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Protein Determination in Food Kjeldahl Method

Introduction The Kjeldahl method involves three stages: digestion, distillation and titration. Protein in Milk In this lab session you will be determining the N content by the Kjeldahl method of a variety of different types of milk. The value for the N content can be used to calculate the protein content. The experimental value for the protein content of these products can be compared with the data on the nutrition panel (remembering that NP data represents average values). When the protein content is derived from the N content (rather than being measured directly), it is assumed that: N comprises 16% of the protein structure, and that Most of the N is present in the form of protein The value of 16% is an average across a range of food proteins. The validity of these assumptions depends on the specific protein source. The first assumption depends on the amino acid profile of a protein and for several protein sources, specific values relating to the %N are available. In the case of rice and soy, the specific factors for converting %N to %protein are 5.95 and 5.71 respectively. Secondly, N may be present in other forms, eg urea, amino acids, nucleotides. In the case of human milk 20-25% of the N is present in a form other than protein (this is referred to as non-protein nitrogen). For cows milk, NPN is approximately 5% of total N. The conversion factor for bovine (cows) mil (6.38) takes this into account so that a separate analysis for NPN doesnt have to be conducted. Procedure Mix Milk sample thoroughly prior to taking sample Place a disposable weighing dish on the balance and tare Add approximately 3 g of the milk sample to the weighing dish RECORD THE EXACT WEIGHT Tip the sample into the digestion tube and then re-weigh the now mostly empty weighing dish so that any sample that was not transferred can be corrected for. RECORD THIS WEIGHT IN YOUR LAB BOOK 5. Label Digestion Tube with permanent marker 6. Add 1 Kjeltab (each tablet contains 1 g Na2SO4 and 10 mg Se) 7. Add 1 glass bead (to prevent bumping) 8. Add 8 mL of digestion acid (conc H2SO4:conc H3PO4 = 100:5) 9. Slowly and carefully add 4 mL H2O2 (35%) 10. Place the tubes in the preheated digester (temp 420C) for 15-30 mins 11. Digestion is complete when the solution is clear 12. Remove tube from digester (but keep in fume cupboard) 13. Allow to cool for 10 mins 14. Dilute your sample with 75 mL H2O (deionised water, measure in measuring cylinder) 15. add 25 mL of 4% boric acid into a 250 mL receiver flask 16. Place your tube in the distilling unit and a receiver flask containing boric acid under the condenser outlet 17. Dispense the alkali (50 mL; 40% 10M NaOH) and start the steam distillation. When the receiver flask volume reaches 125 mL, turn off the steam 18. Titrate the NH3 content with 0.1 N HCl 19. The end point is grey, but then quickly turns light pink. Record the volume at the time it reaches the grey end point. 1. 2. 3. 4.

Calculations: We want to determine the amount of ammonia that was released from the digested sample during the titration. We can determine this from the amount of HCl that was used to titrate the sample to endpoint. The mL of acid (HCl) needed to titrate multiplied by the normality (or normal concentration = 0.1N) will give you the equivalents of ammonia. This is based on the formula ( = / rearranged to = ) The n(NH3) is equal to the n(N) that was in the digested sample

(3) = 0.1 () = 0.1

The mass of N can then be determined by multiplying by the molecular weight of N (14.007) This is based on the formula (n=m/M rearranged to m=n x M)

The percentage N can then be obtained by dividing this mass by the mass of the sample and multiplying by 100

() = 0.1 14.007

We then need to convert the %N to % protein by multiplying by the correct f factor for the milk used

% =

0.1 14.007 100 ()

% = %