JPH09325130A - Method for analyzing anionic compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To analyze low concentration anionic compound having a high complex formation ability with metal by using, as a migration buffer liquid, an anionic material having a high complex formation ability to a metal contained in capillary. SOLUTION: White gold electrodes 14 and 15, cathode side and anode side respectively, of a high voltage generator 13 are immersed in buffer solution baths 11 and 12, respectively. A capillary 16 comprises a molten silica and a polyimide coated layer formed on its outside wall surface, and its start part and end part are submerged in the solution baths 11 and 12, respectively. The coating layer on the liquid bath 12 side of the capillary 16 is peeled off, for forming a detection part 20, and a UV detector is provided. In the solution baths 11 and 12, a buffer liquid having a high complex formation ability to the metal in silica such as 2, 6-pyridine dicarboxylic acid, nitrilotriacetic acid, etc., is held. Thus, when anionic compound sample having a high complex formation ability is analyzed, the buffer liquid and the metal in silica form a complex for masking, thus the sample does not form a complex together with the metal, high precision analysis becoming possible.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a capillary filled with an electrophoretic buffer solution, a high voltage is applied to both ends of the capillary, and a metal such as an organic acid and an inorganic ion introduced from one end of the capillary is applied. The present invention relates to an analytical method for analyzing an anionic compound having a high complex forming ability.

[0002]

2. Description of the Related Art As is well known, the capillary electrophoresis method using capillaries is an analytical means having the highest separation ability at present, for measuring a very small amount of sample of nanoliter (nl) to picoliter (pl). Is being watched as.

FIG. 9 is a block diagram of a conventional capillary electrophoresis apparatus. In the figure, 1 is a first buffer solution tank in which the migration buffer solution is stored, and 2 is a second buffer solution tank in which the migration buffer solution is stored.

The cathode 4 of the high voltage generator 3 is the first
It is immersed in the buffer solution tank 1, and the anode (anode) 5 is immersed in the second buffer solution tank 2. 6 is, for example, a capillary such as fused silica, the starting end of which is the first buffer tank 1,
The end portions are respectively dipped in the second buffer tank 2.

On the side of the second buffer tank 2 of the capillary 6,
A UV on-column detector 7 is provided as a detector.
In such a configuration, when the high voltage generator 3 applies a predetermined voltage to the anode 4 and the cathode 5, the migration buffer solution in the first buffer solution tank 2 passes through the capillary 6 by the electrophoretic force and the electroosmotic flow. And moves in the direction of the second buffer tank 2.

Here, when the sample is injected from the starting end of the capillary 6, it moves in the direction of the second buffer tank 2 by the electrophoretic force and the electroosmotic flow. The moving speed of the sample differs depending on the ionic radius and ionicity of the hydrated sample, and the sample moves in a band shape. As a result, the sample component moving in the capillary 6 has a moving time according to the difference in ionic radius and ionicity of the hydrated sample, which is detected by the UV on-column detector 7.

[0007]

In the capillary electrophoresis apparatus having the above-mentioned structure, when the sample is an anionic compound which does not have a strong UV absorbing property such as an organic acid or an inorganic anion, the sample is shown in FIG. 10 (a). As shown, electroosmotic flow (hereinafter, EOF
Since it runs in the opposite way, it will take time to analyze,
It could not be detected.

Therefore, in JP-A-6-94677, quaternary amines such as cetyltrimethylammonium bromide are added to an electrophoretic buffer such as phthalic acid, trimellitic acid and chromic acid, as shown in FIG. 10 (b). Thus, a method of reversing EOF is disclosed.

In such a method, the running buffer solution
The background is constant due to UV absorption,
When a substance that does not have strong UV absorption passes through it, the absorbance decreases and a negative peak appears. The signal of this negative peak is inverted and the positive peak is detected.

By the way, generally, the fused silica capillary 6 contains impurities. FIG.
It is a figure which shows the metal ion concentration in the fused silica capillary 6 measured by the inductively coupled plasma mass spectrometer (ICP-MS). From this figure, the fused silica capillary 6 has iron (Fe), Al (aluminum), Na (sodium), and Cr.
It can be seen that metals such as (chromium) and Mg (magnesium) are included as impurities.

Here, the above-mentioned phthalic acid, trimellitic acid, chromic acid or the like is used as the migration buffer, and the sample is an organic acid (eg, oxalic acid, citric acid, malic acid or ethylenediaminetetraacetic acid) or an inorganic anion. In the case of an anionic compound having a high complex-forming ability, such as iron and aluminum, which are contained as impurities in the fused silica of the capillary 6, when the sample concentration becomes a low efficiency of about 50 ppm or less, these anions are used. There is a problem that a sample cannot be analyzed because it forms a complex with an ionic compound.

The present invention has been made in view of the above problems, and an object thereof is to provide an analysis method and an analysis device for a low-concentration anionic compound having a strong complex-forming ability with a metal.

[0013]

The above problems can be solved by the following inventions. (1) The first invention A capillary is filled with an electrophoretic buffer solution, a high voltage is applied to both ends of the capillary, and an anionic compound having a high complex-forming ability with a metal introduced from one end of the capillary is formed. In the analysis method, the migration buffer solution is an anionic compound analysis method, which is an anionic substance having a high complex-forming ability with respect to the metal contained in the capillary.

Since the migration buffer is an anionic substance having a high complex-forming ability with respect to the metals contained in the capillaries, it strongly forms a complex with the metals contained in the capillaries and masks these metals. I will end up.

Therefore, even if the sample has a high complex-forming ability with the metal, the sample does not form a complex with the metal, and high-precision analysis is possible even when the sample has a low concentration. (2) Second invention In the first invention, the method for analyzing an anionic compound is such that the material of the capillaries is fused silica and the migration buffer is 2,6-pyridinedicarboxylic acid.

Metals contained in the fused silica used for the capillaries include iron and aluminum. On the other hand, 2,6-pyridinedicarboxylic acid is
It has a high degree of complex stability against aluminum etc.,
When used as a running buffer, these metals mask.

Therefore, even if the sample has a high complex-forming ability with the metal, the sample does not form a complex with the metal, and high-precision analysis is possible even if the sample has a low concentration. (3) Third invention In the first invention, the method for analyzing an anionic compound is such that the material of the capillary is fused silica and the migration buffer is nitrilotriacetic acid.

Metals contained in the fused silica capillary include iron, aluminum and the like. on the other hand,
Nitrilotriacetic acid has a high complex stability with respect to iron, aluminum, etc., and masks these metals when used as a running buffer.

Therefore, even if the sample has a high complex-forming ability with the metal, the sample does not form a complex with the metal, and high-precision analysis is possible even when the sample has a low concentration.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION Next, an embodiment of the present invention will be described with reference to the drawings. 1 is a configuration diagram of an electrophoretic device according to an embodiment of the present invention, and FIG. 2 is a cross-sectional view of a capillary in FIG.

In FIG. 1, 11 is a first buffer solution tank in which the migration buffer solution is stored, and 12 is a second buffer solution tank in which the migration buffer solution is stored.
It is a buffer tank. The cathode side platinum electrode 14 of the high voltage generator 13 is immersed in the first buffer solution tank 11, and the anode side platinum electrode 15 is immersed in the second buffer solution tank 12.

A capillary 16 is immersed in the first buffer solution tank 11 at the start end and in the second buffer solution tank 12 at the end. The capillary 16 of the present embodiment includes a fused silica 17 and a polyimide coating layer 18 formed on the outer wall surface of the fused silica 17.

On the side of the second buffer tank 12 of the capillary 16, the coating layer 18 is peeled off to form a detection section 20. The detector 20 is provided with a UV detector 21 including a UV and diode array detector.

There are 2,6 in the first and second buffer tanks 11,12.
-A running buffer such as pyridinedicarboxylic acid or nitrilotriacetic acid is stored. FIG. 3 is a diagram showing complex stability constants of a compound used as an organic acid or a migration buffer with respect to a metal. For example, it can be seen that 2,6-pyridinedicarboxylic acid has a high complex formation with iron, aluminum, sodium and magnesium contained in the fused silica 17.

Therefore, when a sample of anionic compound having a high complex forming ability such as an organic acid (for example, oxalic acid, citric acid, malic acid or ethylenediaminetetraacetic acid) or an inorganic anion is analyzed, a running buffer solution is used. A certain 2,6-pyridinedicarboxylic acid and a metal (iron, aluminum, sodium, magnesium) contained in the capillary 16 form a complex,
The metal contained in the capillary 16 is masked, the sample does not form a complex with the metal, and highly accurate analysis is possible even when the sample has a low concentration.

[0026]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Detailed specifications of the apparatus having the structure shown in FIG. 1 will be described below. (1) Capillary 16 75 μm inner diameter, 350 μm outer diameter, 80.5 cm total length, effective length (length from the first buffer tank 11 to the detection unit 20) 72 cm (2) Buffer 5 mM 2,6-pyridinedicarboxylic acid, 0.5mM cetyltrimethylammonium bromide pH5.6 (3) Applied voltage -25kV (4) Preconditioning Run the running buffer for 4 minutes (5) Temperature 20 ℃ (6) Sample injection method Using pressure method, 2 at 50mbar Injected for 2 seconds (7) UV detector 21 Detected by indirect absorption method at a measurement wavelength of 200 nm and a reference wavelength of 350 nm [Experiment 1] The results of analysis of the following organic acid standard solutions having organic acid anions are shown in FIG.

1. Chloride ion 2. Sulfate ion 3. Oxalate ion (oxalate) 4. Formate ion (formate) 5. Malate ion (malate) 6. Citrate ion (citrate) 7 Succinate ion 8. Pyruvate ion (pyruvate) 9. Acetate ion (acetate) 10. Lactate ion (lacate) 11. Phosphate ion (phosphate) 12. Pyroglutamate (pyroglutamate) As shown in FIG. , 2,6-pyridinedicarboxylic acid buffer, the reproducibility of migration time (relative standard deviation) was within 0.13%, and the reproducibility of area (peak area) (relative standard deviation) was 0.6 to 2.6. % Was very good. [Experiment 2] Using 2,6-pyridinedicarboxylic acid as a buffer solution, an organic acid in beer diluted 5-fold with water was analyzed, and the results are shown in FIG. Oxalate ions and citrate ions, which easily form complexes with iron and aluminum, can be analyzed. [Experiment 3] An inorganic anion solution having the following composition was analyzed, and the results are shown in FIG. 7.

. [0028] 1 chloride ion (Cl ^-) 2. sulfate ion (SO4 ^2-) 3. chlorate ion (ClO ^3-) 4. bromate ion (BrO ^3-) 5. chlorite ions (ClO ^{2 -} ) 6. Hypophosphite (PO ₂ ^3- ) 7. Iodite (IO ^3- ) 8. Phosphite (PO ₃ ^3- ) 9. Phosphate (PO ₄ ^3- ) This method Now, it has become possible to measure these inorganic anions. [Experiment 4] 2,6-pyridinedicarboxylic acid (PDC) was used as a running buffer, and phthalic acid and trimellitic acid, which are often used as conventional organic acids, were used. The results were shown in FIGS. 8 (a) and 8 (b).

When 2,6-pyridinedicarboxylic acid is used as the running buffer, all organic acid ions are detected as described in Experiment 1. On the other hand, when phthalic acid and trimellitic acid were used as the migration buffer, oxalate ion of peak No.3 and citrate ion of No.6 were not detected when the concentration of organic acid was 50 mg / l or less. Furthermore, the peak of malate ion of No. 5 also became smaller, confirming the effectiveness of 2,6-pyridinedicarboxylic acid.

[0030]

As described in detail above, according to the anionic compound analysis method of the first invention, an anionic substance having a high complex-forming ability with respect to the metal contained in the capillary in the migration buffer solution is used. By doing so, the migration buffer strongly forms a complex with the metal contained in the capillary and masks these metals.

Therefore, even if the sample has a high complex-forming ability with the metal, the sample does not form a complex with the metal, and high-precision analysis is possible even when the sample has a low concentration. According to the anionic compound analysis method of the second invention, in the first invention, the material of the capillary is fused silica,
The running buffer was 2,6-pyridinedicarboxylic acid. Metals contained in the fused silica used for the capillaries include iron and aluminum. On the other hand, 2,6
-Pyridinedicarboxylic acid has a high complex stability number against iron, aluminum, etc., and masks these metals.

Therefore, even if the sample has a high complex-forming ability with the metal, the sample does not form a complex with the metal, and high-precision analysis is possible even if the sample has a low concentration. According to the anionic compound analysis method of the third invention, in the first invention, the material of the capillary is fused silica,
The running buffer was nitrilotriacetic acid.

Metals contained in the fused silica capillary include iron, aluminum and the like. on the other hand,
Nitrilotriacetic acid has a high complex stability against iron, aluminum, etc. and masks it.

Therefore, even if the sample has a high complex-forming ability with the metal, the sample does not form a complex with the metal, and high-precision analysis is possible even if the sample has a low concentration.

[Brief description of drawings]

FIG. 1 is a configuration diagram of an electrophoretic device according to an embodiment of the present invention.

2 is a cross-sectional view of the capillary in FIG.

FIG. 3 is a diagram showing complex stability constants of an organic acid and a compound used as a migration buffer with respect to a metal.

FIG. 4 is a diagram showing the results of Experiment 1.

FIG. 5 is a diagram showing the results of Experiment 1.

FIG. 6 is a diagram showing the results of Experiment 2.

FIG. 7 is a diagram showing the results of Experiment 3.

FIG. 8 is a diagram showing the results of Experiment 4.

FIG. 9 is a configuration diagram of a conventional capillary electrophoresis device.

FIG. 10 is a diagram illustrating a problem of a conventional capillary electrophoresis device.

FIG. 11 is a diagram showing a metal ion concentration in a fused silica capillary.

[Explanation of symbols]

 11 First Buffer Tank 12 Second Buffer Tank 13 High Voltage Generator 14,15 Electrode 16 Capillary 20 Detector 21 UV Detector

Claims (3)

[Claims] 1. An anionic compound having a high complex-forming ability with respect to a metal introduced from one end of the capillary, wherein the capillary is filled with an electrophoretic buffer, and a high voltage is applied to both ends of the capillary. In the method for analysis, the migration buffer is an anionic substance having a high complex-forming ability with respect to the metal contained in the capillary, and the method for analyzing an anionic compound. 2. The method for analyzing an anionic compound according to claim 1, wherein the material of the capillaries is fused silica, and the migration buffer is 2,6-pyridinedicarboxylic acid. 3. The method for analyzing an anionic compound according to claim 1, wherein the material of the capillaries is fused silica and the migration buffer is nitrilotriacetic acid.