Reference no: EM131960750 , Length: word count:800
Analysis of Protein and Fat Content
INTRODUCTION
The four weeks of laboratory sessions will provide you with experience in standard food analysis techniques, used for nutrition information panels and nutrition claims. In your second report, you will prepare a nutrition information panel for your food, as according to FSANZ regulations.
PRACTICAL 1
MATERIALS AND METHODS
Note: Weighing should be carried out to at least three decimal places.
Accurate labelling of glassware is imperative for ease of identifying your samples over the different practical classes. There are four items that must be recorded:
a) Name as initials for both partners i.e. PP & FD.
b) Bench initial i.e. C
c) Practical day, date, time i.e. Monday 2/8 9am
d) Sample i.e. Biscuit / blank
e) Analysis type i.e. B&D, Kjeldahl
(i) Nitrogen and protein determination by the Kjeldahl method
This method is based on James (1999).
Note: this procedure is very hazardous. You must follow safety protocol at all times. Do not operate equipment or handle reagent without supervision from a demonstrator.
Each pair of students will analyse a single sample of their food. Each bench will analyse one blank. Please wait until the end of the practical to complete your calculations and enter results into the iPads.
PLEASE NOTE: Steps 1-5 have been prepared by laboratory technician (therefore you will not be performing these tasks). The demonstrator will demonstrate each of these steps.
1. 1g of representative sample was placed onto ashless filter paper and weighed. The weight of your sample was recorded and will be provided to you by the demonstrator. Record this sample weight in the iPad form.
2. The sample was folded into a pellet and dropped it into a labelled Kjeldahl digestion tube. A blank tube was also prepared - this contains everything found within the sample tube EXCEPT the food sample.
3. This step needs to be done under fume hood. To both tubes, 15 mL of concentrated sulphuric acid was added (HAZARDOUS-wear gloves and safety glasses), along with two Kjeldahl catalyst tablets. (HAZARDOUS-wear gloves and wash hands after use.)
4. The two digestion tubes were then placed into the Kjeldahl digestion block, in which the sample reaches a temperature of 420 °C and is digested for 30-45 minutes. This digestion step converts nitrogen in the food to ammonium sulphate.
5. The sample and blank digestion tubes were then removed from the digester and allowed to cool for approximately 30 minutes. All previously prepared samples can be found in the racks in the fume cupboard.
You are now able to begin the laboratory work, for this session.
6. Make sure that you are wearing gloves and safety glasses. Take two conical flasks and label correctly (one is for the sample per pair, and one for the blank per bench). Place 50 mL saturated boric acid solution and 10 drops of Congo Red indicator into each separately labelled conical flask. Carefully carry both conical flasks to the demonstrator at the Distillation unit.
7. Your demonstrator will now place the conical flask on the shelf beneath the condenser, in the Kjeldahl distillation unit. The digestion tube containing the sample is then placed into the automated Kjeldahl distillation unit.
8. With assistance from the demonstrator, start the distillation process. In the Kjeldahl unit, water and concentrated sodium hydroxide solution (HAZARDOUS-wear gloves) are automatically added. This converts the ammonium sulphate to ammonia.
The sample is then steam distilled. The ammonia-containing distillate generated will be collected in the flask (containing the boric acid and indicator).
9 Steps 6-8 are then repeated using the blank digestion tube. Retain your conical flasks for further analysis.
10 Place the blank conical flask on the white tile under the burette filled with HCl (aq). Record the initial burette volume. Titrate the blank ammonium borate/indicator solutions using 0.1M HCl (aq) to a blue/purple end-point.
THINK: Why do we expect the blank sample to require only a small amount of hydrochloric acid? Record the final reading of the burette. Repeat this step with the sample.
11 Perform the ‘Calculations for Protein Determination' to calculate the amount of protein in your food sample. Submit your results to the iPad form. The final percentage of protein will be used to compare your results in your practical report.
(ii) Determination of Fat Content by Modified Bligh and Dyer Method
1 Working in pairs, prepare a representative sample of the food provided on your bench.
2 Accurately weigh out (to three decimal places) an amount close to 5 g of sample into a labelled quickfit conical flask (no need to record weight of flask). Make sure you ‘zero' the conical flask on the scales before weighing your sample. Record the weight of this sample in your notebook for calculations in the next practical class.
3 Add 50 mL chloroform: methanol (2:1) to the flask in the fume cupboard (HAZARDOUS
- use fume cupboard, ensure no naked flames in vicinity, wear gloves, do not breathe vapour).
4 In the fume cupboard, macerate your sample/solvent mixture with the overhead homogeniser (medium speed for 30 seconds). Fit the stopper and leave in the fume cupboard. This will start to extract the lipid from the sample.
CALCULATIONS FOR PROTEIN DETERMINATION
1. Calculate how much acid was added, in moles, using
2. Calculate the weight of the Nitrogen in the sample, using
3. Calculate the percentage of Nitrogen in your food sample.
4. Calculate the percentage of protein in your food sample.
PRACTICAL 2
MATERIALS AND METHODS
Note: Weighing should be carried out to at least three decimal places. Accurately label your glassware. There are four items that must be recorded:
a) Name as initials for both partners i.e. PP & FD.
b) Bench initial i.e. C
c) Day, date, time i.e. Monday 2/8 8am
d) Sample i.e. Biscuit / blank
e) Analysis type i.e. Soxhlet, B&D
With round bottom flasks (RBF), label as either Soxhlet or Bligh and Dyer.
(ii) Determination of Fat Content by Modified Bligh and Dyer Method (continued)
Each pair of students will analyse a single sample of their food and appropriate blank.
PLEASE NOTE: Steps 1-4 were completed in Practical 1, and steps 5-6 were completed by a lab technician.
5 The sample was filtered through a pleated Whatman no.1 filter paper into a 250 mL separating funnel in the fume cupboard.
6 Next, 50 mL of 0.9% saline (salt water) was added, and the sample shaken vigorously and left to stand until practical session 2, by which time the lower lipid-containing chloroform layer will have separated from the water/methanol layer.
You are now able to begin the laboratory work, for this session.
7 Accurately weigh a labelled 250mL round-bottomed flask (to three decimal places) and record the weight in the appropriate iPad form (wt B&D flask). (Zero a cork ring on the analytical balance, and then place your round bottom flask on the cork ring to measure just the flask.)
8 With the assistance of your demonstrator, run off the lower lipid/chloroform phase into the weighed flask.
9 Remove the chloroform with the rotary evaporator with the assistance of your demonstrator (HAZARDOUS-ensure no naked flames in vicinity, wear gloves, do not breathe vapour). Complete drying at 60°C in an oven for half an hour.
10. Cool down the flasks in desiccator or put them in a tray and cover them with paper tissue, until they get cool.
10 Record the final weight (wt B&D flask+fat), and calculate the weight of the extracted fat. Complete the calculations and enter your results into the iPad form at the end of the class.
(iii) Determination of Fat Content by Soxhlet Method
Adapted from James, C. S. (1999). Analytical Chemistry of Foods. Gaithersburg, USA: Aspen Publishers.
1 The demonstrator will set up the Soxhlet extractor in the fume cupboard. Select a previously dried 500mL round-bottomed flask containing a few boiling chips (BC, pre- dried) from the desiccator and weigh the flask to three decimal places (wt Sox flask). Record data.
2 Wearing gloves, accurately weigh out 2-3 g of a representative sample of food (previously dried by the demonstrator) into a pre-dried fat-free extraction thimble in desiccator. Record data (see demonstrator for data on % moisture of original sample).
3 Plug the extraction thimble lightly with oil-free cotton wool, label using pencil, and place thimble into the extractor.
4 Your demonstrator will add 150 mL petroleum allowing it to siphon over once (HAZARDOUS - use fume cupboard, ensure no naked flames in vicinity). The demonstrator will then add more petroleum ether until the barrel of the extractor is half full.
5 Replace the condenser, and your demonstrator will turn on the flow of cold water. After ensuring that the joints are tight, the demonstrator will place the apparatus on a round- bottom flask (RBF) heater. The solvent should boil gently. Allow the system to siphon over at least ten times.
6 After a minimum of ten siphoning cycles, the demonstrator will carefully dismantle the apparatus (with your help). The residual solvent in the distillation flask (which now contains the extracted fat) is removed by heating on the RBF heater. Leave a small amount of petroleum ether in the flask so the fat does not burn (HAZARDOUS - use fume cupboard, ensure no naked flames in vicinity). The extraction thimble and contents are left to dry in fume cupboard (the food residue will be used for dietary fibre analysis). Complete drying at 60 °C in a drying oven for half an hour.
7 Record the final weight (wt Soxhlet flask+fat), and calculate the weight of the extracted fat (wt B&D extracted lipid). Complete the calculations and enter them into the iPad form at the end of the class.
Attachment:- Assignment.rar