JPH0850120A - Liquid chromatograph - Google Patents

Abstract

PURPOSE:To enhance the measuring accuracy of a liquid chromatograph provided with a concentration column for carrying out the concentrating treatment of a measuring sample. CONSTITUTION:A liquid chromatograph is provided with a separation column 16 for separating a measuring sample, a flow-passage control means 14 for supplying the measuring sample together with a solvent to the separation column 16, a detecting means 17 for analyzing the supplied measuring sample, sample injection means 15, 22, 22a for taking in the measuring sample and for supplying this to the flow-passage control means 14, and a concentration column 30 for concentrating the measuring sample supplied together with the solvent. The content volume of the concentration column 30 is made to 2.0 milliliters or less, and also a column filler consisting of porous carrier coated with a silicone polymer having Si-R (R is a hydropholic group) combination and Si-R' (R' is a hydrophilic group) combination, is filled inside the concentration column 30.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a liquid chromatography apparatus, and more particularly to a liquid chromatography apparatus having a concentration column for concentrating a sample to be measured.

[0002]

Liquid chromatography devices are widely used as chemical analysis means for separating and quantifying chemical substances.
In particular, a liquid chromatography apparatus using a semi-micro column having an inner diameter of 1 to 2 mm has an advantage that it can provide highly sensitive, high resolution and highly accurate analysis results, and active research has been made.

Here, attention is paid to a concentration column that is installed in a liquid chromatography apparatus and is used for performing a concentration treatment of a sample solution. A column packing material is filled inside the concentration column, and the sample solution is concentrated by the column packing material. For example, when a liquid chromatography device is used for separation and analysis of a mixed sample of various substances such as serum, since serum, which is a biological component, contains a large amount of protein component, this protein component is adsorbed on the column packing material. The concentration action is reduced. For this reason, conventionally, in order to prevent the concentration effect of the column packing material from decreasing, pretreatment for removing the protein from the sample has been performed in advance.

However, such a pretreatment operation requires a lot of time and labor and has a problem that the analysis accuracy is lowered. Therefore, in recent years, column packing materials have been provided which can directly inject a protein component-containing sample into the apparatus without performing these deproteinization operations and concentrate the injected protein component-containing sample.

These improved column packings have a structure in which porous glass or silica gel is used as a carrier, and substances having different characteristics are provided on the inner surfaces of the pores. If this column packing material is used, the proteins in serum are macromolecules, so they do not enter the pores, and pass through the column without being adsorbed by the hydrophilic outer surface (outer surface of the pores), and relatively small drug The molecules such as will be adsorbed on the hydrophobic inner surface (inner surface of the pore).

Specific examples of such a column packing material include those described in JP-A-60-56256. In this column packing material, a protein is coated on the outer surface of silica to which octadecylsilyl (ODS) groups are chemically bonded. The protein for coating is made of bovine serum albumin or the like, and the column packing material is obtained by adsorbing and denaturing the protein on ODS-bonded silica.

However, among the above-mentioned column packing materials, the protein-coated ODS silica packing material may cause elution of adsorbed and denatured protein over a long period of use, and a column with high separation efficiency can be obtained. There is a problem in terms of durability and separability, such as being unable to do so.

In order to improve such a problem, as described in JP-A-61-65159 and JP-A-1-123145, a hydrophobic group is provided on the inner and outer surfaces of the porous carrier. Introduce. The self-confidence is a macromolecule, and only the hydrophobic groups on the outer surface are cleaved by using an enzyme such as silica that cannot penetrate into the pores. Then, a hydrophilic group is introduced on the outer surface. Therefore, a method for obtaining a column packing material has also been developed.

Specifically, in the method described in JP-A-61-65159, porous silica having a glyceryl propyl group introduced is used as a starting material, and an oligopeptide is bonded thereto via carbonyldiimidazole, The phenylalanine side chain on the outer surface is cleaved by performing hydrolysis using carboxypeptidase A, which is a protein hydrolase. As a result, glycyl-phenylalanylphenyl-alanine remains as a hydrophobic ligand on the inner surface of the column packing material, and hydrophilic glycyl-glycerylpropyl groups are formed on the outer surface.

On the other hand, according to the method described in JP-A-1-123145, octanoyl chloride is reacted in the presence of triethylamine with porous silica having an aminopropyl group introduced as a starting material to give hydrophobic properties through an amide bond. In this method, a group is introduced, and then an acyl group on the outer surface is hydrolyzed by a polymyxin acylase, and an amino group on the outer surface is reacted with glycidol to make it a hydrophilic group.

[0011]

However, as the column packing material for the concentration column 30 arranged in the liquid chromatography device, the one disclosed in JP-A-61-65159 or JP-A-1-123145 is used. When used, since the column packing disclosed in each publication utilizes an enzymatic reaction, there is a problem that the manufacturing structure of the column packing becomes complicated and the characteristics of the obtained column packing tend to vary. there were.

Further, in the conventional liquid chromatography device, since the inner volume of the concentration column is as large as 2.0 ml or more, the supplied sample solution is rather diluted in the concentration column, There is a problem that the detection accuracy is lowered. This problem greatly affects the detection accuracy especially when the injected sample amount is small.

The present invention has been made in view of the above points, and by using a concentration column that is relatively easy to obtain and has good concentration efficiency, and by making the inner volume of the concentration column small. An object of the present invention is to provide a liquid chromatography device that solves the above problems.

[0014]

In order to solve the above problems, according to the present invention, a separation column for separating a sample to be measured mixed in a solvent supplied by a pump, and the separation column between the pump and the separation column are provided. The sample is supplied with the solvent from the pump and the sample to be measured is supplied from the sample injection pipe, and the sample to be measured is supplied to the separation column together with the flow path control means and separated by the separation column. The sample to be measured is supplied, the detection means for analyzing the supplied sample to be measured, the sample holding mechanism for supporting a plurality of containers storing the sample to be measured, and the sample to be measured from one of the plurality of containers. Concentration treatment of the sample to be measured, which is provided between the sample injection means for selectively collecting the measurement sample and supplying it to the flow path control means and the flow path control means and the separation column, and the solvent to be measured. To concentrate The column for concentration is provided with an internal volume of less than 2.0 ml, and the column for concentration has Si—R (R is a hydrophobic group) bond and Si—R ′ ( R'is characterized in that it is filled with a column packing composed of a porous carrier coated with a silicone polymer having a hydrophilic group) bond.

Further, the hydrophilic group R'in the column packing may be a hydrophilic group having a hydroxyl group. Further, the hydrophobic group R in the column packing may be a hydrocarbon residue having 1 to 18 carbon atoms.

[0016]

In the liquid chromatography device configured as described above, the content volume of the concentrating column is less than 2.0 milliliters, and the inner volume is small. Since it does not occur, and therefore the concentration process can be performed reliably, the detection accuracy can be improved.

The column packing material packed in the concentration column is composed of Si-R (R is a hydrophobic group) bond and Si-R.
It consists of a porous carrier coated with a silicone polymer having R '(R' is a hydrophilic group) bond, and the column packing of this structure has a part of its outer surface hydrophilic, so that proteins and the like are adsorbed. In addition, since it is stable, and is excellent in separation ability, it is possible to reliably perform the concentration treatment. Furthermore, since no enzyme or the like is used, a stable concentration treatment can be realized also by this.

[0018]

Next, an embodiment of the present invention will be described with reference to the drawings. 1 to 3 are schematic configuration diagrams of a liquid chromatography apparatus that is a first embodiment of the present invention.

The liquid chromatography apparatus according to this embodiment has a sample holder 21 having a plurality of sample containers 21a formed therein.
And the sample to be measured is held in each sample container 21a. Each of the sample containers 21a is typically about 200 μm.
It has a capacity of l. The sample injection tube 22 is movably held by the robot 22a.
The sample injection tube 22 is moved to the position P ₁ by the operation of 2a, and is subsequently moved down, so that the sample injection tube 22 is inserted into one selected sample container 21a among the plurality of sample containers 21a. Subsequently, the sample to be measured is sucked into the sample injection tube 22 by operating the syringe 15.

Reference numeral 14 in the drawing denotes a first switching valve 14 configured as a six-way valve, and the first switching valve 14 has a concentrating column 30 and a sample injection which are essential parts of the present invention. End 14 for receiving the tip of tube 22
x is connected. The concentration column 30 is a relatively small column having an internal volume of less than 200 microliters, and is filled with a column packing material described later. Since the sample to be measured supplied in the concentration column 30 is concentrated, the sample to be measured concentrated is supplied to the separation column 16 and the detector 17, which will be described later, so that the inspection accuracy can be improved. You can

The end portion 14x serves as an injection port for the sample to be measured.
Is the part to be measured and is measured in the sample injection tube 22.
For the sample, insert the sample injection tube 22 into the end 14x and
It is injected into the end 14x by operating the ring 15.
Be done. At this time, the sample injection tube 22 is moved by the robot 22a.
Position P corresponding to the end 14x ₂Is moved horizontally to
It is inserted into the end 14x by moving vertically.
It The sample solution injected from the sample injection tube 22 is
The first switching valve 14 is in the state shown in FIGS.
When it is in the ready state, it is supplied into the concentration column 30 for concentration processing.
It is configured to be opened.

In the liquid chromatography apparatus according to this embodiment, the sample outlet of the detector 17 is connected to the second switching valve 23, which is a hexagonal valve. The second switching valve 23 selectively separates the sample to be separated, which is separated by the separation column 16 and detected by the detector 17 and then discharged, into either the waste liquid reservoir 18 or the sample injection pipe 22. Plays the function of supplying. In the state shown in FIGS. 1 and 3, the sample to be separated discharged from the detector 17 is sent to the waste liquid reservoir 18 as shown by the arrow.

On the other hand, when the sample to be separated is fed to the sample injection pipe 22, as shown in FIG. 2, the sample is flow path 15b formed in the joint 15a provided in the syringe 15.
And is supplied to the sample injection tube 22 through. The flow path 15b is
Both the sample supplied from the syringe 15 and the sample supplied from the valve 23 are T-shaped so as to be sent to the sample injection tube 22.

Further, the illustrated apparatus includes a separation column 16
A container 24 for holding a cleaning liquid for cleaning the piping system cooperating therewith is provided, and the cleaning liquid is supplied from the container 24 to the pump 2
It is supplied to the second switching valve 23 via 5. FIG.
In the state shown in FIG. 3 and FIG. 3, the cleaning liquid is sent to the sample injection pipe 22 through the joint 15a to clean it. Sample injection tube 22
When cleaning the needle, the robot 22a moves the needle 22 to the position P.
It is moved to a waste liquid collection position (not shown) different from ₁ or P ₂ .

FIG. 2 shows a state in which the second switching valve 23 is rotated in the apparatus shown in FIG. In the illustrated state, the sample to be separated discharged from the detector 17 passes through the valve 23 along the arrow and is sent to the needle 22 via the flow path 15b in the joint 15a. On the other hand, the cleaning liquid from the container 24 is the second
Of the switching valve 23 of FIG. as a result,
The robot 22a causes the sample injection tube 22 to move to the sample holder 2
By moving the sample 1 to a position corresponding to the appropriate sample container 21a, the separated sample discharged from the detector 17 can be collected in the sample container 21a.

Especially when a semi-micro column having an inner diameter of 1.0 to 2.0 mm is used as the chromatographic separation column 16, the total flow rate of the solvent is at most 50 to 200 μl /
Since it is very small, the increase in the volume of the sample to be separated due to the dilution of the sample with the solvent does not pose a problem, and the sample to be separated can be sufficiently collected in the sample container 21a having a volume of several hundred μl.

FIG. 3 shows a state in which the first switching valve 14 has rotated. In the state shown, the end 14
The supply of the sample to be measured from x to the upper end supply port of the concentration column 30 is blocked, and instead the solvent is supplied by the pump 11 to the upper end supply port of the concentration column 30. Further, the lower end outlet of the concentration column 30 is connected to the separation column 16. Therefore, the measured sample concentrated in the concentration column 30 is supplied to the separation column 16, and the measured sample separated by the separation column 16 is subsequently supplied to the detector 17 to perform a predetermined analysis process. .

FIG. 4 shows a liquid chromatography apparatus according to the second embodiment of the invention. In the liquid chromatography apparatus according to the present embodiment, a concentration column 40 for performing primary separation is arranged between an autosampler 41 and a 6-valve-switching valve (hereinafter referred to as a switching valve) 42 configured as a hexagonal valve. There is.

The concentration column 40 is a column whose internal volume is less than 2.0 milliliters, and will be described later like the concentration column 30 arranged in the liquid chromatography apparatus of the first embodiment. The column packing material is filled. Since the sample to be measured supplied from the autosampler 41 is concentrated in this concentration column 40,
The concentrated sample to be measured is supplied to the separation column 43 and the detector 44, which will be described later, so that the inspection accuracy can be improved.

Further, the switching valve 14, the syringe 15, the sample injection tube 22 and the other constituent elements that function together with these constituent elements described in the first embodiment are incorporated in the autosampler 41. . Further, the pump 45A and the pump 45B include the switching valve 42.
In cooperation with the above, a measurement sample supply system that supplies the measurement sample to the concentration column 40, the separation column 43, and the detector 44 is configured.

According to the configuration shown in FIG. 4, the sample to be measured together with the solvent (mobile phase A) supplied by the pump A,
It is sent from the auto sampler 41 to the concentration column 40 and subjected to concentration processing. Then, the sample to be measured concentrated in the concentration column 40 is supplied to the separation column 43 for performing a separation process together with the solvent (mobile phase B) supplied by the pump 45B. At this time, mobile phase A and mobile phase B
Is switched by the switching valve 42 described above.

The sample to be measured is conveyed to the separation column 43.
And is subjected to predetermined analysis processing on the sample to be measured in the detector 44. Further, the liquid chromatography apparatus configured as described above includes a controller 46 configured by a computer for controlling the analysis process. The controller 46 controls the driving of the auto sampler 41, the switching valve 42, and the pumps 45A and 45B.

The liquid chromatography device employing the above-mentioned two-column system is advantageous in that the treatment of the sample to be measured can be eliminated as compared with the conventional analyzer.
This system is commonly used as a sample treatment fully system.

Next, the switching operation of the switching valve 42 performed by the control processing of the controller 46 will be described. The switching valve 42 controls the flow path of the sample to be measured formed in the liquid chromatography device to the first state and the second state by the control processing of the controller 46.
The state is switched to the state of.

When the switching valve 42 is in the first state, the sample to be measured which is pumped together with the mobile phase A from the autosampler 41 by the pump pressure generated by the pump 45A is switched after passing through the concentration column 40. The sample to be measured is supplied to the valve 42, and the sample to be measured further advances toward a waste liquid storage tank (not shown) along a line shown by a solid line in the figure. At this time, only the substance to be analyzed contained in the sample to be measured is captured by the concentration column 40 and concentrated. At the same time, the mobile phase B is also supplied to the switching valve 10 by the pump pressure generated by the pump 45B and flows toward the separation column 43 along the line shown by the solid line in FIG.

On the other hand, when the switching valve 42 is brought into the second state by the control processing of the controller 46, the sample to be measured concentrated in the concentration column 40 follows the line shown by the broken line in FIG. 4 for analysis. It is supplied to the flow separation column 43. On the other hand, the mobile phase B supplied from the pump 45B is configured to flow out to a waste liquid storage tank (not shown) along a line indicated by a broken line in the figure.

Here, the packing material packed in the concentration columns 30 and 40 will be described below. In this example, as the packing material packed in the concentration columns 30 and 40, Si—R (R is a hydrophobic group) bond, and Si—R ′.
A column packing composed of a porous carrier coated with a silicone polymer having a bond (R 'is a hydrophilic group) is used. By using the column packing of this configuration,
For example, when serum or the like containing a protein component is used as the sample to be measured, the protein separation ability can be enhanced, and thus the concentration treatment can be performed reliably and stably. Hereinafter, a specific configuration of the column packing material used in this example will be described.

As the porous carrier used in this example, for example, any powder generally used as a carrier for liquid chromatography, such as silica gel, alumina, glass beads (for example, porous glass), zeolite, and hydroxyapatite. Alternatively, graphite or the like can be used. Further, it is also possible to use a composite powder, for example, a fine inorganic powder, for example, silica gel, titanium dioxide or hydroxyapatite coated on the surface of a synthetic resin such as polyamide, acrylic resin or polyvinyl alcohol. .

The average particle size of the porous carrier is 2 to 200.
μm, specific surface area 200-300m ²/ G, 40-12
Those having 0Å pores are preferred. Particularly suitable porosity
As a porous carrier, it has pores of 60-80Å and has a specific surface area.
Is 400-600 m²Spherical shape with a particle size of 3 to 50 μm / g
Rui is a crushed silica gel.

The silicone compound having a Si--H group used in the present invention has the following general formula 1

[0041]

Embedded image

(R in chemical formula 1¹, R²And R³Are independent of each other
Is a hydrogen atom or at least one halogen atom
Hydrocarbons having 1 to 10 carbon atoms which may be substituted with
Group, but R¹, R², R³Are hydrogen atoms at the same time
There is no such thing. Also, R^Four, R ^Five, R⁶Water independent of each other
A primary atom or at least one halogen atom
A hydrocarbon group having 1 to 10 carbon atoms which may be replaced
is there. a is an integer of 0 or 1 or more, and b is 0 or 1 or more.
Is an integer above and c is 0 or 2, provided that c is 0
In some cases, the sum of a and b is an integer of 3 or more.
At least one).

The silicone compound of the above chemical formula 1 consists of two groups. The first group corresponds to the case where c = 0 in the above formula 1, and the following general formula 2

[0044]

Embedded image

(In the formula 2, R ¹ , R ² , R ³ , a and b have the same meanings as described above, but preferably R ¹ , R ² and R ³
Is a hydrocarbon group having 1 to 10 carbon atoms which may be independently substituted with at least one halogen atom, and the sum of a and b is 3 or more). Is.

Typical examples of this compound are as follows.

[0047]

[Chemical 3]

[0048]

[Chemical 4]

The compounds represented by the above chemical formulas 3 and 4 are
Each can be used alone or in the form of a mixture thereof. In each of the formulas 3 and 4, n (or a + b) is preferably 3 to 7. Since the boiling point decreases as the value of n decreases, the amount of evaporation and adsorption on the carrier increases. In particular, trimers and tetramers are particularly suitable because they are easily polymerized due to their steric properties.

Specific examples of the cyclic silicone compound represented by the formula 2 include dihydrogen hexamethylcyclotetrasiloxane, trihydrogen bentamethyl cyclotetrasiloxane, tetrahydrogen tetramethylcyclotetrasiloxane, and dihydrogen octamethylcyclo. Mention may be made of pentasiloxane, trihydrogenheptamethylcyclopentasiloxane, tetrahydrogenhexamethylcyclopentanesiloxane, pentahydrogenpentamethylcyclopentasiloxane and the like.

The second group of silicone compounds of the above formula 1 corresponds to the case of c = 2 in the above formula 1, and has the following general formula 5

[0052]

Embedded image

(In the formula 5, R ¹ , R ² , R ³ , R ⁴ , R ⁵ ,
R ⁶ , a and b have the same meanings as described above, and c is 2, but preferably R ^{1 to} R ⁶ are each independently of each other substituted with at least one halogen atom having 1 carbon atom.
10 to 10 hydrocarbon groups). As a typical example of this compound, the following general formula 6

[0054]

[Chemical 6]

Examples thereof include compounds represented by: Specific examples of the linear silicone compound represented by the above formula 5 include 1,1,1,2,3,4,4,4-octamethyltetrasiloxane, 1,1,1,2,3,4,5 , 5,
5-nonamethylpentasiloxane, and 1,1,1,
2,3,4,5,6,6,6-decamethylhexasiloxane etc. can be mentioned. The silicone compound represented by the general formula 1 is brought into contact with the porous carrier in a gas phase state or a liquid phase state.

The contact in the gas phase (gas phase treatment) is carried out, for example, using a closed container at a temperature of 120 ° C. or lower, preferably 100 ° C. or lower, preferably 200 mmHg or less, more preferably 100 mmHg or less. A method of contacting the vapor of a silicone compound in a molecular state on the surface of a carrier,
It can be carried out by a method of bringing the mixed gas of the silicone compound and a carrier gas into contact with the carrier at a temperature of 120 ° C. or lower, preferably 100 ° C. or lower. Examples of the silicone compound suitable for this gas phase treatment include tetrahydrotetraethylcyclotetrasiloxane and tetrahydrotetramethylcyclotetrasiloxane.

On the other hand, the contact in the liquid phase (liquid phase treatment) is
For example, 1 to 50% by weight of a silicone compound solution dissolved in benzene, dichloromethane, chloroform or the like, which is a volatile solvent capable of dissolving the silicone compound, particularly hexane, is added to the carrier in an amount of 0.01 parts by weight of the silicone compound. It may be added to the carrier in an amount of 1 part by weight. In this case, it is preferable to add under stirring.
The surface polymerization of the silicone compound on the surface of the carrier can be carried out by leaving the carrier after the contact treatment at a temperature of 50 to 200 ° C. for 2 hours or more or stirring.

Since this surface polymerization is promoted by the action of the surface active sites of the carrier itself, it is not necessary to add a catalyst. Here, the "active site" means a siloxane bond (Si-O).
-Si) or a site capable of catalyzing the polymerization of a silicone compound having a Si-H (hydrosilyl) group,
For example, it means an acid point, a base point, an oxidation point, or a reduction point.
The surface polymerization is carried out until the active sites on the carrier surface are covered with the silicone polymer film. When the activity of the carrier itself is very weak, an alkali catalyst such as sodium hydroxide or potassium hydroxide may be added to the carrier before or after the contact treatment.
Polymerization may be carried out after adding an alkyl metal catalyst such as dibutyltin or the like such as lylium hydroxide, ammonium hydroxide or calcium hydroxide, as appropriate.

There are two types of structures of the silicone polymer coating on the surface of the carrier. That is, in a silicone polymer in which polymerization occurs by cleavage and recombination of a siloxane bond (—Si—O—Si), —Si—O—Si
Having only a chain structure of units, while the polymerization is H ₂ O or O
_When caused by a cross-linking reaction between hydrosilyl bonds (Si-H) in the presence of ₂ ,

[0060]

[Chemical 7]

It is derived from

[0062]

Embedded image

The silicone polymer will contain a network of units. The above two different types of polymerization are
Depending on the type of carrier and the reaction conditions (temperature, catalyst, etc.), there are cases where they proceed independently, and cases where both types of polymerization proceed simultaneously. And the degree of polymerization also varies. As described above, in the present invention, since a low molecular weight silicone compound is brought into contact with a carrier, the silicone compound penetrates into the inside of the pores of the carrier and is adhered or adsorbed on substantially the entire surface of the powder to polymerize, A very thin coating of silicone polymer (3 Å to 30 Å coating) is formed on the carrier, leaving the porosity of the carrier substantially intact. This porosity is not substantially impaired by the subsequent addition of vinyl compounds and the like.

The molecular weight (weight average molecular weight) of the silicone polymer formed on the surface of the carrier by the above polymerization reaction is 1
More than 50,000. However, in the case of a silicone compound, as it is polymerized by polymerization, it becomes less soluble in water or an organic solvent, and it is not possible to extract the polymer to measure the molecular weight, and the state of being coated on the carrier surface It is also not possible to measure the molecular weight of the polymer at.

Then, the polymer at each stage of the progress of the polymerization was extracted with chloroform, and the molecular weight of the polymer was determined in terms of polystyrene. It was confirmed that there was a maximum of 150,000 polymers. Therefore, the molecular weight of the polymer sufficiently polymerized to a state where it is not extracted with chloroform is 1
It can be said to be 50,000 or more, but it is difficult to confirm the molecular weight in more detail.

Hydrophobic group By the way, unreacted Si-H groups remain in the silicone polymer coated on the surface of the carrier. This Si-H
By reacting the group with a hydrocarbon having a vinyl group in the molecule, a silicone polymer having a Si—C bond can be obtained. As the vinyl compound,
For example, the general formula

[0067]

[Chemical 9]

(In the formula, R ₈ and R ₉ are each independently a hydrogen atom, an alkyl group having 1 to 40 carbon atoms, a cycloalkyl group or a cycloalkenyl group having 4 to 8 carbon atoms, or 1 to 20 carbon atoms. Of the aryl group which may be substituted with an alkyl group of).

The vinyl compound represented by the above general formula is R
Ethylene in which ₈ and R ₉ are both hydrogen atoms, a vinyl compound in which one of R ₈ and R ₉ is a hydrogen atom and the other is a substituent other than a hydrogen atom, for example, an α-olefin compound,
Symmetrical vinyl compound R ₈ and R ₉ are both the same substituents other than a hydrogen atom, or R ₈ and R ₉ may be either asymmetric vinyl compound are different substituents other than hydrogen.

Preferred vinyl compounds are those in which R ₈ and R ₉ are independently hydrogen atom;
To 20 alkyl groups such as 1-hexyl group, 1-octyl group, 1-decyl group, 1-dodecyl group, 1-hexadecyl group, or 1-octadecyl group; cyclohexyl group or cyclohexenyl group; phenyl group or naphthyl group; Alternatively, it is a vinyl compound which is a phenyl group or a naphthyl group substituted with a lower alkyl group having 1 to 4 carbon atoms.

R ₈ is a hydrogen atom, R ₉ is an ethyl group,
Hexyl group, hexadecyl group, or an addition of the vinyl compound is a phenyl group, each C ₄ of the conventional chemically bonded filler - type, C ₈ - type, C ₁₆ - to obtain the equivalent of a type or phenyl type be able to.
The reaction between the vinyl compound and the silicone polymer-coated powder is, for example, 50 to 300 in the presence of a solvent.
It can be carried out by contacting at ℃, gas phase or liquid phase for 2 hours or more. Suitable catalysts are white metal catalysts, ie compounds of ruthenium, rhodium, palladium, osmium, iridium or platinum. In particular, palladium compounds and platinum compounds are preferred.

This reaction can be confirmed by measuring a diffuse reflection spectrum using an FT-IR device. Ie 2
Regarding the absorption of Si—H group at 160 cm ^−1, the absorption intensity was significantly reduced by the addition of the vinyl compound, and the absorption intensity was changed to 280
New absorption based on the alkyl group 0cm ^-1 ~3000cm ^-1 is appearing. Therefore, the reaction rate is calculated by calculating the ratio of the absorption intensities.

[0073] In order to obtain a hydrophilic group present embodiment to such a packing material for liquid chromatography, it is necessary to change the hydrophilic part of the SiH groups on the surface of the silicone polymer coated support obtained as described above . Here, as the hydrophilic group, tetraol or the like shown in Chemical formula 10 below is used.

[0074]

[Chemical 10]

The tetraol is synthesized as follows.

[0076]

[Chemical 11]

Here, when diglycerin is changed to polyglycerin, a polyol is formed, and these can also be used as the hydrophilic group of the present invention. Further, as the hydrophilic group, it is also preferable to use polyoxyethylene allyl ether shown in Chemical formula 11 below. The polyoxyethylene allyl ether can be synthesized by adding ethylene oxide to allyl alcohol as shown in Chemical formula 11.

The filler obtained as described above is different in type from the conventional filler characterized by the chemical bond type, and the usable pH range is extremely wide as 2 to 10, so that it cannot be used in the conventional filler. It can be used even with an alkaline solvent that has not been obtained, and is very safe. Further, when quantifying a drug or a metabolite in a biological component such as serum using the packing material according to the present embodiment using a liquid chromatography device, even if the biological component is directly injected without complicated pretreatment, the brightness is high. Analysis is possible.
The filler obtained by the present invention is a complete polymer coat type and does not use a Lewis acid. Therefore, basic substances such as 2-ethylpyridine and N, N′-dimethylaniline can be eluted.

Hydrophobization-Hydrophilicization Method The filler according to this example can be produced by a method of first hydrophobizing a porous carrier coated with a silicone polymer and then hydrophilizing it. . That is, FIG.
As shown in (A), the surface of the porous carrier 10 is coated with the silicone polymer 12 by the method described above. At this time, the —SiH group of the simple substance of silicone is used for polymerization, but not all of it is consumed.
Then, as shown in FIG. 5B, the —SiH group remaining in the silicone polymer 12 is reacted with the hydrophobic group R having a double bond to form the —SiR group 14. In addition, here, all the -SiH groups of the silicone polymer 12 are -S.
If it becomes an iR group, it is not possible to introduce a hydrophilic aqueous group later, so the addition amount of the hydrophobic group R or the reaction condition is determined according to the purpose.

Next, as shown in FIG. 5C, the unreacted -SiH group of the silicone polymer 12 is reacted with the hydrophilic group R '(indicated by ◯-in the figure) 16 having a double bond to make it hydrophilic. To form the —SiR ′ group of Therefore, the surface of the silicone polymer 12 has a so-called mixed function structure modified with the hydrophobic group R and the hydrophilic group R ′,
Specific elution characteristics can be obtained by the modification ratio of both groups. In the hydrophobization-hydrophilization method, since the introduction of the hydrophobic group, which particularly has a great influence on the retention, can be easily controlled, the retention size can be adjusted by adjusting the introduction amount of the hydrophobic group. There are advantages.

Hydrophilization-hydrophobicization method The filler according to this example can also be produced by a method of first hydrophilizing a porous carrier coated with a silicone polymer and then hydrophobizing the porous carrier. . That is, FIG.
As shown in (A), the surface of the porous carrier 10 is coated with the silicone polymer 12. Then, as shown in FIG. 6B, -SiH remaining in the silicone polymer 12
The group and a hydrophilic group R ′ (shown by ◯ − in the figure) 16 having a double bond are reacted to form a —SiR ′ group 16. In addition,
Here, all the SiH groups of the silicone polymer 12 are
When the SiR ′ group 16 is formed, the subsequent introduction of the hydrophobic group cannot be performed, and therefore the addition amount of the hydrophilic group R ′ or the reaction condition is determined according to the purpose.

Next, as shown in FIG. 6C, the unreacted -SiH group of the silicone polymer 12 is reacted with the hydrophobic group R having a double bond to form a hydrophilic -SiR group 14. Since this method of making hydrophilic-hydrophobic makes hydrophilic first, its hydrophilicity is high and protein adsorption is small. The hydrophilization reaction is desirably performed in water, and the silica gel covered with the silicone polymer is water repellent, and the reaction proceeds from the outer surface of the pores. Therefore, it is considered that the outer surface of the pores is relatively more hydrophilic than the inner surface of the pores. It is considered that hydrophobization occurs relatively more inside than outside.

Simultaneous Treatment Method for Making Hydrophobic and Hydrophilic The filler according to this example can also be produced by a method in which the porous carrier coated with a silicone polymer is made hydrophilic and hydrophobic at the same time. That is, FIG. 7 (A)
As shown in, the surface of the porous carrier 10 is coated with the silicone polymer 12. Then, as shown in FIG. 7 (B), the remaining —SiH group of the silicone polymer 12,
A hydrophobic group R having a double bond at the terminal and a hydrophilic group R ′ having a double bond at the terminal (shown by ○ -in the figure) are reacted to give a hydrophilic —SiR group 14 and a hydrophobic —SiR. 'Set as base 16.

This method of simultaneously treating hydrophobicity and hydrophilicity has the advantage that the reaction may be carried out once. A compound having a relatively hydrophobic group (such as styrene) is replaced with a hydrophilic compound (tetraol,
It is possible to obtain the intended filler by reducing the amount of addition thereof than (polyol, etc.) and by varying the reactivity (the compound of the hydrophobic group is not bulkier than the compound of the hydrophilic group and is considered to be higher than the reactivity). it can. As a reaction solvent, alcohol (styrene and tetraol can be mixed at the same time) is desirable.

Hydrophobization-Epoxidation-Hydrophilicization Method The filler according to the present example is a method in which a porous carrier coated with a silicone polymer is first hydrophobized and then an epoxy compound having an epoxy group is introduced. Then, a hydrophilic group is bonded to the terminal epoxy group to make it hydrophilic. That is, as shown in FIG. 8A, the surface of the porous carrier 10 is coated with the silicone polymer 12 by the method described above.

Then, as shown in FIG. 8B, the —SiH group remaining in the silicone polymer 12 is reacted with the hydrophobic group R having a double bond to form the —SiR group 14. In addition, all of the silicone polymer 12-
If the SiH group becomes a -SiR group, the subsequent introduction of the hydrophilic group cannot be carried out. Therefore, the addition amount of the hydrophobic group R or the reaction condition is determined according to the purpose.

Next, as shown in FIG. 8C, the unreacted -SiH group of the silicone polymer 12 is reacted with the epoxy compound 18 having a double bond and an epoxy group. Therefore, the epoxy compound is bonded to the silicone polymer at the double bond end and has an epoxy group. Next, FIG.
As shown in (D), a hydrophilic group (indicated by a circle in the figure) 20 is reacted to form a hydrophilic SiR ′ group 16.

In this method of making hydrophobic-epoxidation-hydrophilic, after introducing a hydrophobic group first, a method such as allyl glycidyl ether (double bond at one end and epoxy group at the other end) is first prepared. It is possible to increase the hydrophilization density by introducing a group that is not bulkier than tetraol and further reacting it with glycerin or diglycerin.
Therefore, the protein recovery rate is high. For example, first introduce a hydrophobic group (phenyl group), then allyl glycidyl ether (having a double bond at one end,
Preferably having an epoxy group at the other end) to form an -OH group such as diglycerin, glycerin or the like.
Those having a hydrophilic group such as a COOH group are bonded to the epoxy group.

The reaction between the hydrophobic group and the terminal double bond such as allyl glycidyl ether and the Si-H group is carried out by using platinum acid or the like as a catalyst (hydrosilylation).
In addition, Lewis acids, quaternary ammonium salts, tertiary amines and the like are used for the reaction of the epoxy group with diglycerin and the like.
Incidentally, after the addition of allyl glycidyl ether, the epoxy ring may be opened in an acidic solution to form the diol type.

FIG. 9 shows an experimental example in which the above-mentioned column packing material was packed in the concentration columns 30 and 40 and an analysis process was performed. FIG. 9 (A) shows an experimental example in which the conventional column packing material is packed in the concentration columns 30 and 40 and an analysis process is performed, and FIGS. 9 (B) and 9 (C) show the above-mentioned book. The experimental example which packed the column packing material which concerns on an Example in the concentration columns 30 and 40, and performed the analysis process is shown.

Further, the experimental result of FIG. 9B shows the result in which the amount of the sample to be measured introduced was 20 microliters, the concentration was 1/20, and the concentration time was 3 minutes. The experimental results show that the amount of the sample to be measured introduced was 80 microliters, the concentration was 1/80, and the concentration time was 3 minutes. Further, in FIGS. 9A to 9B, the peak of the analysis object is indicated by S in the figure.

As is clear from the figure, the peak of the analytical object was found in the experimental example in which the analysis treatment was carried out using the column packing material according to the present embodiment as compared with the conventionally used column packing material. It can be seen that (indicated by S) appears steeply, and the peaks (indicated by P) due to the components (protein, etc.) not retained are reduced. This is because the column packing material according to the present example does not adsorb proteins and the like because the outer surface of the column packing material is partly hydrophilic and is excellent in separation ability. Further, as is clear from the above description, since the column packing material according to the present example does not use an enzyme or the like, it is possible to realize a stable concentration treatment also by this, and therefore it is considered that good analysis results can be obtained. To be

[0093]

As described above, according to the present invention, the inner volume of the concentration column is less than 2.0 ml and the inner volume is small. Therefore, since the concentration process can be performed reliably, the detection accuracy can be improved.

The column packing material packed in the concentration column is composed of Si-R (R is a hydrophobic group) bond and Si-R bond.
It consists of a porous carrier coated with a silicone polymer having R '(R' is a hydrophilic group) bond, and the column packing of this structure has a part of its outer surface hydrophilic, so that proteins and the like are adsorbed. In addition, since it is stable, and is excellent in separation ability, it is possible to reliably perform the concentration treatment. Furthermore, since no enzyme or the like is used, a stable concentration treatment can be realized also by this.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram of a liquid chromatography apparatus that is a first embodiment of the present invention.

FIG. 2 is a schematic configuration diagram for explaining the operation of the liquid chromatography apparatus that is the first embodiment of the present invention.

FIG. 3 is a schematic configuration diagram for explaining the operation of the liquid chromatography device that is the first embodiment of the present invention.

FIG. 4 is a schematic configuration diagram of a liquid chromatography apparatus that is a second embodiment of the present invention.

FIG. 5 is an explanatory diagram of a process of making a column packing material hydrophobic-hydrophilic for use in a liquid chromatography apparatus which is an embodiment of the present invention.

FIG. 6 is an explanatory diagram of a process for making a column packing material hydrophobic-hydrophilic for use in the liquid chromatography apparatus which is an embodiment of the present invention.

FIG. 7 is a process explanatory diagram of a simultaneous hydrophilic treatment method for a column packing used in a liquid chromatography apparatus which is an embodiment of the present invention.

FIG. 8 is a process explanatory diagram of hydrophobicization-epoxidation-hydrophilicity of a column packing material used in the liquid chromatography apparatus which is one example of the present invention.

FIG. 9 is a diagram for explaining the effect of the present invention.

[Explanation of symbols]

 11, 25, 45A, 45B Pump 14 First switching valve 14x Sample injection port 15 Syringe 15a Joint 15b Flow path 16,43 Separation column 17,44 Detector 18 Waste liquid reservoir 21 Sample holding mechanism 21a Sample container 22 Sample injection tube 23 Second switching valve 30, 40 Separation column 41 Autosampler 42 Switching valve

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Osamu Shirota 1050 Shinba-cho, Kohoku-ku, Yokohama-shi, Kanagawa Stock company Shiseido Research Institute (72) Inventor Yu Otsu 1050, Shinba-cho, Kohoku-ku, Yokohama-shi, Kanagawa Stock company Shiseido Research Center (72) Inventor Michihiro Yamaguchi 1050 Shinba-cho, Kohoku-ku, Yokohama-shi, Kanagawa Shiseido Research Institute Co., Ltd. (72) Inventor Hisao Tsuruta 8-47, Fukakusa-Nishiura-cho, Fushimi-ku, Kyoto Prefecture Kyoto Medical equipment Within the corporation

Claims (3)

[Claims] 1. A separation column for separating a sample to be measured mixed in a solvent supplied by a pump, the separation column being disposed between the pump and the separation column, and being supplied with the solvent from the pump. While supplying the sample to be measured from the sample injection pipe and supplying the sample to be measured together with the solvent to the separation column, the sample to be measured separated by the separation column is supplied. A detection means for analyzing the supplied sample to be measured, a sample holding mechanism carrying a plurality of containers storing the sample to be measured, and selectively selecting the sample to be measured from one of the plurality of containers. Concentration for collecting the sample to be collected and supplying it to the flow path control means, and for concentrating the sample to be measured which is provided between the flow path control means and the separation column and is supplied together with the solvent. And a column for The content of the column for use is less than 2.0 ml, and the Si-R (R is a hydrophobic group) bond,
And a column packing material composed of a porous carrier coated with a silicone polymer having a Si-R '(R' is a hydrophilic group) bond, the liquid chromatography apparatus being configured. 2. A hydrophilic group R ′ in the column packing material.
The liquid chromatography apparatus according to claim 1, wherein the is a hydrophilic group having a hydroxyl group. 3. The hydrophobic group R in the column packing is
3. The liquid chromatography device according to claim 1, wherein the hydrocarbon residue has 1 to 18 carbon atoms.