Determination of Protein Content SMK Negeri 13 Bandung

Protein in Human Body  Protein has many function in human body: - Energy source - Tissue and cell reparation and development - As antibody and hormone and enzym sinthesys - As leveling substance for acid and base balance in cell  In human body, protein through a cycle in which protein decomposed to smaller component (amino acid/peptide). Amino acid and peptide form new protein molecules to replaced the broken one. (There is no life long protein used)

Protein in Food  According to the source, 2 kinds of protein known: - Animal Protein (fish, meat, egg, milk) - Plant Protein (Beans, rice, wheat and several fruits)  Protein content in food stuff are various

Protein  One of big biomolecules besides polysacharide, lipid and polynucleotid which are the main former of live creature  Protein is a polymer of about 20 amino acid linked by the peptide bonding

 Amino acid is the main molecule of protein former/compiler  Amino acid is an organic substance with carboxyl (COOH) and amine (NH 2 ) group. Amino acid structure in general is a C atom linked to 4 groups/atom: amine (NH 2 ), gugus carboxcyl (COOH), hydrogen atom (H), and one rest group (R, residue). R group distinguish an amino acid with another. Amino Acid

AMINO ACID STRUCTURE CC

PEPTIDE BONDING  Peptide bonding is amide bonding which is link 2 amino acids. One peptide has end-N amino acid with free NH 3 + group and end-C amino acid with free COO - group.

ANALYSIS OF PROTEIN CONTENT  The determination of N developed by Johan Kjeldahl (Danish chemist), that’s why it called Kjeldahl’s method  The Kjeldahl method of nitrogen analysis is the worldwide standard for calculating the protein content in a wide variety of materials ranging from human and animal food, fertilizer, waste water and fossil fuels.

Kjeldahl’s Method Step Destruction The central basis in this procedure is the oxidation of the organic compound using strong sulfuric acid Destilaton The nitrogen, from the amine groups, is converted to ammonium ion, and converted to ammonia gas Titration The ammonia gas than distilled and trapped in acid. Excessive acid then titrated by standard basic solution

Diagram of Kjeldahl’s Method

Kjeldahl’s Apparatus

Kjeldahl’s Steps Decomposition DistillationTrapping Titration

Calculation Nitrogen calculationProtein calculation  It is possible to calculate the amount of crude protein in the sample. Although there are differences between different samples, the amount of "crude protein" (CP) can be found by multiplying the percent Nitrogen by a factor (usually 6.25).  CP = %N x

Digestion  Weighing out approximately 1 gm of the sample containing protein, and placing the sample into a digestion flask, along with ml of concentrated sulfuric acid (H 2 SO 4 ).  Adding seven grams of potassium sulfate and a catalyst, usually copper.  Bringing the digestion tube/flask and mixture to a "rolling boil" (about 370 o C to 400 o C) using a heating a block.  Heating the mixture in the tube/flask until white fumes can be seen, and then continuing the heating for about mins, the solution should be clear.  Cooling the tube/flask and cautiously adding 250 mls of water.

Distillation  Raising the pH of the mixture using sodium hydroxide (45% NaOH solution). This has the effect of changing the ammonium (NH 4 + ) ions (which are dissolved in the liquid) to ammonia (NH 3 ), which is a gas.  Separating the nitrogen away from the digestion mixture by distilling the ammonia (converting it to a volatile gas, by raising the temperature to boiling point) and then trapping the distilled vapors in a special trapping solution of about 15 ml HCl (hydrochloric acid) in 70 ml of water.  removing the trapping flask and rinsing the condenser with water so as to make sure that all the ammonia has been dissolved.

Titration  Adding an indicator dye to the acid/ammonia trapping solution.  Putting a standard solution of NaOH (sodium hydroxide) into the buret slowly adding small amounts of the sodium hydroxide solution to the acid solution with the dye.  Watching for "endpoint" has been reached and that now all the acid has been neutralized by the base.  Performing a calculation to find the amount of ammonia, and thus nitrogen.

Calculation  Moles ammonia represent moles nitrogen  gms nitrogen = moles nitrogen x atomic mass (gN = molesN x )  %nitrogen = (gms nitrogen / gms sample) x 100 %N = (gN / gS) x 100