Method for quantitatively detecting L-cysteine

Abstract

The invention relates to a method for quantitatively detecting L-cysteine, which is characterized in that a Na2SO3-KBrO3-K4Fe (CN) 6-H2SO4 reaction system, namely a BSF reaction system, is used as a detection solution, and quantitative analysis of L-cysteine is realized according to different responses, namely different induction times, of the system to L-cysteine with different concentrations. The quantitative analysis method for L-cysteine has the characteristics of high accuracy, easiness in operation, convenience, rapidness and the like.

Description

Technical Field

[0001] The invention relates to an analysis and detection method, specifically to establishing a "Na2SO3-KBrO3-K4Fe(CN)6-H2SO4" BSF clock reaction system, and realizing a quantitative analysis method for L-cysteine ​​according to the different responses of the system to different concentrations of L-cysteine, i.e., different induction times, belonging to the field of analytical chemistry. Background Art

[0002] The molecular formula of L-cysteine ​​is C3H7NO2S. It is a non-essential amino acid and an isomer of cysteine. It has important uses in medicine and food. L-cysteine ​​can be used as a bread fermentation promoter, a preservative, an antioxidant and stabilizer for milk powder and juice, and a nutritional additive for pet food. It can be used in natural juice to prevent the oxidation and browning of VC; it has a detoxifying effect on propylene and aromatic acid poisoning. It also has the effect of preventing radiation damage.

[0003] In medicine, cysteine ​​is mainly used in liver medicine, detoxification medicine, expectorant medicine and other medicines. Cysteine ​​and its derivatives can be used for liver poisoning and detoxification, antipyretic and analgesic, ulcer treatment, fatigue recovery, infusion and comprehensive amino acid preparations, especially for expectoration; it has the effect of treating bronchitis and expectoration.

[0004] The determination of L-cysteine ​​mainly adopts instrumental analysis methods, such as high performance liquid chromatography (HPLC) and high performance liquid chromatography ultraviolet detection (HPLC-UV). There are also reports on the use of fluorescence, spectrophotometry and other determination methods. Chromatography has the advantages of high sensitivity and good accuracy, but the analysis sensitivity is reduced for samples with complex matrices. Therefore, it is very necessary to find a detection and analysis method with good detection effect and simple and fast operation.

[0005]

[0006] Structural formula (I) L-cysteine Summary of the invention

[0007] The present invention aims to provide a new quantitative detection method for L-cysteine, that is, a method for quantitatively detecting L-cysteine ​​using the "Na2SO3-KBrO3-K4Fe(CN)6-H2SO4" BSF clock system as a detection solution. The method is a standard curve (working curve) method developed based on the sensitive response of the BSF clock system to L-cysteine. Specifically, the "Na2SO3-KBrO3-K4Fe(CN)6-H2SO4" BSF clock reaction system is used as a detection solution to record the pH change over time; when the BSF clock reaction starts, a series of L-cysteine ​​sample solutions of different concentrations to be detected are added to the BSF clock system in equal volumes; according to the different induction times generated by the system when the concentrations of the solutions to be detected in the BSF clock system are different, the quantitative detection of the L-cysteine ​​samples to be detected is achieved.

[0008] According to the relationship between the concentration of L-cysteine ​​in the BSF clock system and the induction time, a working curve was established, in which the abscissa is the concentration of L-cysteine ​​in the BSF clock system and the ordinate is the induction time t. When the concentration of L-cysteine ​​in the system is 1.25×10 -3 mol / L to 6.25×10 -3 When the induction time t is between 0.174 and 0.176 mol / L, there is a linear relationship between the induction time t and the concentration of L-cysteine, based on which the quantitative detection of L-cysteine ​​in the sample can be achieved.

[0009] The difference between the present quantitative detection method and the prior art lies in that the present invention uses the "Na2SO3-KBrO3-K4Fe(CN)6-H2SO4" BSF clock system as the detection solution, and the system responds differently to different concentrations of L-cysteine, i.e., the induction time is different, thereby realizing the quantitative analysis of L-cysteine.

[0010] The concentration range of L-cysteine ​​in the test solution (BSF clock system) was 1.25×10 -3 mol / L to 6.25×10 -3 mol / L.

[0011] When L-cysteine ​​was detected in the detection solution (BSF clock system), the temperature of the BSF clock system was controlled at 22±0.5°C.

[0012] Using the above BSF clock system, the concentration range in which L-cysteine ​​can be detected is the optimal concentration range determined experimentally. Within this concentration range, the induction time has a good response to the change in L-cysteine ​​concentration, and the linear correlation coefficient is large. In addition, the concentration range of each component in the detection solution (BSF clock system) is shown in Table 1, and the optimal concentration of the detection solution (BSF clock system) obtained after multiple experiments is shown in Table 2: Table 1: Concentrations of components in the BSF clock system <![CDATA[Na2SO3(mol / L)]]> <![CDATA[KBrO3(mol / L)]]> <![CDATA[K4Fe(CN)6(mol / L)]]> <![CDATA[H2SO4(mol / L)]]> 0.01-0.09 0.02-0.07 0.01-0.05 0.01-0.05 Table 2: Optimal concentrations of components in the BSF clock system <![CDATA[Na2SO3(mol / L)]]> <![CDATA[KBrO3(mol / L)]]> <![CDATA[K4Fe(CN)6(mol / L)]]> <![CDATA[H2SO4(mol / L)]]> 0.07750 0.06500 0.02075 0.01050 The specific experimental steps are as follows: 1. Prepare the test solution (BSF clock system) according to the concentration range specified in Table 1, and its temperature is controlled at 22±0.5℃; insert the prepared working electrode (pH composite electrode, Leici, E-331) into the solution, and connect the other end of the working electrode to the computer through the potential / temperature / pH comprehensive tester (Jiaxing Disheng Electronic Technology Co., Ltd., ZHFX-595). Open the chemical signal acquisition and analysis program in the computer to set the acquisition time and sampling speed, and quickly click the start button to monitor the pH of the solution. The computer records the curve of the pH change over time, that is, the BSF clock spectrum. When the substance needs to be detected, the substance to be detected is quickly added at the same time as the BSF clock system reaction starts, and the BSF clock spectrum of pH change over time is recorded in the same way.

[0013] The basic parameters of the BSF clock spectrum include: Induction time: the time required from the start of the BSF clock system reaction to pH stabilization.

[0014] pH jump range: the pH corresponding to the start of the pH jump in the system to the pH corresponding to the end of the pH jump.

[0015] Establishment of working curve between L-cysteine ​​concentration in detection solution and pH induction time Using distilled water as solvent, cysteine ​​solutions with concentrations of 0.25 mol / L, 0.5 mol / L, 0.75 mol / L, 1 mol / L, and 1.25 mol / L were prepared as sample solutions. At the start of the BSF clock system reaction, 200 μL of the sample solutions of different concentrations were added to 40 mL of the BSF clock system using a pipette, so that the L-cysteine ​​concentration in the system was 1.25×10 -3 mol / L to 6.25×10 -3mol / L; the change in the response of the BSF clock system is the induction time, recorded as t; when the L-cysteine ​​concentration in the system is different, the induction time t of the BSF clock system is also different; the L-cysteine ​​concentration in the system is used as the horizontal axis and t is used as the vertical axis to draw a graph; when the L-cysteine ​​concentration in the system is between 1.25×10 -3 mol / L to 6.25×10 -3 mol / L, the induction time t of the BSF clock system was in a linear relationship with the concentration of L-cysteine, and a working curve was obtained.

[0016] Quantitative detection of L-cysteine By adding a test sample of unknown concentration into the detection solution BSF clock system at the beginning of the BSF clock system reaction, the corresponding induction time (t) of the BSF clock system can be measured. According to the corresponding relationship between t and concentration on the working curve, the concentration of L-cysteine ​​in the detection system can be obtained, and then the concentration of L-cysteine ​​in the test sample can be calculated. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the change in pH value of the test solution (BSF clock system) over time when no sample to be tested is added in Example 1.

[0018] Figure 2 In Example 1, 1.25×10 -3 mol / L L-cysteine, and the pH value of the detection solution (BSF clock system) changes with time.

[0019] Figure 3 In Example 1, 2.5×10 -3 mol / L L-cysteine, and the pH value of the detection solution (BSF clock system) changes with time.

[0020] Figure 4 This is the working curve between pH induction time t and L-cysteine ​​concentration in Example 1.

[0021] Figure 5 This is a graph showing the change in pH value of the test solution (BSF clock system) over time when no sample to be tested is added in Example 2.

[0022] Figure 6 In Example 2, 3.75×10 -3 mol / L L-cysteine, and the pH value of the detection solution (BSF clock system) changes with time.

[0023] Figure 7 In Example 2, 5×10-3 mol / L L-cysteine, and the pH value of the detection solution (BSF clock system) changes with time.

[0024] Figure 8 This is the working curve between pH induction time t and L-cysteine ​​concentration in Example 2.

[0025] Fig. 9 This is a graph showing the change in pH value of the test solution (BSF clock system) over time when no sample to be tested is added in Example 3.

[0026] Fig.10 In Example 3, 1.25×10 -3 mol / L L-cysteine, and the pH value of the detection solution (BSF clock system) changes with time.

[0027] Fig.11 In Example 3, 6.25×10 -3 mol / L L-cysteine, and the pH value of the detection solution (BSF clock system) changes with time.

[0028] Fig.12 This is the working curve between pH induction time t and L-cysteine ​​concentration in Example 3. DETAILED DESCRIPTION Example 1

[0029] The "Na2SO3-KBrO3-K4Fe(CN)6-H2SO4" BSF clock system was used as the detection solution to quantitatively analyze L-cysteine. Equal volumes of L-cysteine ​​sample solutions with different concentrations were added to the BSF clock system to establish a working curve (such as a linear relationship) between the L-cysteine ​​concentration and the induction time in the detection system, so as to achieve the purpose of detecting the L-cysteine ​​concentration in the BSF clock system, and then calculate the concentration of L-cysteine ​​in the test sample.

[0030] (1) Preparation of test solution First, use distilled water to prepare 0.31 mol / L Na2SO3 solution, 0.26 mol / L KBrO3 solution, 0.083 mol / L K4Fe(CN)6 solution and 0.042 mol / L H2SO4 solution respectively. Into a 50 mL small beaker, add 10 mL of 0.31 mol / L Na2SO3 solution, 10 mL of 0.26 mol / L KBrO3 solution, 10 mL of 0.083 mol / L K4Fe(CN)6 solution, and 10 mL of 0.042 mol / L H2SO4 solution in sequence to ensure that the concentrations of each component in the "Na2SO3-KBrO3-K4Fe(CN)6-H2SO4" BSF clock system are Na2SO3 0.07750 mol / L, KBrO3 0.06500 mol / L, K4Fe(CN)6 0.02075 mol / L, and H2SO4 0.01050 mol / L. The total volume is 40 mL, and the temperature is controlled at 22°C.

[0031] At the same time, distilled water was used as solvent to prepare a series of L-cysteine ​​sample solutions with different concentrations.

[0032] (2) Obtaining the BSF clock spectrum The graph of the pH value of the prepared test solution changing with time is recorded by a computer equipped with a chemical signal acquisition and analysis program (without adding the test sample), such as Figure 1 As shown. The pH induction time was 350 s, which was used as a blank control. Two other groups of test solutions were prepared with the same concentration of each component as the above test solution. For one group, at the beginning of the reaction, 200 μL of 0.25 mol / L L-cysteine ​​sample solution was added to 40 mL of BSF clock system, so that the concentration of L-cysteine ​​in the test solution was 1.25×10 -3 mol / L, the addition of L-cysteine ​​shortened the induction time to 316s. Figure 2 As shown; for the other group, at the beginning of the reaction, 200 μL of 0.5 mol / L L-cysteine ​​sample solution was added to 40 mL of BSF clock system, so that the concentration of L-cysteine ​​in the detection solution was 2.5×10 -3 mol / L, the addition of L-cysteine ​​made the induction time become 282s, such as Figure 3 shown. Figure 2 , Figure 3 It was confirmed that different concentrations of L-cysteine ​​in the detection solution led to different induction times for the BSF clock system to appear. -3 mol / L to 6.25×10 -3mol / L, the different induction times of the BSF clock system caused by different concentrations can be observed.

[0033] (3) Quantitative detection A working curve was established based on the relationship between the concentration of L-cysteine ​​in the detection system and the induction time, such as Figure 4 As shown in the figure, the horizontal axis is the concentration of L-cysteine ​​in the BSF clock system, and the vertical axis is the induction time t. When the concentration of L-cysteine ​​in the detection system is 1.25×10 -3 mol / L to 6.25×10 -3 mol / L, the induction time and the concentration of L-cysteine ​​have a linear relationship, and the linear equation is t=-29783c+351.57, R 2 = 0.9968. Based on this, the quantitative detection of L-cysteine ​​in the sample can be achieved. Example 2

[0034] (1) Preparation of test solution First, use distilled water to prepare 0.3 mol / L Na2SO3 solution, 0.25 mol / L KBrO3 solution, 0.083 mol / L K4Fe(CN)6 solution and 0.041 mol / L H2SO4 solution respectively. Into a 50 mL small beaker, add 10 mL of 0.3 mol / L Na2SO3 solution, 10 mL of 0.25 mol / L KBrO3 solution, 10 mL of 0.083 mol / L K4Fe(CN)6 solution, and 10 mL of 0.041 mol / L H2SO4 solution in sequence to ensure that the concentrations of each component in the "Na2SO3-KBrO3-K4Fe(CN)6-H2SO4" BSF clock system are Na2SO3 0.07500 mol / L, KBrO3 0.06250 mol / L, K4Fe(CN)6 0.02075 mol / L, and H2SO4 0.01025 mol / L. The total volume is 40 mL, and the temperature is controlled at 22°C.

[0035] At the same time, distilled water was used as solvent to prepare a series of L-cysteine ​​sample solutions with different concentrations.

[0036] (2) Obtaining the BSF clock spectrum The graph of the pH value of the prepared test solution changing with time is recorded by a computer equipped with a chemical signal acquisition and analysis program (without adding the test sample), such as Figure 5As shown. The pH induction time was 352 s, which was used as a blank control. Two other groups of test solutions were prepared with the same concentration of each component as the above test solution. For one group, at the beginning of the reaction, 200 μL of 0.75 mol / L L-cysteine ​​sample solution was added to 40 mL of BSF clock system, so that the concentration of L-cysteine ​​in the test solution was 3.75×10 -3 mol / L, the addition of L-cysteine ​​shortened the induction time to 235 s. Figure 6 As shown; for the other group, at the beginning of the reaction, 200 μL of 1 mol / L L-cysteine ​​sample solution was added to 40 mL of BSF clock system, so that the concentration of L-cysteine ​​in the detection solution was 5×10 -3 mol / L, the added L-cysteine ​​makes the induction time become 199 s, such as Figure 7 shown. Figure 6 , Figure 7 It was confirmed that different concentrations of L-cysteine ​​in the detection solution led to different induction times for the BSF clock system to appear. -3 mol / L to 6.25×10 -3 mol / L, the different induction times of the BSF clock system caused by different concentrations can be observed.

[0037] (3) Quantitative detection A working curve was established based on the relationship between the concentration of L-cysteine ​​in the detection system and the induction time, such as Figure 8 As shown in the figure, the horizontal axis is the concentration of L-cysteine ​​in the BSF clock system, and the vertical axis is the induction time t. When the concentration of L-cysteine ​​in the detection system is 1.25×10 -3 mol / L to 6.25×10 -3 mol / L, the induction time has a linear relationship with the concentration of L-cysteine, and the linear equation is t=-29669c+351.38, R 2 =0.9964. Based on this, the quantitative detection of L-cysteine ​​in the sample can be achieved. Example 3

[0038] (1) Preparation of test solution First, use distilled water to prepare 0.33 mol / L Na2SO3 solution, 0.27 mol / L KBrO3 solution, 0.082 mol / L K4Fe(CN)6 solution and 0.041 mol / L H2SO4 solution respectively. To a 50 mL beaker, add 10 mL of 0.33 mol / L Na2SO3 solution, 10 mL of 0.24 mol / L KBrO3 solution, 10 mL of 0.082 mol / L K4Fe(CN)6 solution, and 10 mL of 0.041 mol / L H2SO4 solution in sequence to ensure that the concentrations of the components in the "Na2SO3-KBrO3-K4Fe(CN)6-H2SO4" BSF clock system are Na2SO3 0.08250 mol / L, KBrO3 0.06750 mol / L, K4Fe(CN)6 0.02050 mol / L, and H2SO4 0.01025 mol / L. The total volume is 40 mL, and the temperature is controlled at 22 °C.

[0039] At the same time, distilled water was used as solvent to prepare a series of L-cysteine ​​sample solutions with different concentrations.

[0040] (2) Obtaining the BSF clock spectrum The graph of the pH value of the prepared test solution changing with time is recorded by a computer equipped with a chemical signal acquisition and analysis program (without adding the test sample), such as Fig. 9 As shown. The pH induction time was 349 s, which was used as a blank control. Two other groups of test solutions were prepared with the same concentration of each component as the above test solution. For one group, at the beginning of the reaction, 200 μL of 0.25 mol / L L-cysteine ​​sample solution was added to 40 mL of BSF clock system, so that the concentration of L-cysteine ​​in the test solution was 1.25×10 -3 mol / L, the addition of L-cysteine ​​shortened the induction time to 315 s. Fig.10 As shown; for the other group, at the beginning of the reaction, 200 μL of 1.25 mol / L L-cysteine ​​sample solution was added to 40 mL of BSF clock system, so that the concentration of L-cysteine ​​in the detection solution was 6.25×10 -3 mol / L, the addition of L-cysteine ​​made the induction time become 169 s, such as Fig.11 shown. Fig.10 , Fig.11 It was confirmed that different concentrations of L-cysteine ​​in the detection solution led to different induction times for the BSF clock system to appear. -3 mol / L to 6.25×10-3 mol / L, the different induction times of the BSF clock system caused by different concentrations can be observed.

[0041] (3) Quantitative detection A working curve was established based on the relationship between the concentration of L-cysteine ​​in the detection system and the induction time, such as Fig.12 As shown in the figure, the horizontal axis is the concentration of L-cysteine ​​in the BSF clock system, and the vertical axis is the induction time t. When the concentration of L-cysteine ​​in the detection system is 1.25×10 -3 mol / L to 6.25×10 -3 mol / L, the induction time and the concentration of L-cysteine ​​have a linear relationship, and the linear equation is t=-29851c+351.62, R 2 =0.9965 Based on this, the quantitative detection of L-cysteine ​​in the sample can be achieved.

Claims

1. A method for quantitatively detecting L-cysteine, characterized in that: Using distilled water as solvent, prepare a solution of L-cysteine, a sample to be tested; The "Na2SO3-KBrO3-K4Fe(CN)6-H2SO4" BSF clock reaction system was used as the detection solution, and the pH change over time was recorded; the temperature of the BSF clock system was controlled within the range of 22±0.5℃; when the BSF clock reaction started, equal volumes of a series of L-cysteine ​​solutions of the test sample with different concentrations were added to the BSF clock system; according to the different induction times generated by the system when the concentrations of the test solution in the BSF clock system were different, quantitative detection of the test sample was achieved: a working curve was established based on the relationship between the concentration of L-cysteine ​​in the test solution in the BSF clock system and the induction time, in which the horizontal axis was the concentration of L-cysteine ​​in the test solution in the BSF clock system, and the vertical axis was the induction time t; when the concentration of L-cysteine ​​in the system was 1.25×10 -3 mol / L to 6.25×10 -3 When the induction time t is between 0.1mol / L and 0.1mol / L, there is a linear relationship between the induction time t and the concentration of L-cysteine, thereby realizing the quantitative detection of L-cysteine ​​in the sample; The molar concentration ranges of the components in the test solution are: Na2SO3 0.01-0.09 mol / L, KBrO3 0.02-0.07 mol / L, K4Fe(CN)6 0.01-0.05 mol / L, H2SO4 0.01-0.05 mol / L.

2. The method according to claim 1, characterized in that: The molar concentrations of the components in the test solution are Na2SO3 0.07750 mol / L, KBrO3 0.06500 mol / L, K4Fe(CN)6 0.02075 mol / L, and H2SO4 0.01050 mol / L.

3. The method according to claim 1, characterized in that: The temperature of the BSF clock system was controlled at 22°C when detecting L-cysteine ​​solution.

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