph apparatus according to claim 4, wherein the step gradient program corresponds to one sample measurement, and the control unit outputs an operating signal to the sample injection unit at the timing when the phase sensor detects the predetermined phase after a time equal to the operation cycle of the plunger has elapsed before the end time of the final step section included in the step gradient program, and at the same time starts the next measurement from the first step section of the step gradient program.
6. The liquid chromatograph apparatus according to any one of claims 1 to 5, wherein the valve control table divides the array of parameters into a group consisting of the first to fifth parameters and a group consisting of the sixth to tenth parameters, with n=10, and further divides one or both of these groups into two to form subgroups, and parameters corresponding to different eluents are assigned to the subgroups.
7. The liquid chromatograph apparatus according to any one of claims 1 to 6, wherein the parameter is a unit vector having dimensions corresponding to each intake channel.
8. A liquid chromatograph apparatus according to any one of claims 1 to 7, for eluting the HbA1c peak.
9. A liquid chromatograph apparatus according to any one of claims 1 to 7, which elutes at least one peak from hemoglobin D, hemoglobin S, and hemoglobin C.