Application of phenol reagent_Kain Industrial Additive

Background and overview^[1]

Phenol reagent is a commonly used reagent for measuring protein. The color reaction with protein includes two steps: the first step is under alkaline conditions, the protein reacts with copper to form a protein-copper complex; the second step is that the protein reacts with copper to form a protein-copper complex. -The copper complex reduces the phosphomolybdic acid-phosphotungstic acid reagent to produce a blue substance. Under certain conditions, the blue intensity is proportional to the amount of protein. This method is a method for determining protein concentration that is widely used in the field of biochemical work. The advantage is that it is easy to operate and has high sensitivity, and can measure 25 to 250 μg of protein; the disadvantage is that this reagent only reacts with tyrosine and tryptophan in proteins, so it has protein-specific effects, that is, different proteins are affected by tyrosine and tryptophan. The color intensity is slightly different due to different acid contents, and the standard curve is not strictly a straight line.

Apply^[2-3]

Application 1:

The existing methods for measuring the formaldehyde content in the air mostly use the enzymatic reaction of a series of enzymes with formaldehyde, and then place the reactants under a UV/visible light analyzer to detect the degree of increase in absorbance at the main wavelength, thereby Calculate the concentration of formaldehyde. The enzyme systems selected in these methods are complex, have a certain detection cost, and the calculation process is relatively confidential, which limits the scope of use. And its accuracy is low, it is a qualitative measurement, and the place of measurement is limited. In view of the deficiencies in the existing technology, the phenol reagent colorimetric method is used to measure the formaldehyde content in the air, which has high sensitivity, good selectivity and easy operation.

The technical solution is: a method for measuring the formaldehyde content in the air, using different concentrations of formaldehyde and phenol reagents to react to generate azine, using a phenol reagent colorimetric method to draw a standard curve, and then in the presence of ferric ammonium sulfate ferric ions, the azine It is oxidized by ferric ions in an acidic solution to form a blue-green compound. According to the color depth, the formaldehyde content in the air is determined by colorimetry, which includes the following steps:

(1) Sampling: Before sampling, the sampled room must be sealed for 24 hours. Sampling must be carried out in a calm climate environment. Use a large bubble absorption tube containing 5 ml of absorption liquid to collect gas at a flow rate of 0.5 liters/minute. 10 liters;

(2) Drawing of standard curve: Take 8 10 ml colorimetric tubes and prepare the standard color sequence according to the following table:

Add 0.40 ml of 1% ferric ammonium sulfate solution into each tube, shake well, and leave it for 15 minutes. Use a 1 cm cuvette to measure the absorbance at a wavelength of 630 nm, using water as a reference. For formaldehyde content (micrograms), draw a standard curve, or use the least squares method to calculate the regression equation of the standard curve:

Y＝bX+a——(a)

(a) where:

Y—(A-A0), that is, the difference between the absorbance of the standard solution (A) and the absorbance of the reagent blank solution (A0);

X—Formaldehyde content, micrograms;

b—the slope of the regression equation;

a—intercept of the regression equation,

(3) Sample measurement: After sampling, move the sample solution into the colorimetric tube, wash the absorption tube with a small amount of absorption solution, and merge the washing solution into the colorimetric tube to make the total volume 5 ml;

(4) Data processing, calculate the concentration C of the formaldehyde solution according to the following formula, unit: mg/ml,

(b) where:

A—Absorbance of sample solution;

A0—Absorbance of reagent blank solution;

b—the slope of the regression equation;

a—intercept of the regression equation;

Vr—converted to the sampling volume under the reference state, in liters.

Application 2:

At present, the commonly used methods for detecting protein include Kjeldahl nitrogen determination method, automatic nitrogen determination method, and double pulse contraction method. The crude protein in the feed is measured at the same time, and the results are analyzed and compared. The results show: The Kjeldahl nitrogen determination method takes a longer time, and the measurement results are relatively stable; compared with the national standard method, the results of the automatic nitrogen determination method are higher and the relative deviation is smaller, but the experimental cost is high; the double pulse contraction method has lower results, The deviation is large, but the operation is simple and efficient, and it is more suitable for rapid measurement in feed mills and other enterprises. Therefore, these shortcomings make the above methods unable to meet the actual production needs of enterprises. Phenol reagents can be used to provide a detection method for protein in pasture feed to solve the problems raised in the above background art.

The detection steps are as follows:�

(1) Weigh and crush the forage feed, put it into a flask, add sodium hydroxide solution, put it in a water bath at 60°C for 5 hours for reaction, take it out and shake it well, add distilled water to dilute it 40 times, centrifuge for 5 minutes, and let it stand;

(2) Take the supernatant and put it into the test tube, add phenol reagent solution A, mix evenly, and place it in a constant temperature water bath at 38°C for 20 minutes; then add phenol reagent solution B, shake and mix immediately, and place in a constant temperature water bath at 38°C Incubate for 30 minutes, measure the absorbance at a wavelength of 750 nm using a 1cm light diameter cuvette, and compare the measurement results with the standard curve to obtain the protein content of the sample.

The standard curve is established as follows:

(1) Reagent preparation

Standard protein solution 0.10mg/mL;

Phenol Reagent A Solution: Mix 3% sodium carbonate solution, 0.2mol/L sodium hydroxide solution, 0.01% copper sulfate pentahydrate solution, and 0.02% potassium citrate solution in equal volumes. The solution will be prepared and used on the same day;

Phenol Reagent Solution B: Prepare a sodium molybdate solution with a concentration of 2.5%, react with concentrated phosphoric acid with a volume fraction of 85%, add lithium sulfate and hydrogen bromide, dilute it 5 times, filter it, and store it in a brown bottle Medium; calibrate before use;

(2) Curve production

Take 8 test tubes, add different amounts of standard protein and distilled water, mix well, add 4mL of phenol reagent solution A to each tube, mix well, and place it at 38°C for 20 minutes; then add 0.4mL of phenol reagent solution B, and shake immediately Mix well, incubate at 38°C for 30 minutes, and measure the absorbance at 750nm using a 1cm light diameter cuvette; draw a standard curve with the protein concentration as the abscissa and the absorbance as the ordinate.

Main reference materials

[1] Dictionary of commonly used Chinese medicine words

[2]CN201710579528.2

[3] CN201610949713.1