AOAC Official Method 956
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F0FFF183CF924A21BAFC19B09921E416 |
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0.02 |
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2 |
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日期： |
2024-7-30 |
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41.1.24,AOAC Official Method 956.04?,Butyric Acid,(Mole Per Cent) in Fat,Column Chromatographic Method,First Action 1956,Final Action,Surplus 1994,A. Apparatus,Chromatographic tube.—Fuse 15 cm section of 38mmod tubing,to 20 cm of 22mmtubing, which in turn is fused to 5 cm of 7mmtubing,with drip tip.,B. Reagents,(a) Silicic acid.—Mallinckrodt Specialty Chemical Co.,No. 2847. Heat in shallow pan or evaporating dish 18 h at 175°C.,Store in desiccator or tightly sealed container.,(b) Bromocresol green–glycol solution.—Dissolve 700 mg,bromocresol green in 700 mL ethylene glycol by warming on,steam bath. Cool, and add ca 200 mL H2O. Prepare 0.1M NH4OH,by diluting ca 6.6 mL NH4OH to 1 L with H2O. Add 40 mL of this,solution to indicator solution and additional H2O to make 1 L.,Store this ink–blue solution in stoppered bottle.,(c) Packing material.—Mix H2SiO3, (a), in ratio of 100 g to ca,95 mL bromocresol green–glycol solution, (b), until homogeneous olive-,green powder is obtained. Small batches may be mixed in mortar;,larger batches, in mechanical mixer. Prepared packing material may be,stored in tightly stoppered container several months.,(d) Hexane-butanol mixture.—Add1 volume n-butyl alcohol to,100 mL volumes n-hexane (commercial grade; Phillips 66 Co.,Specialty Chemicals, PO Box 968, Borger, TX 79008-0968, or,equivalent).,(e) Isopropanol–KOH solution.—Dissolve 25 g KOH pellets in,400 mL isopropanol by warming and swirling on steam bath. Decant,supernate alcoholic solution from the small amount of aqueous,solution clinging to bottom of flask. Cool and decant supernate,isopropanol–KOH solution, which contains ca 50 mg KOH/mL.,Store in refrigerator.,(f) Potassium hydroxide solution.—Approximately 0.05M. Dilute,60 mL isopropanol–KOH solution, (e), with 440 mL,isopropanol and 500 mL methyl alcohol. Store in amber or “Lifetime,Red” Pyrex bottle.,(g) Thymol blue solution.—Dissolve 300 mg thymol blue in 25 mL,0.05Malcoholic KOH solution, (f), and add 75 mL isopropanol.,C. Preparation of Fatty Acid Solution,(a) Saponification.—Transfer 0.5–0.7 g well-mixed, melted fat,to 20 ′ 150 mm t e s t tube with l i p , and add 5 mL,isopropanol–KOH solution, (e), and boiling chip. Place tube to,depth of 5 cm in boiling H2O bath 20 min to saponify fat, and evaporate,isopropanol, leaving solid soap. Stopper, and analyze within,48 h.,(b) Determination of quantity of acid required to hydrolyze,soap.—Add 5 mL isopropanol–KOH solution, (e), to 10 mL H2O,and 2 drops thymol blue solution, (g), in small beaker or flask. Add,H2SO4 (2 + 1) dropwise, with constant stirring, until initial blue color,turns to yellow, orange, and finally red. This volume H2SO4, measured,with same dropper, is subsequently used to hydrolyze the soap.,(If top of chromatographic column turns blue on addition of fatty,acid solution, use of more H2SO4 to hydrolyze soap is indicated; top,of chromatographic column should be yellow.),(c) Hydrolysis of soap and extraction of fatty acids.—Add to,soap, while cooling tube in coldH2O, number of dropsH2SO4 (2+1),indicated in (b). Break up lumps in bottom of tube with glass stirring,rod. After mass in tube is thoroughly mixed, yellow mixture of fatty,acids clinging to viscous aqueous layer of K2SO4 should be obtained.,Add 10 mL hexane–butyl alcohol solution to tube and thoroughly,mix with glass rod. Aqueous phase should cling to white,precipitate of K2SO4, allowing easy decantation of solvent solution,of fatty acids. This solution of acids is ready for chromatography.,D. Preparation of Chromatographic Column,Overlay 35 g packing material with hexane–butyl alcohol solution,in mortar. Mix with pestle to form slurry. Place small glass wool,plug loosely in constricted end of column and gently tamp into place,with glass rod. Place finger over constricted end of column and add,hexane-butyl alcohol solution until reservoir is half full. Using teaspoon,underlay prepared slurry beneath solvent. Jiggle spoon up,and down along side of reservoir, and let flocculent slurry settle to,bottom of column.,After adding all packing material, remove finger and let solvent,flow out and packing material settle. Apply 5–10 lb (34.5–68.9 kPa),air pressure to top of column to speed up flow of solvent and facilitate,uniform packing of slurry. Release pressure just before last portion,of solvent sinks into column. If column looks uniformly packed,it is ready for use; if not, add more hexane-butyl alcohol solution to,reservoir and again apply pressure as before. Prepared column,should have flow rate of ca 3.5 mL/min without use of pressure. If,flow rate is <3 mL/min, add more bromocresol green-glycol solution,to packing m……
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