JPS613062A - Amino acid analysis - Google Patents

Abstract

PURPOSE:To enable separation and analysis of protein-hydrolyzed amino acid and amino acid of biological liquid in a short time even with a single column, by converting the ion exchange resin to a lithium type from a sodium type using an acid liquid containing ammonium ion. CONSTITUTION:Conversion of the cation exchange resin can be done easily to a lithium type from a sodium type utilizing different nature of ammonia shown when in acid and alkali. In other words, the cation exchange resin is used in a sodium type to separate protein-hydrolyzed amino acid and then, is converted to an ammonium type by feeding an acid liquid containing ammonium ion (e.g. ammonium chloride and ammonium sulfide) to a column. Next, the column is fed with an alkaline liquid containing lithium ion (e.g. lithium hydroxide and lithium chloride) to convert the cation exchange resin to a lithium type and subsequently, amino acid of biological liquid is separated in the acid condition.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to an amino acid analysis method, and particularly to a method suitable for separating and analyzing both protein hydrolyzate amino acids and biological fluid amino acids.

[Background of the invention]

Amino acid analysis methods can be roughly divided into two methods depending on the measurement target: protein hydrolyzate amino acid analysis method (hereinafter referred to as H method) and biological fluid amino acid analysis method (hereinafter referred to as F method). The H method uses a Na dispersion cation exchange resin column and an elution buffer containing sodium ions (Na'') to analyze a protein-constituting amino acid sample that has been pre-hydrolyzed in about 0.5 to 1 hour. The F method uses an Ll-type cation exchange resin column and an elution buffer containing lithium ions (Li") to elute amino acids present in biological fluids such as blood and urine, or animal and plant tissue extracts. 2-4
Analyze by time.

These two analytical methods differ in the cation clusters contained in the elution buffer used. The type of cation in the separation column also differs accordingly. Since it is not easy to convert the cation type of the separation column in conventional amino acid analyzers, it is necessary to use an amino acid analyzer dedicated to the H method and F method, or to replace the dedicated separation columns for each. I was using it.

The present inventors attempted to use one separation column for both H method and F method separation analysis, but in order to change the ion exchange resin in the separation column from sodium type to lithium type,
It took 8 hours. As shown in Figure 2, this is due to the pressure of the buffer solution containing sodium ions in the cation exchange resin made of a copolymer of styrene and divinylbenzene in the separation column. This means that it takes a long time to change the cation exchange resin to a lithium type buffer and create conditions that allow the F method to be carried out. Note that the horizontal axis in FIG. 2 indicates the time after the start of conversion to proceed to the F method after the end of the H method.

[Purpose of the invention]

An object of the present invention is to provide an amino acid analysis method that allows conversion from protein hydrolyzate amino acid separation conditions to biological fluid amino acid separation conditions in a short time even with a single separation column. .

[Summary of the invention]

The present invention utilizes the fact that ammonia exhibits different properties when it is acidic and alkaline, and facilitates the conversion of a cation exchange resin from a sodium type to a lithium type. That is, in the present invention, after separating protein hydrolyzate amino acids using a cation exchange resin in the sodium form, an acidic solution containing ammonium ions is supplied to the separation column to convert the cation exchange resin into the ammonium form. , Next, an alkaline liquid containing lithium ions is supplied to the separation column to convert the cation exchange resin into a lithium type, and then biological fluid amino acids are separated in an acidic state.

[Embodiments of the invention]

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

FIG. 3 shows the overall flow path configuration of the device of this embodiment.

Amino acid elution buffer (la to ljl is 1011 depending on the type of amino acid analysis method (H method and F method above) and analysis stage
A liquid is selected from among them by a solenoid valve (2a to 2j) that automatically opens and closes, and the liquid is sent by a pump (5) via a manifold (3) and a sampler (4). The amino acid sample is injected from the sampler (4) into a stream of elution buffer;
After being pressurized by pump (5) K, it is sent to separation column (6).

Separation column (6) K with an inner diameter of 4 m and a length of 150 m+ is filled with a cation exchange resin with an ion exchange capacity of 2.22;'IJ equivalent/mt, and the injected amino acid sample is chromatographically separated into each component. At this time, the separation column (6) is kept at a predetermined temperature by a heat insulating tank (13)K provided as its jacket, and (12) is its circulating water temperature control device. The elution buffer containing amino acid components eluted from the separation column (6) is mixed with the reaction reagent for amino acid detection in the mixer (9), which is a confluence section, and then transferred to the reaction tank (10).
) Mixing of amino acids and reaction reagents.

As the reaction progresses, the reaction products are detected by the detector (11), and the quantitative analysis of amino acids continues. An example of the reaction reagent used here is ninhydrin reagent.

The pre-adjusted ninhydrin solution is stored in a storage tank (7) and pumped 9 to a mixer (9) by a pump (8). In this case, a visible spectrophotometer is selected as the detector (11), and by measuring the concentration of a colored reaction product between amino acids and ninhydrin, the amino acid concentration in the sample can be determined.

Table IK shows the contents of the elution buffers 1a to 1j in the above channel configuration. Elution buffers are selected in the order of 1a→1j depending on the stage of amino acid analysis.

Here, 1a to 1d are used for H method analysis, and 1g to 1j are used for F method analysis. The reason why multiple elution buffers are assigned to a single analytical method is that a stepwise method is adopted in which the elution buffer is switched midway through the chromatographic separation step in order to perform the chromatographic separation effectively. by.

In this example, a strongly acidic cation exchange resin made of polystyrene-divinylbenzene copolymer is used as the ion exchange resin packed in the separation column.

FIG. 1 will be explained with reference to Table 1. The horizontal axis in FIG. 1 indicates the time after the start of liquid conversion for proceeding with the F method after the end of the H method. In this example, the final buffer for component separation of method H (p
After the separation step with the H5,9 solution (e.g. ttrium citrate buffer), the ammonium chloride buffer
It is fed to a separation column (6). Hydrochloric acid or sulfuric acid is poured into this ammonium chloride buffer, and the pH is maintained at 6 or below, particularly preferably at an acidic level of I)H of 2 to 4. Substances that supply ammonium ions (NH4'') include ammonium chloride, ammonium sulfate, and trimethylamine.

Triethylamine and the like can be used.

In Figure 1, it takes about 20 minutes for the ion exchange resin to convert from the IJium form to the ammonium form, but this is when a 2M ammonium chloride buffer is used, and the concentration If the value is further increased, the exchange capacity will be increased, and the conversion to the ammonium form will be accelerated.

After flowing the ammonium chloride buffer for 30 minutes, the separation column (6) is fed with a lithium hydroxide solution at pH 14 for about 15 minutes. Also in this case, the higher the lithium ion concentration, the shorter the time for the ion exchange resin to convert from ammonia type to lithium type. Substances that can provide lithium ions under alkaline conditions include lithium chloride, lithium sulfate, etc. in addition to lithium hydroxide, and these are used together with lithium hydroxide. The ammonium ion expelling solution preferably has an alkaline pH of 8 or higher, and particularly preferably has a pH of 12 or higher to bring about good results for ammonium removal.

The ion exchange resin in the separation column is converted to lithium form by supplying lithium hydroxide solution, so F
Lithium citrate buffer for the method is supplied. After the separation column is stabilized, separation of the sample by method F can be started approximately 50 minutes after the completion of method H. The supply start time of the lithium hydroxide solution in FIG. 1 may be set earlier.

In this example, a stepwise eluent supply method is employed for the separation of H method and F method, but the present invention can also be applied to a gradient eluent supply method.

With this example device, only the H method analysis can be repeated. In this case, the buffer solutions shown in Table 1 (Xa to IC
) are sequentially supplied to separate the components, then the separation column is regenerated with the regenerating solution (1d), and the buffer solutions (la to lc) are flowed again.

On the other hand, when switching from H method to F method, regeneration liquid (1d)
is not supplied, and after the buffer solution (la-IC) is distributed, the buffer solution (1e) and ammonium expelling solution (1f) are supplied, and then the F method buffer solution (Ig ~ 11) is supplied to the separation column. be done. When performing subsequent separation by the H method, the regeneration liquid (1d) is then supplied to a separation column to convert the ion exchange resin into the IJ form. Furthermore, when only method F is repeated, the supply of the buffer solution (Ig-ti) and the supply of the regeneration solution (IJ) are repeated alternately.

Incidentally, the difficulty in converting the Na type to the Li type of the cation exchange resin is determined by the affinity of the cation feed for the cation exchange resin. The affinity of Na+ for cation exchange resins is approximately twice that of LP (Reference: Chemistry Handbook Basic Edition ■).

1408 (1966)), Li3 adsorbed on a cation exchange resin is easily replaced by Na+, but conversely, Na+ adsorbed on a cation exchange resin is difficult to be replaced by Li+. Therefore, when switching from method F to method H, simply changing the elution buffer from the final elution buffer (11) of method F to the first elution buffer (1a) of method H allows the separation column (6) to easily It changes from type to Na type. On the other hand, the Na type separation column (6) uses the first elution buffer fi of the F method.
g) will not easily become KLI type by just distributing it, and 4
It takes ~8 hours.

According to the method of this example, the Na deaf cation exchange resin is converted to the Li type in a short time, and in addition to the ease of conversion from the Li type to the Na type described above, the H method
F method interconversion in a short time with a single separation column (6)
This is possible with an amino acid analyzer equipped with In the amino acid analyzer shown in Figure 3, in addition to the elution buffers 1a to 1d and 1g to IJ used in the H method and F method, the Na
) and ammonia NHs removal liquid (1f), and on-off valves (2e, 2f) attached to each are equipped. N
The a+ removal solution was a 0.01M hydrochloric acid solution containing 2M ammonium chloride, and the ammonia removal solution was 1.
A 0M lithium hydroxide aqueous solution is used in each case. After the H method analysis is completed, the electromagnetic pulp (2e) is opened and the ammonium chloride buffer (1e) is introduced into the separation column (6). The separation column (6) is under acidic conditions due to the hydrochloric acid.

By the way, the affinity for cation exchange resin is NH4°:
> Na ”) Because it is the order of LP (Reference: Chemistry Handbook Basic Edition II, 1408 (1966)), Na
The type cation exchange resin is easily replaced by N H4, and the separation column (6) once becomes N HJ type.

It was adsorbed on the separation column (6). The total amount of Na9 is Z
When the value was set at 22 milliequivalents/mt×1.88mt=4.18 milliequivalents and a 2M NH4+ solution was passed through at a flow rate of 0.4 mt/mt for 30 minutes, Na'' was completely removed.

The time required can be shortened by increasing the concentration of the NH4 solution or by increasing the flow rate. To convert the NH4 type cation exchange resin to the Li type, use the electromagnetic valve (2e).
After closing the electromagnetic pulp (2f), the 1.0M lithium hydroxide aqueous solution is allowed to flow through the separation column (6).

By flowing 1.0M lithium hydroxide, the separation column (6)e immediately becomes alkaline. Under these conditions, NH4'' is neutral ammonia NH3
As a result, it loses its affinity for cation exchange resins.

As a result, the cation exchange resin becomes LL type within a short time.

When the separation column (6) becomes Li type, if the elution buffer for F method (1 g) is allowed to flow by opening the valve (2 g), F
Formal amino acid analysis can be performed. In this example, the time required from the start of flowing liquid 1f until the F method analysis becomes executable is 15 minutes, and the total time required from the end of the H method to the start of the F method is 45 minutes. This is reduced to 115 hours compared to the 4 to 8 hours required by the method of FIG. By shortening the required time, a ninth-order effect can be expected.

(1) The time required to change the analytical method has been shortened to 45 minutes, which is equivalent to one normal amino acid analysis.
An automatic amino acid analyzer that performs H method-F method analysis using a single separation column becomes possible.

(2) The amount of reagents required for switching the amino acid analyzer from H method to F method is significantly reduced.

(3) Under the same conditions other than the elution buffer, H method and F method
measurements can be made.

(4) It becomes easier to perform two analytical methods, H method and F method, on one type of amino acid sample, and the probability of identification errors, which is a weak point of chromatography, is reduced by mutual comparison of analysis results. I can do it.

By the way, in amino acid analysis measurements, NH3 molecules contained other than the elution buffer are likely to be adsorbed to the cation exchange resin, resulting in fluctuations in the background level, which makes it difficult to increase the sensitivity of amino acid analysis. be. At the same time, NHs adsorbed on the column resin is washed away with a concentrated alkaline solution. Therefore, when a 1.0 M lithium hydroxide solution is used to convert an N H4 type resin to a Li type resin, there is little possibility that N H3 molecules will remain in the separation column.

No problem will arise if an acidic hydrochloric acid solution containing a tertiary amine such as trimethylamine at a concentration of 2M is used instead of the ammonium chloride buffer (1e). That is, trimethylamine and its acidic form trimethylammonium exhibit behavior in the separation column (6) corresponding to NHa and NH,+, respectively. However, on the other hand, tertiary amines do not react with coloring reagents used in amino acid analysis methods, such as the above-mentioned ninhydrin. Therefore, even if tertiary amine remains in the separation column (6), it is detected by the detector (11).
) will not be detected.

[Effects of the Invention] According to the present invention, the ion exchange resin in the separation column can be converted from the sodium type to the lithium type within a short time, so even a single separation column can convert protein hydrolyzed amino acids and biological fluid amino acids. It can provide a practical method for separating and analyzing both.

[Brief explanation of the drawing]

Figure 1 is a diagram to explain the principle technology of the present invention, Figure 2 is a diagram to explain the conversion from sodium type to lithium type when the present invention is not applied, and Figure 3 is a diagram to explain the conversion from the sodium type to the lithium type when the present invention is applied. FIG. 2 is a diagram showing a flow path system of an amino acid analyzer. 1a-1c...Elution buffer for H method, ld, lj...
Regenerating solution, le...ammonium chloride buffer, lf...
・Ammonium expelling solution, 1a to 1j... Elution buffer for F method, 2a to 2j... Solenoid valve, 4... Sampler, No. 7z' JO (07, f between IF+ (minutes) σ cow r- 1 (minute) Mio, (27

Claims (1)

[Claims] 1. In an amino acid analysis method that uses a separation column filled with a cation exchange resin to separate and analyze protein hydrolyzate amino acids and biological fluid amino acids, the above cation exchange resin is used in the sodium form to separate protein hydrolyzate amino acids. After that, an acidic solution containing ammonium ions is supplied to the separation column to convert the cation exchange resin into an ammonium form, and then an alkaline solution containing lithium ions is supplied to the separation column to convert the cation exchange resin. An amino acid analysis method characterized by converting into lithium form and then separating amino acids from biological fluids under acidic conditions.