JP2009222398A - Liquid chromatograph apparatus and liquid chromatography - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a liquid chromatograph apparatus for separating and quantifying a measuring target component in a column for liquid chromatography with high precision even in a case that specimens of various concentrations are prepared. <P>SOLUTION: In the liquid chromatograph apparatus 1, a concentration measuring instrument 17, to which a specimen is guided, is provided on the upstream side of a separation column 16 and, in a case that the concentration of the supplied specimen in the concentration measuring instrument 17 is out of a predetermined concentration range suitable for separation in the separation column 16, the liquid insertion amount of at least one of the specimen 5 and a diluting liquid 11 is changed to adjust diluting magnification and the specimen of a predetermined concentration range is certainly supplied to the separation column 16. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a liquid chromatograph apparatus and liquid chromatography for separating a measurement target component such as hemoglobin, for example, and more specifically, a liquid provided with means for adjusting a sample concentration upstream of a separation column. The present invention relates to a chromatographic apparatus and a liquid chromatography using a liquid chromatographic apparatus.

  Conventionally, various components to be measured have been separated and measured by liquid chromatography using a liquid chromatograph apparatus. For example, when measuring hemoglobin in blood, separation and measurement of hemoglobin are performed by the following procedure. First, prepare whole blood. A sample is obtained by adding a diluent to the prepared whole blood. By adding a diluent, the analyte concentration is adjusted and hemolysis is performed. In this manner, a specimen adjusted to an appropriate concentration and hemolyzed can be obtained. Separation of hemoglobins is performed by feeding this specimen to a separation column of a liquid chromatograph apparatus. Thereafter, the separated hemoglobin is measured.

Such an analysis method by liquid chromatography is disclosed, for example, in Patent Document 1 below.
JP 2000-171454 A

  For example, when measuring hemoglobin by liquid chromatography, the measurement accuracy tends to decrease depending on the concentration of the specimen. That is, there is a limit to the performance of the separation column provided in the liquid chromatograph device, and there is a problem that the measurement accuracy is lowered when a sample having a very high concentration is fed. Therefore, for example, when trying to quantify the measurement target component using the absolute calibration curve method or the internal standard method, the measurement target component may not be accurately quantified.

  The object of the present invention is to solve the above-mentioned drawbacks of the prior art and to separate samples in a concentration range according to the performance of the separation column used even when samples of various concentrations are prepared. And providing a liquid chromatography using the liquid chromatograph and the liquid chromatograph capable of analyzing the component to be measured with high accuracy by liquid chromatography.

  A liquid chromatograph apparatus according to the present invention is a liquid chromatograph apparatus for analyzing a measurement target component in a sample by liquid chromatography, the sample supply means for supplying a sample containing the measurement target component, and the sample A first flow path through which a specimen is supplied from a supply means; a diluent supply means connected to the first flow path for supplying a diluent to the first flow path; and the first flow And the separation column connected to the downstream side of the sample supply unit and the diluent supply unit, the sample supply unit, the diluent supply unit, and the separation column. A second flow path branched from the flow path, flow path switching means for switching between the first flow path and the second flow path, and the second flow path, Concentration measuring hand for measuring the concentration of the specimen And when the sample concentration measured by the concentration measuring means is outside a predetermined concentration range, the flow path switching means is switched so that the sample flows through the second flow path, and the sample supply means and / or When the diluent supply means is driven to bring the analyte concentration within a predetermined concentration range, and the analyte concentration measured by the concentration measuring means is within a predetermined concentration range, the flow path switching means is And a control means for switching to the first flow path and flowing the sample through the separation column to separate the measurement target component.

  As the concentration measuring means, an optical concentration measuring device is preferably used. As the optical concentration measuring apparatus, an apparatus for measuring absorbance or an apparatus for measuring fluorescence can be used. In either case, the analyte concentration can be measured in a non-contact manner. Therefore, the structure of the concentration measuring means can be simplified.

  Further, as the concentration measuring means, a means for measuring the concentration of the specimen by measuring electric resistance or electric impedance is preferably used. In this case, since it is only necessary to insert an electrode into the second flow path, the analyte concentration can be measured with a simple structure.

  In the present invention, when adjusting the sample concentration within the predetermined concentration range, the sample concentration may be adjusted by driving the diluent supply means to change the amount of the diluent, or the sample supply means is driven. The sample concentration may be adjusted by changing the sample supply amount. Moreover, these two types of means may be used in combination.

  In the liquid chromatograph apparatus of the present invention, various components to be measured are analyzed. However, when the component to be measured is hemoglobin, it is preferable because hemoglobin in blood can be separated and measured with high accuracy. .

  The liquid chromatography according to the present invention is performed using a liquid chromatograph apparatus including a separation column, and the step of measuring the concentration of a sample containing a measurement target component and the concentration of the sample are for separation A step of adjusting the sample to be within the predetermined concentration range when the concentration is outside the predetermined concentration range according to the performance of the column; and the concentration of the sample is within the predetermined concentration range In this case, the method further comprises a step of separating the measurement target component in the sample by the separation column.

  By using the liquid chromatograph apparatus according to the present invention, when the concentration of the sample from the sample supply means is outside the predetermined concentration range suitable for measurement in the separation column, the sample supply is performed according to the liquid chromatography of the present invention. The supply amount of the specimen and / or diluent from the means and / or the diluent supply means is changed, and the specimen is supplied to the separation column in a state where the specimen concentration is within a predetermined concentration range. Therefore, regardless of the concentration of the sample prepared first, the measurement target component in the sample can be separated and measured with high accuracy in the separation column.

  Hereinafter, the present invention will be clarified by describing specific embodiments of the present invention with reference to the drawings.

  FIG. 1 is a schematic block diagram of a liquid chromatograph apparatus according to an embodiment of the present invention.

  The liquid chromatograph apparatus 1 includes a sample suction nozzle 3 and a pump 4 as the sample supply means 2. The sample suction nozzle 3 is inserted into a sample container 13 in which the sample 5 is stored. By driving the pump 4, the sample 5 is sucked from the sample suction nozzle 3 and supplied to the first flow path 6. A pump 8 and a nozzle 9 are connected to the first flow path 6 via a valve 7. The pump 8 and the nozzle 9 constitute a diluent supply means 10. The tip of the nozzle 9 is inserted into the diluent 11. The diluent 11 is stored in the container 12.

  The valve 7 is a three-way valve, and is configured to pass the specimen 5 and the diluent 11 downstream of the first flow path 6. Further, by changing the liquid feeding speed of the pump 4 and the pump 8, the ratio of the sample 5 and the diluent 11 supplied to the first flow path 6 is changed. That is, it is possible to change the concentration of the specimen 5 supplied to the first flow path 6.

  In the present embodiment, the sample supply unit 2 is positioned upstream of the diluent supply unit 10, but conversely, the diluent supply unit 10 may be arranged upstream of the sample supply unit 2.

  In the first flow path 6, a flow path switching valve 14 is connected downstream of the sample supply means 2 and the diluent supply means 10. In the flow path switching valve 14, the second flow path 15 is branched from the first flow path 6. That is, by switching the flow path switching valve 14, the liquid sent from the upstream side can be flowed to the downstream portion of the first flow path 6 or the second flow path 15.

  In the first flow path 6, a separation column 16 is connected downstream of the flow path switching valve 14. In the separation column 16, the component to be measured is separated by liquid chromatography when the specimen is fed. The liquid that has passed through the separation column 16 is discarded after the measurement is performed.

  On the other hand, a concentration measuring device 17 as a concentration measuring means is connected to the second flow path 15. The concentration measuring device 17 is composed of an appropriate analyzer that measures the concentration of the supplied sample. In the present embodiment, as shown in FIG. 2, the concentration measuring device 17 is composed of an absorbance measuring device having a light source 17a and a light receiving cell 17b. A measurement cell 15 a for measuring the light irradiated from the light source 17 a is configured in a part of the second flow path 15.

  Instead of the absorbance measurement device, a device that measures fluorescence from the specimen may be used. In the case of using an optical concentration measurement device such as an absorbance measurement device or a measurement device using fluorescence, since the measurement can be performed in a non-contact manner on the specimen, the structure of the channel can be simplified.

  However, as the concentration measuring apparatus, an apparatus that measures the analyte concentration by measuring the electrical resistance or impedance of the analyte may be used. In that case, it is necessary to arrange a plurality of electrodes in the specimen. However, since the electrical resistance and impedance can be easily measured, the measuring device can be miniaturized.

  In the liquid chromatograph apparatus 1, in order to control the pump 4, the pump 8, the flow path switching valve 14, the separation column 16, and the concentration measuring device 17, a control device 18 connected thereto is provided.

  Each of these devices is driven by the control device 18, and the component to be measured can be measured with high accuracy from specimens of various concentrations by liquid chromatography according to the present invention. This will be described more specifically with reference to the flowchart of FIG.

  In this embodiment, a blood sample is used as the sample, and the hemoglobin ratio HbA1c (%) in the blood sample is measured. In this embodiment, a sample obtained by adding a good buffer to whole blood, diluting whole blood 200 times, and hemolyzing was prepared as the first sample.

  Similarly, a good buffer solution was prepared as the diluent 11.

  First, in step S <b> 1 of FIG. 3, the control device 18 drives the pump 4 of the sample supply means, sucks the sample from the sample suction nozzle 3, and sends the liquid to the first flow path 6. In the initial state, the flow path switching valve 14 is set to flow the supplied liquid to the second flow path 15. Accordingly, the sample 5 that has been sent is guided to the second flow path 15 via the flow path switching valve 14. In step S2, the concentration measuring device 17 is driven by a command from the control device 18, and the concentration of the sample is measured. The obtained specimen concentration is given to the control device 18.

  The control device 18 stores in advance a sample concentration range suitable for the separation column 16 used as a predetermined concentration range. The predetermined concentration range is previously input by the user into the control device 18 according to the separation column 16 to be used and stored.

  In step S <b> 3, the control device 18 determines whether or not the concentration of the sample given from the concentration measuring device 17 is outside the predetermined concentration range.

  If the measured sample concentration is outside the predetermined concentration range, a dilution factor for diluting the sample concentration measured by the control device 18 to be within the predetermined concentration range is calculated in step S4. Next, in step S5, the control device 18 changes the liquid feeding speed of the pump 4 and / or the pump 8 so as to realize the above dilution ratio, and the injection amount of the dilution liquid 11 and / or the sampling amount of the specimen 5 are changed. Change. In this way, the specimen within the predetermined concentration range is supplied to the first flow path 6. Then, the process returns to step S2, and the analyte concentration is again measured by the concentration measuring device 17. If the measured analyte concentration is outside the predetermined concentration range, the process returns to step S2 again through steps S4 and S5.

  On the other hand, if the measured analyte concentration is within the predetermined concentration range, the flow path switching valve 14 is switched based on a command from the control device 18 in step S6. As a result, the sample sent from the upstream side of the flow path switching valve 14 is guided not to the second flow path 15 but to the downstream side of the first flow path 6, that is, to the separation column 16. In step S7, separation / measurement by the separation column 16 is performed.

  Therefore, in the measurement method of the present embodiment, the sample is automatically diluted so as to be within the concentration range of the sample suitable for the separation column 16, that is, within the predetermined concentration range. Accordingly, in the separation column 16, a sample having a concentration suitable for the separation column is reliably supplied, and measurement by liquid chromatography is performed. Therefore, even when various specimens are prepared, the component to be measured can be separated and quantified with high accuracy in the separation column 16.

  Next, specific experimental examples will be described.

  FIG. 4 is a diagram showing an example of a chromatogram showing hemoglobins separated from a specimen when the liquid chromatograph apparatus 1 is used.

  As is well known, in liquid chromatography, each component is adsorbed in a separation column, but depending on the elution power, a separation solution having a different elution power and flowing through the separation column is different for each component. Each component adsorbed on the separation column is eluted. Since the elution order varies depending on the components, the chromatogram shown in FIG. 4 is obtained. And each component can be quantified according to the peak area of the chromatogram of FIG.

  First, for comparison, the ratio of hemoglobin A1c in the sample A and the sample B was measured using a normal liquid chromatograph apparatus. Sample A and sample B are whole blood collected from different healthy individuals. The separation column used was a fructogel SO3- (M) strong cation exchanger (40-90 μm) and a packed stainless steel column (φ30 × 100 mm) manufactured by Merck. As shown in Table 1 below, multiple types of specimen solutions were prepared by diluting specimen A and specimen B 10 times, 20 times, 30 times, 50 times, 75 times, 100 times, 150 times, and 200 times, respectively. The ratio (%) of hemoglobin A1c (HbA1c) was measured using the sample solutions at various dilution ratios. The results are shown in Table 1 and FIG.

  As is clear from Table 1 and FIG. 5, in Sample A, if the dilution rate is 30 times or more, the range is 4.6 to 4.62%, whereas if the dilution rate is 20 times, 4%. It can be seen that when the dilution ratio is 10 times, it is as high as 5.21% at 0.85%. In specimen B, the dilution ratio is approximately constant at 5.21% or 5.22% when the dilution ratio is 75 times or more, but when the dilution ratio is 50 times or less, the ratio of HbA1c is 5.52%, 5.67%, It turns out that it is quite high with 5.94% and 6.4%.

  Therefore, in both cases of specimen A and specimen B, when diluted at a relatively high magnification, the ratio of hemoglobin A1c is obtained with high accuracy, but when the dilution magnification is low, that is, when the specimen concentration is high. It can be seen that the ratio (%) of hemoglobin A1c is not obtained correctly and is shifted to a higher value side.

  Therefore, it can be seen that when the separation column using the Merck gel is used, it is desirable to set the dilution ratio of the specimen to 75 times or more. In that case, it is understood that the ratio of hemoglobin A1c can be measured with high accuracy in both the specimen A and the specimen B.

  Therefore, in the liquid chromatograph apparatus 1 of the above embodiment, when the separation column using a Merck gel is used as the separation column 16, the concentration at which the dilution factor is 75 times or more as the predetermined concentration range of the specimen. It can be seen that if the range is set, the ratio (%) of hemoglobin A1c can be obtained with high accuracy.

  The predetermined concentration range may be appropriately set according to the performance of the separation column. In consideration of the type of specimen to be measured, it is preferable to set a dilution factor that is measured with high accuracy in the separation column, that is, a predetermined concentration range.

  In the above embodiment, the ratio of hemoglobin A1c in the blood sample was determined, but the present invention can be used for measurement of various components to be measured using liquid chromatography.

1 is a schematic block diagram of a liquid chromatograph apparatus according to an embodiment of the present invention. It is a top view which shows an example of the density | concentration measuring apparatus in the liquid chromatograph apparatus of embodiment of FIG. It is a flowchart for demonstrating the measuring method by the liquid chromatography which concerns on one Embodiment of this invention. In one Embodiment of this invention, it is a figure which shows the chromatogram for demonstrating each hemoglobin isolate | separated and measured from the blood sample. It is a figure which shows the result of having calculated | required hemoglobin A1c (%) by diluting the sample A and the sample B with various dilution magnifications in the conventional liquid chromatograph apparatus prepared for the comparison.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Liquid chromatograph apparatus 2 ... Specimen supply means 3 ... Specimen suction nozzle 4 ... Pump 5 ... Specimen 6 ... 1st flow path 7 ... Valve 8 ... Pump 9 ... Nozzle 10 ... Diluent supply means 11 ... Diluent 12 ... Container 13 ... Sample container 14 ... Flow path switching valve 15 ... Second flow path 15a ... Measurement cell 16 ... Separation column 17 ... Concentration measuring device 17a ... Light source 17b ... Light receiving cell 18 ... Control device 19 ... Supply device 21 ... Elution Liquid supply device

Claims (7)

A liquid chromatograph for analyzing components to be measured in a specimen by liquid chromatography,
A sample supply means for supplying a sample containing a component to be measured;
A first channel through which a sample is supplied from the sample supply means;
A diluent supply means connected to the first channel for supplying the diluent to the first channel;
A separation column connected to a downstream side of the sample supply means and the diluent supply means in the first flow path;
A flow path of a second flow path branched from the first flow path between the sample supply means and the diluent supply means and the separation column;
Channel switching means for switching between the first channel and the second channel;
A concentration measuring means connected to the second flow path for measuring the concentration of the specimen;
When the sample concentration measured by the concentration measuring means is outside a predetermined concentration range, the flow path switching means is switched so that the sample flows through the second flow path, and the sample supply means and / or the dilution is performed. When the liquid supply means is driven to bring the sample concentration within a predetermined concentration range, and the sample concentration measured by the concentration measuring means is within a predetermined concentration range, the flow path switching means is set to the first concentration range. And a control means for separating the measurement target component by flowing the sample through the separation column.   The liquid chromatograph according to claim 1, wherein the concentration measuring means is an optical concentration measuring device.   The liquid chromatograph apparatus according to claim 1, wherein the concentration measuring unit is an apparatus that measures the concentration of a specimen by measuring electric resistance or impedance.   The liquid according to any one of claims 1 to 3, wherein when the analyte concentration is outside a predetermined concentration range, the diluent supply means is driven and the analyte concentration is adjusted by changing the amount of the diluent. Chromatographic device.   The liquid chromatography according to any one of claims 1 to 3, wherein when the sample concentration is outside a predetermined concentration range, the sample supply means is driven and the sample concentration is adjusted by changing the sample supply amount. Graph device.   The liquid chromatograph apparatus according to any one of claims 1 to 5, wherein the measurement target component is hemoglobin. A liquid chromatography that separates a measurement target component in a sample by a liquid chromatograph device including a separation column,
Measuring the concentration of the sample containing the measurement target component;
Adjusting the sample to be within the predetermined concentration range when the concentration of the sample is outside the predetermined concentration range; and
And a step of separating a measurement target component in the sample by the separation column when the concentration of the sample is within the predetermined concentration range.

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