Amino acid content measurement experiment

["Collaborating Expert | Ziyi Yang, M.S.", "Pharmacy Fudan University"], ["Reviewed by | Dr. Tao Yu", "Biochemistry Molecular Biology University of Chinese Academy of Sciences"]

Summary

The Kjeldahl method is a commonly used chemical analytical method for determining the nitrogen content of organic and inorganic samples. It was originally invented and named by Danish chemist Johann Kjeldahl in 1883. The method is mainly suitable for determining the nitrogen content in samples of proteins, meat, feed, soil, fertilizers, and so on.

Principle

The principle of the Kjeldahl method is based on the quantitative reaction of ammonia, in which nitrogen in an organic matter sample is converted to ammonia gas by digestion and distillation steps and quantitatively determined by titration.

Due to the widespread presence of nitrogen in many biological and environmental samples, Kjeldahl nitrogen determination has become a common method for determining the nitrogen content of samples. Concentrated sulfuric acid and a catalyst are added to the sample, mixed thoroughly, and then heated and digested to decompose the carbon and hydrogen in the sample, which is oxidized to carbon dioxide and water, and the organic nitrogen in the sample is converted to ammonium sulfate.

Alkaline distillation to free ammonia, absorbed with boric acid and then titrated with sulfuric acid or hydrochloric acid standard titration solution, according to the consumption of acid multiplied by the conversion factor, that is, the content of amino acids.

Appliance

The method is widely used in the fields of food, agriculture, and environmental sciences to determine the protein content of samples, to assess the nitrogen content of fertilizers, to monitor the nitrogen content of soils, and to study nitrogen cycling in biochemical and environmental processes.

Operation method

Determination of amino acid content--Kjeldahl method

Principle

The principle of the Kjeldahl method is based on the quantitative reaction of ammonia, in which nitrogen in an organic matter sample is converted to ammonia gas by digestion and distillation steps and quantitatively determined by titration. Due to the widespread presence of nitrogen in many biological and environmental samples, Kjeldahl nitrogen determination has become a common method for determining nitrogen content in samples. Concentrated sulfuric acid and a catalyst are added to the sample, mixed thoroughly, and then heated and digested to decompose the carbon and hydrogen in the sample, which is oxidized to carbon dioxide and water, and the organic nitrogen in the sample is converted to ammonium sulfate. Alkaline distillation to free ammonia, absorbed with boric acid and then titrated with sulfuric acid or hydrochloric acid standard titration solution, according to the consumption of acid multiplied by the conversion factor, that is, the content of amino acids.

Materials and Instruments

Samples, concentrated sulfuric acid, sodium hydroxide

Distillation apparatus (distillation flask, condenser, etc.), trap bottle

Acid-base indicator, buret, buret holder

Buret fixture, boric acid solution

Move

1. Preparation:

Ensure that the laboratory is safe and wear appropriate protective equipment such as lab coat, gloves and goggles.

Prepare the required materials: samples, concentrated sulfuric acid, sodium hydroxide, acid-base indicator, burette, burette holder, distillation unit, and trap bottle.

2、Sample digestion:

Put the accurately weighed sample into the digestion bottle.

Add an appropriate amount of concentrated sulfuric acid, taking care to avoid overdose (potassium sulfate and copper sulfate can be added appropriately as a catalyst to accelerate protein decomposition).

Heat the digestion bottle and use appropriate heating equipment and conditions for digestion. Be careful to control the temperature and avoid spillage and splashing.

3, Distillation:

Transfer the digestion products to the distillation flask and add appropriate amount of sodium hydroxide solution.

Install the condenser and trap flask to ensure a good seal.

Heat the distillation flask so that the ammonia gas produced passes through the condenser and is collected in the catch bottle.

4. Neutralization and Titration:

Dissolve the ammonia in the trap bottle in 10 ml of an acid solution of boric acid solution, a drop of acid-base indicator may be added to assist in determining the point of neutralization.

Titrate using an acid solution of known concentration, dropping slowly until the solution changes color significantly, indicating completion of neutralization.

Record the volume of acid solution used in the titration to calculate the nitrogen content of the sample.

5. Calculate the result:

Calculate the nitrogen content in the sample based on the concentration and volume of the acid solution used in the titration and the mass of the sample.

The following formula can be used to calculate the nitrogen content in the sample:

Nitrogen content (%) = (Concentration of acid solution × Volume of acid solution × Equivalent number of nitrogen atoms) / Mass of sample

where

Acid solution concentration is the concentration of the acid solution used for the titration, and the unit can be moles/liter or grams/liter, depending on the specific experiment.

Acid Solution Volume is the volume of the acid solution used in the titration, which can be in liters or milliliters, depending on the value recorded in the experiment.

Equivalent number of nitrogen atoms is the number of equivalents of nitrogen in a chemical reaction, which for the Kjeldahl method is 1.

Sample mass is the mass of the sample used for the experiment and can be in grams or milligrams, depending on the mass used in the experiment.

Caveat

When performing the Kjeldahl ammonia method experiment, here are some things to keep in mind to avoid making mistakes or causing the experiment to fail:

1. Safety Attention:

The Kjeldahl ammonia method uses strong acids such as concentrated sulfuric acid and requires safe handling and wearing of appropriate protective equipment such as lab coat, gloves and goggles.

Acidic solutions and ammonia are irritating and toxic, keep the laboratory well ventilated and avoid direct contact with these substances.

2. Sample Digestion:

Control the temperature and time of the digestion process and avoid excessive temperature and long digestion time to prevent decomposition of the sample or other unwanted reactions from occurring.

Ensure that digestion bottles are well sealed to avoid gas leakage during the digestion process.

3. Distillation:

Ensure that the distillation unit is well sealed to avoid leakage of ammonia or entry of outside air.

In order to prevent trace amounts of ammonia in the water from escaping by heat and affecting the measurement results, so the water in the water vaporizer should be kept acidic.

Be careful to control the distillation rate, too fast a distillation rate may result in ammonia not being completely trapped.

4. Neutralization and titration:

Use of appropriate acid-base indicators ensures accurate determination of the titration endpoint.

(The following are some common acid-base indicators and their applications:

√ Phenolphthalein indicator:

Color change range: pH 8.2~10.0

Suitable for titration of strong acids and weak bases, such as titration of sulfuric acid and ammonia.

√ Violet blue indicator (ibuprofen blue):

Color change range: pH 4.6~6.0 (red-blue)

Suitable for titration of weak acids with weak bases, e.g., titration of acetic acid with ammonia.

√ Methyl orange indicator:

Color change range: pH 3.2~4.4 (red-yellow)

Suitable for the titration of strong acids and strong bases, such as the titration of hydrochloric acid and sodium hydroxide.

√ Acid-base violet indicator:

Color change range: pH 0.0~1.6 (red-violet)

Suitable for titration of strong acids and bases, e.g., titration of sulfuric acid and sodium hydroxide.

√ Bromophenol Blue Indicator:

Color change range: pH 3.0~4.6 (yellow-blue)

(Suitable for titration of acidic solutions with neutral solutions.)

Slow titration of acid solution for titration, especially near the end of titration, requires careful handling to avoid overdosing.

5. Data recording and calculations:

Maintain accurate experimental records, including data on acid solution concentration, titration volume, etc., for subsequent calculations and analysis.

Pay attention to the consistency of units and make sure that the same units are used in calculations.

6. apparatus and reagents:

The reagent solutions used should be prepared in ammonia-free distilled water.

Ensure that the instruments and reagents used are of good quality and are stored according to the specified storage conditions.

Calibrate instruments such as distillation apparatus and burets to ensure their accuracy and reliability.

Common Problems

1.Incomplete digestion or spillage problems:

Problem: Samples are not completely digested or the reaction overflows during the digestion process.

Solution: Control the digestion temperature and time to ensure that the sample is completely digested, avoiding too high temperature and too long time. Also, use proper digestion vessel and appropriate amount of digestion reagent to avoid reaction overflow.

2. Problems with ammonia collection:

PROBLEM: Ammonia cannot be completely collected or is leaking.

SOLUTION: Ensure that the distillation unit is well sealed and check that all connections are tight. Use a suitable trap bottle and condenser to control the distillation rate and avoid ammonia leakage.

3. Problems determining titration endpoints:

Problem: Difficulty in determining the titration endpoint, especially when the color change is not obvious.

Solution: Choose a suitable acid-base indicator according to the nature of the sample and the titration conditions. If the color change is not obvious, you can use the method of adding drop by drop and slowly observe the color change to accurately determine the titration end point.

4. Problem of abnormal or inaccurate experimental results:

Problem: The experimental results are not as expected or the data are inaccurate.

Solution: Check the accuracy of the experimental operation, such as the accuracy of the sample quality, reagent dosage and concentration. Ensure the accuracy of instrumentation, such as the calibration of the distillation apparatus and the accuracy of the burette. Double-check that the experimental steps were performed correctly and refer to the lab guide or professional resources for verification.

5. Safety:

PROBLEM: A safety issue or accident occurs during an experiment.

SOLUTION: Always follow laboratory safety practices and procedures. Wear appropriate protective equipment and keep the laboratory well ventilated. Assess potential risks and take necessary precautions before experimentation.

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