SU1397808A1 - Method of analyzing arginine - Google Patents

Abstract

This invention relates to analytical chemistry, in particular to the determination of an arginine (Arg) B mixture of amino acids. In order to increase the specificity of the determination and simplify the process, the analyzed solution is preliminarily neutralized to pH 6.9-7.1, then chloroform is added. The mixture obtained is treated with a solution of bromothiol blue with pH 4.0, and stirred until the color is stable. After separation of the phases, the chloroform layer is separated, a part of it is treated with a 0.1 M solution of NaOH, and after separation of the mixture, the aqueous layer is photometric. The percentage of open Arg added is in the range of 94.4-100%, 6 tab. (L

Description

co; o

oo

The invention relates to biochemical research methods and can be used in the determination of arginine in a mixture of amino acids, for example, in Alveein drug mixture or artificial nutrient media.

The purpose of the invention is to increase the specificity of the determination and simplify the method.

The method is carried out as follows.

Reagents: a 0.05% aqueous solution of methyl bromide with a pH of 4.5 (0.01 M HCl is used to bring the pH to 4.5); chloroform; 0.1 M sodium hydroxide solution.

The mixture of amino acids Alvezin is adjusted in pH to pH 7.0 (a 0.01 M solution of HCl or a 0.01 M solution of sodium hydroxide is used). Samples are taken with an approximate arginine content of 0.12-0.6 µmol, 3 ml of chloroform and 2 ml of a 0.05% solution of bromthymol blue with a pH of 4.5 are added. Mix the samples by inverting the tubes (40 pretreatments). After complete separation of the phases, take 2 mL of the yellow-colored solution from the chloroform layer of the Lower Phase and transfer it to a friend of the tube containing 3 mL of a 0.1 M solution of 1 patch and add After complete stratification, the blue-colored upper layer is removed and photometted at 590 nm in a cuvette with a light path of 1 cm on a spectrophotometer or colorimeter against a blank sample (which is also prepared instead of a blend). and amino acids are treated with the same volume of water).

The dependence of the sensitivity of the method for the determination of arginine on the pH values of the solution of bromtimol blue is given in Table 1.

A 0.05% solution of bromthymol blue should have a pH value in the range of 4.0–4.6, since in this range of pH values the maximum sensitivity of the method is achieved (Table 1).

pH of solution 3.0 3,8 4,0 4,4 4,4 4,4 4,8 5,0 6,0 Optical density

0.05 0.24 0.37 0.30 0.40 0.38 0.24 0.23 0.20

Mixing a mixture of amino acids, 40 chloroform and a solution of bromothymol blue leads to the formation of a constant color of the chloroform layer, which indicates a complete extraction of the hydrophobic arginine complex and 45 bromthymol blue into chloroform. This operation can be controlled visually by the absence of an increase in the yellow color of the chloroform layer with mixing.

Mixing is carried out by inverting the tubes, avoiding foaming of the samples, since this leads to confusion of the results. It was established experimentally that for the complete extraction of the arginine-bromthymol complex

Table I

blue to chloroform requires no more than 40 tube inversions. It was also found that the hydrophobic complex arginine-bromothymol blue completely goes into the chloroform layer and in the event that the samples, without mixing, leave for a day or more.

The specificity of the determination of arginine by the proposed method was tested with respect to other amino acids at various RP values of the solution of bromthymol blue (Table 2). From Table 2 it follows that, under the indicated conditions of determination, the method is almost completely specific for arginine.

Note,

Amino acid solutions are adjusted with 0.01 M sodium hydroxide solution to pH 7.0. For the study, an amino acid containing 0.6 µmol of each amino acid is sampled.

1397808

Table 2

513

The PocnpOH4Bo, r, HMOCTh method for the determination of arginine was tested with paoal heat detectors of the same sample (0; 7 µmol) arginine (tab. 3). The obtained data testify from sufficiently good metrological parameters of the method of argie determination. (low p. brss data in parallel; y x xepen).

T a b l and d a 3

  physical density nDoP parameters

R p ri; and hp n not: X-Srean

LLmetichesk echo. S is the mean quadrotic error. V is the coefficient of variation.

The sensitivity of the method for determining arginine under such conditions is 0.12 µmol arginine. Linear relationship between the amount of arginine and onTH4ecKO t races density. The trore is stored in KOH-I concentrations of 0.12-0.6 µmol (Table 4), which is sufficient for constructing a calibration curve.

The possibility of determining arginic acid in the presence of other amino acids was also tested on an artificially prepared mixture of amino acids and the medicinal mixture of amino acids Alresin, to which known quantities of arginines were added. The definition of arginine in the drug mixture Alvezin (the drug, as indicated in the formulation, contains 3.3 g / l of arginine) is presented in table 5. T a b l and c a 5

3.3 g / l

3.15 g / l

95, D /

Determination of arginine in a mixture of amino acids by adding known amounts of arginine to an artificial mixture of amino acids (200 μmol of lysine, nor-valine, cysteine, ornithine, tryptophan, alanine, histidine, valine, serine, nor-leucine, glutamic acid, phenyl-alanna, asparagine, leu-p, 1Ha, ileoleiina, glutamine, aspartic acid, glipine, proline, treg.n n methionine) or in the drug-algae of amino acids Alvezin can be found in the table.6.

The research results show that the proposed method opens arginine in the Alvezin drug mixture in quantities that coincide with those indicated in the recipe, the percentage of discovery of added arginine is within 94, D-100%.

Thus, the proposed method is simpler and more specific than the known one.

Claims (1)

Invention Formula The method for determining arginine by treating the sample solution with a color reagent followed by T a b l and c a ft photometry the obtained colored solution, characterized in that, in order to increase the specificity of the determination and simplification of the method, the analyzed solution is preliminarily neutralized to a pH of 6.9-7.1, chloroform is added, bromothymol blue with pH 4.0 is used as a color reagent 4.6, the mixture is stirred until the color is stable, after separation of the phases, the chloroform layer is separated, a portion of it is treated with a 0 M sodium hydroxide solution, and after separation of the mixture, the aqueous layer is photometric.