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33.2.26 AOAC Offi Official Method 989.05 Fat in Milk Modified Mojonnier Modified Ether Extrac Extraction tion Method First Action Action 1989 Final Fi nal Action Action 1992 IDF–ISO–AOAC Method A. Prin Princi ci ple ple
Fat is extracted ex tracted with mixture mixture of ethers from known weight of milk. Ether extract extract is decanted decanted into preweighed dry weighing weigh ing dish, and ether is evap orated. Extracted Extracted fat is dried to constant constant weight. Re sult is ex press ed as % fat by weigh t. B. Ap pa para ratus tus
(a) Flask.— Mojonnier-style Mojonnier-style ether extrac extraction tion flask with volume of 21–23 mL in lower bulb plus neck at bo ttom of flask. Flask should have smooth, round opening at top th at will seal when closed with cork. ( b ) Weighing dishes.— Metal, M etal, 8.5–9.5 cm diam diam e ter and 4.5–5.5 cm tall; or 250 mL glass beakers. beakers. (c) Cal Caliibra bration tion weights.— Class Class S, standard standard cali cali bration bration weights to verify verify balance balance accu accuracy racy within weight range to be used for weighing weigh ing empty flasks and flask plus test portion and weighing weighing empty dishes and dish plus fat. (d) An Ana alyt lytiical balance.— balance.— Readability Readability 0.0001 g. Accu Accuracy racy on veriifi ver fica cation tion within 0.0002 g. Check peri period odiically and whenever whenever balance bal ance is moved or cleaned. Keep record re cord of balance balance cali cali bration bration checks. (e) Des Desic icca cator.— tor.— Room R oom tem per peraature. For cooling cool ing weighing weighing dishes after after prelim prelimiinary and final final drying. drying. Use coarse desiccant (mesh size 6–1 6) that contains mini minimum of fine parti particles cles and that changes color when moisture moisture is absorbed. (f ) Tongs.— For For handling handling weighing weighing dishes. ( g) Hot pla te. — Steam S team bath or other heating heat ing device. device. For evapo evap ora ration tion of ether at 100°C. Carry out evap ora ration tion in hood. (h) Corks.— High High qual ity natu natural cork stop pers (size 5) for flasks. Soak corks in H2O several several hours to im prove seal. (i) Vac Vacuum oven maintain maintaining ing Vacuum uum or forced air oven.— Vacuum tem per peraature of 70 –75°C at at 50.8 cm (20 in.) of vacuum, or forced air oven maintain maintaining ing tem per peraature of 100 1°C. ( j ) Wa Watt e r b a t h f o r t e m p m p er i n g t e s t s a m p m p l es p r i o r t o weighing.— weigh ing.— With W ith thermom thermomee ter and device device to maintain maintain milk tem per peraature of 38 1°C. C. Reagents Reagents
(a) Ethyl ether.— ACS ACS grade, perox peroxide ide free. No resi res idue on evapora evapo ration. tion. (b) Pe Petro troleum leum ether.— ACS ACS grade, boiling boiling range 30 –60°C. No resiidue on evapo res evapora ration. tion. (c) Am Ammo monium nium hydrox hydroxide.— ide.— Concen Concentrated, ACS grade, specific gravity grav ity 0.9. (d) Ethyl alco alcohol.— hol.— 95%. 95%. No resi residue on evapo evapora ration. tion. (e) Dis Distilled tilled water.— water.— Free Free of oil and mineral mineral residue. (f ) Phenolphthalein indi indica cator.— tor.— 0.5% 0.5% (w/v) in al co cohol. hol. D. Deter Determi mina nation tion
(a) Weighing test portion.— Pre Pre pare by tem per pering ing milk to 38°C as in 925.21 ( see see 33.2.02). 33.2.02). Weigh empty flask with clean, dry cork stop per. Re move s top per. Pipe t ca 1 0 g m ilk in to fla sk.
Place stop per in flask. Weigh t o n ear earest est 0.1 mg. Check balance balance zero be tween test portions. (b) Weighing dishes.— Num ber clean weigh ing dishes and predry under under same condi conditions tions that will be used for fi nal drying drying after after fat extrac extraction. tion. Be sure that all surfaces surfaces where weighing weigh ing dishes will be placed (i.e., hot plate, des desic icca cator, tor, etc.) are clean and free of par tic ul ates . At end of ove n dry drying, ing, place dishes in room tem per peraature desic desicca cator tor and cool to room tem per peraature. On same day as fat extrac extraction, tion, (c), weigh dishes to nearest nearest 0 .1 mg and re cord weights. Check balance balance zero after after weighing weighing each dish. Protect Protect weighed dishes from contam contamiina nation tion with extra extrane neous ous matter. matter. (c) Fat extrac extraction. tion. —To —To test portion in flask add 1.5 mL NH4OH and mix thoroughly. thoroughly. NH4OH neutral neutralizes izes any acid present present and dissolves dis solves ca sein. Add 3 drops drop s of phenolphthalei phenolphthalein n in di dica cator tor to help sharpen visual visual ap pear pearance ance of inter interface face between between ether and aqueous aqueous layers lay ers during during extrac extraction. Add 10 1 0 mL 95% alco al cohol, stopper with H2O-soaked cork, and shake flask 15 s. s . For first ex trac traction, tion, add 25 mL ethyl ether, stop per with cork, and shake shake flask very vig vigor orously ously 1 min, releas re leasing ing built-up pressure pressure by loosen loosening ing stop per as nec neces essary. sary. Add 25 mL petro petroleum ether, stop per with cork, and re peat vigor orous ous shaking shak ing for 1 min. Centri Centrifuge fuge flasks at ca 600 rpm for 30 s to obtain obtain clean separa ration tion of aqueous (bright pink) and ether phases. Decant ether solu lution tion into suitable suitable weighing weighing dish pre pared as in (b). When ether solu solution tion is de canted into dishes, d ishes, be careful careful not to pour over any sus pended solids solids or aqueous aqueous phase into int o weigh ing dish. Ether Eth er can be evapo evap orated at 100°C from dishes while conduct conduct ing sec second ond extrac ex traction. tion. For second second ex trac traction, add 5 mL 95% alco alcohol, hol, stop per with cork, cork, and shake vig orously orously 15 s. Next, add 15 mL ethyl ether, re place cork, and shake flask vigor orously ously 1 min. Add 15 mL petro petroleum ether, stop per with cork, and re re peat vigor orous ous shaking shaking for 1 min. Cen tri trifuge flasks at ca 600 rpm for 30 s to obtain obtain clean separation of aque aqueous ous (bright pink) and ether phases. If inter in terface face is below neck of flask, add H2O to bring level ca half way up neck. Add H 2O slowly down inside in side surface surface of flask so that there is mini min imum distur distur bance bance of sepaara sep ration. tion. Decant Decant ether solu solution tion for second second extrac extraction tion into same weighing weigh ing dish u sed for first ex traction. For third extrac extraction, omit ad addi dition of 95% al alco cohol hol and repeat pro cedure proce dure used for second sec ond extrac extraction. tion. Com plete ly evap evapo orate solvents sol vents in hood on hot plate at 100°C (avoid spatter spattering). ing). Dry extracted ex tracted fat in weighing weigh ing dish to constant constant weight in forced air oven at 100 1°C (30 min) or in vacuum vacuum oven at 70 –75°C at >50.8 cm (20 in.) of vacuum vac uum for 7 min. Remove Remove weighing weighing dishes from oven and place in desic desiccator cator to cool to room tem per peraature. Re cord weight of each weighing dish plus fat. Run pair of reagent blanks each day tests are conducted. conducted. To run reagent re agent blank, re place milk test portion with 10 mL H2O and run test as above. Record Record weight of any dry resi residue col lected and use value in calcu calcula lation. tion. Re agent blank should be <0.0020 g res resiidue. If reagent re agent blanks for set of tests are nega negative use nega negative num ber in calcu cal cula lation. tion. ( Note: Note: To subtract subtract nega negative num ber [av [aver erage age weight blank residue] in equation equation below, below, add it to [(weight dish + fat) weight dish].) Nega Negative blank usually usually indi indicates that dishes were not com pletely dry at start of deter de termi mina nation tion or that balance balance cali bration bration shifted be tween weighing weigh ing of empty pans and pans plu s fat. Cause of negaative blanks should be iden neg identi tified fied and corrected. corrected.
Fat, % = 100 [(weight dish fat) (weight dish)] average weight blank residue weight test portion Maximum recommended difference between du plicates is <0.03% fat. At 3.6% fat: sr = 0.015%; RSDR = 0.512%; sR = 0.020%; r value = 0.044%; RSDr = 0.396%; R value = 0.056% Reference: JAOAC 71, 898(1988). Revised: March 1996