JPH02193068A - Chromatography separation method and chromatography apparatus - Google Patents

Abstract

PURPOSE:To enable a chromatography separation of sulfocysteine by using a separation column packed with a basic anion exchange resin and an acid eluate. CONSTITUTION:A separation column 5 packed with a strong basic anion exchange resin is arranged in a thermostatic cell 6. Solution tanks 1 and 2 hold citric acid buffers different in concentration of sodium and these buffers are sent to the column 5 singly or being mixed through a feed liquid pump 3 and an injector 4. A ninhydrine solution of a liquid tank 7 fed with a nitrogen gas 9 and a buffer of a liquid tank 8 are mixed in the same amount to be sent to a mixer 12. The mixture is converged with a column elution by the mixer 12 to which a reagent for amino acid color formation is fed beforehand and passes through a reaction coil 13 to detect absorbance with an absorption photometer 14. Then, a detection signal is processed with a data processor 15 and a chromatogram display and the results of quantitative calculation are outputted thereby enabling chromatography separation of sulfocysteine satisfactorily.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a chromatographic separation method and a chromatographic apparatus, and particularly to a method and apparatus suitable for separating sulfocystine from other components.

[Conventional technology]

Among the substances derived from cystine, which is a type of amino acid, cystic acid, derivatized carboxymethylcysteine, aminoethylcysteine, etc.

It has been separated and analyzed by chromatography using a mixture sample containing other amino acid components.

These cystine-derived components are analyzed by chromatography under the same conditions as those for ordinary amino acid samples.

That is, an eluent is supplied to a separation column packed with a strongly acidic cation exchange resin, a coloring reagent is mixed downstream of the separation column, and measurement is performed using an absorbance detector.

[Problem to be solved by the invention]

Substances related to cystic acid include cysteine sulfinic acid and sulfocystine, but sulfocystine could not be separated and detected using the conventional amino acid separation method described above.

It is an object of the present invention to provide a chromatography method capable of separating sulfocystine from other components.

Another object of the present invention is to provide a chromatographic separation method that can effectively separate cysteine sulfinic acid, cystic acid, and sulfocystine from each other even if they are contained in a sample.

Another object of the present invention is to provide a chromatographic separation method and an analytical device that can separate cysteine-related three components and other amino acid components together in a sample of a mixture of amino acid components.

[Means to solve the problem]

In the present invention, sulfocysteine is chromatographically separated using a separation column filled with a basic anion exchange resin and an acidic eluent.

(Function) Cystic acid, cysteine sulfinic acid and sulfocystine are represented by the following chemical formula.

Cystic acid N H2HOa S −
CHz -CH-COOH cysteine sulfinic acid
NH4 I HO25-CHx-CH-COOH Sulfocystine NH2 HOaS-3-CH2-CH-COOH Conventional amino acid separation methods use a cation exchange resin column, so the ion exchange resin has -8Oa- On the other hand, 18 Oa'' of the above-mentioned components is repelled and these components are eluted quickly. Therefore, it seems that separation was difficult.

The basic anion exchange resin used in the present invention is based on the sulfonic acid groups, etc. of the above-mentioned components, such as < -5Oa- and -8Oa-.
It works to capture anions such as z-. Therefore, separation is possible due to the ion exchange action on the surface of the ion exchange resin. On the other hand, when other amino acid components are placed in an acidic solution, the dissociation of -COOH is suppressed and -N H derived from the amino group is suppressed.
Because of the formation of s◆, the more acidic the eluent, the faster it elutes from the separation column. If elution conditions are appropriately selected, sulfocystine and cystic acid can be eluted without interference from other amino acid components.

〔Example〕

An embodiment of the present invention will be described with reference to FIGS. 1 to 3.

In FIG. 1, a stainless steel separation column 5 is disposed in a constant temperature bath 6 maintained at a predetermined temperature (for example, 50° C.).The separation column 5 has an inner diameter of 4.6 nm and a length of 8 nm.
A 0wa cylinder is filled with a strong basic anion exchange resin. This filler is an anion exchange resin in which quaternary ammonium is chemically bonded to a copolymer of styrene and divinylbenzene.
It is commercially available as "i3013-N".

In the eluent tank 1, the sodium concentration is O, OSmo Q
A first citrate buffer with a pH of 3.12 and a pH of 3.12 is contained in the eluent tank 2, and a second citrate buffer with a sodium concentration of 1.2 moQ/Q and a pH of 4.88 is contained in the eluent tank 2. Contains citrate buffer. The first and second buffer solutions are supplied alone or in a mixture at a rate of 0.25 mQ/wi by the liquid pump 3.
The liquid is sent to the separation column 5 via the injector 4 at a flow rate of n. An example of the composition of the citrate buffer is shown in Table 1.

Table 1 An amino acid coloring reagent is supplied to the mixer section 12 downstream of the separation column 5. Nitrogen gas 9 is supplied to the liquid tank 7.8 via a pressure reducing valve 10. The same amounts of the ninhydrin solution in the liquid tank 7 and the buffer solution in the liquid tank 8 are mixed, and the mixed liquid is sent to the mixer section 12 by the pump 11 at a flow rate of 0.3 mQ/win. In the mixer section 12, the column eluate and the coloring reagent solution are combined and mixed, and this mixed solution has a temperature of 1.
The colored reaction solution passes through the reaction coil 13 adjusted to 20°C, and passes through the flow cell of the spectrophotometer 14 at wavelength 5.
Absorbance at 70 nm is detected.

The detection signal is processed by the data processing device 15, and a chromatogram display and a quantitative calculation result of the component content are output.

Using the chromatographic apparatus for amino acid analysis shown in Figure 1, the ratio of mixing the second buffer to the first buffer shown in Table 1 was changed over time according to the gradient program shown in Figure 2, and cysteine sulfinic acid and cysteine An example of a chromatogram obtained by chromatographic separation of a mixture sample containing acid and sulfocysteine is shown by the solid line in FIG. The dashed chromatogram in FIG. 3 shows the separation of a standard amino acid mixture sample under the same elution conditions. Thus, sulfocystine elutes later than cysteine sulfinic acid and cystic acid and can be detected by one separation. A 1:1 mixture of the first buffer and the second buffer was supplied as the eluent for 5 to 15 minutes after the start of the analysis, and the pH of this mixture was 4.42, and the sodium concentration was 0. .64moQ/Q.

Next, the eluent used when the separation column is filled with a strongly basic anion exchange resin will be explained.

FIG. 4 is a diagram showing the separation of an amino acid mixture sample when sodium sulfate (NazSOa) is used as an eluent. The eluent used had a pH of 5.0 and a sodium sulfate concentration of 0.21110Q/Q and 0.511IoQ/Q. In FIG. 4, a is the peak of cysteine sulfinic acid, b is the peak of cystic acid, and C is the peak of sulfocystine.

df are the peaks of other common amino acid components.

The peaks a, b, and c become difficult to separate from other amino acid components as the sodium concentration increases.

Cystine sulfinic acid a, cystic acid.

In order to separate sulfocystine C from each other, it is preferable to use a citrate buffer as an eluent.

The citric acid buffer usually contains citric acid and sodium citrate, but may further contain sodium chloride.

FIGS. 5 and 6 show the retention times of each peak when a mixture sample containing three components a, b, and c was separated using a buffer solution consisting of citric acid and sodium citrate. FIG. 5 shows the case where the sodium concentration is low, and FIG. 6 shows the case where the sodium concentration is high. When the sodium concentration is 0.02 mol/n or higher, separation and elution of the three components can be achieved within one hour. When the sodium concentration is 0.02moQ/Q, the pH is 3.
The pH at 93°0.05moQ/Q is 3.87.0
．． The pH at 1rlo Q/Q is 3.80. The higher the sodium concentration, the shorter the elution time of the three components a, b, and Q, but it gradually becomes difficult to separate them from other amino acid components. The peak of sulfocystine C can be separated from other components if the sodium concentration is 1.0 mon/fi or less. A suitable sodium concentration in the eluent for separating and eluating the three components a, b, and a from other amino acid components in a mixture sample containing standard amino acids is 0.02 to 0.1 vaoQ/Q. eg in FIG. 6 are other amino acid components.

Figure 7 shows that the sodium concentration in the citrate buffer is 0.
．． a, b, a when keeping 1noQ/Q constant and replacing part of the sodium citrate with sodium chloride
It shows changes in the elution status of three components.

If the sodium concentration is constant, the elution time will remain approximately constant even if sodium chloride is increased. By the way, when the sodium chloride concentration is ON, the pH is 3.80, 0.025.
The pH in the case of N is 3.80.

pH in case of 0.050 N is 3.78, 0.075
The pH for N is 3.76.

FIG. 8 is a chromatogram obtained by applying the present invention using only the first citrate buffer shown in Table 1. The sulfocystine peak is eluted later than other components.

Figure 9 is a chromatogram shown for comparison with the present invention, in which a strongly acidic cation exchange resin was used as the separation column, and a citrate buffer with a sodium concentration of 0.1 mQ/Q was used as the eluent. This is what I got. Although a three-component mixture sample was used, cysteine sulfinic acid 8 elutes the slowest, and sulfocystine C is not separated from cysteic acid.

〔Effect of the invention〕

According to the present invention, sulfocystine, which has conventionally been considered difficult to separate from other components, can be successfully separated by chromatography.

[Brief explanation of the drawing]

FIG. 1 is a flow path configuration diagram of a liquid chromatograph apparatus for explaining an embodiment of the present invention, FIG. 2 is a diagram showing an example of a gradient program used in the embodiment of FIG. 1, and FIG. A diagram showing an example of a chromatogram obtained in the example of FIG. 1,
Figure 4 is a diagram for explaining the separation state when sodium sulfate is used as the eluent, and Figures 5 and 6 are diagrams for explaining the separation state when citrate buffer is used as the eluent. Figure 7 is a diagram showing the change in elution time when changing the sodium chloride concentration in the citrate buffer,
Figure 8 is a diagram showing an example of a chromatogram when a single citrate buffer is used, and Figure 9 is an example of a chromatogram analyzed using a cation exchange resin column for comparison with the present invention. FIG. 1.2... Buffer solution, 5... Separation column, 7... Ninhydrin solution, 12... Mixer section, 14... Photometer, a... Cystine sulfinic acid, b... Cystine Acid, C...Sulfocysteine. Figure Figure Elution ratio time (M,) Figure Elution time (min,) Figure Figure Elution time (in order) Figure Figure Sodium chloride (N) Figure Elution time (Kaku,)

Claims (1)

[Claims] 1. In a chromatography separation method in which an amino acid mixture sample is introduced into a separation column and an eluent is supplied to the separation column, a basic anion exchange resin is used in the separation column, and citric acid is used as the eluent. A chromatographic separation method characterized by separating sulfocysteine using a buffer solution. 2. The separation method according to claim 1, wherein the basic anion exchange resin is a copolymer of styrene and divinylbenzene bound to quaternary ammonium. . 3. The chromatographic separation method according to claim 1, wherein the citric acid buffer contains citric acid and sodium citrate. 4. The chromatographic separation method according to claim 1, wherein the citric acid buffer has a pH of 3.0 to 4.5. 5. The chromatographic separation method according to claim 1, characterized in that the concentration of the eluent is changed during elution. 6. A chromatography method for separating and eluting an amino acid mixture sample using a separation column, characterized in that the elution of sulfocysteine from the separation column is delayed compared to the elution of other amino acid components from the separation column. Separation method. 7. The chromatographic separation method according to claim 6, wherein the eluent supplied to the separation column is a buffer containing sodium citrate or a buffer containing sodium sulfate. 8. In the separation method according to claim 6, a citric acid buffer is supplied to the separation column, the sodium concentration in this buffer is set to 0.02 to 0.7 mol/l, and cysteine sulfinic acid, cysteic acid and sulfocysteine. 9. In a method of chromatographically separating a sample containing cysteic acid and sulfocysteine using a separation column, the separation column is filled with a basic anion exchange resin and the eluent is acidified. Characteristic chromatographic separation method. 10. In the separation method according to claim 9, the eluent supplied to the separation column has a sodium concentration of 0.02.
A chromatographic separation method characterized in that the concentration is ~1.0 mol/l. 11. A separation column filled with a basic anion exchange resin, and a device for supplying a citrate buffer to the separation column;
A sample introduction section provided on the upstream side of the separation column, a reagent supply device that supplies a reagent for coloring amino acid components to a flow path downstream of the separation column, and a detector for detecting the colored amino acid components. Chromatography equipment equipped with.