AOAC970_51
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ID： |
B730A65AE38C4735BC8760CB5987479F |
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文件大小(MB)： |
0.02 |
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页数： |
2 |
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文件格式： |
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日期： |
2013-4-11 |
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购买： |
文本摘录（文本识别可能有误，但文件阅览显示及打印正常，pdf文件可进行文字搜索定位）：
41.1.45,AOAC Official Method 970.51,Fats (Animal) in Vegetable Fats,and Oils (Determination of Cholesterol),Gas Chromatographic Method,First Action 1970,Final Action 1974,(Sterol fraction of some vegetable oils contains small amounts of,cholesterol. Sterol fraction of palm oil may contain considerable,amount of cholesterol.),A. Preparation of Sample,Saponify and extract unsaponifiable matter from 2.5 ± 0.01 g fat,as in paragraph 1 and 2, 933.08 (see 41.1.39). Discard aqueous solutions.,Transfer ether extract to 250 mL beaker and evaporate to dryness,on steam bath under N2. Dissolve unsaponifiable matter in,4–5 mL CHCl3, transfer to 4 dram vial, and evaporate to dryness.,Rinse beaker with three 3 mL portions CHCl3, taking special care to,dissolve any material on sides of beaker. Transfer rinsings to vial,and evaporate to dryness under N2. Store samples in freezer.,Isolation of Sterols by Thin-Layer Chromatography,B. Reagents and Apparatus,(a) TLC plates.—Prepare from silica gel PF 254 + 366 or HF,254 + 366 (Brinkmann Instruments, Inc., or equivalent) as in C, or,use precoated plates available as “Uniplate” (precoated with silica,gel HF 254 + 366, 500 mmthick; No. 2112, available as special order,upon request) from Analtech, Inc., or “Quanta Gram” (precoated,with silica gel PK5F with fluorescent indicator, 500 mm thick;,No. 4851-830) from Whatman, Inc.,(b) Chloroform.—Distilled in glass (Burdick and Jackson Laboratories,Inc., or equivalent).,(c) Ethyl ether.—Anhydrous, ￡0.01% alcohol (Fisher Scientific,Co. E-138, or equivalent).,(d) Petroleum ether.—Distilled in glass, bp 30–60°C (Burdick,and Jackson Laboratories, Inc., or equivalent).,(e) b-Sitosterol standard solution.—3 mg/mL. Weigh 30.0 mg,b-sitosterol standard (Aldrich Chemical Co., Inc., No. S340-2) into,10 mL volumetric flask and dilute to volume with ethyl acetate.,Commercial material is mixture of campesterol (earlier eluting component),and b-sitosterol.,(f) Thin layer chromatographic apparatus.—Glass plates,20 ′20 cm (ca 8 ′8 in.); Desaga/Brinkmann applicator; mounting,board; spotting template; micro-syringe, 10 mL; desiccating storage,cabinet, Fisher 8-645-6; storage rack, SGA Scientific Inc.,C-4116-3; developing tank (Thomas-Mitchell tank now unavailable);,longwave 15 watt UV lamp (use with UV-absorbing eyeglasses),or Chromato-Vue cabinet equipped with one or two 15 watt,lamps and, preferably, C-50 longwave transilluminator (Ultra-,Violet Products, Inc.); or equivalents.,(g) Thin layer plate scraper.—Optional; adapt from sealing tube,with fritted disk (Corning Glass Works 39580, 30M). See Figure,970.47?(see 49.2.25).,C. Preparation of Plates,Align 5 matching 20 ′20 cm glass plates on mounting board, and,just before coating, wipe plates with tissue dampened with alcohol to,remove any dust or fingerprints. Adjust applicator to deliver 0.5mm,thick layer. Weigh 45 g silica gel into 500 mL Erlenmeyer, add,130 mL H2O, shake vigorously 25–30 s, and pour into applicator.,Immediately coat plates with silica gel suspension and let plates rest,undisturbed until gelled (0.5–1 h). Dry coated plates 32 h at 110°C,and store in desiccating cabinet until just before use.,D. Thin-Layer Chromatography,Line developing chamber with blotting paper and add 100 mL,ether–petroleum ether (1 + 1) to chamber. Cover chamber and,equilibrate 2 h.,Draw line across plate 17 cm from bottom and ca 1 cm from each,side. Spot 10 mL b-sitosterol standard solution, (e), at point 2 cm,from bottom edge and 3 cm from 1 side of plate. Dissolve,unsaponifiable matter, A, in 200 mL CHCl3 and spot entire sample in,10 mL portions on imaginary line 2 cm from bottom edge of plate so,that spot centers are 0.75 cm apart. Rinse vial with ca 100 mL CHCl3,and spot rinse solution in equal portions on top of sample spots.,Immediately insert plate into equilibrated chamber (position plate,to expose coated surface to maximum chamber volume); cover,chamber and seal with tape. Withdraw plate from chamber when solvent,front reaches 17 cm stop line. Evaporate solvent and view plate,under longwave UV light in darkened room. Mark off sterol band,(same Rf, 0.2–0.3, as b-sitosterol standard) with needle, and remove,sterol band as follows (do not remove b-sitosterol standard): Scrape,off sterol band with square end of stainless steel spatula (Fisher,No. 14-375-10, or equivalent) into 100 mL beaker and transfer with,20 mL CHCl3 to 70 mm top diameter funnel containing folded,12.5 cm diameter filter paper (S&S 588, or equivalent). Extract sterols,with five 10 mL portions CHCl3 and evaporate combined filtrate,to near dryness on steam bath under N2. Transfer residue to 3 dram,vial (scre……
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