AOAC966_13.pdf

AOAC Official 966.13 Vanillin and Ethyl Vanillin in Vanilla Extract Paper Chromatographic First Action 1966 Final Action 1967_AOAC.rar

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AOACOfficialMethod966.13VanillinandEthylVanillininVanillaExtractPaperChromatographicMethodFir.rar
AOAC Official Method 966.13 Vanillin and Ethyl Vanillin in Vanilla Extract Paper Chromatographic Method First Action 1966 Final Action 1967_AOAC
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vanillin vanilla extract Ethylvanillin
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AOAC Official 966.13 Vanillin and Ethyl Vanillin in Vanilla Extract Paper Chromatographic First Action 1966 Final Action 1967_AOAC.rar,vanillin,vanilla,extract,Ethylvanillin
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36.2.08AOAC Official Method 966.13Vanillin and Ethyl Vanillinin Vanilla ExtractPaper Chromatographic MethodFirst Action 1966Final Action 1967A. Reagents and Apparatus(a) Mobile solvent.Cyclohexane (practical)ethyl ace-tatemethyl alcohol (100 + 30 + 20).(b) Immobile solvent.10% Dimethylformamide (DMF) inether.(c) Sodium carbonate solution.Dissolve 4 g Na2CO3in H2Oand dilute to 1 L.(d) Chromatographic paper.Whatman No. 3MM, 8 8 in.(e) Chromatographic tank.Mitchell tank and equipment,970.52G(a) (see 10.1.01).(f) Spotting pipet.10 L.(g) Longwave ultraviolet light.B. Preparation of Standard CurvePrepare solutions of vanillin and ethyl vanillin in 35% alcoholcontaining 0.10, 0.15, 0.20, 0.30, and 0.40 g/100 mL. Draw parallellines on chromatographic paper 2.5 and 3.7 cm above bottom edge,using hard pencil. Apply one 10 L spot of each solution on the2.5 cm line, keeping spots 2.5 cm apart and starting 5 cm from leftside of paper. Use separate papers for vanillin and ethyl vanillincurves.Usesamemicropipetforallspottings,rinsingthoroughlybe-fore each application. Let spots air dry, without heat. Handle papercarefully near edges to avoid high blanks.Meanwhileplace100mLH2Oinbottomofchromatographictankcontaining one trough. Fill trough with mobile solution, cover tank,andseal.Letstand15min.Dippaperintoimmobilesolventfromtopdownto3.7cmline,leavingbottom3.7cmofpaperfreefromimmo-bile solvent. Do not permit solvent to reach spots. (Dipping canreadily be done by use of shallow pan containing solvent.) Air drypaperafewmin,removesealfromtank,andplacepaperintankwithbottom edge dipping into mobile solvent. Reseal tank and develop2 h, even though solvent front reaches top before end of this period.Remove paper and air dry. Do not expose developed paper to air1h.Ifdelayisnecessary,placepaperinjarandstoreinrefrigerator.Expose paper to NH3fumes for few min by placing paper inwide-mouth half-gallon (2 L) jar containing small beaker with NH3on bottom, and capping jar. Examine paper under longwave UVlight and outline dark blue areas with soft pencil. Ethyl vanillin willshowhigherRfvaluethanvanillin.Removemarkedareaswithscis-sors and cut each into smaller pieces before placing them in 50 mLErlenmeyers. Cut out 2 blanks from side of paper, each approxi-mately equal in area to developed spots. Use side area for theseblanks, away from spotted areas and their developed rise.Pipet 10 mL Na2CO3solution into each flask, swirl, and letstand 1015 min, with frequent swirling. Centrifuge or filterthrough rapid paper, discarding first portion of filtrate. Deter-mine A at 348 nm, using Na2CO3solution as reference. Also ob-tainaverageAofthe2blanksandsubtractfromstandardAbeforeplotting standard curve.C. DeterminationIf vanilla extract contains 0.4 g vanillin/100 mL, dilute belowthis level with 35% alcohol. Make one 10 L spotting on the 2.5 cmline with same micropipet used to prepare standard curves. Proceedas above and determine vanillin and ethyl vanillin by comparisonwith
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