JPS58210563A - Analysis of strongly acidic amino-acid and its apparatus - Google Patents

Abstract

PURPOSE:To perform readily qualitative and quantitative determination of strongly acidic amino-acid in a sample, by passing the sample containing amino acid to go then with buffer through strongly basic anion exchange resin, and making the effluent develope color or fluorescence. CONSTITUTION:A sample containing amino-acid such as deproteinated blood sample is injected from a sample-introducing part 2 of a septum value and the like, and passed with buffer 12 through a column 3 which is filled with strongly basic anion exchange resin. Strongly acidic amino-acids are held by the column 3, and then eluted gradually, thus makes it sufficiently possible to isolated and quantify cysteic acid, cysteinesulifinic acid, which have not been isolated and quantified satisfactorilly so far. Each amino-acid component leaving the column 3 is mixed with fluorescece emitting agent 52 or the like at a mixing part 6, and its fluorescence emitting intensity and the like are determined with fluorescence photometer 7 or the like. Thus it becomes possible to analyse successively a sample containing strongly acidic amino-acids.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method and apparatus for analyzing strongly acidic amino acids. More specifically, the present invention relates to a simple method and apparatus for separating and analyzing strongly acidic amino acids, which have been difficult to separate and analyze in the past.

Conventionally, an amino acid analysis method is known in which an amino acid-containing sample is passed through a cation exchange resin column together with a buffer and then subjected to absorption or fluorescence analysis.

However, in such a method, strongly acidic amino acids such as taurine were hardly retained on the column #1, making it difficult to separate them.

Regarding this point, there is a proposal to retain strongly acidic amino acids on the column by adjusting the buffer to a pH of 2 or less, but in this case, other amino acids are retained even more strongly on the column and remain even after analysis. /I/ Potash washing and long-term column aging (7μ After washing with potash, it is necessary to flow an acidic buffer through the force ram for a certain period of time (processing), making it particularly unsuitable for continuous analysis.

This invention has been made to solve these problems, and is based on the strong acidic amino acid M'f! : Simple sweeping j The purpose of the present invention is to provide a strongly acidic amino acid analysis method that allows accurate separation and continuous analysis without further post-processing, and an analytical device suitable for carrying out the method.

The inventors of this invention discovered that by using an anion exchange resin, which is the complete opposite, in place of the cation exchange resin that is common in conventional amino acid analysis, strongly acidic amino acids can be
aII! After discovering that it is possible to separate the fucolumn without the need for post-treatment, we have reached this goal.

Thus, the present invention is characterized in that the amino acid-containing sample is passed through an anion exchange ladle together with a buffer, and then the effluent is colored or fluoresced to determine the quality or quantification of strongly acidic amino acids in the amino acid-containing sample. A method for analyzing strongly acidic amino acids is provided. Further, a buffer supply flow path connecting the sample introduction part tme to the anion exchange resin column, an effluent tTL path extending to the anion exchange resin column, an effluent flow path and a color former or fluorescent agent supply flow are provided. A strongly acidic amino acid analyzer is provided, which is comprised of a mixing section that connects to the mixing section, and an absorption or fluorometer that is connected to the mixing section.

The anion exchange resin used in the method of this invention is preferably a strongly basic anion exchange resin. Specifically, a resin in which a quaternary ammonium group is chemically bonded to a styrene-divinylbenzene 11 carrier is preferable. Can be mentioned. In addition, as the buffer for supplying the amino acid-containing sample to the column, known acidic to strongly acidic buffers such as phosphate,
Buffers such as citrate and acetate can be applied as is.

On the other hand, the liquid flowing out from the column is sequentially treated to develop color or fluoresce, preferably every 7 lactations, and the intensity of the color or fluorescence is continuously measured using an absorptiometer or fluorometer. The amino acids can be qualitatively or quantitatively determined. For the above-mentioned coloring treatment, it is appropriate to add a certain amount of a solution containing a coloring agent capable of coloring amino acids; for example, a well-known ninhydrin solution is used. on the other hand,
Similarly, for fluorescence treatment, it is appropriate to add a certain amount of a post-label fluorescence reagent to the effluent.

The most preferred fluorescent reagent is an ortho-phthalaldehyde (hereinafter referred to as oph) solution. It has been known to use OPA solutions for amino acid analysis, but in the conventional method it was necessary to extremely increase the salt concentration and pH of the OPA solution buffer.
A solution, for example F'! , a salt concentration of about 0.3 to 0.5 mol and a pH of about 9 to 10.5 can be used.

According to the method of the present invention, for example, cysteine, acid, and hiscysteine μfinic acid, which are particularly difficult to separate among strongly acidic amino acids, can be separated.

Therefore, by paralleling conventional amino acid analysis,
Qualitative or quantitative determination of various amino acids is possible.

The apparatus of the present invention will be described in detail below according to the accompanying drawings.

explain.

FIG. 1 L1 is a configuration explanatory diagram showing an example of a strongly acidic amino acid analyzer of the present invention. In the figure, the device invented by Jin is
A buffer supply channel (1) equipped with a sample introduction part (2) such as a septum or sampler and connected to an anion exchange resin column (3), and an effluent channel (4) extending to the column (3).
), a coil flow path-shaped mixing section connecting the flow channel and the fluorescent agent supply channel (5), and a fluorescence measurement device equipped with a flose/l' connected to the mixing section. The above-mentioned anion exchange resin column (3) is a strong base anion exchange resin in which a quaternary ammonium compound is chemically added to a simple substance of polystyrene-boriginyl μbenzene. Combined charger, light-adjusted power 2m (technique 8A
-07152504; manufactured by Shimadzu Corporation). In addition, (12) indicates a buffer consisting of 0.05M KH, PO, solution, and the supply amount is 0.5ml/i
Adashi, (52) ti O,08960 represents a fluorescent agent consisting of a boric acid-sodium oxalate solution containing PA, and the amount supplied is 0.3 d1 min. Also, (11) and (5
x) indicates a metering pump and C'il) indicates a recorder.

In the above configuration, an amino acid-containing sample obtained by removing protein from blood, for example, is injected from the sample introduction part (2) and carried to the column (3) K together with the buffer (12). In the column (3), the basic, neutral, and acidic amino acids in the sample first reach the sample and quickly flow out in this order, but the strong acidic amino acids are retained in the L column and gradually elute, so that each strong acidic amino acid is It separates and flows out with other amino acids with sufficient retention time. Next, each of these separated phases is sufficiently fluoresced by OPA in the mixing section (6), and then sent to a fluorometer (7) where the fluorescence intensity of each is measured, and based on this value and retention time, strong acid Each component of the amino acid is qualitatively and/or quantitatively determined. The column is maintained under suitable acidic conditions throughout the analysis, and there is no need for post-treatment such as alkaline washing of the column even in continuous analysis.

As shown in the above specific examples, it can be seen that according to the method and apparatus of the present invention, strongly acidic amino acids can be easily separated and analyzed.

FIG. 2 shows an analysis example (chromatogram) of an amino acid mixture containing cystic acid and cysteine sulfinic acid using the apparatus of the present invention. As described above, it can be seen that according to the method and apparatus of the present invention, strong acidic amino acids can be separated well. In addition, in the figure, A means taurine, B means cysteine sulfinic acid, and C means cystic acid. The equipment used was as shown in the specific example, and the conditions were as follows: mobile layer: monopotassium phosphate;
Column solution (0.05M), column pressure: 4oKg/
crls reaction solution; 0.08960PA-containing buffer solution (flow rate fi 0.3rd/min), reaction coil; inner diameter 0.5mm x 2000mm, fluorescence photometer 1FLD-1
, column temperature: 50°C.

[Brief explanation of the drawing]

FIG. 1 is a configuration explanatory diagram showing an example of an apparatus suitable for implementing the strongly acidic amino acid analysis method of the present invention, and FIG. 2 is a chromatogram showing an example of measurement when Jin's invention tl- is applied. . (1)...Buffer supply channel, (2)...
・Sample introduction part, (3)... Anion exchange resin power 2
(4)...Effluent channel, (5)...Fluorescent agent supply channel, (Su...Mixing section, (7)...
...Fluorescence Oki 1 meter, (11) (51) ... Metering pump, (12) ... Buffer, (52) -
...Fluorescent agent, (71) ...Recorder. 3 Procedures rti, i, E 轡August 77, 1981 special r Device 3, relationship with the case of the person making the amendment Patent applicant address 3, Kawaramachi-dori Nijo-shita Roots Funato-cho, Nakagyo-ku, Kyoto City
Address 78 Name (199) Shimadzu Corporation Representative Setsu Yokoji Male 4, Agent 530 Address 1-3 Quarter One Building 6, 5-J Nishitenma, Kita-ku, Osaka, subject to amendment

Claims (1)

[Claims] (v) Qualitative or quantitative determination of strongly acidic amino acids in an amino acid-containing sample by passing the amino acid-containing sample together with a buffer through an anion exchange resin and causing the effluent to develop color or fluorescence. A strongly acidic amino acid analysis method characterized in that: (2) the method according to claim 1IJ4, wherein the anion exchange resin is a strongly basic anion exchange resin; (3) an anion exchange resin column provided with a sample introduction section; a buffer supply channel connected to the anion exchange resin column, an effluent channel extending to the anion exchange resin column, a mixing section connecting the effluent channel and the color former or fluorescent agent supply channel; A strongly acidic amino acid analyzer comprising a connected absorbance or fluorometer. The device according to item 3.