Determination the content of cysteine in food by potassium dichromate spectrophotometry

. In this paper, a new method for the determination of cysteine by potassium dichromate spectrophotometry is established. In the H 2 SO 4 medium, potassium dichromate has strong oxidation. Potassium dichromate can react with the hydrosulfuryl(-SH) in cysteine, which makes potassium dichromate fade, and the fading degree of potassium dichromate is proportional to the amount of cysteine added. Therefore, the content of cysteine can be quantitatively determined by determinating the decrease value of absorbance. The various influencing factors for the determination the content of cysteine by potassium bichromate spectrophotometry are investigated. Under the optimal conditions, the content of cysteine in food is determined, and the results are satisfactory.


Introduction
Cysteine(Cys，the structure is shown in Figure 1) is the only reducing amino acid with sulfhydryl in the amino acid. It is one of the essential and important amino acid for the human body, and it is widely used in the fields of food, cosmetics and medicine. In biological metabolism, cysteine is involved in the reduction process of cells and has the role of regulating and protecting the liver, the imbalance of cysteine metabolism can lead to a series of functional diseases of the immune system. Therefore, the studies on the determination method of cysteine is of great significance in life science. Fig. 1 The structure of cysteine According to the literature, the determination methods of cysteine mainly include spectrophotometry[1-2], colorimetric analysis [3][4], electrochemical analysis[5-6], electrochemiluminescence analysis[6], fluorescence analysis [8][9], flow-injection analysis [10][11], etc.
In this paper, a new method for the determination the content of cysteine by potassium dichromate spectrophotometry is established. In the presence of H 2 SO 4 , potassium bichromate can oxidize cysteine, which makes the absorbance of potassium dichromate decreases. The decrease value of absorbance is proportional to the * Corresponding Author: wxrong5093@sohu.com amount of cysteine added. The relationship between the decrease value of absorbance ∆A and the cysteine concentration is △A = 0.0161+2.2861C(mg/mL) in the range of 0.002400 ～ 0.1680 mg/mL. Therefore, the content of cysteine can be quantitatively determined by determinating the decrease value of absorbance. Under the optimal conditions, this method is used to determine the content of cysteine in food, and the results are satisfactory.

Equipment and reagents
UV-2401 UV-visible spectrophotometer and 722S spectrophotometer are used.

Methods
In a 25mL comparison tube, 7.00 mL K 2 Cr 2 O 7 solution, 4.00 mL H 2 SO 4 solution and 5.00 mL cysteine standard solution or appropriate amount of the cysteine sample solution are added. Then the solution is diluted to the mark with distilled water and mixed well. This is the determination solution. In another 25mL comparison tube, the blank solution(7.00 mL K 2 Cr 2 O 7 solution + 4.00 mL H 2 SO 4 solution) is prepared in the same way. The two BIO Web of Conferences 59, 01012 (2023) https://doi.org /10.1051/10. /bioconf/20235901012 ICBB 2023 solutions are reacted for 25 min at 85℃ in water both and cool down using the running water for 5 minutes. After placing 5 min, the absorbance(A 1 ) of the blank solution and the absorbance(A 2 ) of the determination solution are measured at 435 nm against water, the decrease value of absorbance △A(A 1 -A 2 ) is calculated.

Absorption spectrum
According to experimental method, the absorption spectrum in the range of 400～500 nm are shown in Figure 2. It can be seen that the maximum absorption wavelength of the blank solution and the determination solution are at 435 nm. So, 435 nm is used for the whole experiment. 1-the blank solution(K 2 Cr 2 O 7 +H 2 SO 4 ); 2-the determination solution (K 2 Cr 2 O 7 +H 2 SO 4 +cysteine) Control conditions: K 2 Cr 2 O 7 solution:5.00 mL; H 2 SO 4 solution: 2.00 mL; cysteine standard solution: 5.00 mL; reaction temperature: room temperature; reaction time:5 min.

Reaction temperature
From table 1, we can see that as the reaction temperature increases, the absorbance(△A) also increases accordingly. The absorbance( △ A) reaches maximum value and is remained essentially unchanged when reaction temperature is 85 ℃ ～ 95 ℃ . Afterwards, the absorbance(△A) decreases when reaction temperature is 100℃. Thus, 85℃ is used.

Reaction time
The effect of the reaction time is showed in table 2. It can be seen from table 2 that the absorbance (△A) increases with the reaction time increases, at first. After the reaction time is 20 min, the absorbance(△A) remains essentially unchanged. So, 25 min is chosen.

Placement time
The datas in Table 3 indicate that the placing time had a minimal effect on the absorbance( △ A). Therefore, 5 minute is applied.

H2SO4 solution dosage
The effect of H 2 SO 4 solution dosage can be seen in Figure  3. The results show that the absorbance (△A) increases as H 2 SO 4 solution dosage increases, then the absorbance(△A) reaches maximum value and is remained basically unchanged when H 2 SO 4 solution dosage is 3.00～5.00 mL. Whereafter, the absorbance(△ A) decreases with the increase of H 2 SO 4 solution dosage. Thus, H 2 SO 4 solution dosage is selected as 4.00 mL.

K2Cr2O7 solution dosage
The effect of K 2 Cr 2 O 7 solution dosage is shows in Figure  4. Form Figure 4, we can see that the absorbance(△A) increases with K 2 Cr 2 O 7 solution dosage increases in the beginning, and then the absorbance( △ A) reaches maximum value when K 2 Cr 2 O 7 solution dosage is 6.00～ 9.00 mL. Hence, 7.00 mL K 2 Cr 2 O 7 solution is used. Fig. 3  curve is showed in Figure 5. The linear equation between the the cysteine mass concentration and the absorbance (△A) is △A=0.0161+2.2861C(mg/mL) in the range of 0.002400~0.1680 mg/mL, and the correlation coefficient is 0.9996.

Determination the content of food grade cysteine
0.0500 g food grade cysteine is weighed and dissolved in bidistilled water, then this solution is transferred into a 100 mL volumetric flask and diluted to the 100.0 mL, mixed well. This is the food grade cysteine sample solution.
In the optimum conditions, 0.50 mL food grade cysteine sample solution are added. According to the proposed method(2.2 method ), the absorbance(A 1 ) of the blank solution and the absorbance (A 2 ) of the determination solution are measured at 435 nm against water, the absorbance( △ A=A 1 -A 2 ) is calculated. The content of food grade cysteine can be calculated based on the absorbance( △A) and the linear equation, and the recovery rates of standard addition are determined. Meanwhile, the content of the food grade cysteine is determined by standard method. The results as show in Table 4. From Table 4, we can see that the content of food grade cysteine is 989.9 mgꞏg -1 by proposed method, and the recovery rates are 96.2%～98.8%. The content of food grade cysteine is 973.5 mgꞏg -1 by standard method.

Conclusion
In this paper, a new method for the determination of cysteine by potassium dichromate spectrophotometry is established. Under the optimal conditions, the content of food grade cysteine is determined by proposed method, and the recovery rates of standard addition are 96.2%～ 98.8%. The determination result by proposed method agree well with the determination result by standard method. This method does not need complicated or expensive equipment, and it does not require expensive reagents also. Therefore, this method has the advantages of simply, rapidness, convenience, low analytical cost and so on. It is obvious that this study had certain practical significance and practical value on establishing a new method for the determination of cysteine.