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1、9.1.01 AOAC Of fi cial Method 986.15 Ar senic, Cad mium, Lead, Se le nium, and Zinc in Hu man and Pet Foods Multielement Method First Ac tion 1986 Fi nal Ac tion 1988 Co dex-AdoptedAOAC Method* A. Prin ci ple Ma te rial is di gested with HNO3 in closed sys tem. Cd and Pb are de ter mined by an odic
2、strip ping voltammetry (ASV). As, Se, and Zn are de ter mined by atomic ab sorp tion spectrophotometry (AAS) af ter gen er a tion of metal hy drides (for As and Se). B. Ap pa ra tus (a) Po laro graph.With an odic strip ping ac ces so ries. Typ i cal op er at ing pa ram e ters for Prince ton Ap plied
3、 Re search Model 174 (current Model 394; ) with hang ing drop Hg elec trode are: scan rate, 5 mV/s; scan di rec tion, +; scan range, 1.5 V; ini tial po ten tial, -0.7 V; mod u la tion am pli tude, 25 mV; op er a tion mode, dif fer en tial pulse; dis play di rec tion, “-”; drop time, 0.5 s; low pass
4、fil ter, off; se lec tor, off; pushbutton, ini tial; out put off set, off; and cur rent range, 510 mA, or as needed. Other in stru ments and elec trodes such as wax im preg nated graph ite may be used ac cord ing to man u fac turers di rec tions. (b) Atomic ab sorp tion spectrophotometer.With Zn, As
5、, and Se hol low cath ode lamps or As and Se electrodeless dis charge lamps, 3 slot, 10 cm Bolling burner head, airC2H2 and H2N2en trained air flames, and deu te rium arc back ground corrector. (c) De com po si tion ves sel.70 mL. See 974.14A (see 9.2.24). (d) Hy dride gen er a tor.See Fig ure 986.1
6、5A. Con structed from fol low ing: (1) Flat bot tom flask.Boro sili cate glass, 50 mL (Corning No. 5160, or equiv a lent). (2) Stop per fit tings.Two-hole (one through cen ter) No. 9 rub ber stop per, fit ted with gas out let tube of 100 mm 18 in. (3 mm) id poly eth yl ene tub ing through cen ter ho
7、le. Place bot tom of gas out let tube through cut off bot tom 1 in. (25 mm) seg ment of 58 in. (16 mm) poly eth yl ene test tube with hole in bot tom so that 3 mm of tube pro trudes through test tube. In sert through sec ond hole 75 mm 18 in. (3 mm) id poly eth yl ene tub ing as N2 in let tube. Seal
8、 bot tom end of tube with burner and then punch sev eral holes at sealed end with 21 gage nee dle. Al ter na tively, pre pare sim i larly 500 mm 116 in. (1.5 mm) id poly eth yl ene tub ing and hold in place in stop per with hole-through sep tum. Con nect other end of tub ing to AA spectrophotometer
9、with 500 mm Tygon tub ing by cut ting auxiliary line at ca 75 mm from mix ing cham ber and at tach ing tub ing. (3) Gen er a tor mount.(Op tional.) 64 mm 0.5 in. (13 mm) id pipe se cured to lab o ra tory ring stand by means of clamp holder. In sert ex ten sion clamp into pipe and at tach an other cl
10、amp to back of clamp to hold clamp in place and to serve as han dle; clamp is now free to ro tate ca 180. At tach rub ber stop per of hy dride gen er a tor to ex ten sion clamp with stiff wire and po si tion just at level of clamp jaws. In op er a tion, place flask of gen er a tor be tween jaws of e
11、x ten sion clamp, in sert stop per firmly into neck of flask, then tighten clamp jaws around neck of flask. Unit can be rap idly and uni formly in verted by ro tat ing han dle on ex ten sion clamp, thus al low ing test solution and so dium borohydride to mix rap idly and reproducibly. (e) Pipets.50
12、and 100 mL Eppendorf micropipets, or equiv a lent. C. Re agents (Use dou ble dis tilled water. Rinse all glass ware with HNO3 1 + 1 fol lowed by thor ough H2O rinse. De con tam i nate di ges tion ves sels by di gest ing with re agents to be used in di ges tion. Rinse thor oughly with H2O. De con tam
13、 i na tion is nec es sary to re duce blanks, es pe cially for Pb, to ac cept able level.) (a) Acids.(1) Ni tric acid.Re dis tilled. (2) Perchloric acid.70%, dou ble vac uum dis tilled. (3) Hy dro chlo ric acid.8M. Di lute 66 mL HCl to 100 mL with H2O. (b) Ni trate so lu tion.Equimolar so lu tion of
14、KNO3 and NaNO3.Dis solve 54.3 g KNO3 and 45.7 g NaNO3 (avail able as Suprapur, Nos. 5065 and 6546, re spec tively, EM Sci ence) in H2O in 200 mL vol u met ric flask, di lute to vol ume, and mix. To fur ther pu rify, add 12 drops NH4OH to 25 mL aliquot and ex tract with 2 mL 10 mg dithizone/mL CCl4 u
15、n til lower sol vent layer is col or less. (c) Mag ne sium so lu tions.(1) Mag ne sium chlo ride so lu tion.37.5 mg/mL. Dis solve to tal of 3.75 g MgO, USP, by add ing small amounts at time to 100 mL 8M HCl. (2) Mag ne sium ni trate so lu tion.75 mg/mL. Mix 3.75 g MgO, USP, with ca 30 mL H2O, slowly
16、 add HNO3 to dis solve (ca 10 mL), cool, and di lute to 50 mL with H2O. (d) So dium borohydride so lu tion.4.0 g NaBH4/100 mL 4% NaOH. (e) Po tas sium io dide so lu tion.Dis solve 20 g KI in H2O and di lute to 100 mL. Pre pare just be fore use. (f) Metal pow ders.Pu rity: 99.99 + % Cd, Pb, Zn; 99.99
17、% Se. (g) Cad mium stan dard so lu tions.(1) Stock so lu tion.1 mg/mL. Dis solve 1.000 g Cd pow der in 20 mL HNO3 (1 + 1) in 1 L vol u met ric flask, and di lute to vol ume with H2O. (2) Working so lu tion.2 mg/mL. Pipet 10 mL stock so lu tion into 2005 AOAC IN TER NA TIONAL Fig ure 986.15A. Hy drid
18、e gen er a tor: 1, poly eth yl ene tub ing; 2, rub ber stop per; 3, flame sealed poly eth yl ene tub ing with holes punched at one end; 4, re agent cup; 5, so dium borohydride so lu tion; 6, test so lu tion; 7, ni tro gen in let from “auxiliary” line of AAS. 2006 AOAC INTERNATIONAL 100 mL vol u met
19、ric flask, and di lute to vol ume with H2O. Pipet 2 mL di luted so lu tion into 100 mL vol u met ric flask and di lute to vol ume with H2O. (h) Lead stan dard so lu tions.(1) Stock so lu tion.1 mg/mL. Dis solve 1.000 g Pb pow der in 20 mL HNO3 (1 + 1) in 1 L vol u met ric flask, and di lute to vol u
20、me with H2O. (2) Working so lu tion.5 mg/mL. Pipet 1 mL stock so lu tion into 200 mL vol u met ric flask and di lute to vol ume with H2O. (i) Zinc stan dard so lu tions.(1) Stock so lu tion.1 mg/mL. Dis solve 1.000 g Zn pow der in 20 mL HCl (1 + 1) in 1 L vol u met ric flask, and di lute to vol ume
21、with H2O. (2) Working so lu tions.0.2, 0.5, 1.0, and 1.5 mg/mL. Pipet 1 mL stock so lu tion into 100 mL vol u met ric flask and di lute to vol ume with H2O. Pipet 2, 5, 10, and 15 mL di luted so lu tion into sep a rate 100 mL vol u met ric flasks, each con tain ing 1 mL HClO4, and di lute to vol ume
22、 with H2O. (j) Ar senic stan dard so lu tions.(1) Stock so lu tion.Dis solve 1.320 g As2O3 in min i mum vol ume 20% NaOH in 1 L vol u met ric flask, acid ify with HCl (1 + 1), and di lute to vol ume with H2O. (2) Working so lu tions.1, 2, 3, 4, and 5 mg/mL. Pipet 10 mL stock so lu tion into 100 mL v
23、ol u met ric flask, and di lute to vol ume with H2O. Pipet 1, 2, 3, 4, and 5 mL di luted so lu tion into sep a rate 100 mL vol u met ric flasks, and di lute to vol ume with H2O. (k) Se le nium stan dard so lu tions.(1) Stock so lu tion.1 mg/mL. Dis solve 1.000 g Se pow der in min i mum vol ume HNO3
24、in 200 mL beaker and evap o rate to dry ness. Add 2 mL H2O and evap o rate to dry ness. Re peat ad di tion of H2O and evap o ra tion to dry ness twice. Dis solve in min i mum vol ume HCl (1 + 9) in 1 L vol u met ric flask, and di lute to vol ume with HCl (1 + 9). (2) Working so lu tions.1, 2, 3, 4,
25、and 5 mg/mL. Pipet 10 mL stock so lu tion into 100 mL vol u met ric flask and di lute to vol ume with H2O. Pipet 1, 2, 3, 4, and 5 mL di luted so lu tion into sep a rate 100 mL vol u met ric flasks and di lute to vol ume with H2O. D. Closed Sys tem Di ges tion (Do not ex ceed man u fac turers spec i
26、 fi ca tions of 0.3 g sol ids with 70 mL ves sel. Pro ceed cau tiously with new or un tried uses. Let such test por tions stand with HNO3 over night or heat on hot plate cau tiously until any vig or ous re ac tion sub sides. Then pro ceed with closed ves sel di ges tion. Open ves sel in hood since n
27、i tro gen ox ides are re leased.) Weigh 0.3 g test portion (dry ba sis) into de con tam i nated de com po si tion ves sel, add 5 mL HNO3, close ves sel with lid, and heat in 150?C oven 2 h. Cool in hood, re move ves sel from jacket, and trans fer con tents to 10 mL vol u met ric flask. Add 4 mL H2O
28、to ves sel, cover with lid, and while hold ing lid tightly against rim, in vert sev eral times, and add rinse to flask. Di lute to vol ume with H2O and mix. E. An odic Stripping Voltammetry (For Cd and Pb.) Pipet aliquot of di gested test so lu tion into de con tam i nated 50 mL Vycor cru ci ble and
29、 add 2 mL ni trate so lu tion, C(b). Con duct re agent blank si mul ta neously. Heat on hot plate at low heat to dry ness; then in crease heat to max i mum (ca 375?C). Ni trate salts will melt and di gest or ganic mat ter in 1520 min. Place cru ci bles in 450?C fur nace to ox i dize any re main ing
30、car bo na ceous mat ter (1020 min). Di ges tion is com plete when melt is clear. Let cool, add 1 mL HNO3 (1 + 1) to so lid i fied melt, and heat on hot plate to dry ness to ex pel car bon ates and ni trites and to con trol acid ity. Dis solve in 5.0 mL HNO3 (0.5 mL/L), warm ing on hot plate to speed
31、 so lu tion. Trans fer to polarographic cell with 5.0 mL H2O. Bub ble O2-free N2 through so lu tion 5 min; then di rect N2 over so lu tion. Set dial for Hg drops at 4 mm di vi sions. Stir so lu tion with mag netic stir rer at con stant and re pro duc ible rate so Hg drop is not dis turbed. Slide se
32、lec tor switch to “Ext. Cell” and mea sure time for 120 s with stop watch. Turn off stir rer and let stand 30 s. Press “Scan” but ton to ob tain peaks cor re spond ing to Cd and Pb at ca -0.57 and -0.43 V, re spec tively, against sat u rated cal o mel elec trode. Add known vol umes of each stan dard
33、 to test so lu tion in cell from Eppendorf pipet. Amounts added should be ca 1 , 2 , etc. of amount metal pres ent ini tially in cell, and each ad di tion should not change orig i nal vol ume sig nif i cantly. Af ter each ad di tion, bub ble N2 through so lu tion briefly and per form de po si tion a
34、nd strip ping op er a tions ex actly as for orig i nal so lu tion. Plot mg metal added on x-axis against peak height on y-axis. Ex trap o late lin ear line to x-axis to ob tain mg metal in cell. mg Metal/g test por tion = MM- g test portion 10 mL aliquot taken where M and M = mg metal from stan dard
35、 curve for test solution and blank, re spec tively. F. Atomic Ab sorp tion Spectrophotometry (For As, Se, and Zn.) (a) Ar senic.Pipet aliquot di gested test so lu tion into de con tam i nated 50 mL round, flat-bottom boro sili cate flask, and add 1 mL Mg(NO3)2 so lu tion, (c)(2). Heat on hot plate a
36、t low heat to dry ness; then in crease heat to max i mum (ca 375?C). Place flask in 450?C fur nace to ox i dize any car bo na ceous mat ter and to de com pose ex cess Mg(NO3)2 (30 min). Cool, dis solve res i due in 2.0 mL 8M HCl, add 0.1 mL 20% KI to re duce As5+ to As,3+ and let stand 2 min. Con du
37、ct re agent blank with test portion. 2005 AOAC IN TER NA TIONAL Fig ure 986.15B. Hy dride gen er a tor and mount con nected to auxiliary line of spectrophotometer. Test tube acid trap con nected be tween gen er a tor and in stru ment is not in cluded in method. 2006 AOAC INTERNATIONAL Pre pare stan
38、dards as fol lows: To six 50 mL flasks (same type as used for test portion) add 2.0 mL MgCl2 so lu tion, (c)(1), and to 5 flasks add 50 mL aliquots of re spec tive work ing stan dard so lu tions so that se ries will con tain 0, 0.05, 0.1, 0.15, 0.20, and 0.25 mg As. (Other amounts may be used de pen
39、d ing on sen si tiv ity of sys tem.) Add 0.1 mL 20% KI to each flask, mix, and let stand 2 min. Con nect gen er a tor to in stru ment as shown in Fig ure 986.15B and ad just pres sures and flows for H2H2-en trained air flame as in Ta ble 986.15. Op er ate in stru ment ac cord ing to man u fac turers
40、 in struc tions, with As lamp in place and re corder set for 20 mm/min. Add 2.0 mL 4% NaBH4 so lu tion to re agent dis penser of gen er a tor, and in sert rub ber stop per tightly into neck of flask con tain ing test solution or stan dard. With sin gle rapid, smooth mo tion, in vert flask, let ting
41、so lu tion mix with test solution or stan dard. (This op er a tion must be per formed reproducibly.) Sharp, nar row A peak will ap pear im me di ately. When re corder pen re turns to base line, re move stop per from flask, and rinse re agent dis penser with H2O from squeeze bot tle; then suck out H2
42、O. Pro ceed with next test solution or stan dard. When se ries is com plete, rinse glass ware thor oughly. Plot cal i bra tion curve of mg As against A, and ob tain mg As in test solution aliquot from this curve. Cor rect for re agent blank. (b) Se le nium.Pro ceed as in (a), us ing Se lamp and stan
43、 dards, but omit ad di tion of KI so lu tion. KI will re duce Se to el e men tal state and cause loss of sig nal. In stead, cover flask with small watch glass and place on steam bath 10 min, and cool to room tem per a ture. (c) Zinc.Pipet 1 mL aliquot di gested test so lu tion into de con tam i nate
44、d 25 mL Erlenmeyer, and add 0.1 mL HClO4. Heat on hot plate to white fumes of HClO4. Solution should be com pletely di gested as in di cated by clear, prac ti cally col or less so lu tion. If solids char, add 0.5 mL por tions HNO3 and again heat to white fumes. Finally, heat just to dry ness but do
45、not bake. Cool, and dis solve res i due in 3.0 mL HClO4 (1 + 99). Op er ate in stru ment in ac cor dance with man u fac turers in struc tions, us ing airC2H2 flame, and mea sure A of test solutions and stan dards, C(i)(2). Di lute test so lu tions with HClO4 (1 + 99), if so lu tion is too con cen tr
46、ated. Plot cal i bra tion curve of mg Zn against A, and ob tain mg Zn in test so lu tion aliquot from this curve. Cor rect for re agent blank. Ref er ence: JAOAC 63, 485(1980). CAS-7440-38-2 (ar senic) CAS-7440-43-9 (cad mium) CAS-7439-92-1 (lead) CAS-7782-49-2 (se le nium) CAS-7440-66-6 (zinc) Re v
47、ised: March 1996 * Adopted as a Co dex Al ter na tive Ap proved Method (Type III) for atomic ab sorp tion spectrophotometry of ar senic in all foods. Adopted as a Co dex Al ter na tive Ap proved Method (Type III) for an odic strip ping voltammetry of cad mium, lead, and zinc in all foods. Adopted as
48、 an Al ter na tive Ap proved Method (Type III) for atomic ab sorp tion spectrophotometry of cad mium and se le nium in nat u ral min eral wa ters. Adopted as a Co dex Ref er ence Method (Type II) for atomic ab sorp tion spectrophotometry of ar senic in nat u ral min eral wa ters. Para graph E: Adopt
49、ed as a Co dex Ref er ence Method (Type II) for colorimetry, dithizone of lead in choc o late. Also adopted as a Co dex Method for an odic strip ping voltammetry of lead in co coa pow ders (co coa) and dry cocoasugar mix tures. Para graph F: Adopted as a Co dex Al ter na tive Ap proved Method (Type III) for atomic ab sorp tion spectrophotometry of ar senic in fruit juices. 2005 AOAC IN TER NA TIONAL Ta ble 986.15. Flow rates and pres sures for ar senic and se le nium de ter mi na tio