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1.
Rhodes-Gotheli method (alkaline ether extraction method) (according to GB 5413.
3-2010)
This method is adopted by the International Organization for Standardization (ISO), the Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO), etc.
, and is an international standard method for the quantification of lipids such as milk and dairy products
.
(1) Principle Use ammonia- ethanol solution to destroy the colloidal properties and fat globule membrane of the milk, so that the non-fat components are dissolved in the ammonia- ethanol solution and the fat is freed.
The alkaline hydrolysate of the sample is extracted with ether and petroleum ether , and the sample is distilled.
Or evaporate to remove the solvent and determine the quality of the extract dissolved in the solvent
.
(2) Reagents Unless otherwise specified, the reagents used in this law are of analytical grade or above, and the water is the third-grade water specified in GB/T6682-2008
.
①Amylase: Enzyme activity ≥1.
5U/mg
.
②Ammonia: The mass fraction is about 25% (ammonia with a higher concentration can be used)
.
③ ethanol : volume fraction of at least 95%
.
④ Ether : does not contain peroxides, does not contain antioxidants, and meets the requirements of the test
.
⑤Petroleum ether : boiling range 30~60℃
.
⑥Mixed solvent: mix diethyl ether and petroleum ether in equal volume and prepare before use
.
⑦Iodine solution: about 0.
1mol/L
.
⑧ Congo red solution (C 32 H 22 Na 6 Na 2 O 6 S 2 ): Dissolve 1 g of Congo red in water and dilute to 100 mL
.
⑨ Hydrochloric acid (6mol/L): Measure 50mL of 12mol/L hydrochloric acid and slowly pour it into 40mL of water, dilute to 100mL, and mix
.
(3) Instruments
①Analytical balance: Sensitivity is 0.
1mg
.
② Centrifuge: It can be used to place liposuction bottles or tubes, and the speed is 500~600r/min
.
③ Electric heating constant temperature water bath
.
④ Oven
.
⑤ bottle liposuction: liposuction bottles with cork or other stopper should not affect the use of a solvent (e.
g.
, silica gel or poly tetrafluoroethylene )
.
Cork stoppers should be immersed in ether first , and then placed in water at or above 60°C for at least 15 minutes, and used after cooling
(4) Operation steps
①Preparation of the fat collection bottle: Add a few zeolite to the dry fat collection bottle and put it in an oven to dry for 1 hour
.
Then cool the fat collection bottle to room temperature and weigh it to the nearest 0.
②Blank test: Use 10mL of water instead of the sample for a blank test
.
③Determination:
a.
Precisely suck (weigh) a certain amount of sample (10.
00mL milk; 1~5g milk powder, dissolved in 10mL 60℃ water) into a liposuction bottle
.
b.
Add 2.
0mL ammonia water, and immediately put the liposuction bottle into a water bath at (65±5)℃ after mixing thoroughly, and heat for 15-20min (take out and shake from time to time)
.
Take out and cool to room temperature
c.
Add 10 mL of ethanol and mix gently but thoroughly to avoid the liquid getting too close to the bottleneck
.
If necessary, add two drops of Congo Red solution
d.
Add 25mL ether , plug the bottle stopper, keep the liposuction bottle in a horizontal position, clamp the extended part of the small ball upwards to the shaker, shake at about 100 times/min for 1 minute, or manually shake , But care should be taken to avoid the formation of permanent emulsions
.
After the liposuction bottle is cooled, carefully open the stopper, rinse the stopper and the bottle neck with a small amount of mixed solvent, so that the rinsing fluid flows into the liposuction bottle
e.
Add 25 mL of petroleum ether, plug the re-wet stopper, and gently shake for 30 seconds as described in d
.
f.
Put the stoppered liposuction bottle into a centrifuge and centrifuge at 500~600r/min for 5min
.
Or let the liposuction bottle stand for at least 30 minutes until the upper layer is clear and clearly separated from the water phase