ANALYTICAL cHEMISTRY - V

MSCM303

Experiment 7

Aim of the Experiment

Part A: To determine the casein content in the given sample of milk/milk powder.

Part B: To determine the total protein content in the given sample of milk/milk powder.

Principle

Part A: Casein content

  • Casein is family of related phospho-proteins. These proteins are commonly found in mammalian milk comprising of 80% of proteins in cow’s milk and between 20% and 45% of proteins in human milk. Sheep and buffalo milk have a highest casein content than other types of milk with human milk having a particularly low casein content.

  • Casein has a wide variety of uses, from being a major compound of cheese, to use as food additive. The most common for of casein is sodium caseinate. As a food source, casein supplies amino acids, carbohydrates and two essential calcium and phosphorous.

  • Composition of casein: Casein contains a high number of proline residues, which do not interact. These are also no disulfide bridges. As a result, it has relatively little tertiary structure. It is relatively hydrophobic, making if poorly soluble in water. It is found in milk as a suspension of particles, called casein micelles, which show only limited resemblance with surfactant type micelles in a sense that hydrophilic parts reside at the surface and they are spherical. However, in sharp contrast to surfactant micelles, the interior of a casein micelle is highly hydrated. The caseins in the micelle are held together by calcium ions and hydrophobic interactions.

  • The isoelectric point of casein is 4.6. Since milk’s pH is 6.6, casein has a negative charge in milk. The purified protein is water soluble. While it is also insoluble in neutral salt solutions, it is readily dispersible in dilute alkalis and in salt solutions such as aqeous sodium oxalate and sodium acetate.

Part B: Total protein content

  • There are two major categories of milk protein that are broadly defined by their chemical composition and physical properties.

  • The primary group of milk proteins are the caseins and make up the largest structures in the fluid portion of milk- the casein micelles. Each casein micelle is roughly spherical and about a tenth of micrometer across. The casein family contains phosphorous and this high phosphate content of casein family allows it to associate with calcium and from calcium phosphate salts. Caseins have an appropriate amino acid composition that is important for growth and development of nursing young. Collectively, they make up around 76%- 86% if the protein in milk by weight.

  • Milk contains dozens of other types of proteins besides casein and including enzymes. These proteins are more water soluble than caseins and do not form larger structures. These proteins remain suspended in whey, remaining when casein coagulate into curd and are collectively called as whey proteins.

  • Other whey proteins are the immunoglobins (anti-bodies found in colostrum) and serum albumins - a serum protein. Whey proteins also include enzymes, hormones, growth factors, nutrient transporters, disease resistance factors and others. Whey protein family consists of approximately 50% beta- lactoglobulins, 20% alpha lactoglobulins, lactoferrins, transferrin and many minor proteins. They also contain a large amount of sulfur containing amino acids. Whey proteins make up approximately 20% of the proteins in milk by weight. Lactoglobulin is the most common whey protein by a large margin.

About the method used in this experiment:

  • Proteins in milk, milk powder, milk tablet can be determined by this method of titration or by Lowry method. In this method the formalin solution blocks the -NH2 group of protein and activates carbonyl group to react with NaOH. The carbonyl group of formalin reacts via condensation reaction and the carboxylic group of the protein is available for the titration with NaOH using phenolphthalein giving a color change from colorless to light pink.

  • Any acidic or basic species present in the sample matrix acts as interfering agent to this method. Peptides, amino acids, amine salts, etc. interfere. So, to remove this interference formalin solution is added which blocks these groups in milk.

Apparatus required

Part A: Cheese cloth, Filter paper, Beaker, pH meter, etc.

Part B: Volumetric flask, Conical flask, Beaker, Funnel, Burette, Pipette, etc.

Chemicals required

Part A: Milk, Milk powder, 1 M HCl solution, 1 M NaOH solution, etc.

Part B: Milk, Milk powder, 50 mL formalin solution (~ 40 % w/w), 0.1 M NaOH solution, 0.1 M Oxalic acid solution, Phenolphthalein (as indicator), etc.

Preparation of reagents

Part A:

  • Sample preparation:

For milk: Take 50 mL of milk in a 100 mL volumetric flask and dilute it upto the mark using distilled water.

For milk powder: Take 5 g of milk powder and dissolve it in water. Make the volume to 100 mL in a 100 mL volumetric flask using distilled water.

  • 1 M HCl solution:

1000 mL 1M HCl solution ≡ 36.5 g HCl

100 mL 1 M HCl solution ≡ ________ g.


Volume = Mass / Density = ________ mL.

Therefore, Take ________ mL of concentrated HCl and dilute it to 100 mL using distilled water.

  • 1 M NaOH solution:

1000 mL 1M NaOH solution ≡ 40 g

100 mL 1 M NaOH solution ≡ ________ g.

Part B:

  • Sample preparation:

For milk: Take 50 mL milk in a 100 mL volumetric flask and dilute it up to the mark using distilled water.

For milk powder: Take 5 g of milk powder and dissolve it in water. Make up the volume to 100 mL in a volumetric flask using distilled water.

  • 0.1 N oxalic acid solution:

1000 mL 1 N oxalic acid ≡ 63 g

100 mL 0.1 N Oxalic acid ≡ ________ g.

  • 0.1 N NaOH solution:

1000 mL 1 N NaOH ≡ 40 g

100 mL 0.1 N NaOH ≡ ________ g.

  • Neutralized formalin solution:

Take 50 mL formalin solution in a beaker and add 1 M NaOH solution till the pH is 7.

Procedure (Part A):

  • Take 50 mL of diluted milk/milk powder sample in a beaker. Add 1 M HCl solution and make the pH to 4.6. Allow the solution to stand for 30 minutes till all the precipitated casein settles down. Filter the solution using cheese cloth.

  • Now, dissolve the precipitates of casein in 1 M NaOH solution. Again add 1 M HCl solution till the pH changes to 4.6. Allow the precipitates to settle down (leave the solution undisturbed for 30 minutes).

  • Filter the precipitates through previously weighed filter paper quantitatively. Add a little of acetone and petroleum ether to dry the ppts. Weigh the filter paper again (with precipitates). Obtain the weight of precipitates by subtraction.

Milk analysis_Casein content_1.mp4
Milk analysis_Casein content_2.mp4
Milk Analysis_Casein_3.mp4
Milk analysis_Casein content_4.mp4

Procedure (Part B):

  • Pipette out 10 mL of the diluted solution of milk / milk powder. To this, add 4 mL of neutralized formalin solution. (Formalin is taken in a beaker (50 mL) and the pH is set to 7 using 1 M NaOH solution to get neutralized formalin).

  • Add 2-3 drops of phenolphthalein indicator to it and titrate it against the standardized 0.1 M NaOH solution from burette.

Milk analysis_Protein content_1.mp4
Milk analysis_Protein content_2.mp4

Observation (Part A)

Observation (Part B)

Calculation (Part A)

Calculation (Part B)

Result (Part A)

  1. Amount of casein in 100 mL milk __________ g.

  2. Amount of casein in 100 g milk powder __________ g.

Result (Part B)

  1. Amount of total protein in 100 mL milk __________ g.

  2. Amount of total protein in 100 g milk powder __________ g.

Reference Material

  1. G H Jeffery, J Bassett, J Mendham and R C Denney, Vogel's Textbook of Quantitative Chemical Analysis, 5th Edition

  2. S. Suzanne Nielsen, Food analysis, 4th Edition

Questions


Developed by

Dr. Viraj Bhanvadia,

Assistant Professor, Chemistry,

viraj.bhanvadia@gsfcuniversity.ac.in