Quantitation of Protein:Micro-Kjeldahl Method
互联网
2003
【
Purpose
】
Study the principle and the operational techniques of micro-Kjeldahl method.
【 Principle 】
The Kjeldahl method measures the nitrogen content of a compound and may be used to determine the protein content of a sample provided that the proportion of nitrogen in the protein is known.
When nitrogenous protein is heated with concentrated sulphuric acid, C、H two elements are oxidized to CO 2 and H 2 O, while nitrogen is oxidized to ammonia which can change into (NH 4 ) 2 SO 4 with sulphuric acid. This process is called digest. Because this digest stage is slow, Potassium or sodium sulphate is usually included to increase the boiling point of the mixture, CuSO 4 is included as catalyst to accelerate the digest reaction.
After digest, concentrated alkali which is added in Kjelgahl apparatus can decompose (NH 4 ) 2 SO 4 in digest solution to ammonia. Ammonia is distilled by steam distillation and absorbed with boric acid of definite quantity and concentration. Ammonia combine with H + in boric acid and depresses the concentration of H + , which causes the color change of indicator. It is titrated with standard hydrochloric acid until it recovers initial concentration of H + . According to the quantity of standard hydrochloric we can determine the content of nitrogen in the compound.
The nitrogen content of protein is usually accepted as 16% of the total weight, namely, 1g nitrogen of protein is equal to 6.25g protein. The quantity of protein of sample is calculated by 6.25 multiply the content of nitrogen determined by Kjeldahl method.
【 Materials 】
1. Apparatus
A suit of Kjeldahl apparatus, Four Kjeldahl flask of 50ml, Pipet, Conical, Test tubes, Several small beadings
2. Reagents
(1) Concentrated sulphuric acid, 30% NaOH solution, 2% boric acid, standard hydrochloric acid (0.01mol/L),
(2) K 2 SO 4 -CuSO 4 mixed powder: Mix K 2 S0 4 and CuSO 4 ·5H 2 O with ratio 3：1 and skive well.
(3) Mixed indicator: Mix 50ml 0.1% alcohol solution of methyl-olefin blueness and 200ml 0.1% methyl red, which is stored in brown flasks.
(4) Sample solution: confect albumin of moggy blood serum of 3mg/ml as sample.
【 Procedures 】
1. Setting up the Kjeldahl apparatus: the method of setting up is showed as below fig.
2. Digest: Take four 50ml Kjeldahl flasks and number them, then, add a beading in each of them. Add 1ml of sample solution and 200mg of catalyst (K 2 SO 4 -C U SO 4 ·5H2O) and 5ml of concentrated sulphuric acid in No.1 and No.2 Kjeldahl flasks. Pay attention to add sample to the bottom of flasks and not to adhere it to flask wall and flask neck. Add 1ml of distilled water and as commensurate catalyst and concentrated sulphuric acid as No.1 flask to No.3 and No.4 Kjedahl flasks which is as blank contrasts. Digest in vent hood .
At the beginning of digest we should control the firepower to prevent the digest solution from rushing to the flask neck. After releasing white smoke of SO 2 decomposed by H 2 SO 4 , enhance the firepower properply and continue to digest until digest solution is clear pale cyan . Cut off fire and place Kjeldahl flasks in vent hood to cool it to room temperature.
Study the principle and the operational techniques of micro-Kjeldahl method.
【 Principle 】
The Kjeldahl method measures the nitrogen content of a compound and may be used to determine the protein content of a sample provided that the proportion of nitrogen in the protein is known.
When nitrogenous protein is heated with concentrated sulphuric acid, C、H two elements are oxidized to CO 2 and H 2 O, while nitrogen is oxidized to ammonia which can change into (NH 4 ) 2 SO 4 with sulphuric acid. This process is called digest. Because this digest stage is slow, Potassium or sodium sulphate is usually included to increase the boiling point of the mixture, CuSO 4 is included as catalyst to accelerate the digest reaction.
After digest, concentrated alkali which is added in Kjelgahl apparatus can decompose (NH 4 ) 2 SO 4 in digest solution to ammonia. Ammonia is distilled by steam distillation and absorbed with boric acid of definite quantity and concentration. Ammonia combine with H + in boric acid and depresses the concentration of H + , which causes the color change of indicator. It is titrated with standard hydrochloric acid until it recovers initial concentration of H + . According to the quantity of standard hydrochloric we can determine the content of nitrogen in the compound.
The nitrogen content of protein is usually accepted as 16% of the total weight, namely, 1g nitrogen of protein is equal to 6.25g protein. The quantity of protein of sample is calculated by 6.25 multiply the content of nitrogen determined by Kjeldahl method.
【 Materials 】
1. Apparatus
A suit of Kjeldahl apparatus, Four Kjeldahl flask of 50ml, Pipet, Conical, Test tubes, Several small beadings
2. Reagents
(1) Concentrated sulphuric acid, 30% NaOH solution, 2% boric acid, standard hydrochloric acid (0.01mol/L),
(2) K 2 SO 4 -CuSO 4 mixed powder: Mix K 2 S0 4 and CuSO 4 ·5H 2 O with ratio 3：1 and skive well.
(3) Mixed indicator: Mix 50ml 0.1% alcohol solution of methyl-olefin blueness and 200ml 0.1% methyl red, which is stored in brown flasks.
(4) Sample solution: confect albumin of moggy blood serum of 3mg/ml as sample.
【 Procedures 】
1. Setting up the Kjeldahl apparatus: the method of setting up is showed as below fig.
2. Digest: Take four 50ml Kjeldahl flasks and number them, then, add a beading in each of them. Add 1ml of sample solution and 200mg of catalyst (K 2 SO 4 -C U SO 4 ·5H2O) and 5ml of concentrated sulphuric acid in No.1 and No.2 Kjeldahl flasks. Pay attention to add sample to the bottom of flasks and not to adhere it to flask wall and flask neck. Add 1ml of distilled water and as commensurate catalyst and concentrated sulphuric acid as No.1 flask to No.3 and No.4 Kjedahl flasks which is as blank contrasts. Digest in vent hood .
At the beginning of digest we should control the firepower to prevent the digest solution from rushing to the flask neck. After releasing white smoke of SO 2 decomposed by H 2 SO 4 , enhance the firepower properply and continue to digest until digest solution is clear pale cyan . Cut off fire and place Kjeldahl flasks in vent hood to cool it to room temperature.
