JPH11304782A - Method for separating sample by liquid chromatograph - Google Patents

Abstract

PROBLEM TO BE SOLVED: To make a peak of each component sufficiently sharp and measure in a short time without adversely influencing separation, by feeding an eluate by a gradient elution method or step elution method, and lowering an elution power of the eluate in the halfway. SOLUTION: This method is useful for separating and determining a saccharified hemoglobin or the like requiring a short-time analysis. A column filled with a cation exchange filler is used for the separation and determination. An eluate is fed in a range of a salt concentration of 20-1000 mM, pH4-9 by a gradient elution method or step elution method. The salt concentration of the eluate is lowered to a range of 5-100 mM, pH 0.1-1 in the halfway, thereby decreasing an elution force of the eluate to separate a sample. Eluates A, B, C, D have different elution forces and can be adapted to be switched at a set time by a solenoid valve 1. The eluate is introduced by a feed pump 2 together with the sample introduced from a sample injection part 3 to a column 4, where each component is detected by a detector 5. Each peak is calculated by an integrator 6.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for separating a sample by liquid chromatography.

[0002]

2. Description of the Related Art When a sample is separated by using a liquid chromatograph, besides a method of using one having a constant solution power as an eluent, the peak of each component is sharpened to improve the degree of separation. For the purpose of shortening the measurement time and the like, the eluent is generally sent by a gradient elution method or a step elution method.

[0003] The gradient elution method is also referred to as a gradient elution method, and is a method of linearly increasing the dissolving power of an eluent from weak to strong in time (see FIG. 1).
This is a method in which a plurality of liquid sending pumps are used to send liquids having different melt outputs and the ratio is continuously changed to change the overall melt output.

[0004] The stepwise elution method is also called a stepwise elution method, and is a method of increasing the dissolving power of an eluent in a stepwise manner (see FIG. 2). Switch the liquids with different melt output by solenoid valve, etc.
This is a method of changing the melting power stepwise.

In these methods, to change the dissolution power of the eluent, for example, the polarity, ionic strength,
pH and the like. (The above conventional techniques are described in the Handbook of High Performance Liquid Chromatography, edited by the Japan Society for Analytical Chemistry, Kanto Chapter, pp. 39-40, 117-119
1985).

[0006] However, when the properties of the components are similar or when elution is required in a short time, if the gradient is too large in the gradient elution method or the dissolution output is too high in the step elution method, , Peaks may overlap between components having similar properties, which may adversely affect the separation. Therefore, in such a case, the slope is loosened or completely eliminated at the point where the peaks overlap, or the degree of increase in the melt output is reduced. However, such measures can reduce the sharpness of the peak and increase the measurement time.
In some cases, the advantages of the gradient elution method or the step elution method were not exhibited.

[0007]

DISCLOSURE OF THE INVENTION The present invention has been made in view of such problems, and an object of the present invention is to provide a gradient elution method or a step elution method for separating a sample by liquid chromatography. Is to provide a separation method in which each component peak is sufficiently sharpened and the measurement can be performed in a short time without adversely affecting the separation.

[0008]

SUMMARY OF THE INVENTION The present invention provides a method for separating a sample by liquid chromatography, in which an eluent is sent by a gradient elution method or a step elution method, and the dissolution output of the eluent is reduced during the separation. This is a characteristic method of separating a sample by liquid chromatography.

Hereinafter, the present invention will be described in detail. In the present invention, an eluent with a low elution power refers to an eluent that delays the retention time of a peak, and an eluent with a high elution power refers to an eluent that shortens the retention time of a peak. To tell. In order to lower the dissolution power of the eluent, for example, in cation exchange chromatography, a method of lowering the salt concentration of the eluent, a method of lowering the pH, and the like can be mentioned. In reverse phase chromatography, a method of increasing the polarity of the organic solvent of the eluent is used, and in normal phase chromatography, a method of reducing the polarity of the organic solvent of the eluent is used.

As a filler for cation exchange liquid chromatography, a material obtained by introducing a carboxyl group or a sulfonic acid group or the like as a functional group into a silica or organic polymer base material can be used. As a filler for anion exchange liquid chromatography, a filler obtained by introducing a tertiary amino group, a quaternary ammonium group, or the like as a functional group into a silica-based or organic polymer-based substrate can be used.
As a buffer for ion exchange liquid chromatography,
Phosphoric acid, citric acid, phthalic acid, boric acid, carbonic acid, etc., and 2- (N-morpholino) ethanesulfonic acid (ME
S), N-2-hydroxyethylpiperazine-N'-2
Good's buffers such as ethanesulfonic acid (HEPES), bis (2-hydroxyethyl) iminotris- (hydroxymethyl) methane (Bis-Tris) can also be used.

As a packing material for reverse phase chromatography, a filler obtained by introducing an octadecyl group, an octyl group, a phenyl group, or the like as a functional group into a silica-based or organic polymer-based substrate can be used. As a solvent of the eluent, any of the solvents generally used for reverse phase chromatography, such as water, acetonitrile, methanol, ethanol, and the like can be used. As a packing material for normal phase chromatography,
Silica gel, alumina and the like can be used. As eluent solvent, benzene, toluene, xylene, hexane, etc.
Any of the solvents generally used for normal phase chromatography can be used.

Although the field of application of the method of the present invention is not particularly limited, it can be used for analysis in the medical field which requires analysis in a short time (for example, separation and quantification of glycated hemoglobin or the like which is used as an index for diagnosing diabetes). Particularly useful.

The case where the method of the present invention is used for separation and quantification of glycated hemoglobin will be described below. For separation and quantification of glycated hemoglobin, a column packed with a cation exchange packing material was used, and the eluent was used at a salt concentration of 20 to 1000 mM, pH4.
The solution is fed by the gradient elution method or the step elution method in the range of ~ 9, and the salt concentration of the eluate is 5-100m
The separation is performed by lowering the dissolution power of the eluent by lowering the M and pH in the range of 0.1 to 1.

FIG. 3 shows an example of the structure of the apparatus when the method of the present invention is carried out by the step elution method. Eluents A, B,
C and D have different melting powers (for example, salt concentration, p
H, polarity, etc.), and is configured to be switched to each eluent at a set time by the electromagnetic valve 1. The eluent is guided to the column 4 together with the sample introduced from the sample injection unit 3 by the liquid sending pump 2, and each component is detected by the detector 5. The area and height of each peak are calculated by the integrator 6.

[0015]

Embodiments of the present invention will be described below. Example 1 Separation and quantification of hemoglobins in a blood sample was performed using the apparatus shown in FIG. A column filled with a cation exchange resin (Micronex A1c HS-IV, manufactured by Sekisui Chemical Co., Ltd.) was used. 170m as eluent A
M, eluent B: 190 mM, eluent C: 150 mM
mM and eluent D were each 330 mM, each of which was a phosphate buffer at pH 6. As a sample, blood collected from a healthy person was hemolyzed and diluted 100-fold using a surfactant (phosphate buffer containing 0.01% by weight of Triton X-100, pH 7). The eluent was switched using the stepwise elution method so that the hemoglobin A1c peak (peak P5 described later) could be separated and quantified from the peak of each hemoglobin in 2 minutes. FIG. 4 shows the eluent switching conditions and the obtained chromatogram. That is, the eluent is eluent A for 0 to 38 seconds, eluent B having a higher elution power for 38 to 58 seconds, eluent C having a lower elution power for 58 to 78 seconds, and eluent C for a lower elution power for 78 to 100 seconds. The eluent D having the highest dissolution output was sent again for 100 to 120 seconds. 415 nm absorption intensity was used for peak detection.

In the chromatogram of FIG.
1 to P3 are hemoglobin A1a (HbA1a) and hemoglobin A1b (HbA1b), and peak P4
Is hemoglobin F (HbF), peak P5 is hemoglobin A1c (HbA1c), and peak P6 is hemoglobin A0 (HbA0).

In the above-mentioned method, the peak P6 is eluted earlier due to an increase in the dissolution output by switching from the eluent A to the eluent B, which hinders accurate detection of the peak P5 which is the HbA1c peak. In order to prevent this, the eluent B is switched to the eluent C having a lower dissolution output.

In order to check the reproducibility, the same sample was repeatedly measured 10 times by this method.
The concentration was calculated. Table 1 shows ten measured values, their average values, and their coefficient of variation (unit%) (standard deviation ％ average value × 100). HbA1c concentration (%) = area of peak P5 / (peak P
(1 area + peak P2 area + peak P3 area + peak P4 area + peak P5 area + peak P6 area)
× 100

Comparative Example 1 Separation and quantification of hemoglobins in the same sample as in Example 1 was performed in the same manner as in Example 1 except that the eluent C in Example 1 was not used. FIG. 5 shows the eluent switching conditions and the obtained chromatogram. That is, the eluent is eluent A for 0 to 38 seconds, eluent B having a higher elution power for 38 to 78 seconds, eluent D having the highest elution power for 78 to 100 seconds, and eluent D having the highest elution power for 100 to 120 seconds. The eluent A was sent again. The effect of raising the dissolution output by switching from the eluent A to the eluent B had an effect, and as indicated by a circle in FIG. 5, a part of the peak P6 eluted earlier and overlapped with the peak P5. I understand. In order to check the reproducibility, the same sample was repeatedly measured 10 times by this method, and the HbA1c concentration was calculated in the same manner as in Example 1. Table 1 shows the ten measured values, the average value and the coefficient of variation.

Comparative Example 2 The procedure of Example 1 was repeated, except that the eluent C in Example 1 was not used, and the eluent B1 (180 mM, pH 6 phosphate buffer) was used instead of the eluent B. Thus, the hemoglobins in the same sample as in Example 1 were separated and quantified. The conditions for switching the eluent and the resulting chromatogram are shown in FIG. That is, the eluent is eluent A for 0 to 38 seconds, eluent B1 for 38 to 78 seconds, and 78 to 100 seconds.
In the second, the eluent D having the highest solution output was sent, and in the 100 to 120 seconds, the eluent A was sent again. Compared to Comparative Example 1, since the eluent A was switched to the eluent B1 having a lower dissolution output than the eluent B, a part of the peak P6 did not elute early, but the peak P5 did not become sharp, As can be seen from FIG. 6, the peak P4 partially overlaps the peak P4 and the peak P6 as circled in FIG. In order to check reproducibility, the same sample was repeatedly measured 10 times by this method.
The HbA1c concentration was calculated in the same manner as described above. Table 1 shows the ten measured values, the average value and the coefficient of variation.

[0021]

[Table 1]

[0022]

The structure of the method for separating a sample by liquid chromatography according to the present invention is as described above. When the method of the present invention is used, the peaks of the respective components are sufficiently sharpened in the gradient elution method or the step elution method. And the separation between component peaks is also good. As a result, performance such as reproducibility can be improved.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram for explaining a gradient elution method.

FIG. 2 is an explanatory diagram for explaining a step elution method.

FIG. 3 is a diagram showing an example of the configuration of an apparatus when performing the step elution method.

FIG. 4 is a diagram showing conditions for switching the eluent of Example 1 and the obtained chromatogram.

FIG. 5 is a diagram showing switching conditions of an eluent of Comparative Example 1 and the obtained chromatogram.

FIG. 6 is a diagram showing switching conditions of the eluent of Comparative Example 2 and the obtained chromatogram.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Solenoid valve 2 Liquid sending pump 3 Sample injection part 4 Column 5 Detector 6 Integrator A, B, B1, C, D Eluent

Claims (1)

[Claims] 1. A sample according to a liquid chromatograph, wherein an eluent is sent by a gradient elution method or a stepwise elution method when a sample is separated by a liquid chromatograph, and the dissolution output of the eluate is reduced in the middle of the eluent. Separation method.