JP3355393B2 - Capillaries with coated inner walls - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a capillary on which a polymer is adsorbed and a method for producing the same.

[0002]

BACKGROUND OF THE INVENTION [0002] Capillary electrophoresis (hereinafter sometimes referred to as CE) is a general term for electrophoresis performed in a capillary having an inner diameter of 100 µm or less. CE has an ultra-high resolution (100,000 to 100,000 theoretical plates)
Stage), high-speed analysis, minute sample volume (several nl), and using a water-based solvent. Utilizing these features, CE is used in various fields such as measurement of drugs, foods, and environmental substances. Among them, protein analysis is one of the fields that may be further developed by CE in the future. However, it is considered that hydrophobic silicon and polar silanol groups are present on the inner wall of the capillary. Therefore, in protein analysis by CE, ionic and hydrophobic interactions between the solute and the inner wall of the capillary are considered. Often, tailing of peaks or irreversible adsorption of solutes on the wall is observed. As a solution, capillaries whose inner walls are coated with a hydrophilic substance are provided.

[0003]

The capillaries coated on the inner wall known at present have many problems such as poor stability, lot difference, insufficient adsorption prevention ability, and high price. Therefore, further techniques for solving these problems are desired.

[0004]

SUMMARY OF THE INVENTION The present invention relates to a capillary in which a ionic polymer having a positive charge and an ionic polymer having a negative charge are alternately adsorbed on the inner wall of the capillary.
Absorbs the ionic polymer having a positive charge several times.
At least one negatively charged ionic polymer.
Capillary adsorbed alternately an ionic polymer having a capillary, characterized in that the times adsorbed, the ionic polymer and the negative charge in a positive charge on the inner wall of the capillary
The ionic polymer having a positive charge several times
Adsorb and carry at least a negatively charged ionic polymer.
Capillary characterized in that it is once adsorbed and the innermost layer is a positively charged ionic polymer, and the ionic polymer having a positive charge and the ionic polymer having a negative charge are alternately adsorbed on the inner wall of the capillary.
Adsorbs positively charged ionic polymer multiple times
Ionic polymer having a negative charge at least once
A capillary , wherein the inner layer is a ionic polymer having a negative charge, and a ionic polymer having a positive charge and an ionic polymer having a negative charge are alternately adsorbed on the inner wall of the capillary.
To adsorb a positively charged ionic polymer multiple times,
Adsorb ionic polymer with negative charge at least once
A method for producing a capillary, wherein before the ionic polymer having a positive charge is adsorbed, the capillary is first rinsed with a strong alkaline solution. .

[0005] The capillary according to the present invention means a capillary for capillary electrophoresis, a capillary used for connection of gas chromatography / mass spectrometry, and the like. Furthermore, infusion analysis of mass spectrometry, capillary electrophoresis / mass spectrometry Application to metrology is also possible. The material of the capillary is usually fused silica glass, and the inner diameter is usually 50μ ~
100μ.

The ionic polymer having a positive charge in the present invention means a polymer having a positive charge dissolved in water or an aqueous organic solvent such as methanol, ethanol, acetonitrile and the like, and specifically, polybrene, chitosan, diethylaminoethyl and the like. Dextran, polyethyleneimine and the like can be mentioned. These substances are usually used alone, but may be used in combination. Also, different materials can be used for each layer.

The ionic polymer having a negative charge in the present invention means a polymer having a negative charge which is dissolved in water or an aqueous organic solvent such as methanol, ethanol, acetonitrile and the like. Specifically, dextran sulfate, heparin, heparin Sulfuric acid, hyaluronic acid, chondroitin sulfate, keratan sulfate, polygalacturonic acid, alginic acid,
Teicuronic acid and the like can be mentioned. These substances are usually used alone, but may be used in combination. Also, different materials can be used for each layer.

In the capillary according to the present invention, when the ionic polymer having a positive charge and the ionic polymer having a negative charge are alternately and adsorbed the same number of times, the innermost layer (the layer in contact with the electrophoresis running solution) has a negative charge. An ionic polymer,
When the number of times of adsorption of the ionic polymer having a positive charge is one more than that of the ionic polymer having a negative charge, the innermost layer is an ionic polymer having a positive charge. Usually, a positively charged ionic polymer and a negatively charged ionic polymer are alternately coated one by one, or a positively charged ionic polymer, a negatively charged ionic polymer, and then a positively charged ionic polymer. The polymer is alternately coated, but both layers can be alternately coated more than once.

In order to produce the capillary according to the present invention, for example, when the ionic polymer having a positive charge and the ionic polymer having a negative charge are alternately provided once each, first, the capillary is washed with a surfactant for cleaning laboratory equipment. Agent, followed by rinsing with 1N sodium hydroxide, rinsing with water, then rinsing with a low concentration of ionic polymer with a positive charge, rinsing with a high concentration of ionic polymer with a positive charge, and immediately By rinsing with an ionic polymer having a negative charge of A capillary having two or more layers of ionic polymer adsorbed thereon can be produced by repeating the above procedure except that rinsing with 1N sodium hydroxide and subsequent water is not performed. Rinsing with 1N sodium hydroxide can also be performed with another strong alkaline solution such as potassium hydroxide.

The concentration of the low-concentration ionic polymer having a positive charge differs depending on the type of the ionic polymer and cannot be determined unconditionally. However, in general, the concentration of the electroosmotic flow does not largely depend on the concentration of the ionic polymer. Of polybrene, for example, 0.1 to 0.5%, preferably about 0.4%. The high concentration of ionic polymer having a positive charge means a high concentration within a concentration range in which the electroosmotic flow does not largely depend on the concentration of the ionic polymer.For example, in the case of polybrene, 3% to 10% is preferable. Is between 5% and 10%.

The concentration of the ionic polymer having a high concentration of negative charge is preferably as high as possible so that it can be dissolved in a solvent. However, in general, when the concentration is high, the viscosity of the solvent increases and it becomes difficult to rinse. It is determined by the balance between the two, and is usually 1% to 5%, for example, about 2% for dextran sulfate and about 3% for heparin.

[0012]

As described above, the capillary which has not been treated with the inner wall has a silanol group on the inner wall, so that the electroosmotic flow depends on the dissociation of the silanol group. As the pH of the electrophoresis liquid decreases, the dissociation of silanol groups is suppressed and the electroosmotic flow decreases, and almost stops at pH 3 or less (Fig. 1, □). The capillary according to the present invention has an electroosmotic flow independent of the pH of the electrophoresis solution when, for example, dextran sulfate is coated on the innermost layer (FIG. 1, mark ●). On the other hand, in a commercially available coat capillary represented by an LPA coat capillary, the electroosmotic flow is suppressed at all pHs (FIG. 1, FIG.
Triangle). Therefore, the inner wall untreated capillary and L
The essential difference between the PA-coated capillary and the capillary according to the present invention is that, in the present example, the capillary according to the present invention has an electroosmotic flow from the anode side to the cathode side under acidic conditions. A remarkable effect is obtained as shown in the following experimental examples (in FIG. 1, the mark ◇ is a capillary coated with polybrene, which is a positively charged ionic polymer. The effective length of the capillary is 20 cm).

Further, by coating the ionic polymer having a positive charge and the ionic polymer having a negative charge a plurality of times, there is a remarkable effect that the service life (useful life) of the capillary is significantly increased. In addition, capillaries coated multiple times have improved chemical stability in all of acidic solutions, alkaline solutions and aqueous organic solvents. That is, even if these solvents are used, the coating does not easily peel off. In addition, capillaries coated multiple times have a high theoretical plate number, and exhibit a remarkable effect especially in the analysis of basic proteins.

Experimental Example 1 Capillary zone electrophoresis under acidic conditions Capillary zone electrophoresis under acidic conditions (capil
Figure 2 shows an example of lary zone electrophoresis (hereinafter abbreviated as CZE).
Shown in Since the electrophoresis running solution has a pH of 2.75, it is considered that the electroosmotic flow is almost stopped in the untreated capillary. As the analytical sample, quinine having a positive charge at pH 2.75, p-toluenesulfonic acid having a negative charge, and neutral phenol were used. In CZE using the untreated capillary, only quinine having a positive charge was detected (FIG. 2, upper panel). Neutral phenol and negatively charged p-toluenesulfonic acid were not detected because the electroosmotic flow was suppressed. three
In the capillary obtained in Example 1, all peaks were detected due to generation of electroosmotic flow (lower figure in FIG. 2). As is clear from the above, the use of the capillary according to Reference Example 1 enabled simultaneous analysis of a positive charge, a negative charge, and a neutral compound under acidic conditions, which was conventionally impossible.

Experimental Example 2 Micellar electrokinetic chromatography under acidic conditions Micellar electrokinetic chromatography
Tic chromatography (hereinafter abbreviated as MEKC) is a mode that enables separation of a compound without charge by adding ionic micelles to a running solution. MEKC under acidic conditions generates a slight electroosmotic flow because SDS is adsorbed on the inner wall of the capillary. A sample with a large capacity ratio (k ') is detected on the anode side, a small sample is detected on the cathode side,
Samples with mobility equal to the reverse direction of the electroosmotic flow are not detected from either. Therefore, simultaneous analysis is not possible,
Alternatively, a significantly longer analysis time is required for possible samples. FIG. 3 shows an experimental example of MEKC under acidic conditions (pH 2.75) using the capillary obtained in Reference Example 1 . Analysis was performed using phenol, 3,5-xylenol and 2-naphthol for the sample, and simultaneous analysis was achieved in a short time. That is, by using the capillary according to Reference Example 1 , simultaneous analysis of neutral compounds under acidic conditions can be performed by ME.
It could be done in a short time with KC.

Experimental Example 3 Measurement of pKa In the measurement of pKa using a conventional capillary with an untreated inner wall, it was difficult to measure a compound having a low pKa value. In other words, since the electroosmotic flow of the untreated capillary becomes smaller as the pH decreases, the electroosmotic flow eventually stops.Therefore, capillary electrophoresis, which calculates the pKa from the difference between the sample and the electroosmotic flow, generates an error in the acidic region. I was Therefore, constant electroosmotic flow occurs even under acidic conditions
PKa measurement was performed using the capillary obtained in Reference Example 1 . Tryptophan was used as a sample, and compared with the values of Ishihama et al. And the literature values. The results are shown in Table 1 (in the table, ML
coated refers to the capillary according to Reference Example 1 , and uncoated
ed means uncoated capillary used as control).

[0017]

[Table 1]

As is clear from the table, the measured values using Reference Example 1 were in good agreement with the literature values. The electropherogram at pH 3.10 is shown in FIG. The electroosmotic flow was detected after 20 minutes in the untreated capillary of the inner wall (upper figure in FIG. 4), but was detected within 4 minutes in the capillary according to Reference Example 1 (lower figure in FIG. 4). In addition, in the capillary according to Reference Example 1 , the peak of the electroosmotic flow is detected with good reproducibility at substantially the same time even at a pH of 3.10 or less, so that the pKa measurement of a compound having a lower pKa value can be performed more accurately. it is conceivable that. In addition, as is apparent from FIG. 4, the capillary according to Reference Example 1 enabled shortening of the analysis time under acidic conditions.

Experimental Example 4 The durability test in continuous analysis was performed using the capillary obtained in Example 1 . Evaluation was made by measuring the electroosmotic flow at pH 3. As a result, the capillary coated with one layer of polybrene used as a control peeled off the coating approximately 20 times, whereas the capillary according to Example 1 was stable 593 times. . This is probably because the ionic interaction between heparin and polybrene is stronger than the ionic interaction between silanol groups on the inner wall of the capillary and polybrene.

[0020]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the present invention is limited thereto.

Reference Example 1 First , in order to remove dirt from the inner wall of the capillary and completely dissociate the silanol groups,
Using a rinsing function of a capillary electrophoresis apparatus (Beckman P / ACE2100), rinsing was performed with 1NNaOH for 30 minutes, and then with water for 15 minutes. Next, it was rinsed with a 0.4% polybrene aqueous solution for 30 minutes and left overnight. After standing overnight, confirm that the electroosmotic flow (eo) has been reversed using a marker (formamide diluent), rinse the 5% aqueous polybrene solution for 5 minutes, and immediately rinse the 2% aqueous dextran sulfate solution for 10 minutes. By doing so, a capillary was manufactured.

Example 1 First , in order to remove dirt from the inner wall of the capillary and completely dissociate the silanol groups, a surfactant for washing laboratory equipment was used for 10 minutes using a rinsing function of a capillary electrophoresis apparatus (Beckman P / ACE2100). , 1N
Rinse with NaOH for 30 minutes, then with water for 15 minutes. Next, a capillary according to the present invention was manufactured by rinsing for 15 minutes in the order of a 10% aqueous solution of polybrene, a 3% aqueous solution of heparin III (purchased from Wako Co., Ltd.), and a 10% aqueous solution of polybrene.

[0023]

[Brief description of the drawings]

FIG. 1 is a graph showing the electroosmotic flow of an untreated inner wall capillary and an inner wall coated capillary. ML means the capillary according to the present invention, uncoated means the uncoated capillary used as a control

FIG. 2 is a diagram showing capillary zone electrophoresis under acidic conditions. MLcoated means the capillary according to the present invention, and uncoated means the uncoated capillary used as a control. The upper right figure shows a state without electroosmotic flow, and the lower right figure shows that electroosmotic flow is flowing in the direction of the arrow.

FIG. 3 is a diagram showing micellar electrokinetic chromatography under acidic conditions. MLcoated means the capillary according to the present invention

FIG. 4 is a diagram showing pKa measurement of tryptophan by capillary electrophoresis. MLcoated refers to the capillary according to the invention, uncoated refers to the uncoated capillary used as a control.

Continuation of front page (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 27/447

Claims (7)

(57) [Claims] 1. A capillary having a positively charged ionic polymer and a negatively charged ionic polymer alternately adsorbed on the inner wall of the capillary, the capillary having a positive charge.
Negatively charged ions that adsorb on-polymer multiple times
A capillary, wherein a hydrophilic polymer is adsorbed at least once . 2. A capillary in which a ionic polymer having a positive charge and an ionic polymer having a negative charge are alternately adsorbed on the inner wall of the capillary.
Negatively charged ions that adsorb on-polymer multiple times
A capillary which adsorbs a hydrophilic polymer at least once, and wherein the innermost layer is an ionic polymer having a positive charge. 3. A capillary in which a ionic polymer having a positive charge and an ionic polymer having a negative charge are alternately adsorbed on the inner wall of the capillary.
Negatively charged ions that adsorb on-polymer multiple times
A capillary which adsorbs a hydrophilic polymer at least once, and wherein the innermost layer is a negatively charged ionic polymer. 4. The ionic polymer having a positive charge is polybrene, chitosan, diethylaminoethyldextran or polyethyleneimine.
Capillary according to item. 5. The method according to claim 1, wherein the ionic polymer having a negative charge is dextran sulfate, heparin, heparin sulfate, hyaluronic acid, chondroitin sulfate, keratan sulfate, polygalacturonic acid, alginic acid or teichronic acid.
3. The capillary according to any one of 3. 6. A capillary in which a ionic polymer having a positive charge and an ionic polymer having a negative charge are alternately adsorbed on the inner wall of the capillary.
Negatively charged ions that adsorb on-polymer multiple times
A method for producing a capillary, comprising adsorbing a hydrophilic polymer at least once . 7. The method for producing a capillary according to claim 6, wherein the ionic polymer having a positive charge is first rinsed with a strong alkaline solution before the ionic polymer is adsorbed.