JPS6367663B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an amino acid analysis method and apparatus, and more particularly to an amino acid analysis method and apparatus that can detect even cyclic amino acids such as proline with high sensitivity. One of the methods for quantitatively analyzing amino acids using a liquid chromatograph is a fluorescence analysis method that uses o-phthalaldehyde (hereinafter abbreviated as OPA) as a fluorescent reagent. In this case, cyclic amino acids such as proline are treated with an oxidizing agent in advance and then reacted with a fluorescent reagent, and the optimal amount of the fluorescent reagent to be used has been reported. (“o-
Phthalaldehydes: Fluorogenic Detection of
Primary Amines in the Picomole Range.
Comparison with Fluorescamine and
Ninhydrin”Proc.Nat.Acad.Sci.USA72, 1975).
In other words, this usage amount was determined experimentally, and is usually used at a concentration of 0.08% for 1 part of the reaction solution obtained by oxidizing the eluent of liquid chromatography.
One part of OPA solution was added. Although this amount was considerably in excess of the amount of amino acids contained in the test sample, the sensitivity for proline and the like was not satisfactory. As a result of examining various conditions, the inventors of this invention found that the sensitivity of cyclic amino acids such as proline can be dramatically improved by increasing the amount of OPA used by more than twice the amount used above. I found it. In addition, the pretreatment process with an oxidizing agent is performed not only on eluents containing only cyclic amino acids such as proline, but also on eluents containing general amino acids, to minimize the reduction in sensitivity of general amino acids. We have discovered a method and device that can improve the sensitivity of cyclic amino acids such as proline. The present invention will be explained in detail below based on Examples and Comparative Examples. Example 1 FIG. 1 shows an example of the amino acid analyzer of the present invention. This amino acid analyzer 1 consists of a liquid chromatograph section 2 that separates amino acid components from an amino acid-containing sample, an oxidizing agent mixing section 7 that mixes a sodium hypochlorite solution into the eluent, a first reaction coil 12, and a first reaction coil 12. a fluorescent reagent mixing section 13 that mixes an OPA solution into the reaction solution flowing out from the reaction coil 1;
It consists of a second reaction coil 18 and a fluorescence detection section 19. The liquid chromatograph section 2 is composed of a step gradient device 3, a mobile phase liquid feed pump 4, a sample introduction device 5, and a separation column 6, as usual, and eluent flows out at a flow rate of about 0.5 ml/min. . The oxidizing agent mixing section 7 consists of an oxidizing agent solution tank 8, an ironing pump 10 connected to the tank 8, a preheating coil 11 whose one end is connected to the ironing pump 10, and the other end of the preheating coil 11. is connected to the inlet portion of the first reaction coil 12, and mixes the oxidizing agent solution 9 into the eluent at a flow rate of about 0.3 ml/min. The reason why we used the straining pump 10 instead of the commonly used plunger pump was because the flow rate of the oxidizing solution 9 was lower than usual, about 0.3 ml/min, in order to avoid diluting the sample with the oxidizing solution 9. However, with a normal plunger pump, the discharge flow rate becomes unstable when the flow rate is set to 0.5 ml/min or less. As a result of being able to reduce the flow rate using the straining pump 10, it has also become possible to obtain a long reaction time with a short reaction coil. Oxidizing agent solution 9 contains 122.1 g of sodium carbonate and 40.7 g of boric acid.
and potassium sulfate 56.4g dissolved in water total amount 3
Commercially available sodium hypochlorite (available chlorine concentration approximately 5%) in carbonate-borate buffer (PH10.2)
It contains 0.1% of As a conventional buffer solution, a boric acid buffer solution has usually been used, but the boric acid buffer solution has problems such as forming precipitates when the temperature decreases and having a weak buffering effect. These problems were solved by using the above carbonate-borate buffer. This will be explained in further detail in Experimental Example 5 below. The first reaction coil 12 is a stainless steel pipe with an inner diameter of about 0.5 mm and a length of about 0.2 m, and is maintained at about 68° C. in a constant temperature bath 20. The fluorescent reagent mixing section 13 basically has the same configuration as the oxidizing agent mixing section 7, and the OPA solution tank 1
The OPA solution 15 is mixed into the reaction liquid flowing out from the first reaction coil 12 at a flow rate of about 0.3 ml/min. The reason why the straining pump 16 is adopted is the same as that stated in the explanation of the oxidizing agent mixing section 7.
OPA solution 15 includes 2.0 g of OPA, 35 ml of ethanol,
Add the above carbonate-borate buffer to 4 ml of 2-mercaptoethanol and 1 ml of 20% Brij35 aqueous solution to make the total volume.
The volume is 500ml. Therefore, the OPA concentration is approximately
It is 0.4%. The reason for using the carbonate-boric acid buffer solution as the buffer solution is the same as the reason stated in the explanation of the oxidizing agent mixing section 7. The second reaction coil 18 is a stainless steel pipe with an inner diameter of about 0.5 mm and a length of about 2 m.
Like the reaction coil 12, it is maintained at about 68°C. The fluorescence detection section 19 is a normal fluorescence detection device. Specific examples of each component of the device 1 include the following. Step gradient device 3; Shimadzu SGR-1A Mobile phase liquid pump 4; Shimadzu LC-3A Sample introduction device 5; Shimadzu SIL-1A Separation column 6; Shimadzu ISC-07/S1504 Mobile phase 21; 0.001 in 0.2N sodium citrate
Contains % n-caprylic acid and 7% ethanol, adjusted to PH3.25 with perchloric acid. Shigo pump 10, 16; Shimadzu PRR-2A Preheating coil 11, 17; Inner diameter 0.8 mm, length 1 m
Stainless steel pipe Fluorescence detection section 19; Shimadzu FLD-1A In the above device 1, 0.2N sodium citrate (PH2.2) containing 10 μg of proline in 1 ml.
When 20μ of the sample was introduced and analyzed, as shown in Figure 2, amino acid analysis was performed with good sensitivity to proline. Comparative Example 1 Analysis was conducted under the same conditions as in Example 1 except for the conditions shown below. Oxidizing agent solution mixing flow rate: approx. 0.5ml/min OPA solution mixing flow rate: approx. 0.5ml/min OPA solution; OPA 0.4g, ethanol 7ml, 2-
The above carbonate-borate buffer was added to 1 ml of mercaptoethanol and 1 ml of 20% Brij35 aqueous solution to make a total volume of 500 ml. First reaction coil; stainless steel pipe with inner diameter of 0.5 mm and length of 0.35 m. Second reaction coil; Stainless steel pipe with inner diameter of 0.5 mm and length of 0.7 m. Temperature of first and second reaction coils: approximately 65°C. The results are shown in Figure 3. Compared to the analysis results using the method and apparatus of the present invention shown in FIG. 2, the detection sensitivity for proline is significantly lower. Experimental Example 1 (Study of OPA concentration range) In the apparatus shown in FIG. 1, relative sensitivity was investigated when changing the concentration of OPA solution. The compositions of OPA solutions with varying concentrations are shown in Table 1.
The results are shown in Figure 4A. FIG. 4B is a graph showing the concentration of OPA solution and relative sensitivity in the apparatus shown in FIG. 1, with the length of the first reaction coil being 0.1 m and the length of the second reaction coil being 1 m. be. From these results, the concentration of OPA solution is 0.2~
It can be seen that 0.5% is preferable. Experimental Example 2 (Study of reaction time with oxidizing agent) In the apparatus shown in FIG. 1, the relative sensitivity was investigated when the length of the first reaction coil was changed. Example 1 for conditions other than the length of the first reaction coil
and the same conditions as shown in Table 1. The results are shown in Figure 5. From this, it can be seen that the reaction time with the oxidizing agent is preferably about 2 seconds to 4 seconds, and the length of the first reaction coil is preferably about 0.1 m to 0.3 m. Experimental Example 3 (Study of reaction time with OPA) In the apparatus shown in Fig. 1, the relative sensitivity was investigated when the length of the second reaction coil was changed. Example 1 for conditions other than the length of the second reaction coil
and the same conditions as shown in Table 1. The results are shown in Figure 6. From this, it can be seen that the reaction time with OPA is preferably about 10 seconds to 40 seconds, and the length of the second reaction coil is preferably about 1 m to 4 m. Experimental Example 4 (Study of Reaction Coil Temperature) In the apparatus shown in FIG. 1, the relative sensitivity was investigated when the temperature of the thermostatic chamber 20 was changed. Conditions other than temperature were the same as those shown in Example 1 and Table 1. The results are shown in Figure 7a. From this, it can be seen that in the apparatus shown in FIG. 1, it is preferable to maintain the temperature of the reaction coils 12, 18 at about 68°C. Figure 7b is a graph of the relationship between reaction coil temperature and relative sensitivity in the apparatus shown in Figure 1, with the length of the first reaction coil being 0.4 m and the length of the second reaction coil being 4 m. It is. Figure 7c is a graph of the relationship between reaction coil temperature and relative sensitivity in the apparatus shown in Figure 1, with the length of the first reaction coil being 0.6 m and the length of the second reaction coil being 6 m. It is. Experimental Example 5 (Study of Buffer Solution) In order to investigate the slowing effect of buffer solution, 6 ml of buffer solution was added to 5 ml of mobile phase for amino acid analysis with different pH and the fluctuation in PH after mixing was measured. The following three types of mobile phases were used. A 0.2N Na ⁺ (sodium citrate) PH3.25 B 0.2N Na ⁺ (〃) PH4.25 C 1.2N Na ⁺ (〃) PH9.00 The carbonate-borate buffer according to this invention (PH10) is used as the buffer solution. Table 2 shows the results using .2), and Table 3 shows the results using the conventional boric acid buffer (PH10.5). This shows that the carbonate-borate buffer according to the present invention has excellent buffering power.

[Table] Note) Add the above to the total amount using the carbonate-borate buffer.
Make it 500ml.

【table】

【table】 [Brief explanation of drawings]

FIG. 1 is a configuration diagram of an embodiment of the device of the present invention;
Figure 2 is a chromatogram when amino acid analysis was performed according to the method of the present invention using the apparatus shown in Figure 1. Figure 3 is a chromatogram when amino acid analysis was performed using the conventional method using a conventional reaction coil. Figure 4 is a graph of relative sensitivity characteristics with respect to OPA concentration.
FIG. 5 is a graph of relative sensitivity characteristics with respect to reaction time with an oxidizing agent, FIG. 6 is a graph of relative sensitivity characteristics with respect to reaction time with OPA, and FIG. 7 is a graph of relative sensitivity characteristics with respect to reaction coil temperature. DESCRIPTION OF SYMBOLS 1... Amino acid analyzer, 2... Liquid chromatograph section, 7... Oxidizing agent mixing section, 8... Oxidizing agent solution tank, 9... Oxidizing agent solution, 10, 16... Straining pump, 11, 17... ...Preheating coil, 12...
...First reaction coil, 13... Fluorescent reagent mixing section, 14... OPA solution tank, 15... OPA solution, 18... Second reaction coil, 19... Fluorescent detection section, 20... Constant temperature bath, 21...mobile phase.

Claims (1)

[Claims] 1. A sample obtained by subjecting an amino acid-containing sample containing at least proline to a liquid chromatograph section.
The eluent containing 10 ^-12 to 10 ^-8 moles of amino acid is reacted with sodium hypochlorite in the first reaction section, and the resulting reaction solution is then added to 1 part (volume) of the reaction solution in the second reaction section. 1. A method for amino acid analysis, which comprises adding 0.2 to 0.5 parts of O-phthalaldehyde solution having a concentration of 0.2 to 0.5% to the phthalaldehyde, causing the reaction, and then subjecting the reaction solution to fluorescence analysis to quantify amino acids. 2 The reaction time in the second reaction section is 10 seconds to 40 seconds.
2. The method of claim 1, wherein: 3. The method according to claim 1, wherein a Na ₂ CO ₃ --H ₃ BO ₃ --K ₂ SO ₄ mixture is used as a solvent in the oxidizing agent solution and the O-phthalaldehyde solution. 4 A liquid chromatograph section that separates the amino acid components of an amino acid-containing sample containing at least proline and flows out the eluent, an oxidizing agent mixing section that mixes a sodium hypochlorite solution into the eluent using a straining pump, inner diameter is 0.5mm and length is 0.1m to 0.3m
A first reaction coil of the first reaction coil, a generation method in which an O-phthalaldehyde solution with a concentration of 0.2 to 0.5% is mixed into the reaction liquid flowing out from the first reaction coil at a flow rate of 0.2 to 0.5 times the flow rate of the reaction liquid using a straining pump. Fluorescent reagent mixing part,
An amino acid analyzer comprising a second reaction coil with an inner diameter of 0.5 mm and a length of 1 m to 4 m and a fluorescence detection section that are connected in this order. 5. The apparatus according to claim 4, wherein the solvent in the oxidizing agent solution and the O-phthalaldehyde solution is a mixture of Na ₂ CO ₃ --H ₃ BO ₃ --K ₂ SO ₄ .