The purpose of the document is to establish a general and uniform method for the determination of fat in dispute milk powder samples.
2.0 SCOPE
This standard testing procedure is suitable for the determination of fat in disputes milk powder samples.
3.0 RESPONSIBILITY
The expert and trained chemist are responsible for following this procedure. The technical manager is responsible for implementing this procedure.
A. DETERMINATION OF FAT, BY MOJONNIER – FAT – EXTRACTION METHOD
4.0 REQUIREMENTS
4.1 Apparatus
4.1.1 Analytical balance
4.1.2 Distillation or evaporation apparatus
4.1.3 Desiccators
4.1.4 Drying oven
4.1.5 Majonnier-type-fat extraction flask
4.1.6 Fat collecting vessels
4.1.7 Wash bottle
4.1.8 Boiling aids
4.2 Glassware
4.2.1 Measuring cylinder – 5ml , 25 ml
4.2.2 Pipette- 10 ml
4.2.3 Tongs
4.2.4 Volumetric flask –100 ml
4.3 Reagent
4.3.1 Ammonia solution— Mass fraction of NH3 approximately 25%
4.3.2 Ethanol
4.3.3 Congo red solution
4.3.4 Diethyl ether-Free from peroxide
4.3.5 Light petroleum.- Boiling point range 30°C to 60°C
4.3.6 Mixed solvent- mix equal volumes of diethyl ether and light petroleum
5.0 PROCEDURE
5.1 First Extraction
5.1.1 For WMP sample take 1 g and for SMP dried Whey, and dried buttermilk. take 1.5 g of the sample by weighing it to the nearest 1mg.
5.1.2 Transfer the sample in mojonnier tube or fat extraction tube.
5.1.3 Add about 10ml of preheated water at a temperature of 65°C + 5°C to the test portion in the fat – extraction tube. Mix thoroughly with the test portion in the small bulb until the test portion is completely dispersed.
5.1.4 Add 2 ml of concentrated ammonia solution to the test portion.
5.1.5 Heat the tube to 65°C + 5°C in the water both for 15min to 20min with occasional shaking.
5.1.6 Add 10ml of ethanol .mix gently but thoroughly by allowing the contents of the fat – extraction tube to flow backward and forwards between the small and large bulb.
5.1.7 Add 25 ml of diethyl ether to the tube, and close the tube with a stopper. Shake the flask vigorously but not excessively, for 1min to avoid the formation of persistent emulsions.
5.1.8 Add 25ml of the light petroleum ether in the tuber and close the tube with a stopper, mix gently for 30 sec.
5.1.9 Allow the closed tube to stand for at least 30min. until the supernatant layer is clear and distinctly separated from the aqueous layer.
5.1.10 Hold the fat – extraction tube by the small bulb and carefully decant as much as possible of the supernatant layer into the prepared fat – collecting vessel. Containing a few boiling aids in the case of a boiling or conical flask, avoid decanting any aqueous layer.
5.2 Second Extraction
5.2.1 Add 5 ml of ethanol to the contents of the fat – extraction tube. Using the ethanol, rinse the inside of the neck of the flask or tube.
5.2.2 Add 15ml of diethyl ether in a tube close the tube with a stopper and mix it gently for 1min.
5.2.3 Add 15ml of light petroleum ether close the tube with a stopper and mix it for 30 sec.
5.2.4 Again allow the closed tube to stand for at least 30min until the supernatant layer is clear and distinctly separated from the aqueous layer.
5.2.5 Collect fat layer in fat collecting vessel or conical flask or beaker.
5.3 Third Extraction
5.3.1 Again add 15ml of diethyl ether to the tube close the tube with a stopper and mix it for 1 min.
5.3.2 Add 15ml of diethyl ether in a tube close the tube with a stopper and mix it for 1 min.
5.3.3 Add 15ml of light petroleum ether.
5.3.4 Close the tube with a stopper and mix it for 30sec.
5.3.5 Again allow the closed tube to stand for 30min until the supernatant layer is clear and distinctly separated from the aqueous layer.
5.3.6 Collect the fat layer in the fat collecting vessel and add a few boiling aids. Evaporation in the water bath.
5.3.7 After evaporation transfer fat –the collecting vessel or conical in the oven for 1hr. (temperature 102°C).
5.3.8 Cool it down in the desiccator fat-collecting vessel.
5.3.9 Weigh the fat – collecting vessel to the nearest 1,0mg
5.3.10 Carry out a blank test simultaneously with the determination same reagent but without a sample.
6.0 CALCULATION
B. DETERMINATION OF FAT USING FAT EXTRACTION TUBES WITH SIPHON
7.0 REQUIREMENTS
7.1 Apparatus
7.1.1 Analytical balance
7.1.2 Distillation or evaporation apparatus
7.1.3 Desiccators
7.1.4 Drying oven
7.1.5 Siphon-type-fat extraction tube
7.1.6 fat collecting vessels
7.1.7 Wash bottle
7.1.8 Boiling aids
7.2 Glassware
7.2.1 Measuring cylinder - 5 ml, 25 ml
7.2.2 Pipette- 10 ml
7.2.3 Tongs
7.2.4 Volumetric flask –100 ml
7.3 Reagent
7.3.1 Ammonia solution— Mass fraction of NH3 approximates 25%
7.3.2 Ethanol
7.3.3 Congo red solution
7.3.4 Diethyl ether-Free from peroxide
7.3.5 Light petroleum.- Boiling point range 30°C to 60°C
7.3.6 Mixed solvent- mix equal volumes of diethyl ether and light petroleum
8.0 PROCEDURE
8.1 Weigh about 1.0g of dried whole milk powder or 1.5g of dried skimmed milk powder in fat- extraction
8.2 tube.
8.3 Add 10ml of water at 65°C + 5°C to the above test portion in the fat extraction tube and add 2 ml
ammonia solution and mix it.
8.4 Heat the tube at 65°C + 5°C in water bath for 15min to 20min with occasional shaking
8.5 Add 10ml ethanol and Mix gently.
8.6 Add 25ml diethyl ether. Close the fat- extraction tube with a cork bung saturated with water or with a stopper of other material wetted with water.
8.7 Shake the tube vigorously with repeated inversion of 1 min.
8.8 Carefully remove the bung or stopper and rinse it and the neck of the tube with a little mixed solvent (diethyl ether and light petroleum)
8.9 Add 25ml light petroleum
8.10 Close the fat extraction tube and shake it gently for 30sec
8.11 Centrifuge the tube for 1min to 5min at the radial acceleration of 80g to 90g.
8.12 Carefully remove the bung or stopper.
8.13 Rinse the neck with mixed solvent.
8.14 Inset a siphon fitting or wash bottle fitting into the fat extraction tube.
8.15 Carefully transfer the supernatant layer out of the fat- extraction tube into fat –the collecting vessel.
8.16 Avoid the transfer of any of the aqueous layers.
8.17 Rinse the outlet of the fitting with a little mixed solvent.
8.18 Collect the rinsing in fat – the collecting vessel.
8.19 Loosen the fitting from the neck of the fat- the extraction tube.
8.20 Slightly raise the fitting and rinse the lower part of its long inner limb with a little mixed solvent.
8.21 Lower and re-insert the fitting and transfer the rinsing to the fat-collecting vessel.
8.22 Rinse the outlet of the fitting with a little mixed solvent and collect rinsing in fat- the collecting vessel.
8.23 If desired remove the solvent or a part of it from the fat – the collecting vessel by distillation or evaporation.
8.24 Again loosen the fitting and add 5ml ethanol to the fat extraction tube.
8.25 Use ethanol to rinse the long inner limb of the fitting.
8.26 Carry out the second extraction by repeating the first extraction steps.
8.27 Instead of 25ml, use only 15ml of diethyl ether and 15ml light petroleum.
8.28 Carry out the third extraction; again use only 15ml diethyl ether and 15ml light petroleum.
8.29 Remove the solvents from fat- the collecting vessel by distillation or by evaporation.
8.30 Heat the fat- collecting vessel with boiling or allow solvent in to escape, for 1hr in a drying oven maintained at 102°C.
8.31 Remove fat- the collecting vessel from the oven and immediately verify whether or not the fat is clear.
8.32 Protect the fat- collecting vessel from dust.
8.33 Allow it to cool to the temperature of the weighing room.
8.34 Note down the weight for 1st reading.
8.35 Heat the fat- collecting vessel with boiling or allow solvent vapor to escape, for 30min in a drying oven maintained at 102°C.
8.36 Cool and reweigh it.
8.37 Record the minimum mass as the mass of fat- the collecting vessel and extracted matter.
8.38 Use water for the blank sample.
9.0 CALCULATION
10.0 REVISION/CHANGE HISTORY
11.0 ATTACHMENTS
· Appendix-1: FLOW CHART FOR FAT, BY MOJONNIER – FAT – EXTRACTION METHOD
· Appendix-2: FLOW CHART FOR FAT, USING FAT EXTRACTION TUBES WITH SIPHON
12.0 REFERENCES
IS 11721:2013
7.0 REQUIREMENTS
7.1 Apparatus
7.1.1 Analytical balance
7.1.2 Distillation or evaporation apparatus
7.1.3 Desiccators
7.1.4 Drying oven
7.1.5 Siphon-type-fat extraction tube
7.1.6 fat collecting vessels
7.1.7 Wash bottle
7.1.8 Boiling aids
7.2 Glassware
7.2.1 Measuring cylinder - 5 ml, 25 ml
7.2.2 Pipette- 10 ml
7.2.3 Tongs
7.2.4 Volumetric flask –100 ml
7.3 Reagent
7.3.1 Ammonia solution— Mass fraction of NH3 approximates 25%
7.3.2 Ethanol
7.3.3 Congo red solution
7.3.4 Diethyl ether-Free from peroxide
7.3.5 Light petroleum.- Boiling point range 30°C to 60°C
7.3.6 Mixed solvent- mix equal volumes of diethyl ether and light petroleum
8.0 PROCEDURE
8.1 Weigh about 1.0g of dried whole milk powder or 1.5g of dried skimmed milk powder in fat- extraction
8.2 tube.
8.3 Add 10ml of water at 65°C + 5°C to the above test portion in the fat extraction tube and add 2 ml
ammonia solution and mix it.
8.4 Heat the tube at 65°C + 5°C in water bath for 15min to 20min with occasional shaking
8.5 Add 10ml ethanol and Mix gently.
8.6 Add 25ml diethyl ether. Close the fat- extraction tube with a cork bung saturated with water or with a stopper of other material wetted with water.
8.7 Shake the tube vigorously with repeated inversion of 1 min.
8.8 Carefully remove the bung or stopper and rinse it and the neck of the tube with a little mixed solvent (diethyl ether and light petroleum)
8.9 Add 25ml light petroleum
8.10 Close the fat extraction tube and shake it gently for 30sec
8.11 Centrifuge the tube for 1min to 5min at the radial acceleration of 80g to 90g.
8.12 Carefully remove the bung or stopper.
8.13 Rinse the neck with mixed solvent.
8.14 Inset a siphon fitting or wash bottle fitting into the fat extraction tube.
8.15 Carefully transfer the supernatant layer out of the fat- extraction tube into fat –the collecting vessel.
8.16 Avoid the transfer of any of the aqueous layers.
8.17 Rinse the outlet of the fitting with a little mixed solvent.
8.18 Collect the rinsing in fat – the collecting vessel.
8.19 Loosen the fitting from the neck of the fat- the extraction tube.
8.20 Slightly raise the fitting and rinse the lower part of its long inner limb with a little mixed solvent.
8.21 Lower and re-insert the fitting and transfer the rinsing to the fat-collecting vessel.
8.22 Rinse the outlet of the fitting with a little mixed solvent and collect rinsing in fat- the collecting vessel.
8.23 If desired remove the solvent or a part of it from the fat – the collecting vessel by distillation or evaporation.
8.24 Again loosen the fitting and add 5ml ethanol to the fat extraction tube.
8.25 Use ethanol to rinse the long inner limb of the fitting.
8.26 Carry out the second extraction by repeating the first extraction steps.
8.27 Instead of 25ml, use only 15ml of diethyl ether and 15ml light petroleum.
8.28 Carry out the third extraction; again use only 15ml diethyl ether and 15ml light petroleum.
8.29 Remove the solvents from fat- the collecting vessel by distillation or by evaporation.
8.30 Heat the fat- collecting vessel with boiling or allow solvent in to escape, for 1hr in a drying oven maintained at 102°C.
8.31 Remove fat- the collecting vessel from the oven and immediately verify whether or not the fat is clear.
8.32 Protect the fat- collecting vessel from dust.
8.33 Allow it to cool to the temperature of the weighing room.
8.34 Note down the weight for 1st reading.
8.35 Heat the fat- collecting vessel with boiling or allow solvent vapor to escape, for 30min in a drying oven maintained at 102°C.
8.36 Cool and reweigh it.
8.37 Record the minimum mass as the mass of fat- the collecting vessel and extracted matter.
8.38 Use water for the blank sample.
9.0 CALCULATION
10.0 REVISION/CHANGE HISTORY
11.0 ATTACHMENTS
· Appendix-1: FLOW CHART FOR FAT, BY MOJONNIER – FAT – EXTRACTION METHOD
· Appendix-2: FLOW CHART FOR FAT, USING FAT EXTRACTION TUBES WITH SIPHON
12.0 REFERENCES
IS 11721:2013
END OF THE DOCUMENT
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