Quantitative method for amino acids using ionization equilibrium

Abstract

The ninhydrin reaction used in the amino acid quantification method has a low yield of Ruhemann's purple and does not correspond to the decomposition amount of amino acids. Also, the reaction rate is easily affected by the liquid temperature and liquid properties and is difficult to control. Due to these factors, the measurement accuracy of amino acid quantification decreases. 【Solution means】To an amino acid (Gly or Ala or Thr) sample solution, a silver nitrate solution with a known concentration is added so that the amount of substance of Ag is in excess of the amount of substance of the amino acid contained therein. Then, a sodium hydroxide solution with a known concentration is dropped into the mixed solution, and the dropping amount of the sodium hydroxide solution at the moment when the brown precipitate of silver oxide can be visually recognized is measured. Using the measurement results obtained by the same procedure as above with a plurality of amino acid sample solutions with known amino acid concentrations, the amino acid concentration is determined using a calibration curve for the known amino acid solution concentration of the dropping amount of the sodium hydroxide solution at the moment when the formation of the silver oxide precipitate can be visually recognized under the temperature conditions. + ​

Description

Technical Field

[0001] The present invention relates to a method for quantifying an amino acid, which comprises adding a silver nitrate solution with a known concentration to a mixed solution such that the amount of substance of Ag is in excess of the amount of substance of the amino acid contained in the amino acid sample solution under any temperature condition, dropping a sodium hydroxide solution with a known concentration into the mixed solution, and calculating the amino acid concentration from the dropping amount of the sodium hydroxide solution at the moment when a brown precipitate of silver oxide can be visually recognized. +

Background Art

[0002] A mixed solution of an amino acid and ninhydrin undergoes a ninhydrin reaction that exhibits a blue-violet color (the coloration of Ruhemann's purple generated by the reaction) upon heating. This coloration is used for the detection of amino acids, proteins, and various peptides. Further, the higher the amino acid concentration, the greater the amount of Ruhemann's purple generated by the ninhydrin reaction and the darker the coloration. Therefore, amino acids can be quantified from the degree of this coloration. Currently, amino acid quantification devices using this principle are also widely utilized.

[0003] The reaction rate of the ninhydrin reaction depends on the amino acid concentration under the same heating conditions and the same ninhydrin concentration. Thus, the higher the concentration of the amino acid solution used, the shorter the time until the moment when the purple coloration can be visually recognized. From this relationship, amino acids can also be quantified from the time until the coloration can be visually recognized.

[0004] Furthermore, a decomposition reaction of an amino acid in which the formation of Ruhemann's purple is suppressed is caused by a ninhydrin reaction to which an iodine solution is added. The decrease in the amount of iodine that coincides with the decomposition amount of the amino acid at this time is determined by iodine titration, and a method for calculating the amino acid concentration from the decrease in the amount of iodine based on the reaction rate of the ninhydrin reaction due to the type and concentration of the amino acid has also been developed and is used as a simple method for quantifying amino acids by iodine titration.

Prior Art Documents

Patent Documents

[0005] ​ [Patent Document 1] Patent No. 7278513

[0006] [Patent Document 2] Patent No. 7566413 Summary of the Invention [Problem to be solved by the invention]

[0007] The yield of Ruhemann's purple produced by the ninhydrin reaction is low and does not correspond to the amount of amino acids decomposed. In addition, the reaction rate of the ninhydrin reaction is easily affected by the temperature and properties of the solution and is difficult to control. Therefore, using the ninhydrin reaction to quantify amino acids has the problem of reducing the measurement accuracy. [Means for solving the problem]

[0008] The present invention relates to a method for detecting the amount of Ag in an amino acid (Gly, Ala, or Thr) sample solution, the amount of Ag being greater than the amount of the amino acid contained in the amino acid sample solution, under any temperature condition. + a sodium hydroxide solution of a known concentration is dropped into a mixed solution to which a silver nitrate solution of a known concentration has been added so that the amount of substance is in excess, the amount of the sodium hydroxide solution dropped is measured at the moment when a brown precipitation of silver oxide can be visually observed, and the amino acid concentration of the amino acid sample solution is determined using a calibration curve, which has been previously prepared using measurement results obtained by the same procedure as described above using amino acid sample solutions of multiple known amino acid concentrations, for the known amino acid solution concentrations at the moment when the formation of the silver oxide precipitation can be visually observed, under the above temperature conditions.

[0009] The present invention relates to a method for detecting the amount of Ag in an amino acid (Gly, Ala, or Thr) sample solution, the amount of Ag being greater than the amount of the amino acid contained in the amino acid sample solution, under any temperature condition. +A silver nitrate solution with a known concentration is added to a mixed solution such that the amount of substance is in excess, and a sodium hydroxide solution with a known concentration is added dropwise. Using the dropwise addition amount x (mL) of the sodium hydroxide solution at the moment when the brown precipitate of silver oxide can be visually confirmed, the concentration [Na + (mol / L) of sodium ions at the moment when the brown precipitate of silver oxide can be visually confirmed is obtained by the following formula (1). [Na + is substituted into the following formula (2), and based on the equilibria represented by the equilibrium constants K 1 、K 2 、K 3 、K 4 , the concentrations of each substance at the moment when the brown precipitate of silver oxide can be visually confirmed are represented by formulas (3), (4), (5), (6), and the mass balance is represented by formulas (7), (8). The amino acid concentration is calculated by solving the system of eight simultaneous equations below. [OH - 0 (x / 1000)=[Na + ···(1) [Na + +[Ag + +[H + =[A - +[OH - +[NO 3 - ···(2) K 1 =[A - / [A ± [OH - ···(3) K 2 =[AgA] / [A - [Ag + ···(4) K 3 =[Ag + [OH - ···(5) K 4 =[H + [OH - ···(6) [A] 0 =[A ± +[A - +[AgA] ···(7) [Ag]​0 =[Ag + +[AgA] ···(8) In the formula, [A] 0 is the initial concentration of amino acid in the previous mixed solution (mol / L), [Ag] 0 is the concentration of the added silver nitrate solution (mol / L), [OH - 0 is the concentration of the dropped sodium hydroxide solution (mol / L), K 1 is the equilibrium constant shown by the reaction of the amino acid zwitterion and OH - , K 2 is the complex formation constant of the amino acid silver complex, K 3 is the solubility product, K 4 represents the ion product of water, and also, [A ± is the concentration of the amino acid zwitterion, [A - is the concentration of the amino acid anion (mol / L), [Ag + is the concentration of silver ions (mol / L), [AgA] is the concentration of the amino acid silver complex (mol / L), [OH - is the concentration of hydroxide ions (mol / L), [H + is the concentration of hydrogen ions (mol / L), [NO 3 - is the concentration of nitrate ions (mol / L), and all of these are the concentrations at the moment when the brown precipitate of silver oxide can be visually recognized.

Advantages of the Invention

[0010] Since the present invention is a method for quantifying amino acids that does not rely on the ninhydrin reaction, it does not require strict control of the liquid temperature and liquid properties, and it is also not necessary to measure the degree of color development of rhodamine purple using an absorptiometer or the like. That is, expensive equipment is not required, and there is an effect that amino acids can be easily quantified only by visual measurement in the same way as the neutralization titration method, the redox titration method, the iodine titration method, etc.

Brief Description of the Drawings

[0011]

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Mode for Carrying Out the Invention

[0012] Amino acid zwitterion A ± When an acid is added, it becomes the amino acid cation A +To amino acid anion A by a base - is TIFF0007691802000002.tif10170 It was considered that amino acids could be quantified by using an amino acid metal complex composed of an amino acid anion and a metal ion.

[0013] At room temperature, 1 mL of 0, 0.04, 0.08, 0.12, 0.16, 0.20 mol / L NaOH solution was added to 10 mL of 0.15 - 0.25 mol / L Gly solution respectively to generate amino acid anions. Then, 10 mL of 0.2 mol / L copper nitrate solution was added, and the color change of the generated Gly-Cu complex was observed. At this time, the conditions were set so that Cu 2+ was contained in an excessive amount with respect to the amount of substance of the amino acid. Also, the concentration of the NaOH solution in the present invention was calculated by titration with an oxalic acid solution of known concentration.

[0014] As a result, the formation of a dark blue Gly-Cu complex was confirmed. It can be seen that the higher the concentration of the NaOH solution added to the amino acid, the greater the amount of amino acid anions generated, and thus the more Gly-Cu complex is generated (Figure 2). Also, the higher the concentration of the amino acid, the more the amount of the generated Gly-Cu complex increased as well. This suggests that the concentration of the amino acid solution can be determined from the color change of this amino acid metal complex. Furthermore, in the following experiments, it was examined in detail using the absorbance at 710 nm, which is the peak wavelength of the Gly-Cu complex, as an index.

[0015] At room temperature, 1 mL of 0, 04 - 0.16 mol / L NaOH solution was added to 10 mL of 0.1 mol / L Gly solution respectively, and then 10 mL of 0.1 mol / L copper nitrate solution was added. The color change of the solution due to the Gly-Cu complex was measured with a visible spectrophotometer at an absorbance of 710 nm.

[0016] The absorbance (710 nm) increased with respect to the concentration of the added NaOH solution and then turned to decrease (Figure 3). The Gly-Cu complex was generated according to the amount of added OH - , and following the preferential action of OH - on Gly, next OH - is Cu2+ and copper hydroxide precipitation occurs. The reason for the decrease in absorbance in Figure 3 is the formation of copper hydroxide precipitation The point where the slope of TIFF0007691802000003.tif917010nm changes is the OH at which copper hydroxide precipitation begins to form - It can be said that it indicates the amount of substance of.

[0017] Furthermore, using Gly solutions of various concentrations, in the same manner as

[0015] , by determining the concentration of the NaOH solution at which copper hydroxide precipitation begins to form from the point where the slope of the absorbance changes, the relationship in Figure 4 was obtained. It can be seen that the higher the concentration of the Gly solution, the higher the concentration of the NaOH solution, and the amino acid concentration can be calculated from the concentration of the NaOH solution at which copper hydroxide precipitation begins to occur. However, to obtain this calibration curve, an absorptiometer is required, and NaOH solutions with various concentrations must be prepared, and the procedure is quite complicated. Therefore, the following method was tried to more simply examine the moment when the precipitation of the hydroxide begins by precipitation titration with a NaOH solution instead.

[0018] Under the temperature condition of 25 °C, 10 mL of 0.02 - 0.1 mol / L amino acid (Gly, Ala, Thr, Val, Ile) solutions were added with 10 mL of 0.1 mol / L copper nitrate solution to make a mixed solution, and then 0.1 mol / L NaOH solution was added dropwise to perform precipitation titration to determine the NaOH solution at the moment when precipitation formation could be visually recognized. Furthermore, using a Gly solution for the amino acid, 0.1 mol / L silver nitrate solution and 10 mL of 0.1 mol / L zinc nitrate solution were added to the mixed solution respectively, and 0.1 mol / L NaOH solution was added dropwise to perform precipitation titration in the same manner. At this time, the condition was set so that the amount of substance of metal ions was in excess with respect to the amount of substance of amino acids.

[0019] As a result, a relationship was obtained in which the higher the concentration of the amino acid, the larger the amount of NaOH solution added dropwise (Figure 5). Also, various types of amino acids were tried, and the same tendency was observed. The concentration of the amino acid solution can be determined from the calibration curve of the amino acid solution concentration with respect to the amount of NaOH solution added dropwise at the moment when this precipitation formation can be visually recognized. Furthermore, even when the metal ion was replaced with Ag + , Zn 2+ , a similar relationship was obtained (Figure 6). The differences in the graphs of Figure 6 are considered to be caused by the differences in the coordination number, solubility product, and complex formation constant of the metal ions. Since both metal ions form amino acid anions and amino acid metal complexes, when a NaOH solution is added dropwise to an amino acid solution containing a metal ion, Ag 2 O, Zn(OH) 2 should originally form an instant brown or white precipitate, but no precipitate forms, and then precipitation occurs when the addition of the NaOH solution is continued. In the target precipitation titration, observing the formation of copper hydroxide precipitate using colored Cu 2+ is less accurate in visually capturing the instant of precipitate formation than observing the change from colorless to brown by adding Ag + . Furthermore, since Ag + is considered to coordinate with amino acid anions in a 1:1 ratio, it is also convenient when developing for the quantification of amino acid mixed solutions. For these reasons, the metal ion used for the formation of amino acid metal ions was set as Ag + .

[0020] Ag + When performing precipitation titration of an amino acid solution added with Ag

[0021] with a NaOH solution, it was compared whether the instant of precipitate formation could be accurately judged visually or when using a turbidimeter. At room temperature, 10 mL of a 0.1 mol / L Gly solution was added with 10 mL of a 0.25 mol / L silver nitrate solution, and 0.1 mL of a 0.1 mol / L NaOH solution was added step by step to measure the turbidity. The turbidity was measured using a portable turbidimeter (AS ONE Corporation TN100IR). At the same time, the instant of precipitate formation was visually judged 34 times to obtain the frequency distribution of the minimum unit addition amount of the NaOH solution that could be judged to have formed a precipitate.The value of when the solution started to become cloudy as indicated by the turbidity meter and the moment of precipitate formation as judged by the naked eye were close (Figure 7). It was confirmed that the moment of precipitate formation can be adequately observed by the naked eye. However, since silver nitrate solution discolors due to exposure to light, it is not recommended to leave the measurement solution for a long time or to titrate in an environment exposed to direct sunlight.

[0022] Figure 8 summarizes the reactions that occur when an amino acid solution containing silver nitrate is titrated with NaOH. + When adding and titrating with NaOH solution, first, A ± A - This equilibrium can be expressed by equation 1. Then, A - Ag + is coordinated to form an amino acid silver complex. The complex formation constant is given by formula 2. Next, A - Since the amount of Ag produced is limited, + finishes coordination, and Ag 2 A precipitation of O occurs. At this time, equation 3, which expresses the solubility product, and equation 4, which expresses the ion product of water, hold true. Since both reactions are in equilibrium, they are not thought to occur simultaneously according to the equilibrium constant, rather than one reaction ending and the next starting. Furthermore, the material balance relationship is equation 5 for amino acids and equation 6 for silver. Add equation 7, which indicates electrical neutrality, and these seven equations express the precipitation titration. In other words, by setting these seven equations simultaneously, the amount of amino acids can be quantified from the amount of NaOH solution dropped at the moment the precipitate forms. Figure 9 summarizes the theory of quantifying amino acids.

[0023] Since it is not possible to calculate an algebraic solution for the seven simultaneous equations mentioned above, a program using Newton's method was created (Figure 10) to calculate a numerical approximation solution. + It is assumed that the amino acid anion forms a 1:1 complex.

[0024] Based on the theory of Fig. 9, verification was carried out with the program configuration of Fig. 10 (Fig. 11). To 2 mL of each amino acid (Gly, Ala, Thr) solution at 0.05 - 0.15 mol / L, 2 mL of 0.25 mol / L silver nitrate solution was added, and it was titrated with 0.05 mol / L NaOH solution to measure the dropping amount of NaOH solution until precipitation formed. The amino acid concentration was calculated and verified using the program for the system of simultaneous equations consisting of the seven formulas. The complex formation constant K AgA was determined in advance by solving the system of simultaneous equations using an amino acid solution with a known concentration.

[0025] As a result, the amino acid solution concentration used for verification and the calculated concentration showed close values in any concentration range (Fig. 12). The relationship between the amino acid concentration at this time and the dropping amount of NaOH solution at the moment when the precipitation of silver oxide can be visually observed is shown in Fig. 13. When an excessive amount of a solution containing Ag + is added to the amino acid solution and NaOH solution is dropped, the amino acid can be quantified from the dropping amount of NaOH solution at the moment when the formation of silver oxide precipitation can be visually observed.

[0026] In this study, in the quantification method using this amino acid metal complex, it was limited to the quantification of one type of amino acid and presented as a new method. Theoretically, even when multiple amino acids are mixed, it is possible to perform the same quantification by preparing as many systems of simultaneous equations as the number of amino acids according to Fig. 7 and programming accordingly.

Industrial Applicability

[0027] The quantitative analysis of amino acids is utilized in various fields. Particularly, it also occupies an important position in the analysis of the amino acid composition of proteins in the medical field and the like. Compared with existing methods, the amino acid quantification method found in the present invention is an epoch-making method that can simply quantify amino acids only by precipitation titration, and there is an expectation that it will become a new option for amino acid analysis methods. Finally, the present invention finds that there is a possibility that new titration methods incorporating ionization equilibrium will be developed in the future in addition to various existing titration methods.

Claims

【Request 1】 Under any temperature condition, an amino acid (Gly, Ala, or Thr) sample solution is added with a larger amount of Ag than the amount of the amino acid contained in the amino acid sample solution. ＋ a sodium hydroxide solution of a known concentration is dropped into a mixed solution to which a silver nitrate solution of a known concentration has been added so that the amount of substance is in excess, the amount of the sodium hydroxide solution dropped is measured at the moment when a brown precipitation of silver oxide can be visually observed, and the amino acid concentration of the amino acid sample solution is determined using a calibration curve, which has been previously prepared using measurement results obtained by the same procedure as described above using amino acid sample solutions of multiple known amino acid concentrations, for the known amino acid solution concentrations at the moment when the formation of the silver oxide precipitation can be visually observed, under the above temperature conditions. 【Request 2】 Under any temperature condition, an amino acid (Gly, Ala, or Thr) sample solution is added with a larger amount of Ag than the amount of the amino acid contained in the amino acid sample solution. ＋ A sodium hydroxide solution of a known concentration is dropped into a mixed solution to which a silver nitrate solution of a known concentration has been added so that the amount of substance is in excess. The sodium ion concentration [Na ＋ ] (mol / L) was calculated using the following formula (1), and [Na ＋ ] into the following formula (2) to obtain the equilibrium constant K １ , K ２ , K ３ , K ４ The amino acid concentration is calculated by solving the following eight simultaneous equations, which are composed of equations (3), (4), (5), and (6) that represent the concentrations of each substance at the moment when the brown precipitation of silver oxide can be visually observed, and equations (7) and (8) that represent the mass balance, based on the equilibrium shown in the following equation: [OH － ] ０ (8 / 1000)=[Na ＋ ]・・・(1) Na ＋ )+[g ＋ )+[R ＋ \[[A － )+ － )+[NO ３ － ] ・・・(2) K １ =[A － ] / [A ± ][OH － ] ・・・(3) K ２ =[A] / [A] － ][A] ＋ ]・・・(4) K ３ =[Ag ＋ ][OH － ] ・・・(5) K ４ =[H ＋ ][OH － ] ・・・(6) [A] ０ =[A ± ]+[A － ]+[AA] ・・・(7) [Ag] ０ =[Ag ＋ ]+[AgA] ・・・(8) In the formula, [A] ０ is the initial concentration of amino acid in the mixed solution (mol / L), [Ag] ０ is the concentration of the added silver nitrate solution (mol / L), [OH － ] ０ is the concentration of the dripped sodium hydroxide solution (mol / L), and K １ is an amino acid zwitterion and OH － The equilibrium constant for the reaction is K ２ is the complex formation constant of the amino acid silver complex, K ３ is the solubility product, K ４ represents the ionic product of water, and [A ± ] is the amino acid zwitterion concentration, [A － ] is the amino acid anion concentration (mol / L), [Ag ＋ ] is the silver ion concentration (mol / L), [AgA] is the amino acid silver complex concentration (mol / L), [OH － ] is the hydroxide ion concentration (mol / L), [H ＋ ] is hydrogen ion concentration (mol / L), [NO ３ － ] is the nitrate ion concentration (mol / L), and all of these are concentrations at the moment when the brown precipitate of silver oxide can be visually observed.

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