Laboratory‎ > ‎ Analysis of Proteins

THEORY

Proteins are polymers of amino acids. Twenty different types of amino acids occur naturally in proteins. Proteins differ from each other according to the type, number and sequence of amino acids that make up the polypeptide backbone. As a result they have different molecular structures, nutritional attributes and physiochemical properties. Proteins are important constituents of foods for a number of different reasons. They are a major source of energy, as well as containing essential amino-acids, such as lysine, tryptophan, methionine, leucine, isoleucine and valine, which are essential to human health, but which the body cannot synthesize. Proteins are also the major structural components of many natural foods, often determining their overall texture, e.g., tenderness of meat or fish products. Isolated proteins are often used in foods as ingredients because of their unique functional properties, i.e., their ability to provide desirable appearance, texture or stability. Typically, proteins are used as gelling agents, emulsifiers, foaming agents and thickeners. Many food proteins are enzymes which are capable of enhancing the rate of certain biochemical reactions. These reactions can have either a favorable or detrimental effect on the overall properties of foods. Food analysts are interested in knowing the total concentration, type, molecular structure and functional properties of the proteins in foods.There are several methods for the determination of proteins in foods.

Kjeldahl method:

The Kjeldahl method was developed in 1883 by a brewer called Johann Kjeldahl. A food is digested with a strong acid so that it releases nitrogen which can be determined by a suitable titration technique. The amount of protein present is then calculated from the nitrogen concentration of the food. The same basic approach is still used today, although a number of improvements have been made to speed up the process and to obtain more accurate measurements. It is usually considered to be the standard method of determining protein concentration. Because the Kjeldahl method does not measure the protein content directly a conversion factor (F) is needed to convert the measured nitrogen concentration to a protein concentration. A conversion factor of 6.25 (equivalent to 0.16 g nitrogen per gram of protein) is used for many applications, however, this is only an average value, and each protein has a different conversion factor depending on its amino-acid composition. The Kjeldahl method can conveniently be divided into three steps: digestion, neutralization and titration.

Principles

Digestion

The food sample to be analyzed is weighed into a digestion flask and then digested by heating it in the presence of sulfuric acid (an oxidizing agent which digests the food), anhydrous sodium sulfate (to speed up the reaction by raising the boiling point) and a catalyst, such as copper, selenium, titanium, or mercury (to speed up the reaction). Digestion converts any nitrogen in the food (other than that which is in the form of nitrates or nitrites) into ammonia, and other organic matter to C02 and H20. Ammonia gas is not liberated in an acid solution because the ammonia is in the form of the ammonium ion (NH4+) which binds to the sulfate ion (SO42-) and thus remains in solution:

N(food) ® (NH4)2SO4 (1)

Neutralization

After the digestion has been completed the digestion flask is connected to a recieving flask by a tube. The solution in the digestion flask is then made alkaline by addition of sodium hydroxide, which converts the ammonium sulfate into ammonia gas:

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

The ammonia gas that is formed is liberated from the solution and moves out of the digestion flask and into the receiving flask – which contains an excess of boric acid. The low pH of the solution in the receiving flask converts the ammonia gas into the ammonium ion, and simultaneously converts the boric acid to the borate ion:

NH3 + H3BO3 (boric acid) ® NH4+ + H2BO3- (borate ion) (3)

Titration

The nitrogen content is then estimated by titration of the ammonium borate formed with standard sulfuric or hydrochloric acid, using a suitable indicator to determine the end-point of the reaction.

H2BO3- + H+ ® H3BO3 (4)

The concentration of hydrogen ions (in moles) required to reach the end-point is equivalent to the concentration of nitrogen that was in the original food (Equation 3). The following equation can be used to determine the nitrogen concentration of a sample that weighs m grams using a xM HCl acid solution for the titration:

Where vs and vb are the titration volumes of the sample and blank, and 14g is the molecular weight of nitrogen N. A blank sample is usually ran at the same time as the material being analyzed to take into account any residual nitrogen which may be in the reagents used to carry out the analysis. Once the nitrogen content has been determined it is converted to a protein content using the appropriate conversion factor: %Protein = F´ %N.

Advantages 

The Kjeldahl method is widely used internationally and is still the standard method for comparison against all other methods. Its universality, high precision and good reproducibility have made it the major method for the estimation of protein in foods.

Disadvantages

It does not give a measure of the true protein, since all nitrogen in foods is not in the form of protein. Different proteins need different correction factors because they have different amino acid sequences. The use of concentrated sulfuric acid at high temperatures poses a considerable hazard, as does the use of some of the possible catalysts The technique is time consuming to carry-out.

Enhanced Dumas method

General Principles:

A sample of known mass is combusted in a high temperature (about 900 oC) chamber in the presence of oxygen. This leads to the release of CO2, H2O and N2. The CO2 and H2O are removed by passing the gasses over special columns that absorb them. The nitrogen content is then measured by passing the remaining gasses through a column that has a thermal conductivity detector at the end. The column helps separate the nitrogen from any residual CO2 and H2O that may have remained in the gas stream. The instrument is calibrated by analyzing a material that is pure and has a known nitrogen concentration, such as EDTA (= 9.59%N). Thus the signal from the thermal conductivity detector can be converted into a nitrogen content. As with the Kjeldahl method it is necessary to convert the concentration of nitrogen in a sample to the protein content, using suitable conversion factors which depend on the precise amino acid sequence of the protein.

Advantages

It is much faster than the Kjeldahl method (under 4 minutes per measurement, compared to 1-2 hours for Kjeldahl). It doesn’t need toxic chemicals or catalysts. Many samples can be measured automatically. It is easy to use.

Disadvantages

High initial cost. It does not give a measure of the true protein, since all nitrogen in foods is not in the form of protein. Different proteins need different correction factors because they have different amino acid sequences. The small sample size makes it difficult to obtain a representative sample.another protein determination methods are;

·        UV-visible spectroscopy

·        Biuret Method

·        Lowry Method

·        Dye binding methods

·        Turbimetric method

PURPOSE: 

In this experiment we determined theamount of protein in food product.for this purpose we used kjeldahl method.because,this method is most convenient and well accepted method.firstly,we converted the nitrogen to the ammonium bisulfate then ammonia was distilled with boric acid to form ammonium.In the titration step ammonium was titrated with hydrochloric acid.

PROCEDURE

·        2 gr of sample was taken into digestion flask

·        10 gr of potassium sulfate was added to increase the boiling point

·        catalyst which was cupper sulfate was added to the digestion flask

·        25 ml of sulphiric acid was added to the flask

·        then,digestion flask was placed on kjeldahl onit(adjusted to 400 °C for 40 min)

·        the digestion flask was heated until clear green color was observed

·        the digestion flask was placed into the machine.the distillation process was done by the machine

·        the ammonium borate solution was titrated with hydrochloric acid

MATERİALS

  • 2 gr sample

  • burette

  • potassium sulfate

  • erlenmeyer flask

  • copper sulfate

  • boric acid

  • sulphiric acid

  • HCl solution

  • digestion flask

  • NaOH solution

  • methyl red as indicator

  • Boiling chips

CALCULATION

We consumed 26,8 ml of hydrochloric acid in order to reach the end point for 2 gr sample

Amount of gr of NH3(gr)  = 26,8ml* 1,4015mg /1000 ml  = 0,0375 mg  = 0,0000375 gr

%protein = 0,0000375 /2 gr *100*6,25 = 0,011

DISCUSSION

In this experiment we determined the amoumt of protein for a given sample by determining the nitrogen content.ıt was found that the amount of proteıns in the sanple was 0,011%.this meant that 0,011% of 100 gr sample was protein.we used kjeldahl method for determining of protein. The Kjeldahl, Dumas and IR methods require very little sample preparation. After a representative sample of the food has been selected it can usually be tested directly. On the other hand, the various UV-visible methods require extensive sample preparation prior to analysis. The protein must be extracted from the food into a dilute transparent solution, which usually involves time consuming homogenization, solvent extraction, filtration and centrifugation procedures. In addition, it may be difficult to completely isolate some proteins from foods because they are strongly bound to other components.in the experiment we used potassium sulfate because the reaction needed high temperature to be completed so potassium sulfate increased the boiling point of the solution.

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