JPH05133946A - Method of separation amino acids - Google Patents

Abstract

PURPOSE:To enable separation of multi-component amino acid efficiently at a high speed without shortening the life of a filler and the apparatus by using a styrene-divinyl benzene copolymer with higher pressure resistance and with a low column pressure and by causing an eluent pass at such a linear speed as to improve separation performance most. CONSTITUTION:A fine particulate styrene-divinyl benzene copolymer which is obtained by the copolymerization of styrene and m-divinyl benzene of D- divinyl benzene at a mixing ratio of below 20%vol. of an isomer, for example, is swelled by tetrachloroethane and sulfonated by action of chlorosulfuric acid to obtain an St-DVB-based ion exchange resin with excellent pressure resistance. The particle size of the exchange resin is preferably about 1-10mum in terms of uniformity of packing, a column pressure and the like. Thus, when an eluent passes through a column packed with the exchange resin at a linear speed of 0.1-0.2cm/sec, multi-component amino acid can be separated efficiently at a high speed.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for separating amino acids, and more particularly, a method for separating amino acids suitable for efficiently and rapidly separating amino acid components in a sample by using a polymer packing having a low column pressure. Regarding

[0002]

2. Description of the Related Art Conventionally, as a polymer packing material for liquid chromatography for separating multi-component amino acids with high performance,
Ion exchange resins in which an ion exchange group is added to a styrene-divinylbenzene copolymer are widely used. The styrene-divinylbenzene copolymer is obtained by suspension-polymerizing styrene and divinylbenzene in an aqueous solution. As divinylbenzene which is a crosslinking agent monomer, about 40% by weight of m-divinylbenzene and about 20% of p-divinylbenzene are used. %, M-ethylvinylbenzene of about 30% by weight, p-ethylvinylbenzene of about 10% by weight, and the like are used as a mixture. In Japanese Patent Laid-Open No. 54-26396,
A mixture of 55% by weight divinylbenzene, 40% by weight ethylvinylbenzene and 5% by weight of other saturated compounds has been used.

In recent years, high-performance separation and high-speed separation have been required in the field of liquid chromatography, and along with this, the particle size of column packing and the flow rate of eluent have been increased. However, the separation column filled with a polymer filler has the disadvantages of higher column pressure and lower pressure resistance than a column filled with a filler such as silica gel, and the conventional styrene-divinylbenzene copolymer system ion When the exchange resin is used as the column packing material, the pores due to the three-dimensional network structure of the packing material are compressed, the life of the packing material is shortened, and the column pressure is increased, which imposes a burden on the device, particularly the liquid delivery pump. There was a big problem.

[0004]

In view of the above problems, the present inventors have proposed a method for synthesizing a styrene-divinylbenzene copolymer having a low column pressure and a packing material for liquid chromatography using the same (special feature: (Heihei 3-2753).
An object of the present invention is to provide a method for separating an amino acid component, which can efficiently separate a multi-component amino acid at high speed using the styrene-divinylbenzene copolymer having a low column pressure.

[0005]

The present invention provides an ion exchange resin obtained by sulfonation of a copolymer of styrene and an isomer having a mixing ratio of 20% by volume or less and divinylbenzene (hereinafter referred to as St- Separation of amino acids in a sample by passing an eluent at a linear velocity of 0.1 to 0.2 cm / sec through a separation column packed with a DVB-based ion exchange resin) Regarding the method.

The St-DVB type ion exchange resin used in the present invention has a mixing ratio of styrene and isomers of 20% by volume.
A fine particle styrene-divinylbenzene copolymer obtained by copolymerizing the following m-divinylbenzene or p-divinylbenzene is swollen with, for example, tetrachloroethane and then sulfonated by the action of chlorosulfuric acid. The ion exchange resin obtained by
It has an ion exchange capacity of / g or more.

The copolymerization of styrene-divinylbenzene copolymer is carried out, for example, in an aqueous solution in which a water-soluble polymer such as polyvinyl alcohol is dissolved as a particle size adjusting agent and a surfactant such as sodium dodecyl sulfate is dissolved as an anti-adhesion agent. , Styrene, divinylbenzene and polymerization initiator,
It is carried out by a suspension polymerization method. In order to obtain a finely divided copolymer having excellent separation performance, the monomer concentration is 100 g or less per 1 liter of water, and the water-soluble polymer concentration is 5.0.
It is preferable that the stirring temperature is set to x10 ^-2 g / ml or more, the stirring speed is set to 1,500 rpm or more, and the polymerization temperature is set to 50 to 100 ° C. As the polymerization initiator, a peroxide such as benzoyl peroxide or tert-butyl peroxide, or an azo compound such as azobisisobutylnitrile or azobiscyclohexanenitrile is used. Usually, 1 wt% or more is added to the monomer. It The degree of crosslinking of the monomers in the copolymer varies depending on the components to be separated, but when used for high-speed separation of amino acids, it is preferably about 5 to 30%.

Since the copolymer thus obtained uses divinylbenzene, which is substantially free of isomers, it will cross-link monomers having almost the same structure, and thus a copolymer having a uniform pore structure will be obtained. A combined product is obtained, and by adding an ion exchange group thereto, an ion exchange resin having a low column inlet pressure and excellent pressure resistance can be obtained. Although the column efficiency (HETP) is improved as the particle size of the ion-exchange resin is smaller, it is preferably 1 to 10 μm, more preferably 2 to 6 μm from the viewpoint of packing uniformity, column pressure and the like. ..

The inner diameter and the length of the column used in the present invention are not particularly limited, but the inner diameter is preferably in the range of 2 to 7 mm, and the length is usually 60 mm or more. The conditions for packing the ion-exchange resin in the column differ depending on the packed resin, but the eluent used for separation is 100 to 200 kg.
It is preferable to perform packing at a column pressure of / cm ² for about 1 hour.

In the present invention, the linear velocity of 0.1 to 0.
It is necessary to pass the eluent at 2 cm / sec. At linear velocities outside this range, high-speed separation, reduction in column pressure, and improvement in separation performance cannot be achieved.

[0011]

EXAMPLES The present invention will be described in detail below with reference to examples. Example 1 Styrene and m-divinylbenzene were subjected to suspension polymerization in an aqueous solution in the presence of polyvinyl alcohol (particle size adjusting agent), sodium dodecyl sulfate (particle adhesion preventing agent), and benzoyl peroxide (polymerization initiator). Then, a copolymer having a degree of crosslinking of m-divinylbenzene of 20% was obtained, and the copolymer was sulfonated to obtain an St-DVB ion exchange resin having a particle size of 3 μm.

This ion exchange resin was packed in a stainless steel column having an inner diameter of 4.6 mm and a length of 60 mm at a pressure of 150 kg / cm ² with an eluent having a Li ⁺ concentration of 1.00 M at pH 4.1. The column pressure of the obtained separation column was pH 2.
8. Li ⁺ concentration 0.09 M, ethanol concentration 3 wt% eluent flow rate 0.35 ml / min (linear velocity 0.12 cm / se
60kg / cm2 when flowing through the column kept at 38 ℃ in c)
^{Was 2} .

Using this separation column, the column temperature is 57 ° C.
The column pressure when the eluent having a pH of 2.8, a Li ⁺ concentration of 0.09 M, and an ethanol concentration of 3% by weight was flowed while changing the linear velocity was measured under the conditions. The result is shown by a circle in FIG. When the linear velocity is 0.1 cm / sec, the column pressure is 4
When the linear velocity was 0 kg / cm ² and the linear velocity was 0.2 cm / sec, the column pressure was 80 kg / cm ² , but when the linear velocity was higher than this, the column pressure rapidly increased.

Further, the column efficiency with respect to the linear velocity (HET
P) was investigated, and the result is shown in FIG.
HETP is constant in the range of up to 0.2 cm / sec,
Since HETP sharply rises at linear velocities exceeding 0.2 cm / sec, it was found that efficient linear separation of amino acids is possible with linear velocities in the range of 0.1 to 0.2 cm / sec.

Comparative Example 1 A St-DVB ion exchange resin was prepared in the same manner as in Example 1 except that commercially available divinylbenzene in which m-divinylbenzene and p-divinylbenzene were mixed was used. After synthesizing and using this, a separation column was prepared and the column pressure was measured by the same method.
^{Was 2} .

Further, using this separation column, the relationship between the linear velocity of the eluent and the column pressure was investigated in the same manner as in Example 1, and the results are shown by the triangle marks in FIG. At a linear velocity of 0.1 cm / sec, the column pressure was 110 kg / cm ² , and it was found that the column pressure increased sharply as the linear velocity increased.

Example 2 Using the separation column prepared in Example 1, high-speed separation of 39 components of biological fluid amino acid was carried out for 80 minutes, and its chromatogram is shown in FIG. 3. It is possible to separate all 39 components. did it. The flow velocity of the eluent at this time is 0.17 cm / linear velocity
In sec, the column pressure was 84 kg / cm ² . Also, detection was carried out at wavelengths of 440 nm and 570 nm by ninhydrin color reaction.

The column temperature starts from 38 ° C., 32 ° C. after 2.8 minutes, 60 ° C. after 14.4 minutes, 4 ° C. after 23.1 minutes.
3 ° C, 50 ° C after 28 minutes, 43 ° C after 29.4 minutes, 3
70 ° C. after 3.6 minutes, 40 ° C. after 66.2 minutes, 65.8
After 70 minutes, the temperature was changed to 70 ° C. The pH of the eluent is 2.
Start at 8 and set to 3.0 after 12.6 minutes, gradually increase to 3.1 by 21 minutes, then 21.1 minutes to 3.6, 2
3.7 after 9.5 minutes, 3.6, 49.1 after 33.7 minutes
It was changed to 3.3 after 5 minutes, 4.1 after 53.6 minutes, and 4.1 after 70.1 minutes.

The Li ⁺ concentration starts from 0.09M and is 1
After 2.6 minutes, the concentration was changed to 0.123M, and the concentration was gradually increased from 12.6 minutes to 0.140M in 21 minutes.
0.239M after 2 minutes, 0.255M after 29.5 minutes, 3
0.721M after 3.7 minutes, 0.454 after 49.1 minutes
M, 1.0 after 53.6 minutes, 0.85 after 60.6 minutes
It was changed to 0M and 1.0M after 70.1 minutes.

The ethanol concentration (% by weight) starts from 3%, 10% after 33.7 minutes, 1.8% after 49.1 minutes,
0% after 53.6 minutes, 0.6% after 60.6 minutes, 70.
It was changed to 0% after 1 minute.

Comparative Example 3 Using the separation column prepared in Comparative Example 1, high-speed separation of biofluid amino acids was carried out in the same manner as in Example 2.
The column pressure became 300 kg / cm ² or more, which exceeded the pressure limit of the apparatus, and analysis was impossible.

[0022]

According to the separation method of the present invention, a styrene-divinylbenzene copolymer having a low column pressure and a high pressure resistance is used, and an eluent is passed through at a linear velocity that can most improve the separation performance. Therefore, it is possible to perform high-performance, high-speed separation of multi-component amino acids without shortening the life of the filler and the device.

[Brief description of drawings]

FIG. 1 is a diagram showing a relationship between an eluent linear velocity and a column pressure.

FIG. 2 is a diagram showing a relationship between an eluent linear velocity and HETP.

FIG. 3 is a diagram showing a chromatogram obtained by rapid separation of 39 amino acid components by the method of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C07C 229/02 8930-4H G01N 30/48 M 8506-2J 30/88 F 8506-2J // C07B 63/00 F 7419-4H

Claims (1)

[Claims] 1. A mixing ratio of styrene and an isomer is 20% by volume.
The amino acid component in the sample is separated by passing the eluent at a linear velocity of 0.1 to 0.2 cm / sec through a separation column filled with an ion exchange resin obtained by sulfonation of a copolymer with divinylbenzene A method for separating amino acids, which comprises: