JPH08129003A - Ultraviolet visible spectrophotometer detector - Google Patents

Abstract

PURPOSE: To suppress the fluctuations of the base line of a chromatogram while using a flow cell composed of a resin. CONSTITUTION: A cylindrical cavity part 14 is formed in a cell body 12 made of an opaque resin and a moving phase into which a sample is injected is allowed to flow through the cavity part and light is projected on the moving phase. The diameter of the cavity part 14 is changed in size on the inlet and outlet sides of projection light to make the diameter of an incident light path smaller than that of an emitting light path to prevent that light is refracted in the liquid within a flow cell to impinge against a wall surface.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a liquid chromatograph,
Particularly, it relates to an ultraviolet-visible spectrophotometer detector used in an inert liquid chromatograph for biotechnology.

[0002]

2. Description of the Related Art In a liquid chromatograph for biotechnology, a highly corrosive solvent containing halogen is used as a mobile phase, and such a mobile phase passes through a flow cell of an ultraviolet-visible spectrophotometer detector. As a material for the flow cell, stainless steel is usually used for a member in contact with the mobile phase (referred to as a “wetted member”). However, in a liquid chromatograph for biotechnology, when stainless steel is used, the stainless steel may be corroded by the mobile phase. In addition, since the metal ions are eluted from stainless steel to denature the sample, the accuracy of analysis decreases. Therefore, it is necessary to use a liquid contact member made of resin for the flow cell of the UV-visible spectrophotometer detector in the liquid chromatograph for biotechnology. Teflon (trademark), ethylene trifluoride,
There are polyether ether ketone (PEEK) and the like.

[0003]

In the analysis by liquid chromatography, when the composition of the solvent in the flow cell changes, the projected light is refracted in the flow cell and strikes the wall surface. When the material of the cell body is resin, the projected light impinges on the wall surface, so that the absorbance by the liquid (the liquid consisting of the components of the sample and the mobile phase) in the flow cell apparently increases. Hereinafter, this point will be described with reference to FIG.

FIG. 2 is a view showing a vertical section of a flow cell of a conventional UV-visible spectrophotometer detector. In this flow cell, a cylindrical hollow portion 54 is formed in a cell body 52 made of a resin, and the mobile phase in which the sample is injected flows into the hollow portion 54 and light (visible light or ultraviolet light) is passed. Is projected. Lenses 56a and 56b are arranged on the light entrance side and the light exit side of the cavity 54, respectively.
The packings 55a and 55b are respectively interposed between the lenses 56a and 56b and the cell body 52, and the lens pressers 58a and 58b having the thread grooves are pressed from the outer side to the inner side.
6b is fixed to the cell body 52.

When parallel light is projected onto the flow cell in such a structure, the parallel light is incident on the lens 56a on the entrance side.
After being focused on one point in the vicinity of the center of the hollow portion 54 by, the light is diffused toward the exit of the hollow portion 54, and again collimated by the lens 56b on the exit side to be emitted from the flow cell to the outside. That is, the parallel light projected on the flow cell exits from the flow cell through the path indicated by the alternate long and short dash line in FIG. 2, and does not hit the wall surface in the cavity 54. The light passing through the flow cell in this way is detected by the photocell 60 arranged on the outlet side of the flow cell, whereby the absorbance of the liquid in the flow cell is measured. However, when the solvent composition in the flow cell is not uniform (for example, when the solvent of the sample injected into the chromatograph passes through the flow cell), the sample solvent and the mobile phase have different refractive indexes in the cavity 54. Since the two liquids coexist, light is refracted at the interface between them. As a result, the light projected on the flow cell strikes the wall surface in the cavity 54 via the path indicated by the solid line in FIG. 2, for example. When the cell body 52 is made of stainless steel or the like and the wall surface is close to a mirror surface, it is reflected even if light hits and is not absorbed by the wall surface. By hitting the wall surface, at least part of the light is absorbed by the wall surface, and the amount of light passing through the flow cell and reaching the photocell 60 is reduced by that amount. This apparently increases the absorbance of the liquid in the flow cell. When the material of the cell body 52 is a resin such as Teflon or ethylene trifluoride that transmits light, the light that hits the wall surface is transmitted through the cell body 52 (when the material is a translucent resin, the wall surface is However, the amount of light reaching the photocell 60 decreases. As a result, the absorbance is apparently increased as in the case of the resin that does not transmit light. When the absorbance is increased in this way, the baseline of the chromatogram drawn based on the output signal of the detector is greatly changed, and a peak P01 (hereinafter, this peak is referred to as a “solvent peak”) as shown in FIG. 3 is generated. Appears.

The above-mentioned solvent peak P01 that appears when the solvent of the sample passes through the flow cell is relatively large, so it may be mistaken for a peak corresponding to the component of the sample (hereinafter referred to as "sample peak"). Solvent peak P
If there is a sample peak near 01, the height and area of the sample peak may not be calculated accurately.

Also in the case of performing gradient analysis, since liquids having different refractive indices coexist in the flow cell, light refracts in the flow cell and the baseline of the chromatogram fluctuates. For example, the mobile phase is A liquid and B liquid.
When the concentration of the B liquid is changed as shown in FIG. 4 (a) in the case of different types, it corresponds to the bent portion of the curve of FIG. 4 (a) (the change in the refractive index is discontinuous at this portion). A large fluctuation of the baseline appears as shown in FIG. Therefore, even when performing the gradient analysis, this variation causes the same problem as described above.

The present invention has been made to solve such a problem, and an object thereof is to suppress the above-mentioned fluctuation of the baseline while using a flow cell made of a resin. It is to provide an ultraviolet-visible spectrophotometer detector that can be used.

[0009]

Means for Solving the Problems The present invention, which has been made to solve the above-mentioned problems, provides a mobile phase which has passed through a column of a liquid chromatograph in a flow cell constituted by using a liquid contact member made of a resin, Flowing the sample, by projecting visible light or ultraviolet light into the flow cell to measure the absorbance, in the ultraviolet visible spectrophotometer detector to detect various components of the sample, the visible light or ultraviolet light of the flow cell Is smaller than the inner diameter on the side where the visible light or the ultraviolet light is emitted.

[0010]

Operation When analyzing a sample by liquid chromatography, the mobile phase and the sample that have passed through the column of the liquid chromatograph are flown into the flow cell of the UV-visible spectrophotometer detector, and light (visible light or UV Light) is projected.

Now, considering the case where the solvent of the sample passes through the flow cell and the case where the refractive index of the mobile phase changes in the case of performing gradient analysis, liquids having different refractive indexes coexist in the flow cell at this time. Light may be refracted at these boundaries and hit the wall surface in the flow cell. Since the material of the wall surface (wetted member) is resin,
When the projected light hits the wall surface in the flow cell, at least part of the light is absorbed by the wall surface (in the case of resin that does not transmit light) or passes through the wall surface (in the case of resin that transmits light), The amount of light emitted from the outlet is reduced accordingly. This apparently increases the absorbance of the liquid in the flow cell. However, in the UV-visible spectrophotometer detector of the present invention, since the inner diameter of the incident side of the projection light in the flow cell is smaller than the inner diameter of the exit side, in the state where the refraction of light does not occur, the projection light and the wall surface in the flow cell There is a space between them, and even if some refraction of light occurs, the projected light passes through the flow cell without hitting the wall surface. This suppresses an increase in absorbance caused by the coexistence of liquids having different refractive indexes in the flow cell.

[0012]

EXAMPLE FIG. 1 is a view showing a longitudinal section of a flow cell of an ultraviolet-visible spectrophotometer detector which is an example of the present invention.
In this flow cell, similarly to the above-described conventional example shown in FIG. 2, a cylindrical cavity portion 14 is formed in the cell body 12 made of resin, and the mobile phase in which the sample is injected is formed in the cavity portion 14. Is emitted and light (visible light or ultraviolet light) is projected. However, in the flow cell of the conventional example, the inner diameter is constant (see FIG. 2), but in the flow cell of the present embodiment, the inner diameter on the inlet side of the projection light in the cavity 14 is smaller than the inner diameter on the outlet side. That is, in this embodiment, the optical path diameter on the incident side in the flow cell is smaller than the optical path diameter on the exit side, and for example, in a flow cell having an optical path length of 10 mm, the inlet side optical path diameter is 1.0 mm and the outlet side optical path diameter is 1.7 mm. It Other parts in this embodiment are the same as those in the conventional example, and the lenses 16a and 16b, the packing 15
a, 15b, lens retainers 18a, 18b, and photocell 20 are lenses 56a, 56 in the conventional example, respectively.
b, packings 55a, 55b, lens retainers 58a, 5
8b corresponds to the photocell 60. In addition, cell body 1
If a resin that transmits light such as Teflon or ethylene trifluoride is used as the material of 2, the light passing through the cell body 12 becomes stray light, and the relationship between the output signal of the detector and the concentration of the component to be detected is linear. Disappear. Therefore, in this embodiment, a resin such as PEEK that does not transmit light is used as the material of the cell body 12.

When a chromatographic analysis is performed using the UV-visible spectrophotometer detector of this embodiment, the mobile phase and the sample that have passed through the column are flowing in the cavity 14 and the external phase is applied to this. Light is projected from. The projected light is parallel light, but like the conventional example, after being converged at one point in the vicinity of the center of the cavity portion 14 by the lens 16a on the entrance side, it is diffused toward the exit of the cavity portion 14 and then exits. The lens 16b on the side again collimates the light and outputs it from the flow cell to the outside. That is, the light projected on the flow cell goes out of the flow cell through the path indicated by the alternate long and short dash line in FIG. 1, for example. Here, since the diameter of the cavity 14 on the entrance side of the projected light is smaller than the diameter of the exit side, usually, a predetermined region near the center of the exit of the cavity 14 (the entrance of the cavity 14, that is, the light entrance Light comes out only from the area where the areas are almost equal. Therefore, unlike the conventional example, liquids with different refractive indices coexist in the flow cell due to the passage of the sample solvent through the flow cell, etc. Even if the light projected from the outside is refracted, the light does not hit the wall surface, You will be able to pass. That is, the light projected from the outside does not hit the wall surface even if it is refracted in the flow cell, and goes out of the flow cell through the path shown by the solid line in FIG. 1, for example.

As described above, when liquids having different refractive indices coexist in the flow cell, light is conventionally refracted and hits the wall surface, which causes an apparent increase in absorbance. Such an increase in absorbance can be suppressed. As a result, the fluctuation of the baseline of the chromatogram due to the passage of the sample solvent in the flow cell is suppressed, and as shown in FIG. 3, the solvent peak P02 (peak shown by the broken line) appearing in the chromatogram is It is much smaller than the solvent peak P01 in the example.

Further, in the present embodiment, even when the gradient analysis is performed, the apparent increase in absorbance due to the coexistence of liquids having different refractive indexes in the flow cell can be suppressed. For example, liquid A (water) and liquid B (methanol)
When monochromatic light having a wavelength of 254 nm is projected onto a flow cell in which a mobile phase consisting of different types is flowing at a flow rate of 1 ml / min to measure the absorbance, as shown in FIG. From 100% to
In the conventional example, a chromatogram as shown by the solid line in FIG. 4 (b) is obtained. As can be seen from these figures, the baseline of the chromatogram in the conventional example is large near the point corresponding to the bent portion of the curve in FIG. 4 (a) (the change in the refractive index becomes discontinuous at this portion). fluctuate. On the other hand, in this embodiment,
Since the light path diameter on the incident side of the flow cell is smaller than the light path diameter on the exit side, light is prevented from hitting the wall surface by refraction, and an apparent increase in absorbance is suppressed. As a result, the chromatogram when the concentration of the B liquid is changed as shown in FIG. 4 (a) becomes as shown by the dotted line in FIG. 4 (b), and the fluctuation of the baseline as in the conventional example can be seen. I will not be able to.

As described above, according to this embodiment, the fluctuation of the chromatogram due to the refraction of light in the flow cell is suppressed, and the baseline is stabilized. This allows
It is possible to avoid misidentifying the solvent peak as a sample peak and to accurately calculate the height and area of the sample peak, so that the analysis accuracy is improved.

In the above embodiment, the flow cell is provided with the lenses 16a and 16b, and the projected light is used for the cavity portion 14.
However, in the case where such a lens is not provided in the flow cell, an optical system for collecting the projection light in the cavity 14 of the flow cell is provided outside the flow cell. Will be placed. Therefore, even if the flow cell is not provided with a lens, the path of the light projected on the flow cell is almost the same as that of the above embodiment by making the optical path diameter on the incident side in the flow cell smaller than the optical path diameter on the exit side. Therefore, the same effect as above can be obtained.

[0018]

According to the present invention, the light projected onto the flow cell passes through the flow cell without hitting the wall surface even if it is refracted to some extent. Therefore, the increase in the absorbance due to the refraction of the light in the flow cell can be suppressed. Therefore, the fluctuation of the chromatogram caused by the coexistence of liquids having different refractive indexes in the flow cell is reduced, and the baseline is stabilized. As a result, the analytical accuracy of the chromatograph is improved, and highly quantitative analysis can be performed.

[Brief description of drawings]

FIG. 1 is a diagram showing a longitudinal section of a flow cell of an ultraviolet-visible spectrophotometer detector which is an embodiment of the present invention.

FIG. 2 is a view showing a vertical section of a flow cell of a conventional UV-visible spectrophotometer detector.

FIG. 3 is a diagram showing a chromatogram based on an output signal of an ultraviolet-visible spectrophotometer detector.

FIG. 4 is a diagram (a) showing a concentration change of a liquid constituting a mobile phase in a gradient analysis, and a diagram (b) showing a chromatogram corresponding to the concentration change.

[Explanation of symbols]

 12 ... Cell body 14 ... Cavities 16a, 16b ... Lens 20 ... Photocell

Claims (1)

[Claims] 1. A mobile phase and a sample which have passed through a column of a liquid chromatograph are caused to flow through a flow cell configured by using a liquid contact member made of a resin, and visible light or ultraviolet light is projected into the flow cell to obtain an absorbance. By measuring the, in the ultraviolet-visible spectrophotometer detector to detect various components of the sample, the visible light of the flow cell or the inner diameter of the side on which the visible light or ultraviolet light is incident is the side on which the visible light or ultraviolet light is emitted. Ultraviolet-visible spectrophotometer detector characterized by being smaller than the inner diameter.