2. Welcome Department of Nutrition and Food Hygiene

3. Experimental Rules Come here on time in white working clothing. Not permitted to eat anything in the lab. Pay high attention to the dangerous or poisonous reagents. Take good care of the apparatus. Keep the classroom quiet. Complete experiment report in English if required. Remember to clean the classroom, wash the apparatus, put everything back after experiment.

4. Whole Experiment Plan Determination of nutrients: Protein Vitamin C Food hygiene Milk Wine Edible oil Food contaminant: nitrite Nutritional evaluation Dietary survey, calculation and evaluation

5. Apparatus Anhydrous potassium sulfate Concentrated sulfuric acid Copper sulfate (10%) Sodium hydroxide solution (50%) Mixed indicator Boric acid solution(1%) 0.1M ammonium sulfate 0.01M standard hydrochloric acid Determination of protein in food by microkjeldahl method Reagents Kjeldahl flask Electric heater Ventilation cabinet Digestion rack Distillation apparatus Glass bead Sucker Buret

6. Determination of protein in food by microkjeldahl method Principle The components of the food is very complex and most of them are insoluble. It is not feasible to measure the protein content directly. Usually, protein contains 16% of nitrogen according to the results of element analysis. protein content nitrogen content So the protein Nitrogen must be changed into ammonium which is easy to measured, then we can get the protein content in the sample.

7. Determination of protein in food by microkjeldahl method Calculation Protein nitrogen Ammonium sulfate (NH 4 ) 2 SO 4 H 2 SO 4 Digestion NaOH Distillation NH 3 Boric acid Ammonium borate (NH 4 ) 2 B 4 O 7 HCl Titration

8. Determination of protein in food by microkjeldahl method

9. Procedure Step 1: wet digestion Step 2: distillation Step 3: titration Step 4: calculation

10. Step 1: Wet Digestion sample: 0.2gram 0.3g K 2 SO 4 2ml CuSO 4 3ml H 2 SO 4 Glass beads Small funnel heating clear 20-30 min Complete digestion 100ml volumetric bottle Kjeldahl flask Blank Recovery: 5ml ( N H 4 ) 2 SO 4

11. Step 1: Wet Digestion Reagent: K 2 SO 4 : increasing boiling point CuSO 4 : catalyst H 2 SO 4 : oxidant Basic reaction: CH 2 (NH 2 )COOH + H 2 SO 4 CH 2 (NH 2 )OH + CO 2 + SO 2 + H 2 O CH 2 (NH 2 )OH + H 2 SO 4 NH 3 + CO 2 + SO 2 + H 2 O 2 NH 3 + H 2 SO 4 (NH4) 2 SO 4

12. Step 2: Distillation Wash the distillation apparatus. Distill some distilled water. 10ml 1% H 3 BO 3 2~3D mixed indicator 5ml dilution 5ml 50% NaOH Taper flask Making sure that the tip of tube under the surface of liquid. inner chamber from funnel more 5 minutes blue green. heat Remove above 1 min Remove

13. Step 2: Distillation Reagent: 1% boric acid mixed indicator 50% sodium hydroxide solution Basic reaction: (NH4) 2 SO 4 + NaOH NH 3 + Na 2 SO 4 + H 2 O 2 NH 3 + 4H 3 BO 3 (NH 4 ) 2 B 4 O 7 + 5H 2 O

14. Step 3: Titration Reagent: 0.01M standard hydrochloric acid Basic reaction: (NH 4 ) 2 B 4 O 7 + 2HCl 2NH 4 Cl + 4H 3 BO 3 End point: blue-purple

15. Step 4: Calculation