Determination Of Protein Content Objective Biology Essay


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Kjeldahl is an analytical method, which is suitable for quantitative determination of nitrogen in the trinegative state in organic compounds. This method was developed in 1833 by Johan Kjeldahl.

In this experiment, we analyzed the protein content, proteins are polymers of amino acids, which are important component of foods, because they are a major source of energy in our daily lives so that they are very essential to human health, also our body cannot synthesize the protein. Therefore, we need intake protein in our diet.

Kjeldahl method which involves 3 main steps, they are Digestion, Distillation, and Titration.

For digestion step, concentrated sulfuric acid was used, also there are in the presence of a catalyst, which helps amine nitrogen are converted to ammonium ions.

Organic N + H2SO4 → (NH4)2SO4 + H2O + CO2 + other sample matrix byproducts

For distillation step, the ammonium ions are converted into ammonia gas by heated and distilled with Sodium hydroxide. The ammonia gas is led into a trapping solution, such as hydrochloric acid, boric acid, which dissolves and becomes an ammonium ion once again.

(NH4)2SO4 + 2NaOH→2NH3 + Na2SO4 + 2H2O

For titration step, the amount of the ammonia that has been trapped in the receiving solution, it is titrated by standard solution to calculate the value of nitrogen.

2NH3 +2HCl → (NH4)2SO4 +H2SO4

There are two types of titration-back titration and direct titration. Both methods are testing for the ammonia which present in the distillate with a color change.

In our experiment, we use the back titration, since the ammonia has captured in a receiving flask with excess of measured standardized acid. The excess of acid which can keep the pH in acidic that means the indicator does not have color change until the solution is titrated with base.

(NH4)2SO4 +H2SO4 +2NaOH →Na2SO4 + (NH4)2SO4 + 2H2O


Analytical balance

Kjeldahl digestion and distillation apparatus-complete with heat source, traps, and block-tin, or equivalent noncorrosive tubing condensers (x1)

Kjeldahl flasks-500 mL. (x1)

Distillate receiving flasks-500 mL, or any convenient size.

Digestion rack (x1)


Burette (x2)

5mL Pipette (x1)

Conical flask (x2)

Chemical used

Sodium hydroxide pellets

Anti-bumping granules

Methyl red indicator

0.5M Hydrochloric standard solution

0.5M Sodium hydroxide standard solution

0.5M Sodium carbonate

0.5M Potassium hydrogen phthalate

Potassium sulphate pellet

Copper(II) sulphate, anhydrous

Methyl red indicator

Sample used

Spiced pork cubes


Standard preparation

Weigh 40g Sodium hydroxide NaOH, and dissolve in water

Dilute in the 100mL volumetric flask and make up with water to the mark.

To prepare 0.5M standard hydrochloric acid

Pipette 4.2mL 37% hydrochloric acid, dissolve in water

Dilute in the 100mL volumetric flask and make up with water to the mark

To prepare 0.5M standard Sodium hydroxide

Weigh 0.9999g NaOH, and dissolve in water

Dilute in the 250mL volumetric flask and make up with water to the mark.

To prepare 0.5M standard Sodium Carbonate

Weigh 0.9999g NaOH, and dissolve in water

Dilute in the 250mL volumetric flask and make up with water to the mark.

To prepare 0.5M standard Potassium hydrogen phthalate

Weigh 0.9999g NaOH, and dissolve in water

Dilute in the 250mL volumetric flask and make up with water to the mark.

Working Procedure

1.000 g of sample was weighed and put into Kjeldahl flask.

About 15 g of K2SO4, 0.04 g of anhydrous CuSO4, 20 mL H2SO4 and 0.5-1.0 g alundum granules were added into Kjeldahl flask.

The flask was placed on the digestion rack in an inclined position and then sample flask was heated at the 5 min boil rate until dense, white fumes clear bulb of flask.

Swirl gently and continue heating an additional 40 min after the liquid has become clear and colorless. The Kjeldahl flasks should be rotated a minimum of three times during the digestion.

About 300 mL of water was added cautiously, and cool to room temperature.

50mL 0.5M standard hydrochloric acid was transferred accurately into receiving flask.

An additional 0.5-1.0 g anti-bumping granules was added to cooled digestion flask.

Mix thoroughly and add 100mL 10M NaOH solution (pellets) to make strongly alkaline. Pour the alkali down the side of the Kjeldahl flask so that it does not mix quickly with the acid.

Immediately connect the Kjeldahl flask to the other end of the condenser tube and thoroughly mix the contents by shaking.

Apply heat at about a 7.5 min boil rate and distill until at least 150 mL of distillate have been collected.

Titrate the contents of the receiving flask with 0.1 M NaOH solution, using 3 or 4 drops of methyl red indicator.

Conduct a blank determination of all reagents simultaneously with the samples and similar in all respects. Correct for blank determined on reagents.

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