Improvements in protein assay devices and precautions for use

Protein is one of the most important indicators of food inspection. Its determination is usually based on the classical Kjeldahl method. The instruments used by the Kjeldahl method include: Azometer, also known as a protein analyzer. Kjeldahl is divided into automatic and semi-automatic nitrogen analyzers. Automatically complete the addition of alkali, boric acid, distillation, ammonia absorption throughout the entire process, plus the volume of boric acid and alkali plus distillation and absorption process time can be set. No manual operation is required for convenience. However, for users who require high experimental results, it is recommended to use a semi-automatic Kjeldahl tester. Because the semi-automatic Kjeldahl nitrogen analyzer can control every step of its own, including time, reagents, etc., it can master the entire process of protein determination. The introduction of the Kjeldahl method in the national standard is relatively straightforward, and there are no notes on what should be noted in the operation. This causes difficulties for the person who has just performed the test. If the individual steps are not properly handled, it will directly affect the test results. Accuracy. Some people think that Kjeldahl distillation equipment is not safe enough, and some people are afraid to operate. Based on the experience of multi-party learning and years of testing, the author summarized the individual steps that were difficult to grasp in the protein determination operation, and modified the detection device slightly to improve the safety of the test and the accuracy of the test results. Has a good reference, is introduced as follows (operation steps refer to GB/T 5009. 522003).

1 Safety modification of steam generator

There are two air pipes on the flask of the steam generator, one is the air pipe leading to the reaction chamber, and the other is a safety air pipe. During the distillation process, the spring clip of the airway leading to the reaction chamber is released. The pressure inside the steam generator flask can be kept basically safe, but after the distillation is completed, the airway leading to the reaction chamber should immediately be clamped with a spring clip. Dead, water vapor generator flask can not be timely cooling, it will make the water vapor generator flask pressure increases rapidly, the safety tube is too short boiling water boiling easily hurt, boiling water splashed on the electric furnace is easy to damage the electric furnace, safety tube is too easy The internal pressure of the steam generator flask is increased, and the steam generator flask easily bursts.

In order to prevent the boiling water from overflowing and the water vapor generator flask burst, place a hole in the plug of the steam generator flask, insert a piece of glass tube (bleed tube), and insert the glass tube into the stopper to expose the rubber stopper. Must not enter the steam generator flask below the liquid level, the upper end connected to a small section of hose (modified steam generator distillation flask rubber stopper with safety tube, air tube, deflation tube each one), clip on the spring clip, distillation When the deflation tube clamp is clamped and the distillation is stopped, the open air tube spring clamp is immediately released after the spring clamp that leads to the air tube of the reaction chamber is clamped, and the effect of balancing the internal pressure of the distillation flask is achieved, so as to prevent the boiling water from overflowing the safety tube. It also prevents the steam generator flask from bursting.

2 Precautions

2. 1 Kjeldahl flask and sample requirements

Weigh 0. 1 ~ 5. 0g sample. If a sample of 1g or more is to be used, a larger Kjeldahl flask is required, preferably 500 mL and 800 to 1000 mL. Such a Kjeldahl flask has a better effect on shortening the ammoniating time, heating uniformity and complete ammoniation. The sample should be uniform. If the solid sample should be finely studied in advance, the liquid sample should be mixed evenly. When the sample is placed in the Kjeldahl flask, do not adhere to the bottleneck. In case the adhesion can be slowly washed down with a small amount of water to avoid incomplete digestion of the sample, resulting in low results.

2. 2 Addition of H2 SO4 and K2 SO4

Decomposition of organic matter requires concentrated H2SO4. The amount of H2SO4 should be determined according to the type and content of organic substances in the sample. If the content of lipid in the sample is high, more H2SO4 is added. In order to increase the decomposition temperature, K2SO4 must be added in an appropriate amount. If too much K2SO4 is added, the temperature will be too high. The resulting ammonium bisulfate will also decompose and release ammonia to cause losses; K2SO4 will add too little, and the decomposition temperature will be too low. Too long. The addition ratio of K2SO4 and H2SO4 is: 1g sample, K2SO4: H2SO4 = 7g: 12mL This ratio is used at home and abroad, and it is recognized. There is also a ratio in the national standard: 1g sample, K2SO4: H2SO4 = 6g: 20mL. The solid state of the sample after digestion and cooling indicates that the amount of K2SO4 is too large or the amount of H2SO4 is insufficient.

2. 3 Catalyst Selection

Used as catalysts are Hg, HgO, Se, selenium compounds, CuSO4, TiO2, Hg and HgO are toxic but the results are good, Se and CuSO4 results are basically the same, TiO2 results are low; for the digestion of cereals are generally used CuSO4 is used as catalyst. The advantage of CuSO4 as catalyst is that it can be used as an indicator of alkaline reaction when distilling ammonia.

2. 4 Selection of Indicators

Mix 1 part methyl red ethanol solution (1g/L) with 5 parts bromocresol green ethanol solution (1g/L) immediately before use. It can also be mixed with 2 parts methyl red ethanol solution and 1 part methylene blue ethanol solution. Whichever mixing indicator is used, it must be mixed when it is used. It is green in alkaline solution, gray in neutral solution and red in acidic solution.

2. The amount of 5 NaOH added

Before the distillation, a sufficient amount of NaO H with a concentration of 40% was added. Only sufficient NaO H was added to allow the ammonia to be fully absorbed by the boric acid to prevent ammonia from escaping from the sample solution, otherwise the measured value was low.

2. 6 The intensity of the heat source

The strength of the heat source and the digestion rate during digestion are related to whether the sample is fully ammoniated. Even if the K2SO4 salt is added too much, if the heat source is weak, it is meaningless. The heat source is too strong to cause loss of ammonia, and the ammonia recovery rate is low. In addition, the size of the Kjell's bottle, the thickness and length of the neck, etc., are also related to the intensity of the heat source. During the entire digestion process, gentle boiling should be maintained so that the firepower can be concentrated at the bottom of the Kjeldahl flask. The fire must not be excessive. Otherwise, the digestion solution can bump and stick the sample to the wall of the nitrogen bottle, and the protein adhered to the wall of the nitrogen bottle. Nitrogen is lost in the absence of sulfuric acid.

2. 7 Degree of digestion

The digestive juice became blue-green and clear and transparent, indicating complete digestion. During digestion, if the solution is not transparent, cool the Kjeldahl flask and add 20% to 3 mL of hydrogen peroxide to promote oxidation and continue digestion for 30 minutes.

2. 8 Absorption Solution Concentration

Boric acid is generally used as an absorbing solution, and ammonia can be completely absorbed at a concentration of 3% or more. Generally, 4% of boric acid is used for safety reasons.

2. 9 Judgment of distillation effect

Whether or not the ammonia was completely distilled off, the ammonia absorption flask was removed, and the distilled droplets were measured using a p H test paper. If not, the ammonia distillation was thorough.