JPH09236580A - Medium for capillary electrophoresis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enhance the separability, reproducibility and reliability while suppressing the adsorption of a sample easily adsorbed on a capillary by adding polyvinylamine to a medium for capillary electrophoresis. SOLUTION: A medium for capillary electrophoresis contains polyvinylamine. Polyvinylamine is a polymer containing a polymerization unit (polymerization unit A) represented by formula I obtained by polymerizing vinylamine and may be a polymer also having a polymerization unit (polymerization unit B) represented by formula II. Concretely, a polymer obtained by partially or wholly hydrolyzing poly(N-vinylalkylamide) is designated. In the formula II, R is an alkyl group such as a methyl group, an ethyl group, an isopropyl group or a t-butyl group. The number of the polymerization units A in polyvinylamine is pref. 10-10000 and the number of the polymerization unit B is pref. 0-10000.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an optical isomer, serum, protein, amino acid,
The present invention relates to a capillary electrophoresis medium used as a filling liquid in a capillary and a sample preparation liquid when separating and analyzing sugars, nucleic acids and the like.

[0002]

2. Description of the Related Art Capillary electrophoresis is a method of performing electrophoresis in a microscopic area such as in a fused silica capillary having a diameter of 200 μm or less, as compared with a slab electrophoresis method which has been widely used in the past. Very little heat, diffusion and convection. Therefore, high-resolution separation can be performed in a short time, and the required sample is a very small amount of several hundreds nl or less, so that new separation of optical isomers, serum, proteins, amino acids, sugars, nucleic acids, etc. It is attracting attention as an analytical technique.

Further, in order to separate these measuring objects accurately, methyl cellulose (M.
Strege and A. Lagu, Anal. Chem., 63,1233 (1991)),
Hydroxypropyl methylcellulose (HE Schwartz
et al., J. Chromatogr., 559,267 (1991)), agarose (P. Bocek and A. Chrambach, Electrophoresis, 1
2,1059 (1991)) and other buffer solutions containing water-soluble polymers and surfactants, and capillaries filled with hydrogels such as polyacrylamide (AS Cohen et a
L., Proc. Natl. Acad. Sci. USA, 85, 9660 (1988)) is widely used.

[0004]

[Problems to be Solved by the Invention] However, in protein separation / analysis, since the material of the capillaries is silica, the sample is easily adsorbed on the inner wall of the capillaries, resulting in poor resolution, poor reproducibility and poor reliability. There is. Therefore, a technique has been proposed in which the inner wall of the capillary is coated with silane coupling-polyacrylamide to suppress adsorption of proteins and the like. But with this method,
There are problems that the coating operation is complicated and that it is difficult to obtain a coating that is sufficiently uniform to obtain a sharp peak.

Therefore, an object of the present invention is to provide a medium for capillary electrophoresis for suppressing the adsorption of a sample that is easily adsorbed on a capillary and improving the resolution, reproducibility and reliability.

[0006]

According to the present invention, there is provided a medium for capillary electrophoresis containing polyvinylamine.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The medium for capillary electrophoresis of the present invention contains polyvinylamine. The polyvinylamine referred to in the present invention is a polymer containing a polymerized unit represented by the following general formula (1) in which vinylamine is polymerized (hereinafter abbreviated as polymerized unit A), and represented by the following general formula (2). The polymer may also have a polymerized unit (hereinafter abbreviated as polymerized unit B). Specifically, poly (N-vinylalkylamide)
A polymer obtained by hydrolyzing a part or all of the above can be mentioned.

[0008]

Embedded image

R represents an alkyl group such as a methyl group, an ethyl group, an isopropyl group, or a t-butyl group. The number of polymerized units A in the polyvinylamine is preferably 10 to 10,000, and the number of polymerized units B is preferably 0 to 10,000.

The number of polymerized units B in the case of having polymerized units B: the number of polymerized units A is 1: 0.0001 to 1: 10,0.
00, preferably 1: 0.1 to 1: 100, more preferably 1: 1 to 1: 100.

The polyvinylamine has a molecular weight of 500.
~ 1,000,000, preferably 1,000-50
30,000, particularly preferably 10,000 to 100,000
Is desirable.

Polyvinylamine is prepared by, for example, polymerizing N-vinylalkylamides such as N-vinylacetylamide and N-vinylisopropylamide by radical polymerization, ionic polymerization, photopolymerization, radiation polymerization or the like to give polyamines. It can be easily obtained by obtaining (N-vinylalkylamide) and further hydrolyzing these.

The radical polymerization is carried out by water or alcohols such as methanol and ethanol, chloroform, methylene chloride, tetrahydrofuran, 1,4-dioxane,
In an organic solvent such as N, N-dimethylformamide, dimethylsulfoxide, acetonitrile or a mixed solvent of water and these organic solvents, under degassing conditions or under an inert gas atmosphere such as nitrogen, argon, carbon dioxide,
The reaction can be carried out in the presence of a radical polymerization initiator at −50 to 200 ° C., preferably 0 to 120 ° C. for 10 minutes to 200 hours, preferably 1 to 24 hours.

As the radical polymerization initiator, it is preferable to use an azo compound and / or an organic peroxide having a 10-hour half-life temperature of 160 ° C. or lower. Specifically, the azo compounds include 2-cyano-2-propylazoformamide, 1,1′-azobis (cyclohexane-1-carbonitrile), 2,2′-azobis (2-amidinopropane) dihydrochloric acid. Salt, 2,2'-azobis (2-methylbutyronitrile), 2,2'-azobisisobutyronitrile, 2,2'-azobis (2,4-dimethylvaleronitrile), 2,2'- Azobis (4-methoxy-2,4-
Dimethylvaleronitrile), 4,4'-azobis (4-
Cyanovaleric acid), dimethyl 2,2'-azobisisobutyrate, 2,2'-azobis (2- (5-methyl-2-imidazolin-2-yl) propane) dihydrochloride, 2,2'-
Azobis (2- (2-imidazolin-2-yl) propane) dihydrochloride, 2,2'-azobis (2- (2-imidazolin-2-yl) propane), 2,2'-azobis (2-methyl -N- (1,1-bis (hydroxymethyl) ethyl) propionamide), 2,2'-azobis (2-methyl-N- (2-hydroxyethyl) propionamide), 2,2'-azobisbutyi Rylamide dihydrate, 2,2'-azobis (2- (hydroxymethyl) propionitrile), 2,2'-azobis (2,4,4-
Trimethylpentane) and the like. As the organic peroxide, benzoyl peroxide, diisoprooyl oxydicarbonate, tert-butyl peroxy-2-ethylhexanoate, tert-butyl peroxypivalate, tert-butyl peroxydiisobutyrate,
Lauroyl peroxide, t-butyl peroxyacetate,
Examples include tert-peroxy octoate and tert-butyl peroxybenzoate. These can be used as a mixture. The amount of the radical polymerization initiator used is preferably 10 parts by weight or less, and particularly preferably 5 parts by weight or less with respect to 100 parts by weight of the polymerizable monomer as a raw material.

Hydrolysis of the poly (N-vinylalkylamide) is carried out by using an acid catalyst such as hydrochloric acid, sulfuric acid, tosylic acid, cation exchange resin, etc .; ammonia, triethylamine, sodium hydroxide, sodium carbonate, sodium hydrogen carbonate, Using a base catalyst such as anion exchange resin,
Water; alcohols such as methanol, ethanol, propanol, butanol; organic solvents such as acetonitrile, tetrahydrofuran, 1,4-dioxane and the like or a mixed solvent thereof, 0 to 120 ° C., preferably 20 to 10 ° C.
At 0 ° C, 1 minute to 200 hours, preferably 10 minutes to 2
It can be carried out by reacting for 4 hours. Also,
Using hydrazine as a catalyst, 50 to 100 ° C., 1 to 48
You may stir for time. Further, (C ₂ H ₅ ) ₃ O ⁺ BF ₄ ⁻ in an organic solvent such as chloroform, methylene chloride or benzene,
It can also be carried out by allowing poly (N-vinylalkylamide) to act on the poly (N-vinylalkylamide) at 0 to 60 ° C. for 30 minutes to 6 hours, and further by acting a weak base such as sodium hydrogen carbonate.

The content of polyvinylamine in the medium for capillary electrophoresis of the present invention varies depending on its molecular weight and hydrolysis rate and is not particularly limited, but it is generally 0.
0001-10 mg / ml, preferably 0.001-
It is preferably 0.1 mg / ml. A desired electroosmotic flow can be obtained by changing the addition amount within this range.

As a component other than polyvinylamine contained in the medium for capillary electrophoresis of the present invention, various buffers applicable to the medium for capillary electrophoresis can be used without limitation, as long as the effects of the present invention are not impaired. Generally, a pH buffer solution having a pH of 1 to 12, preferably 3 to 10, such as a phosphate buffer solution, a borate buffer solution, a citrate buffer solution, an acetate buffer solution, a carbonate buffer solution, or a Tris buffer solution is used together. be able to. In addition, polysaccharides such as dextran, hyaluronic acid and hydroxymethyl cellulose, synthetic polymers such as polyvinyl alcohol, polyethylene glycol and polyvinylpyrrolidone may be contained.

As the capillaries to be filled with the medium for capillary electrophoresis of the present invention, those commercially available for capillary electrophoresis or gas chromatography can be used without limitation. Regarding the size of the capillary, the inner diameter is usually 1 to 200 μm, preferably 50 to 1
00 μm, length is usually 1 cm to 10 m, preferably 10
Those having cm to 1 m can be preferably used. If the length is less than 1 cm, sufficient separation cannot be achieved, and 10
When it exceeds m, it is not preferable because it requires a long time for separation.

As a method of using the medium for capillary electrophoresis of the present invention, after the inside of the capillary, the electrode tank and the sample tank are filled with the medium, electrophoresis can be performed by conventionally known operations and conditions. .

The voltage during the electrophoresis is 1 to 30 k.
V, preferably 5 to 20 kV is desirable. As a method of injecting the sample, a conventionally known method such as a gravity method or a migration method can be used. The temperature during electrophoresis does not need to be specifically controlled, but it is preferable to maintain constant temperature conditions to obtain higher reproducibility, and to raise the temperature during electrophoresis to improve resolution and shorten measurement time. The temperature may be lowered or lowered.

The substance that can be electrophoresed using the medium for capillary electrophoresis of the present invention is a water-soluble or water-dispersible substance, and is not particularly limited, but among them, as a substance that exhibits excellent effects. Include a compound having a high pKa or a protein having a high pI value in an aqueous environment. In the prior art, these substances having a high pKa or pI were easily adsorbed because the inner wall of the capillary was negatively charged, and the reproducibility and reliability of the measurement results were reduced.

[0022]

EFFECT OF THE INVENTION Since the medium for capillary electrophoresis of the present invention contains polyvinylamine, it suppresses adsorption to the inner wall of the capillary even for a sample that is easily adsorbed on the capillary and is difficult to separate by capillary electrophoresis in the past. Gives excellent reproducibility. Further, polyvinylamine exhibits various interactions such as an electrical interaction, a hydrophobic interaction, a hydrogen bond or a molecular sieving effect with a test sample, so that excellent separation ability can be provided.
Therefore, highly reliable electrophoresis can be performed.

[0023]

The present invention will be described in more detail with reference to the following examples.
The present invention is not limited to these.

[0024]

[Synthesis Examples 1-1 to 1-4] 5 g (58.8 mmol) of N-vinylacetylamide (hereinafter abbreviated as NVA) was dissolved in 45 ml of distilled water, and the amount of VA-04 shown in Table 1 was dissolved therein.
4 (trade name, manufactured by Wako Pure Chemical Industries, Ltd., 2,2′-azobis (2- (2-imidazolin-2-yl) propane))
Was added, the mixture was degassed and sealed, and then polymerized at 60 ° C. for 24 hours. After completion of the reaction, reprecipitation was performed in methanol to obtain polyNVA. The molecular weight was determined using GPC (developing solvent; THF, standard sample; polymethylmethacrylate). The results are shown in Table 1.

[0025]

[Table 1]

[0026]

[Synthesis Examples 2-1 to 2-4] 3 g of polyNVA (molecular weight 100,000) obtained in Synthesis Example 1-4 was dissolved in 100 ml of 2N-HCl aqueous solution, and subjected to hydrolysis treatment under heating under reflux. 20 ml was sampled at every predetermined time shown in Table 2, ultrafiltered, and then freeze-dried to obtain a powdery sample.
The hydrolysis rate of the obtained sample was determined by ¹ H-NMR.
Table 2 shows the results.

[0027]

[Table 2]

[0028]

[Synthesis Examples 3-1 to 3-3] 1 g of the polyNVA obtained in Synthesis Examples 1-1 to 1-3 was respectively added to a 2N-HCl aqueous solution 30.
It was dissolved in ml and refluxed for 40 hours. After ultrafiltration, freeze-drying was performed to obtain polyvinylamine in powder form.

[0029]

Example 1 Polyvinylamine (molecular weight: about 50,100) obtained in Synthesis Example 2-4 was added to a 20 mM acetate buffer (p.
It was dissolved in H3.7) at a concentration of 0.01 mg / ml to obtain a medium for capillary electrophoresis. Capillary electrophoresis was performed using this medium under the following conditions. The results obtained are shown in FIG. The sample injection was performed at 25 mV for 3 seconds.

Specimen sample; Mixture of ribonuclease, myoglobin, cytochrome C, and lysozyme Electrophoresis conditions: Device: JASCO Corporation CE-900 capillary inner diameter; 100 μm capillary outer diameter; 375 μm capillary length; 65 cm Effective capillary length; 50 cm Applied voltage 26kV (400V / cm) temperature; 30 ± 0.5 ° C detector; UV (200nm)

[0031]

Comparative Example 1 Capillary electrophoresis was performed under the same conditions as in Example 1, using only 20 mM acetate buffer (pH 3.7). The obtained results are also shown in FIG.

[0032]

[Comparative Example 2] Similar to Example 1, except that Synthesis Example 1-
Capillary electrophoresis was performed using the poly NVA obtained in 4. The obtained results are also shown in FIG.

From the above results (FIG. 1), 1. With the conventional buffer alone, the sample (protein) was adsorbed on the inner wall of the capillary, and no peak was detected.
(Comparative Example 1). 2. When poly-NVA was used, the adsorption of the sample was suppressed, but the separation of the sample was insufficient. 3. On the other hand, in the system to which the polyvinylamine of the present invention was added, the adsorption of the specimen was suppressed and, at the same time, a high resolution was obtained. It is clear that.

[0034]

Reference Example 1 The polyNVA or polyvinylamine obtained in Synthesis Examples 1-4 and 2-1 to 2-4 were dissolved in an acetate buffer (20 mM, pH 3.7) to a concentration of 0.1 mg / ml. Then, the electroosmotic flow was measured using a 1% phenol solution as a measurement marker. The results are shown in FIG. In addition,
The sample injection was performed at 10 kV for 3 seconds. It is clear that the desired electroosmotic flow can be obtained by changing the hydrolysis rate of polyNVA as shown in FIG.

[0035]

[Reference Example 2] The polyvinylamines obtained in Synthesis Examples 3-1 to 3-3 and 2-4 were mixed with acetate buffer (20 mM, pH 3.
In 7), use the ones dissolved at various concentrations shown in the figure,
The electroosmotic flow was measured using a 1% phenol solution as a measurement marker. The results are shown in FIG. It is clear that the desired electroosmotic flow can be obtained by varying the polyNVA concentration and molecular weight as shown in FIG.

[0036]

Reference Example 3 The electroosmotic flow was measured with the polyvinylamine obtained in Synthesis Example 2-3 dissolved in buffer solutions having pH 3.7 and pH 8.6 at various concentrations shown in the figure. FIG. 4 shows the results. The electroosmotic flow decreased as the concentration increased, and the electroosmotic flow could be reversed in a wide range of pH 3.7 to 8.6. From the results of FIG. 4, it is clear that the desired electroosmotic flow can be obtained by changing the concentration.

From the results of Reference Examples 1 to 3, it is understood that the electroosmotic flow can be set to a desired value by using the medium for capillary electrophoresis of the present invention.

[Brief description of drawings]

1 (a), 1 (b) and 1 (c) are diagrams showing capillary electrophoresis charts in Example 1, Comparative Example 1 and Comparative Example 2, respectively.

FIG. 2 is a diagram showing the results of Reference Example 1.

FIG. 3 is a diagram showing the results of Reference Example 2.

FIG. 4 is a diagram showing the results of Reference Example 3.

Front page continuation (72) Inventor Mitsuru Akashi 2-14-6, Kotokujidai, Kagoshima City, Kagoshima Prefecture (72) Inventor Akio Kishida 3-8-2, Kotokujidai, Kagoshima City, Kagoshima Prefecture (72) Inventor, Yoshinobu Baba Hyogo 1-1-18-402 Tamon, Tarumi-ku, Kobe, Japan (72) Kenichi Ito 2-45-16-301 Arata, Kagoshima-shi, Kagoshima Prefecture (72) Shio Kimura 3-3-1 Karato, Kagoshima-shi, Kagoshima Prefecture (72) 72) Inventor Yasuhiko Inami 631-1 Funabiki, Kiyotake-cho, Miyazaki City, Miyazaki Prefecture

Claims (1)

[Claims] 1. A medium for capillary electrophoresis, which comprises polyvinylamine.