JPH1123555A - Differential refraction factor detector for liquid chromatograph - Google Patents

Abstract

PROBLEM TO BE SOLVED: To make stable measurement with no loss of a sample by inserting a stirring chamber with the capacity specific times the capacity of a sample cell between the sample cell and a reference cell, diluting the eluate from the sample cell, and utilizing it as a reference solution. SOLUTION: A sample injected from a sample injection port 6 is separated into components by a separation column 8, and the components are guided into the sample cell 10 of a differential refraction factor detector for detection. The components sent out from the sample cell 10 are branched at the ratio of about 99:1, for example, at a passage branch section 12, and the component concentration in one passage is set to about 1/100. This diluted component is further diluted and uniformalized by a stirring chamber 1 9 with the capacity about 10 times the capacity of the sample cell 10 and is sent to a reference cell 21. The component concentration in the reference cell 21 is largely diluted, and the component can be used as a reference solvent with no problem. When the sample cell 10 and the reference cell 21 are connected in series, the same stability can be obtained as the stability available when two liquid feed pumps 4 are used for a liquid feed, and a low-cost device structure can be obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a differential refractive index detector for a liquid chromatograph, and more particularly to a liquid chromatograph for detecting a component by sending a solvent to a sample cell and a reference cell by a single liquid feed pump. The present invention relates to a differential refractive index detector for use.

[0002]

2. Description of the Related Art In a differential refractive index detector for a liquid chromatograph having a sample cell and a reference cell, a stable baseline can be obtained by flowing the same solvent through the sample cell and the reference cell, and stable measurement can be performed. it can. However, in order for the same solvent to flow through the sample cell and the reference cell, a liquid pump is required for each, which not only makes the chromatographic apparatus expensive, but also reduces the number of movable parts such as check valves that frequently cause trouble. Requires more frequent maintenance.

For this reason, the solvent sent by one liquid sending pump is branched before reaching the sample cell, and the pipe in front of the reference cell is filled with the solvent, and the branched solvent (to be measured) (Including some of the components) to the piping to flush the filled solvent into a reference cell.

[0004]

However, since the ratio of branching depends on the resistance of the branch pipe, if the solvent viscosity changes, the diversion ratio will change. In particular, in size exclusion chromatography, since the viscosity of the polymer substance to be measured varies depending on the degree of polymerization, the flow separation ratio depends only on the solvent alone, the solvent containing the component, and the degree of polymerization of the various components contained. Changes subtly. In the liquid chromatography method, components are identified by the elution time of the chromatogram peak, and each component is quantified based on the area and height of the peak. give.

For example, in size exclusion chromatography, when the flow rate (elution volume) from a column changes by 1%,
There is also a report that an error of 20% or more occurs in the measurement results (average molecular weights of various components identified as described above) (DDBly, HJ Stoklosa, JJkirkland and WWYau, An
al. Chem. 47 (11), 1810 (1975)).

[0006] Furthermore, since the solvent branches, a small amount of a solvent containing a part of the components flows to the reference cell side, so that all of the samples used for the measurement are not used for detection.

[0007] In order to solve this problem, the solvent sent by one liquid sending pump is branched after the sample cell, a pipe in front of the reference cell is filled with the solvent, and after passing through the sample cell. It is conceivable that the solvent is branched and the solvent in the pipe is flushed.However, components that are not sufficiently stirred and not diluted will be sent to the reference cell in accordance with the pipe capacity and branch ratio from the branch flow path to the reference cell. If the measurement is performed for a long time, a drift or a negative peak occurs in the baseline of the chromatogram.

[0008] Therefore, in the above two methods, it is necessary to replace the solvent in the flow path on the reference cell side after the branch pipe at regular time intervals. When measuring, time for stabilization was required in addition to the measurement.

In order to solve this problem, it has been attempted to simply connect the sample cell and the reference cell in series. However, a back pressure is applied to the sample cell by the reference cell, the connection pipe, and the outlet pipe. It is difficult to stabilize the baseline. In addition, since the detection signals are inverted between the sample cell and the reference cell, there are two conflicting peaks, a normal peak detected by the sample cell and a negative peak detected by the reference cell delayed by the piping capacity connecting both cells. Since the peak is obtained, the analysis of the measurement result becomes complicated. For this reason, in the case where the time required for elution is longer than the time required for the solvent to pass through the sample cell volume, the connection piping volume of both cells, and the reference cell volume, it cannot be detected as a peak shape suitable for measurement. .

The present invention has been made in view of the problems of the prior art,
A liquid chromatography differential refractive index detector that enables the solvent to be sent to the sample cell and reference cell with a single liquid sending pump, detects the injected sample without loss, and enables more stable continuous measurement. To provide.

[0011]

Means for Solving the Problems The present inventor has conducted studies to solve the conventional problems as described above, and as a result, has completed the present invention. That is, the present invention provides a liquid chromatograph in which a stirring chamber having a volume of at least 10 times the sample cell volume is inserted between the sample cell and the reference cell in order to dilute the eluate from the sample cell and use it as a reference solution. Differential refractive index detector.

In the differential refractive index detector according to the present invention, since one of the branch flow paths provided after the sample cell is connected to the waste liquid tank, the pressure in the sample cell is controlled by the back pressure by the connection pipe and the waste liquid tank. It is determined by the atmospheric pressure inside and is kept almost constant. Further, the other of the branch channels also has a diluting effect for reducing sample components flowing to the reference cell side. For example, if a 99: 1 branch is performed between the waste liquid tank side and the reference cell side, only the concentration of 1/100 of the component detected in the sample cell flows to the reference cell side.

The components in the solvent branched to the reference cell side in the branch channel are sent to a stirring chamber having an appropriate volume,
It is further diluted and homogenized in the stirring chamber. Since it is preferable that the stirring chamber can stir instantaneously and uniformly, a dynamic mixer that dynamically stirs (dilutes) is particularly preferable.

Here, considering the component concentration in the solvent sent from the stirring chamber to the reference cell, when the component concentration in the chamber is uniformly stirred instantaneously, the component concentration sent to the reference cell and the concentration in the stirring chamber are considered. Are equal, and the concentration change can be calculated by the following equation.

[0015]

(Equation 1)

Here, x (t) is the component concentration in the stirring chamber at time t and the component concentration flowing out of the stirring chamber, and x0 is the initial component concentration in the stirring chamber (accordingly, it becomes 0 at the start of measurement). It is. V0 is the volume of the stirring chamber, v is the amount of the inflowing solvent, a is the component concentration in the inflowing solvent, and "exp" is exponential. For example, the volume of the stirring chamber is set to 1 ml, filled with water, and the organic solvent is flowed at 1 ml / min from the liquid sending pump.
Assuming that the branching ratio between the waste liquid side and the reference cell side is 99: 1, that is, 10 μl of the organic solvent per minute flows into the stirring chamber, the organic solvent concentration in the stirring chamber after 60 minutes is as follows. Can be calculated.

[0017]

(Equation 2)

That is, the solvent (water) in the stirring chamber is replaced by 45.12% (451.2 μl) in 60 minutes.

Under the same conditions, the 1% sample concentration was 50 μm.
l, assuming that the component elutes from the column with a spread of 10 minutes, and after 6 injections every 10 minutes, ie 60
Calculate the component concentration in the stirring chamber after one minute. First,
The component concentration at the time of elution from the column was 1%, 50μ
1 component spreads for 10 minutes, that is, 10 ml.
It can be calculated as 05 mg / ml. And then 1/100
To 0.5 μg / ml. According to the above formula, the replacement rate of the stirring chamber is 45.12%, so it can be calculated as 0.2256 μg / ml. That is, the component concentration in the stirring chamber after 60 minutes is 0.00002225.
Calculated as 6%. This is compared with the refractive index concentration coefficient dn / dc of standard polystyrene frequently used in GPC = 0.195.
When converted to an RI signal from ml / g, 4.4 × 10 ^-8 R
IU. In actual chromatography, there are regions where the components are contained and regions where the components are not contained, so that the influence on the baseline is even smaller than this calculated value.
It can be seen that the influence on the baseline can be suppressed to this extent, so that it can be sufficiently used as a reference solution.

In the above calculation, the split flow ratio was 99: 1, the volume of the stirring chamber was 1 ml, that is, the sample cell (usually 10 μm).
l) was calculated as 100 times, but if the volume of the stirring chamber is 100 μl, that is, 10 times that of the sample cell, the effect on the baseline signal can be calculated as 4.4 × 10 ⁻⁷ RIU. As described above, it is preferable that the volume of the solution in the stirring chamber be at least 10 times the volume of the sample cell, and more preferably at least 100 times.

Since the solvent from the stirring chamber is sent to the reference cell and then to the waste liquid tank, the pressure in the reference cell is almost constant by the back pressure by the connecting pipe to the waste liquid tank and the atmospheric pressure in the waste liquid tank. Will be kept. The back pressure of the sample cell and the reference cell can be made equal by using pipes of the same size (diameter) for connection between the sample cell and the waste liquid tank and between the reference cell and the waste liquid tank.

The degree of dilution and homogenization can be freely controlled by changing the branching ratio in the branch channel and the solution volume in the stirring chamber.

[0023]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail based on an embodiment shown in the drawings.

FIG. 1 is a schematic view of a liquid chromatograph apparatus having a differential refractive index detector for a liquid chromatograph according to the present invention. The solvent 2 in the solvent reservoir 1 is sucked into the liquid sending pump 4 via the pipe 3, and supplied to the separation column 8 via the pipe 5, the sample inlet (six-way valve) 6, and the pipe 7.
The eluate from the column 8 enters the sample cell 10 of the differential refractive index detector via the pipe 9, and passes through the pipe 11 to the flow path branching section 12.
Branch into two flow paths. One of the solvents branched in the flow path branching section 12 is discharged into a waste liquid tank 23 via a pipe 13, an electromagnetic valve 14, and a pipe 15. The solvent branched to the other flow path enters the reference cell 21 of the differential refractive index detector via the pipe 16, the needle valve 17, the pipe 18, the stirring chamber 19, and the pipe 20. The electromagnetic valve 14 is open at the time of measurement, and the ratio of branching at the flow path branching portion 12 depends on the degree of throttle of the needle valve 17. The solvent from the reference cell 21 is drained to a waste tank 23 via a pipe 22.

The sample injected from the sample injection port 6 is separated into each component in the separation column 8 and introduced into the sample cell 10 of the differential refractive index detector and detected. The components that have come out of the sample cell 10 are branched at a constant ratio in a flow path branching section 12 via a pipe 11. For example, when branched at a ratio of 99: 1, the component concentration in one of the flow paths is 1 / the value of that detected in the sample cell.
It will be diluted to 100. The diluted components are further diluted and homogenized in the stirring chamber 19 and sent to the reference cell 21 via the pipe 20.

As a result, the concentration of the components flowing through the reference cell 21 is adjusted to a concentration that does not adversely affect the use of the sample concentration as a reference solvent when the sample concentration is normally measured, by dilution in the branch part of the flow path and dilution in the stirring chamber. Can be. When the concentration of the non-target component is higher than the concentration of the target component, a concentration gradient due to the non-target component may naturally occur on the reference cell side, and may be observed as a drift on the chromatogram. , Needle valve 1
By narrowing down 7 and reducing the amount of the solvent diverted from the flow path branching section 12 to the stirring chamber side, it is possible to reduce the drift.

The electromagnetic valve 14 is provided for solvent exchange. When the electromagnetic valve 14 is closed and the needle valve 17 is opened, all the solvent from the liquid feed pump 4 flows into the stirring chamber 19 and the reference cell 21. Therefore, solvent exchange can be easily performed.

FIGS. 2 and 3 show that the THF solvent was 1.0 ml / ml.
2 is a record of the output of the differential refractive index detector when flowing at a flow rate of 1 min. FIG. 2 is a recording record when the sample cell and the reference cell are directly connected, and FIG. 3 is a differential refractive index of the present invention. It is an output record by a detector. 2 and 3 that a stable baseline was obtained by the flow channel of the present invention.

FIGS. 4 and 5 show chromatograms of standard polystyrene measured by incorporating a differential refractive index detector into a liquid chromatograph. FIG. 4 shows a chromatogram when a sample cell and a reference cell are directly connected. And FIG.
Is a chromatogram obtained by the differential refractive index detector of the present invention. FIGS. 4 and 5 show that a chromatogram suitable for measurement cannot be obtained only by directly connecting the sample cell and the reference cell.

In the present invention, when 50 μl of standard polystyrene having a concentration of 0.001% was repeatedly injected, the measurement result was that the reproducibility of the weight average molecular weight was around 0.5% in CV value and 5 × 10 ^{−7 in} baseline drift. About RIU / h was obtained, and measurement stability equivalent to the configuration using two liquid feeding pumps was obtained.

[0031]

According to the present invention, in the sample cell, all of the sample subjected to the measurement passes while maintaining the separation ability separated by the column, but the reference cell does not affect the detection and has the same liquidity. The sample components diluted by the solvent will pass through. It is empirically recognized that, when a liquid having passed through another column or branch is led to the reference cell as in the conventional method, the liquid does not have the same liquid property due to a problem such as a column or branch. Not only this problem can be avoided, but also in a configuration in which the sample cell and the reference cell are connected in series, the same stability as sending the solvent to each of the sample cell and the reference cell with two pumps can be obtained. An inexpensive device configuration becomes possible. Further, since it is not necessary to separately send a solvent to the reference cell, the amount of solvent consumption can be suppressed.

[Brief description of the drawings]

FIG. 1 is a view showing a differential refractive index detector for a liquid chromatograph according to the present invention.

FIG. 2 is a diagram illustrating a state of a baseline signal when a sample cell and a reference cell are simply connected in series.

FIG. 3 is a diagram illustrating a state of a baseline signal in a differential refractive index detector illustrated in FIG. 1;

FIG. 4 is a diagram showing a chromatogram of standard polystyrene measured by connecting a sample cell and a reference cell in series.

FIG. 5 is a diagram showing a chromatogram of standard polystyrene measured by the differential refractive index detector shown in FIG. 1.

[Description of sign]

 1 Solvent reservoir 2 Solvent 3 Piping 4 Liquid sending pump 5 Piping 6 Sample injection port 7 Piping 8 Separation column 9 Piping 10 Sample cell 11 Piping 12 Flow branch 13 Piping 14 Solenoid valve 15 Piping 16 Piping 17 Needle valve 18 Piping 19 Stirring Chamber 20 piping 21 reference cell 22 piping 23 waste liquid tank

Claims (4)

[Claims] 1. A differential refractive index detector for detecting a change in a refractive index difference between a sample cell and a reference cell, wherein an eluate from the sample cell is diluted and used for use as a reference solution between the sample cell and the reference cell. A differential refractive index detector for a liquid chromatograph, in which a stirring chamber having a volume at least 10 times the sample cell volume is inserted. 2. The differential refractive index detector for a liquid chromatograph according to claim 1, further comprising a branch flow path for equalizing the pressure difference between the sample cell and the reference cell, and an air damper. 3. The differential refractive index detector for a liquid chromatograph according to claim 1, further comprising a temperature adjusting mechanism for adjusting the temperature in the stirring chamber to the temperature in the solvent sump or the column thermostat. 4. The differential refractive index detector for a liquid chromatograph according to claim 1, wherein the volume of the solution in the stirring chamber is at least 100 times the volume of the sample cell.