JP2912232B2 - Analysis method for anionic compounds - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method in which a capillary is filled with an electrophoresis buffer, 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. The present invention relates to an analysis method for analyzing an anionic compound having a high complexing ability.

[0002]

2. Description of the Related Art As is well known, the capillary electrophoresis method using a capillary is intended to measure a very small amount of a sample, such as nanoliter (nl) to picoliter (pl), and is an analytical means having the highest separation capability at present. It is attracting attention.

FIG. 9 is a configuration diagram of a conventional capillary electrophoresis apparatus. In the drawing, reference numeral 1 denotes a first buffer tank in which an electrophoresis buffer is stored, and 2 denotes a second buffer tank in which an electrophoresis buffer is stored.

The cathode (cathode) 4 of the high voltage generator 3 is the first
The anode (anode) 5 is immersed in the second buffer solution tank 2 while being immersed in the buffer solution tank 1. Reference numeral 6 denotes a capillary made of, for example, fused silica, the starting end of which is in the first buffer solution tank 1,
The end portions are immersed in the second buffer solution tank 2, respectively.

[0005] On the second buffer solution tank 2 side 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 electrophoresis buffer in the first buffer solution tank 2 passes through the capillary 6 by electrophoretic force and electroosmotic flow. To the second buffer tank 2 direction.

Here, when a sample is injected from the beginning of the capillary 6, the sample moves in the direction of the second buffer tank 2 by electrophoretic force and electroosmotic flow. The sample moves at different speeds depending on the ionic radius and ionicity of the hydrated sample, and the sample moves in a band. As a result, the sample component moving through the capillary 6 has a movement time corresponding to the difference in ionic radius and ionicity of the hydrated sample, and this is detected by the UV on-column detector 7.

[0007]

In the capillary electrophoresis apparatus having the above structure, when the sample is an anionic compound which does not have strong UV absorption, such as an organic acid or an inorganic anion, etc., FIG. As shown, electroosmotic flow (hereinafter, EOF)
Electrophoresis, the analysis takes longer,
Could not be detected.

In Japanese Patent Application Laid-Open No. Hei 6-94677, a quaternary amine such as cetyltrimethylammonium bromide is added to an electrophoresis buffer such as phthalic acid, trimellitic acid or chromic acid, as shown in FIG. Thus, a method of inverting the EOF is disclosed.

In such a method, the electrophoresis buffer
The background is constant due to UV absorption,
When an object that does not have strong UV absorption passes, the absorbance decreases and a negative peak occurs. The signal of this negative peak is inverted and detected as a positive peak.

In general, the fused silica capillary 6 contains impurities. FIG.
FIG. 3 is a diagram showing a metal ion concentration in a fused silica capillary 6 measured by an inductively coupled plasma mass spectrometer (ICP-MS). From this figure, the fused silica capillary 6 contains iron (Fe), Al (aluminum), Na (sodium), Cr
It can be seen that metals such as (chromium) and Mg (magnesium) are contained as impurities.

Here, the above-mentioned phthalic acid, trimellitic acid, chromic acid or the like is used as the electrophoresis buffer, and the sample is made of an organic acid (for example, oxalic acid, citric acid, malic acid or ethylenediaminetetraacetic acid) or an inorganic anion. In the case of an anionic compound having a high complexing ability, such as an iron compound, a metal such as iron or aluminum contained as an impurity in the fused silica of the capillary 6 when the sample concentration becomes low, about 50 ppm or less, There is a problem that a sample cannot be analyzed by forming a complex with the ionic compound.

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

[0013]

The above object can be attained by the following invention. (1) First invention A capillary is filled with an electrophoresis buffer solution, a high voltage is applied to both ends of the capillary, and a metal introduced from one end of the capillary and an anionic compound having a high complex-forming ability are used. In the method for performing an analysis, it is a method for analyzing an anionic compound in which the electrophoresis buffer is an anionic substance having a high complexing ability with respect to a metal contained in the capillary.

Since the electrophoresis buffer is an anionic substance having a high ability to form a complex with the metal contained in the capillary, it forms a strong complex with the metal contained in the capillary and masks these metals. I will.

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

Metals contained in the fused silica used for the capillary include iron, aluminum and the like. On the other hand, 2,6-pyridinedicarboxylic acid is used for these iron,
It has a high degree of complex stability with respect to aluminum, etc.
The use of the electrophoresis buffer masks these metals.

Therefore, even if the sample has a high ability to form a complex with a metal, the sample does not form a complex with the metal, and high-precision analysis can be performed even at a low concentration of the sample. (3) Third invention In the first invention, the method for analyzing an anionic compound in which the material of the capillary is fused silica and the electrophoresis buffer is nitrilotriacetic acid.

The 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 and the like, and masks these metals when used in an electrophoresis buffer.

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

[0020]

Next, an embodiment of the present invention will be described with reference to the drawings. FIG. 1 is a configuration diagram of an electrophoresis apparatus according to one embodiment of the present invention, and FIG. 2 is a cross-sectional view of the capillary in FIG.

In FIG. 1, reference numeral 11 denotes a first buffer tank in which an electrophoresis buffer is stored, and 12 denotes a second buffer in which an electrophoresis buffer is stored.
This is a buffer tank. The platinum electrode 14 on the cathode side of the high-voltage generator 13 is immersed in the first buffer solution tank 11, and the platinum electrode 15 on the anode side 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 end. The capillary 16 according to 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 capillary 16 on the side of the second buffer solution tank 12, the coating layer 18 is peeled off, and a detection section 20 is formed. The detection unit 20 is provided with a UV detector 21 including a UV and a diode array detector.

The first and second buffer tanks 11 and 12 contain 2,6
-An electrophoresis buffer such as pyridinedicarboxylic acid or nitrilotriacetic acid is stored. FIG. 3 is a diagram showing a complex stability constant of a compound used for an organic acid or an electrophoresis 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 analyzing a sample of an organic acid (for example, oxalic acid, citric acid, malic acid or ethylenediaminetetraacetic acid) or an anionic compound having a high complexing ability such as an inorganic anion, the analysis buffer 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 high-precision analysis is possible even when the sample has a low concentration.

[0026]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The detailed specifications of the apparatus having the structure shown in FIG. 1 will be described below. (1) Capillary 16 inner diameter 75 μm, outer diameter 350 μm, total length 80.5 cm, effective length (length from first buffer solution tank 11 to detection unit 20) 72 cm (2) Buffer solution 5 mM 2,6-pyridinedicarboxylic acid, 0.5 mM cetyltrimethylammonium bromide pH 5.6 (3) Applied voltage -25 kV (4) Preconditioning Run electrophoresis buffer for 4 minutes (5) Temperature 20 ° C (6) Sample injection method Injection for 2 seconds (7) UV detector 21 Detected by the indirect absorbance method at a measurement wavelength of 200 nm and a reference wavelength of 350 nm [Experiment 1] The results of analyzing an organic acid standard solution having the following organic acid anions are shown in FIG.

1. Chloride ion 2. Sulfate ion 3. Oxalate ion 4. Formate ion 4. Formate 5. Malate ion 6. Malate 6. Citrate ion 7. Succinate ion 8. Pyruvate 9. Acetate 10. Lactate 11. Phosphate 12. Pyroglutamate As shown in Fig. 5. When using 2,6-pyridinedicarboxylic acid buffer, the reproducibility of transfer time (relative standard deviation) is within 0.13%, and the reproducibility of area (peak area) (relative standard deviation) is 0.6 to 2.6. % Was very good. [Experiment 2] Using 2,6-pyridinedicarboxylic acid as a buffer, 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 a complex with iron or aluminum, can be analyzed. [Experiment 3] An inorganic anion solution having the following composition was analyzed, and the results are shown in FIG.

1. Chloride ion (Cl ⁻ ) 2. Sulfate ion (SO4 ²⁻ ) 3. Chlorate ion (ClO ³⁻ ) 4. Bromate ion (BrO ³⁻ ) 5. Chlorite ion (ClO ^{2 -)} 6. hypophosphite ion (PO ₂ ^3-) 7. iodate (IO ^3-) 8. phosphite ion (PO ₃ ^3-) 9. phosphate ion (PO ₄ ^3-) this method Now, these inorganic anions can be measured. [Experiment 4] The electrophoresis buffer used was 2,6-pyridinedicarboxylic acid (PDC) and phthalic acid and trimellitic acid, which are commonly used for organic acids. The test was carried out with a liquid, and the results are shown in FIGS.

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

[0030]

As described in detail above, according to the method for analyzing an anionic compound of the first invention, an anion substance having a high ability to form a complex with a metal contained in a capillary by using an electrophoresis buffer. As a result, the electrophoresis buffer strongly forms a complex with the metals contained in the capillary and masks these metals.

Therefore, even if the sample has a high ability to form a complex with a metal, the sample does not form a complex with the metal, and high-precision analysis can be performed even at a low concentration of the sample. According to the anionic compound analysis method of the second invention, in the first invention, the material of the capillary is a fused silica,
The running buffer was 2,6-pyridinedicarboxylic acid. Examples of the metal contained in the fused silica used for the capillary include iron, aluminum, and the like. Meanwhile, 2,6
-Pyridinedicarboxylic acid has a high complex stability to iron, aluminum and the like, and masks these metals.

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

The metals contained in the fused silica capillary include iron, aluminum and the like. on the other hand,
Nitrilotriacetic acid has a high degree of complex stability with respect to iron, aluminum and the like, and is masked.

Therefore, even if the sample has a high ability to form a complex with a metal, the sample does not form a complex with the metal, and high-precision analysis can be performed even at a low concentration of the sample.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of an electrophoresis apparatus according to one embodiment of the present invention.

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

FIG. 3 is a diagram showing a complex stability constant of a compound used for an organic acid or an electrophoresis 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 shows 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 apparatus.

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

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

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 11 1st buffer tank 12 2nd buffer tank 13 High voltage generator 14, 15 electrode 16 Capillary 20 Detector 21 UV detector

Claims (3)

(57) [Claims] 1. An anionic compound having a high ability to form a complex with a metal introduced from one end of the capillary by applying a high voltage to both ends of the capillary by filling the capillary with an electrophoresis buffer. A method for performing an analysis, wherein the electrophoresis buffer is an anionic substance having a high complexing ability with respect to a metal contained in the capillary. 2. The method according to claim 1, wherein the material of the capillary is fused silica, and the electrophoresis buffer is 2,6-pyridinedicarboxylic acid. 3. The method for analyzing an anionic compound according to claim 1, wherein the material of the capillary is fused silica, and the electrophoresis buffer is nitrilotriacetic acid.