AOAC Official Method 981
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96154D8E6FB24987BC8C2E0B93E7B397 |
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0.11 |
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2 |
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日期： |
2016-2-28 |
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46.2.08,AOAC Of fi cial Method 981.13,Cresidine Sulfonic Acid,Schaeffer’s Salt, 4,4￠-(Diazo-amino)bis(5-methoxy-,2-methyl-benzenesulfonic Acid),and 6,6￠-Oxybis(2-naphthalenesulfonic Acid),in FD&C Red No. 40,Liquid Chromatographic Method,First Ac tion 1981,Fi nal Ac tion 1982,A. Prin ci ple,Aque ous so lu tion of dye is an a lyzed by ion ex change LC us ing,gra di ent elu tion.,B. Apparatus,(a) Liq uid chromatograph.—With gradient elution capability,and au to matic sam pler, e.g., DuPont Model 830 with Model 838,programmable gradient accessory or DuPont Model 850, either,chromatograph with Model 834 au to matic sam pler with 20 mL,sam pling loop. Op er ating con di tions: chart speed 0.5 cm/min; eluant,flow rate 0.6 mL/min; temperature ambient. Gradient profile for,Model 838 pro gram ma ble gra di ent ac ces sory: pro gram,Figure 981.13A, is scribed on pro gram charts pro vided and run at time,set ting of 40 min with 10 min equil i bra tion pe riod (only pri mary,eluant flow ing) be tween each gra di ent run. Gra di ent pro file for,DuPont Model 850: seg ment 1, 0–18% in 16 min; seg ment 2, 18–62%,in 6 min; seg ment 3, 62% for 18 min; with 12.7 min equil i bra tion,pe riod (only pri mary eluant flow ing) be tween each gra di ent run.,(b) De tec tors.—(1) DuPont 254 nm de tec tor (low pres sure Hg,source) with at ten u a tion set at 0.08 AUFS. (2) DuPont Model 835,multiwavelength de tec tor (me dium pres sure Hg lamp source and,325–385 nm fil ter) with at ten u a tion set at 0.04 AUFS or DuPont UV,spectrophotometer de tec tor set at 382 nm and 0.04 AUFS.,(c) Col umn.—DuPont Zipax SAX col umn 100 cm ′ 2.1 mm id,(Cat. No. 851950 405). Con di tion new col umn by heat ing 90 h at,50°C with 0.01M Na2B4O7 flow ing at 1000 psi and then let ting it rest,2 weeks un used.,(d) Spectrophotometer.—Vis i ble and UV range.,C. Re agents,(a) Eluants.—(1) Pri mary eluant, 0.01M Na2B4O7. (2),Sec ond ary eluant, 0.2M NaClO4 in 0.01M Na2B4O7.,(b) Standard solutions.—(1) Cresidine sulfonic acid (CSA), ca,0.18 mg/mL H2O. (2) Schaeffer’s Salt (SS), ca 0.22 mg/mL H2O.,(3) 6,6￠-Oxybis(2-naphthalenesulfonic acid) (DONS), ca 1.00 mg/mL,H2O. (4) 4,4￠-(Diazoamino)bis(5-methoxy-2-methylbenzenesulfonic,acid) (DMMA), ca 0.10 mg/mL 1% aque ous NaOH. De ter mine,ex act con cen trates of stan dard so lu tions (1)–(3) from UV spec tra of,di luted aliquots in H2O; de ter mine con cen tra tion of stan dard,so lu tion (4) from vis i ble spec tra of di luted aliquots in 1% NaOH.,Ap prox i mate a (mg/mL, 1 cm) are: (1) 47 at 250 nm, (2) 292 at,231 nm, (3) 179 at 238 nm, and (4) 61.3 at 426 nm.,D. Suit abil ity Test,Pre pare test so lu tion con tain ing 1% FD&C Red No. 40 and,rel a tive to FD&C Red No. 40, 0.2% CSA, 0.3% SS, 0.1% DMMA,and 0.5% DONS. Set pa ram e ters and run blank gra di ent (no,in jec tion) and test so lu tion. Com pare chromatogram to that of,Fig ure 981.13B. If sim i lar res o lu tion is not at tained, ad just,op er at ing pa ram e ters to those needed to re solve these com pounds.,E. Cal i bra tion,For each cal i brat ing so lu tion, dis solve 0.250 g FD&C Red No. 40,(free of CSA, SS, DMMA, and DONS) in ca 50 mL H2O, add 10 mL,0.1M Na2B4O7, add aliquots in di cated in Ta ble 981.13, and di lute to,100 mL with H2O. Cal cu late con cen trates of com pounds be ing,de ter mined as weight % of FD&C Red No. 40. Cal cu late %,con cen tra tion in cal i bra tion so lu tions as:,C = V ′ C￠ ′ 0.4,where V = vol ume stan dard so lu tion taken; C￠ = con cen tra tion,stan dard so lu tion as de ter mined spec tro pho to met ri cally in mg/mL;,and 0.4 = 100(%)/250 (mg). Mea sure ar eas of peaks for CSA, SS,and DONS, us ing 254 nm de tec tor, and for DMMA, us ing 382 nm,de tec tor. Ob tain ar eas from in te gra tor or by mul ti ply ing peak height,by peak width at half peak height. Plot % con cen tra tion of each,com pound against peak ar eas. Use cal i bra tion so lu tions 1–5 to,de ter mine re ten tion times of the 4 com pounds.,F. De ter mi na tion,Weigh 0.250 g test sample, add 10 mL 0.1M Na2B4O7, then add ca,50 mL H2O, and, when dis solved, di lute to 100 mL with H2O.,Chromatograph test so lu tions in ter spersed with cal i brat ing,so lu tions, and com pare chromatograms. Iden tify by re ten tion times,and mea sure ar eas of peaks cor re spond ing to the 4 com pounds. Use,cal i bra tion plots to de ter mine % CSA, SS, DMMA, and DONS.,Ref er ences: JAOAC 64, 324(1981); 66, 400(1983).,CAS-6471-78-9 (cresidine sulfonic acid),CAS-135-76-2 (Schaeffer’s salt),Figure 981.13A. Gra di ent pro file pro gram for,sep a rat ing in ter me di ates and re ac tion by-products from,FD&C Red No. 40 by LC.,2006 AOAC INTERNATIONA……
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