JPH04249736A - Concentrating vessel of eluate for infrared spectral measurement - Google Patents

Abstract

PURPOSE:To enable highly-sensitive measurement of a sample of a minute amount by educing a solute locally, regarding a concentrating vessel of an eluate which receives the eluate flowing out of a column of a high performance liquid chromatograph(HPLC) and concentrates the eluate by evaporating a solvent thereof. CONSTITUTION:A sample holding recession 28 of which the area of a cross section turns small toward the bottom is formed in a base 26, the surface of the bottom has a light-reflecting property and the surface of the sample holding recession 28 is coated with a water-repellent substance 30.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an eluate concentration container for receiving eluate flowing out from a column of a high performance liquid chromatograph (HPLC) and concentrating the eluate by evaporating its solvent.

0002

2. Description of the Related Art Techniques for identifying and determining the structure of components of a sample separated by HPLC using infrared spectroscopy are gradually being used. Although this technique has been established as a general method in gas chromatography/infrared spectroscopy, it has not yet been technically established as a general method in HPLC/infrared spectroscopy. The reason is that HPLC
Mobile phase solvents generally have a number of fairly strong absorption bands in the infrared region, so the infrared absorption spectrum of the separated components contained in a small amount in the eluate is often hidden by the absorption of the solvent. This is because it is difficult to detect separated components (solutes) from the eluate while the eluate is in its original state.

[0003] Several methods have therefore been tried to eliminate the influence of this solvent. There are currently two technically established methods:

(1) Difference spectrum method This is a method in which the eluate from HPLC is introduced into a flow cell and subjected to infrared spectroscopy to obtain a difference spectrum from a previously measured spectrum of the solvent.

[0005] However, even in this method, the influence of the solvent cannot be avoided, and in particular, if the solvent has a strong infrared absorption band, it is impossible to detect the infrared spectrum of the solute in that wavelength range. . In addition, by reducing the thickness of the flow cell,
When the influence of infrared absorption of the solvent is reduced, the separated components are also reduced, making it impossible to detect the infrared spectrum of the solute.

(2) Mobile phase removal method Therefore, the following method has been proposed (Japanese Patent Application No. 1983).
-147393, U.S. Patent No. 4,841,145)
.

As shown in FIG. 9A, a sample holding recess 12 is formed in a sampling plate 10 whose surface reflects infrared rays, and an eluent 14 from an HPLC column is dropped into the sample holding recess 12. Then, the solvent of the eluate 14 is evaporated, and the solute 16 is precipitated as shown in FIG. 9B. When the solute 16 is irradiated with infrared IR, the infrared IR passes through the solute 16, is reflected by the sample holding recess 12, and further passes through the solute 16 and comes out. The intensities of the incident infrared IR and the outgoing infrared IR are detected as a function of wave number to obtain an infrared spectrum.

This method has the advantage that it is not subject to limitations on the type of solvent or the measurement wavelength range.

[0009]

However, in the case of trace analysis, that is, when the concentration of the solute 16 in the eluate 14 is extremely low, or in order to avoid remixing the components separated by the column, it is necessary to collect the If the amount of eluate 14 to be used is small, the amount of sample at the infrared IR irradiation point is not sufficient.
High-sensitivity measurements cannot be made.

One of the reasons for this is that the solute 16 is deposited over the entire surface of the sample holding recess 12 that is in contact with the eluate 14. This problem may be solved by making the sample holding recess 12 conical, but it cannot actually be solved. This is due to the following reasons.

(2) When the eluate 14 is dropped into the sample holding recess 12, the solute 16 begins to precipitate from the contact area between the surface of the eluate 14 and the surface of the sample holding recess 12.

(2) Since a nucleus is necessary for the growth of a crystal, solute 16 is precipitated using the first solute precipitated as a nucleus.

■ The sampling plate 10 is made of metal as a material that reflects infrared rays, but the metal surface has a small contact angle with the liquid as shown in FIG. 9B, and the eluate 1
The periphery of number 4 spreads out thinly. For this reason, it tends to be deposited where it was first deposited.

Therefore, only a small amount of solute 16 is deposited on the bottom surface of the sample holding recess 12, and the sample precipitation is concentrated on the side surfaces of the sample holding recess 12.

Note that if the eluate 14 is dropped onto a flat surface, this is out of the question since the eluate 14 will spread more than if the eluate 14 is dropped into the sample holding recess 12.

On the other hand, an infrared microscope is used to measure a minute amount of sample. In this case, since the area of the sample irradiated with infrared rays is approximately 100 μm×100 μm, it is preferable to localize the solute 16 within approximately 100 μm×100 μm.

Furthermore, in infrared spectroscopy, the absorbance increases as the sample thickness increases, so it is necessary to make the sample thickness as large as possible. For this purpose, it is necessary to prevent the sample from spreading.

Based on the analysis of the problems by the present inventors, the purpose of the present invention is to evaporate the solvent of the eluate flowing out from HPLC and locally precipitate the solute, thereby increasing the concentration of a trace amount of sample. An object of the present invention is to provide an eluate concentration container for infrared spectrum measurement that enables sensitivity measurement.

[0019]

[Means for Solving the Problems and Their Effects] In order to achieve this objective, a sample holding recess whose cross-sectional area becomes smaller as it approaches the bottom surface is formed in the base material, and the bottom surface has light reflective properties. Provided is an eluate concentration container for infrared spectrum measurement according to the present invention, in which the surface of the sample holding recess is made of a water-repellent material.

[0020] The eluate collected in this eluate concentration container is
When the solvent evaporates, it condenses at the bottom of the sample holding recess, resulting in concentration.

[0021] The sample holding recess has, for example, a flat bottom surface, and the area of the bottom surface is approximately the area of a convergent cross section of infrared rays emitted from an infrared microscope.

[0022] With this configuration, it is possible to receive the specularly reflected light of the infrared irradiation light, and most of the sample can be present in the infrared convergence section, making it possible to perform highly sensitive measurements of trace amounts of samples. Become.

[0023] The base material is, for example, a shiny metal,
The sample holding recess is coated with a water-repellent material.

[0024] This configuration is easy to produce, so
Eluate concentration containers are cheaper.

[0025] The base material is, for example, a water-repellent material,
The bottom surface of the sample holding recess is made of shiny metal.

In the case of this configuration, since the metal surface is not coated with a water-repellent substance, infrared absorption of the coating substance and chemical reaction with the coating substance are not a problem, and it can be applied to a wide range of samples. .

[0027] The base material is, for example, a shape memory alloy in which the bottom of the sample holding recess is conical, and the state in which the bottom is flat is memorized.

In the case of this configuration, the sample is locally concentrated and precipitated at the conical bottom of the sample holding recess, and moreover, during infrared spectrum measurement, the eluate concentration container is heated to return it to its memorized shape. , can receive specularly reflected light of irradiated infrared rays, and can perform high-sensitivity measurements.

[0029] For example, a plurality of the sample holding recesses are formed on the base material at predetermined intervals.

[0030] With this configuration, samples can be collected intermittently and continuously into the eluate concentration container. Also, in some cases, while collecting the sample, at the same time,
Infrared spectrum measurement can be performed on the precipitated sample within the sample holding recess.

The eluate concentration container described above is used for reflection measurement, and a sample holding recess whose cross-sectional area becomes smaller as it approaches the bottom is formed in the base material of the infrared transmitting material, and the surface of the sample holding recess is Transmission measurement becomes possible by using an eluate concentration container in which the material is water-repellent.

[0032]

Embodiments Hereinafter, embodiments of the present invention will be explained based on the drawings.

Prepare a conical cup 18, a hemispherical cup 20, or a boat-shaped cup 22, as shown in FIGS. 1(A), (B), and (C), in which the cross-sectional area becomes smaller as it approaches the bottom surface. do. Even if the eluate flowing out from HPLC is collected using these, the solute spreads and precipitates as described above, so there is no concentration or condensation effect. So, these 1 cup
The surfaces of the recesses 8, 20, and 22 are coated with a water-repellent material. In this way, when the solvent in the collected eluate is evaporated, it condenses in the center of the recesses of the cups 18, 20, and 22, thereby performing concentration.

Next, infrared microscopic measurement of the condensed sample will be explained.

There are two methods: one is to take out the condensed sample for measurement, and the other is to directly measure the sample in the cup. However, since the purpose of sample concentration is to measure a trace amount of sample, it is difficult to remove the sample from the cup, and it is necessary to directly measure the sample in the cup.

[0036] There are two methods for measuring the sample inside the cup: a reflection method and a transmission method. First, the reflection method will be explained.

1). Reflection method (1) First embodiment FIG. 2 shows an eluate concentration container 24 used in the reflection method. This eluate concentration container 24 has a rectangular parallelepiped base material 26 and a conical sample holding recess 28 formed therein.

The base material 26 is preferably made of metal, particularly aluminum or stainless steel with a polished surface, in order to increase the reflectance and perform highly sensitive measurements.

The center bottom of the sample holding recess 28 is flat in order to receive the specularly reflected light of the irradiation light.

Furthermore, the surface of the sample holding recess 28 is coated with a water-repellent substance 30.

The water-repellent material 30 is preferably a material whose surface free energy is smaller than the surface tension of the sample to be collected. This is because the water-repellent effect increases, so-called wetting phenomenon does not occur, and the sample becomes difficult to precipitate. As such a coating material, for example, fluorinated resin, polyethylene, silicone oil, etc. are preferable. When the coating material itself exhibits infrared absorption in the infrared measurement wavelength range, it is preferable to keep the thickness of the coating material within the range of a monomolecular layer to several molecular layers.

[0042] In such an eluate concentration container 24, HPL
The eluate 32 flowing out from C is collected and, depending on the volatility of the eluate 32, is air-dried, heated, depressurized, or passed with dry gas to evaporate the solvent in the eluate 32 and remove the solute. 32A is precipitated.

At this time, the solute 34A is also precipitated in the surrounding area other than the flat bottom part of the sample holding recess 28, but compared to the conventional method shown in FIG.
Since the solute 2 aggregates, the solute 32A is locally precipitated at an overwhelmingly high rate on the flat bottom surface of the sample holding recess 28.

Next, as shown in FIG. 3, the eluate concentration container 24 is placed opposite the Cassegrain objective 34a of the infrared microscope 34. A Fourier infrared spectrophotometer (FT-IR) 36 is connected to the infrared microscope 34, and infrared IR from the Fourier infrared spectrophotometer 36 is transmitted to the infrared microscope 34.
The solute 32A deposited on the flat bottom of the sample holding recess 28 is convergently projected through the right half of the Cassegrain objective mirror 34a of No. 4. Therefore, the diameter of this bottom flat part is ideally about the diameter of the convergence circle of the projected infrared IR (100 to 2
00 μm) is desirable. The infrared IR passes through the solute 34A and is reflected at the flat bottom of the sample holding recess 28. The infrared IR passes through the solute 32A again and is focused on the left half of the Cassegrain objective 34a, and is directed to the Fourier infrared spectrophotometer 36. and the infrared spectrum is measured.

(2) Second Embodiment For the normal eluate 32, the eluate concentration container 24 described above is used.
However, if the eluate 32 chemically reacts with the coating material or if the water-repellent material 30 exhibits large absorption in the measurement wavelength range, the water-repellent material 30 cannot be used. Therefore, in such a case, an eluate concentration container 38 as shown in FIG. 4 is used.

The eluate concentration container 38 has a rectangular parallelepiped base material 40 and a conical sample holding recess 42 formed therein.

The base material 40 is a water-repellent material, such as a fluorinated resin. Fluorinated resin has low light transmittance even if the plate thickness is small, so it is not suitable for the transmission method described below.

The center of the base material 40 has a diameter of 100 to 20 mm.
A through hole with a diameter of about 0 μm is formed, and a metal wire 44 whose tip end surface is polished flat is inserted into the through hole.

When manufacturing such an eluate concentration container 38, care must be taken to ensure that the tip of the metal wire 44 does not protrude from the bottom of the sample holding recess 42. This is because, if this protrusion exists, the solute 34A will precipitate between the peripheral surface of the tip end of the metal wire 44 and the bottom of the sample holding recess 42, and the sample condensation rate will decrease.

If this point can be cleared, since the metal wire 44 is not coated with a water-repellent substance, infrared absorption of the coating substance and chemical reaction with the coating substance will not be a problem, and it can be applied to a wide range of samples. Can be done.

(3) Third Embodiment In order to increase the sample condensation rate, it is better to make the cup conical in shape and not provide a flat portion at the bottom. However, unless a flat portion is provided, specularly reflected light of the irradiated light cannot be received, and high sensitivity measurement cannot be performed. Therefore, in order to solve this problem, an eluate concentration container 46 as shown in FIG. 5 is used.

As shown in the same figure (B), the eluate concentration container 4
6, a sample holding recess 48 is press-molded from a plate-shaped shape memory alloy. At this time, at the bottom of the sample holding recess 48,
The shape of a flat part with a small area is memorized.

[0053] When collecting the eluate 32,
As shown in the figure, the shape is made without this flat part. When the solvent of the eluate 32 has evaporated and condensation has been completed, the sample holding recess 48 is heated to bring it into the state shown in FIG. 3(B).

[0054] As a result, the solute 34A is transferred to the sample holding recess 4.
The conical bottom of 8 allows for highly sensitive measurement by locally concentrating and receiving specularly reflected light of the irradiated light.

If the sample holding recess 48 is formed small, the eluate concentration container 46 can be returned to a flat plate. In this case, the opening angle of the Cassegrain objective 34a and the cone angle of the sample holding recess shown in FIG. Since there is no need to consider the relationship between
Handling becomes very convenient during measurement.

(4) Fourth Embodiment FIG. 6 shows an eluate concentration container 50 used for intermittent continuous measurement.

The rectangular plate-shaped eluate concentration container 50 has a large number of sample holding recesses 50a formed in a matrix. For example, each sample holding recess 50a is coated with a water-repellent substance 30 as shown in FIG. 2, or a metal wire 44 as shown in FIG. 4 is fitted. This eluate concentration container 50 is placed on an X-Y stage 52, and is
The eluate flowing out from the discharge pipe 54 is accommodated by being driven intermittently in the direction and the Y direction.

(5) Fifth Embodiment FIG. 7 shows an eluate concentration container 56 which is also used for intermittent and continuous measurements.

The eluate concentration container 56 has a disc shape, and has a large number of sample holding recesses 56a formed at the edge along the outer periphery. For example, each sample holding recess 56a is coated with a water-repellent substance 30 as shown in FIG. 2, or a metal wire 44 as shown in FIG. 4 is fitted. The eluate concentration container 56 is placed on a rotation stage 58, and is intermittently and continuously rotated at the pitch angle θ of the sample holding recess 56a.

In this configuration, while the eluate is flowing down from the discharge pipe 54, the sample holding recess 5 is simultaneously
Infrared microscopic measurements can be performed on the precipitated sample within 6a.

2). Transmission method In the case of transmission method, there is no need to consider specular reflection, so
The flat bottom portion of the sample holding recess 28 as shown in FIG. 2(B) becomes unnecessary. Therefore, the sample condensation rate can be made higher than in the case of the reflection method.

However, it is necessary to minimize the thickness of the plate serving as the base material on which the sample holding recess is formed, to reduce light scattering within this plate, and to increase light transmittance. Suitable materials for this plate include ZnSe, KRS-5, etc., which have low infrared absorption. In particular, since KRS-5 itself is water repellent, there is no need to coat it with a water repellent material.

[0063]

Effects of the Invention As explained above, according to the eluate concentration container for infrared spectrum measurement according to the present invention, a sample holding recess whose cross-sectional area becomes smaller as it approaches the bottom is formed in the base material. Since the bottom surface is light reflective and the surface of the sample holding recess is a water-repellent material, the eluate collected in this eluate concentration container will condense and concentrate at the bottom of the sample holding recess when the solvent evaporates. This method has the excellent effect of allowing infrared analysis to be carried out, and greatly contributes to expanding the scope of application of infrared analysis.

If the sample holding recess has a flat bottom surface and the area of the bottom surface is approximately the area of the convergent cross section of the infrared rays emitted from the infrared microscope, the specularly reflected light of the infrared irradiation light can be received. In addition, since most of the sample can be present in the infrared convergence section, it is possible to perform high-sensitivity measurement of a trace amount of sample.

[0065] If the base material is made of a shiny metal and the sample holding recess is coated with a water-repellent substance, the production becomes easy and the eluate concentration container can be provided at a low cost.

If the base material is made of a water-repellent material and the bottom surface of the sample holding recess is made of a shiny metal, the metal surface is not coated with a water-repellent material, so that the infrared absorption of the coating material and the chemical interaction with the coating material are prevented. This method has the advantage that reaction is not a problem and can be applied to a wide range of samples.

If the bottom of the sample holding recess is made of a conical shape memory alloy and the flat state of the bottom is memorized,
The sample is locally concentrated and precipitated at the conical bottom, and by heating the eluate concentration container to return it to its memorized shape during infrared spectrum measurement, it is possible to receive the specularly reflected light of the irradiated infrared rays. This brings about the effect that high-sensitivity measurement can be performed.

[0068] By forming a plurality of sample holding recesses on the base material at predetermined intervals, it is possible to collect samples intermittently and continuously into the eluate concentration container, and in some cases, it is possible to collect samples in a continuous manner. However, on the other hand, it is also possible to perform infrared spectrum measurement on the precipitated sample within the sample holding recess.

The eluate concentration container described above is used for reflection measurement, and a sample holding recess whose cross-sectional area becomes smaller as it approaches the bottom is formed in the base material of the infrared transmitting material, and the surface of the sample holding recess is If the eluate concentration container is made of a water-repellent substance, the same concentration effect as described above can be obtained and the permeation measurement becomes possible.

[Brief explanation of the drawing]

FIG. 1 is a perspective view showing the types of sample holding recess shapes of an eluate concentration container for infrared spectrum measurement.

FIG. 2 shows an eluate concentration container for infrared spectrum measurement of the first embodiment, (A) is a perspective view, and (B) is a B-B of (A).
FIG.

FIG. 3 is a state diagram of infrared microscope measurement.

FIG. 4 is a sectional view of an eluate concentration container for infrared spectrum measurement according to a second embodiment.

FIG. 5 is a cross-sectional view of the eluate concentration container for infrared spectrum measurement of the third example, in which (A) shows the sample collection state;
B) shows the state of sample precipitation.

FIG. 6 is a perspective view showing an eluate concentration container for infrared spectrum measurement according to a fourth embodiment, which is used for intermittently continuous measurement.

FIG. 7 is a perspective view showing an eluate concentration container for infrared spectrum measurement according to a fifth embodiment, which is used for intermittently continuous measurement.

FIG. 8 is a cross-sectional view of a conventional sampling plate 10, in which (A) shows a sample collection state and (B) shows a sample deposited state.

[Explanation of symbols]

28, 42, 48, 50a, 56a Sample holding recess 3
2 Eluate 32A Solute 18, 20, 22 Cup 24, 38, 46, 50, 56 Eluate concentration container 26
, 40 base material 30 water-repellent substance 34 infrared microscope 34a Cassegrain objective mirror 36 Fourier infrared spectrophotometer 44 metal wire

Claims (7)

[Claims] Claim 1: A sample holding recess (28, 42, 48, 50a) whose cross-sectional area becomes smaller as it approaches the bottom surface.
, 56a) is formed on the base material (26, 40), the bottom surface has light reflective properties, and the surface of the sample holding recess is coated with a water-repellent material (3).
0.0, 40) An eluate concentration container for infrared spectrum measurement. 2. The sample holding recess (28, 42, 48
2. The eluate concentration container for infrared spectrum measurement according to claim 1, wherein the container has a flat bottom surface, and the area of the bottom surface is approximately the area of a convergent cross section of infrared rays emitted from an infrared microscope. 3. The base material (26) is a shiny metal, and the sample holding recess (28) is coated with a water-repellent material (30).
The eluate concentration container for infrared spectrum measurement described above. 4. The base material (40) is a water-repellent material, and the bottom surface of the sample holding recess (42) is a shiny metal. Eluate concentration container for infrared spectrum measurement. 5. The base material includes the sample holding recess (48
) is made of a conical shape memory alloy, and a flat state of the bottom is memorized.
The eluate concentration container for infrared spectrum measurement described above. 6. The sample holding recess (50a, 56a)
The eluate concentration container for infrared spectrum measurement according to any one of claims 1 to 5, wherein a plurality of are formed on the base material at predetermined intervals. 7. A sample holding recess, the cross-sectional area of which becomes smaller as it approaches the bottom surface, is formed in a base material of an infrared transmitting material, and the surface of the sample holding recess is made of a water-repellent material. Eluate concentration container for external spectrum measurement.