JP2024101969A - Amino-acid quantification method using ninhydrin reaction rate - Google Patents

Abstract

To provide address such a problem that a degree of purple coloration observed in a ninhydrin reaction that is determined by an amount of Ruhemann's purple formed, depends on an amino-acid concentration, in other words, the amino acids can be quantified from the degree of coloration represented by the generated Ruhemann's purple formed by reacting all of the amino acids used in the ninhydrin reaction, however, at this time, it is necessary to use an expensive instrument such as a visible spectrophotometer and use the absorbance as an index for the degree of coloration; and to provide a quantification method using a simple measurement capable of quantifying an amino acid from a degree of coloration.SOLUTION: An amino-acid quantification method includes: mixing a ninhydrin solution of known concentration kept at a temperature condition T0 and a solution of one type of amino acids (Gly, Trp or Ala) at the temperature condition; simultaneously therewith, in a process of heating the mixed solution to a temperature condition Tm, measuring a time x from immediately after warming the mixed solution until a moment coloration originating from Ruhemann's purple generated by the ninhydrin reaction can be visible; and calculating an amino-acid concentration based on a correspondence table consisting of amino-acid concentrations corresponding to heating times t.SELECTED DRAWING: Figure 17

Description

The present invention relates to a method for quantifying amino acids, which comprises heating a mixed solution of an amino acid and ninhydrin to cause a ninhydrin reaction, focusing on the color development time until the moment when the color development can be visually recognized, and calculating the amino acid concentration from the color development time based on the ninhydrin reaction rate resulting from the type and concentration of the amino acid.

When a ninhydrin solution is added to an amino acid and heated, the dye Ruhemann's purple is produced, giving it a bluish purple color. This reaction is called the ninhydrin reaction, and is used to detect amino acids, proteins, and various peptides. In addition, the higher the amino acid concentration, the greater the amount of Ruhemann's purple produced by the ninhydrin reaction, resulting in a darker color, and the amount of amino acid can be quantified from the degree of this color. Currently, amino acid quantification devices that use this principle are widely used.

McMurry Introduction to Organic Chemistry 4th Edition Written by JOHN MCMURRY p. 471-478

The degree of purple coloration observed in the ninhydrin reaction is determined by the amount of Ruhemann's purple produced, and therefore depends on the amino acid concentration. In other words, all of the amino acids used in the ninhydrin reaction are reacted, and the amino acids can be quantified from the degree of coloration of the resulting Ruhemann's purple. In this case, the degree of coloration must be measured using an expensive instrument such as a visible spectrophotometer, with absorbance as an index, and using this degree of coloration in a quantitative method for amino acids is a challenge when considering a simple measurement.

A certain reaction time is required to react all the amino acids used in the ninhydrin reaction. During this time, reduced ninhydrin, an intermediate product in the ninhydrin reaction, is easily oxidized in the air, and it is difficult to accurately convert the degree of Ruhemann's purple coloration into the amino acid concentration.

It was found that the time from immediately after heating a mixed solution of an amino acid and ninhydrin to the moment when the color change due to the ninhydrin reaction can be visually observed varies depending on the type of amino acid (Gly, Trp, Ala, Val, Leu, Ile, Asp, Met, His, Phe) and also on the amino acid concentration, even for amino acid solutions of the same concentration.

In the present invention, a ninhydrin solution of known concentration maintained at temperature condition _T0 and a solution of one kind of amino acid (Gly, Trp, or Ala) at the same temperature condition are mixed and heated to temperature condition _Tm at the same time, the time x is measured from immediately after the mixed solution is heated to the moment when coloration due to Ruhemann's purple generated by the ninhydrin reaction can be visually recognized, and distilled water of the same volume as the mixed solution is previously heated from temperature condition _T0 to Tm in the same manner as the heating method described above. _m , a unit time having an upper limit of 1 second is defined, the temperature of the distilled water is continuously measured from immediately after heating at the maximum time in a time period separated by the unit time, and an amino acid concentration is calculated based on a correspondence table containing amino acid concentrations corresponding to a heating time t from a relational equation which holds when an accumulated amount of absorbance from immediately after heating to the time x, which is indicated by the amount of Ruhemann's purple produced per unit time and calculated from a rate of increase in absorbance (570 nm) proportional to a ninhydrin reaction rate, reaches 0.010, which is the minimum absorbance value at which the color of the ninhydrin reaction can be visually recognized.

The present invention measures the amount of amino acids by measuring the time from immediately after heating a mixed solution of amino acids and ninhydrin in the ninhydrin reaction to the moment when coloration becomes visible, and does not require the degree of coloration as an indicator. In other words, it has the effect of eliminating the need for expensive equipment and simply quantifying amino acids by visual measurement, similar to neutralization titration, oxidation-reduction titration, iodometry, etc.

Furthermore, the present invention is a quantitative determination method that combines the reaction rate in the early stage of the reaction with visual confirmation of coloration, and has the effect of making it possible to similarly measure the concentration of reactants in reactions in which the reaction product turns colored.

FIG. 1 illustrates the ninhydrin reaction. FIG. 1 shows the reaction mechanism of the ninhydrin reaction. FIG. 1 is a diagram showing the change over time in a ninhydrin reaction and the definition of the color development time until the moment when color development can be visually recognized. FIG. 1 is a graph showing the relationship between the concentration of ninhydrin solution and the color development time. FIG. 13 is a graph showing the relationship between the concentration of an amino acid (Gly) solution and the color development time. FIG. 1 shows the color development time of various amino acids. FIG. 1 is a graph showing the absorbance at 420 to 720 nm of ninhydrin reaction solutions using various amino acid solutions. FIG. 13 is a graph showing the absorbance at 420 to 720 nm of ninhydrin reaction solutions at various concentrations of Gly solutions. FIG. 1 is a graph showing the absorbance (570 nm) of a ninhydrin reaction solution at the moment when the color change due to the ninhydrin reaction was visually observed. This figure shows that when the rate of production of Ruhemann's purple is replaced with the rate of increase in absorbance (570 nm), the integral value up to the coloring time becomes equal to the reference value of absorbance (570 nm) at which coloring can be visually recognized. FIG. 1 is a graph showing the change in absorbance (570 nm) versus ninhydrin reaction time (warming time). FIG. 1 shows an Arrhenius plot (Gly, Trp, Ala). FIG. 1 is a diagram showing the activation energy and frequency factor (frequency coefficient) that determine the reaction rate constant in the ninhydrin reaction of each amino acid (Gly, Trp, Ala). FIG. 1 is a graph showing a relational expression by which the cumulative amount of absorbance (570 nm) indicated by the amount of Ruhemann's purple produced per unit time from immediately after heating in the ninhydrin reaction until the color development time is equal to the reference value of absorbance for visual color recognition. FIG. 1 shows a method for quantifying amino acids using ninhydrin reaction rate. FIG. 15 is a diagram showing a calculation formula reflecting the relational expression of FIG. 14 . FIG. 13 is a diagram showing an example of a correspondence table between color development time and amino acid solution concentration. FIG. 13 is a diagram showing the results of verification in which a Gly standard solution of known concentration was used and the Gly solution concentration calculated based on a correspondence table from the measured color development time. FIG. 13 is a diagram showing the concentration of a Gly solution calculated by changing the reference value of absorbance (570 nm) for visual color recognition using a Gly standard solution of known concentration.

The ninhydrin reaction is a reaction in which a mixed solution of ninhydrin and amino acids turns purple when heated (Figure 1), and is known as a method for detecting amino acids. The reaction mechanism is as follows: First, amino acids are decomposed into _NH3 and other substances by the oxidizing action of ninhydrin, and ninhydrin itself is converted into reduced ninhydrin. Next, unreacted ninhydrin and reduced ninhydrin undergo a condensation reaction via _NH3 , producing Ruhemann's purple, which turns the color (Figure 2).

An example of the change over time in the ninhydrin reaction in which 10 mL of a 0.02 mol/L ninhydrin solution and 10 mL of a 0.02 mol/LGly solution, each prepared at 25° C., were mixed and simultaneously heated in a water bath at 70° C., is shown in FIG. 3. The moment of coloring was visible at 51 s. The time from immediately after heating until the coloring became visible was defined as the coloring time x.

When 10 mL of 0.02 mol/LGly solution was used as the amino acid, 10 mL of ninhydrin solutions of various concentrations were mixed and heated from 25°C to 75°C, and the relationship with the color development time was examined. The higher the concentration of the ninhydrin solution, the shorter the color development time (Figure 4). When the concentration of the glycine solution was changed under the same conditions, the higher the concentration of the amino acid solution, the shorter the color development time (Figure 5). In other words, it is considered that the color development time is determined by the ninhydrin reaction rate.

In addition, 10 mL of ninhydrin solutions of each concentration prepared at 25°C was mixed with 10 mL of 0.02 mol/L amino acid solutions, and the color development time of the ninhydrin reaction, which was simultaneously heated at 75°C in a water bath, varied depending on the type of amino acid (Figure 6). This difference in color development time is thought to be due to differences in the ninhydrin reaction rate.

Based on this coloration time, we consider a method for quantifying amino acids. First, we define the absorbance at the moment when the coloration of the ninhydrin reaction is visually observed. When 10 mL of 0.01 mol/L ninhydrin solution and 10 mL of 0.02 mol/L amino acid solution, each prepared at 25°C, were mixed and simultaneously heated in a water bath at 70°C for 5 to 10 minutes, the absorption spectrum of the ninhydrin reaction solution in the visible region of 420 to 720 nm was examined. A peak due to Ruhemann's purple was observed at 570 nm for all amino acids (Figure 7).

In addition, 10 mL of 0.01 mol/L ninhydrin solution prepared at 25°C was mixed with 10 mL of glycine solution of each concentration, and simultaneously heated at 70°C for 10 minutes. The absorption spectrum of the ninhydrin reaction solution in the visible region of 420 to 720 nm was examined, and the intensity at 570 nm changed depending on the amino acid concentration (Figure 8). The absorbance at 570 nm was used as an index of the amount of Ruhemann's purple produced.

Furthermore, the standard value of absorbance at 570 nm at which the color of the ninhydrin reaction can be visually recognized was determined as follows. Histidine, which undergoes a ninhydrin reaction even at room temperature, was used as an amino acid, and 10 mL of a 0.01 mol/L ninhydrin solution and 10 mL of a 0.02 mol/L histidine solution were mixed at room temperature, and a part of the solution was used to measure the absorbance at 570 nm. At the same time, visual observation of the color was performed, and the absorbance at 570 nm at the moment when the color was visually recognized was measured 50 times (FIG. 9). The most common absorbance was 0.010. Considering the average value of 0.0102 and the frequency distribution, the color of the ninhydrin reaction can be visually recognized with an absorbance of 0.010 at 570 nm as the standard value. As will be described later, it was found that there was no significant difference in the calculated amino acid concentration even with an absorbance of 0.009 and an absorbance of 0.011 (FIG. 19).

The production rate v of Ruhemann's purple can be expressed by formula 1 in FIG. 10. From the reaction formula in FIG. 2, Ruhemann's purple is produced from two molecules of ninhydrin and one molecule of amino acid, and from experiments, it can be empirically shown by the order of formula 1 in FIG. 10. Here, v is replaced with the rate of increase V of absorbance. V is proportional to v, so formula 2 in FIG. 10 holds, and in the short reaction time until the moment of coloration, the ninhydrin and amino acid solution concentrations approximate the initial concentrations before the reaction, so formula 3 in FIG. 10 holds. The liquid temperature also changes with respect to the heating time t. Since V also changes with this change in liquid temperature, V becomes a function V(T) of the liquid temperature T. However, the liquid temperature T is a function of the heating time t. Here, by integrating V with the heating time t, the relationship of formula 4 in FIG. 10 is obtained, and the left side becomes the reference value of coloration, 0.010. In other words, if ck is obtained and the reaction rate formula is completed, the amino acid can be quantified.

The change in absorbance (570 nm) versus reaction time (heating time) in the ninhydrin reaction using Gly as the amino acid is shown (Figure 11). The slope of this function represents the rate of increase V in absorbance, and ck for each reaction temperature is calculated using equation 3 in Figure 10 to obtain an Arrhenius plot (Figure 12). Arrhenius plots obtained from experiments are also shown for Trp and Ala. Ea and cA calculated from the slope and intercept of this Arrhenius plot are summarized in a table (Figure 13). This allows the reaction rate equation to be determined.

Here, it is assumed that the heating time is divided into n terms in 0.1 second increments up to the coloring time, and the liquid temperature also changes from _T0 to _Tn (FIG. 14). However, since the liquid temperature does not change much with an upper limit of 1 second, the unit time can be arbitrarily determined within the range. The absorbance (570 nm) of the Ruhmann purple generated in a unit time is determined, for example, from immediately after heating to 0.1 s by formula 6 in FIG. 14, and the absorbance (570 nm) of the Ruhmann purple generated in each section is determined in the same manner. The cumulative amount of absorbance (570 nm) indicated by the amount of Ruhmann purple generated in each unit time up to the coloring time is equal to the reference value (570 nm) at which coloring can be visually recognized, 0.010, and the amino acid concentration can be calculated using formula 8 in FIG. 14 by modifying formula 7 in FIG. 14. Formula 8 in FIG. 14 shows the relational expression described in claim 1.

10 mL of ninhydrin solution prepared at 30°C and 10 mL of amino acid solution were mixed and heated at 60°C at the same time, and the measured value of the color development time of the ninhydrin reaction and the data showing the change in the liquid temperature of the distilled water at the maximum time of 0.1 seconds per unit time of the same volume of distilled water as the mixed solution under the same measurement method and heating conditions were used to compare the concentration of a Gly standard solution of known concentration with the concentration calculated by the method found in the present invention (Figure 15). Figure 16 shows the calculation method for an example in which a 0.1 mol/LGly standard solution was used for the verification. Using the constants of the ninhydrin reaction rate equation specific to each amino acid obtained in advance and the concentration of the ninhydrin solution used in the measurement, the amino acid concentration for the heating time t can be calculated from the liquid temperature at the heating time t according to the relational equation of Equation 8 in Figure 14. Figure 17 shows a correspondence table of amino acid concentrations for the heating time t in the above example. This correspondence table shows an example of the correspondence table described in claim 1. From the verification experiment, the color development time was measured to be 26.5 s, and from the correspondence table, the amino acid concentration can be calculated to be 0.104 mol/L.

Similarly, verification was performed using Gly standard solutions of various concentrations. The concentrations of the Gly standard solutions used in the verification and the calculated concentrations were close to each other (FIG. 18). The concentrations of 0.05 mol/L Trp and Ala standard solutions were also close to each other. The amino acid quantification method discovered in the present invention allows amino acids to be quantified simply by measuring the coloration time. Finally, the reference value for coloration was set to 0.010, and although there is variation in the reference value for visual absorbance from the frequency distribution in FIG. 9, a reasonable measurement was obtained from FIG. 19, and visual confirmation of coloration is sufficiently reliable.

Quantitative analysis of amino acids is used in various fields, including the food industry. In particular, it plays an important role in the analysis of amino acid composition of proteins. In contrast to existing methods, the method for quantifying amino acids discovered in this invention can quantify amino acids in a short time by simply heating the solution and measuring the color development time, and is expected to be useful in various fields.

Claims (1)

A ninhydrin solution of known concentration kept at temperature condition _T0 and a solution of one kind of amino acid (Gly, Trp, or Ala) kept at the same temperature condition are mixed and heated to temperature condition _Tm at the same time. In the process, the time x from immediately after the mixed solution is heated to the moment when the color caused by Ruhemann's purple generated by the ninhydrin reaction can be visually recognized is measured. In advance, a volume of distilled water equal to that of the mixed solution is heated from temperature condition _T0 to Tm in the same manner as in the heating method described above. _m , a unit time having an upper limit of 1 second is determined, the temperature of the distilled water is continuously measured from immediately after heating at the maximum time in a time period separated by the unit time, and an amino acid concentration is calculated based on a correspondence table containing amino acid concentrations corresponding to a heating time t from a relational equation which holds when an accumulated amount of absorbance from immediately after heating to the time x, which is indicated by the amount of Ruhemann's purple produced per unit time and calculated from a rate of increase in absorbance (570 nm) proportional to a ninhydrin reaction rate, reaches 0.010, which is the minimum absorbance value at which the color of the ninhydrin reaction can be visually recognized.