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AOAC Official 955.31 Vanillin Ethyl Vanillin and Coumarin in Vanilla Extra

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AOAC Official 955.31 Vanillin, Ethyl Vanillin, and Coumarin in Vanilla Extract Chromatographic Separation First Action 1955 Final Action_AOAC.rar,AOAC Official 955.31 Vanillin, Ethyl Vanillin, and Coumarin in Vanilla Extra

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36.2.10AOAC Official Method 955.31Vanillin, Ethyl Vanillin,and Coumarin in Vanilla ExtractChromatographic Separation MethodFirst Action 1955Final ActionA. Apparatus(a) Spectrophotometer.Capable of determining A at 270 and325 nm. Adjust to high sensitivity to utilize slit width 3 mm above light path.(c) Chromatographic tube.14 od 450 mm.B. Reagents(Samebatchofisooctanemustbeusedtopreparesolventmixtureand all dilutions for set of determinations.)(a) Silicic acid.Reagent grade “100-mesh” powder, suitableforchromatography.DetermineSiO2contentasfollows:Accuratelyweigh ca 1 g silicic acid into weighed Pt crucible. Ignite in furnace15 min at 615C, cool in desiccator, and reweigh. Calculate % SiO2(z) in silicic acid. Calculate weight silicic acid (x) required for 5.8 gcolumnas:x=3.384100/z.Volume(mL)H2O(y)requiredforcol-umn is 5.80 x.(b) Isooctane.Practical grade 2,2,4-trimethylpentane, 99.5 +%, bp 98100C.(c) Isooctanechloroform solvent mixture.Add 40 mL CHCl3to 1 L isooctane and mix. Store in airtight bottle. (Do not use rubberstopper.) Solution contains ca 3.85% CHCl3.(d) Coumarin standard solution.1 mg/mL. Accurately weigh100 mg coumarin into 100 mL volumetric flask, dissolve in 50 mLCHCl3, and dilute to volume with isooctane.(e) Ethyl vanillin standard solution.Prepare as in (d), usingethyl vanillin.(f) Vanillin standard solution.Prepare as in (d), usingvanillin.C. Determination of AbsorptivitiesPipet 1 mL coumarin standard solution into 100 mL volumetricflask, add 3.4 mL CHCl3, dilute to volume with isooctane, and mix.Determine A at 270 and 325 nm against solvent, (c), as reference inthe1cmsilicacells.Calculatea(g/L;1cm)forcoumarinat270andat 325 nm as: a = 100A.Determine a for ethyl vanillin and vanillin similarly.D. Preparation of Chromatographic ColumnPacksmallcottonwadinbottomofdrychromatographictube.Tox g silicic acid in mortar add y mL H2O from buret, mix thoroughlyandquicklytouniformpowderyconsistencywithpestle,andimme-diately add 25 mL solvent, (c). Mix and rapidly pour slurry throughfunnelintotube.Rinsemortarandfunnelwithsmallvolumesolvent.Remove any air bubbles formed by stirring with long thin glass rod.Pack column with ca 13.8 kPa (2 lb/sq in.; 10.4 cm Hg) air pressureuntil bottom of meniscus of free solvent just touches top surface ofsilicicacidbutouterpartofmeniscusisstillclearlyvisible.Immedi-ately release pressure. (Important: If column channels or cracks,discard. During packing and thereafter, keep column vertical.Tipping ruins column for further use although it may appear nor-mal.) Carefully add 15 mL solvent down side of tube with aid ofglassrodsocolumnisnotdisturbed.Drivesolventthroughcolumn.Washed column is now ready for calibration.E. Calibration of ColumnPipet and combine 1 mL of each standard solution, (d)(f), in25 mL volumetric flask. Dilute to volume with isooctane and mix.Pipet2mLsolutiondownonesideofchromatographictubeontotopof column. Drive solution into column with ca 2 lb/sq in. (10.4 cmHg) air pressure and collect eluate in 10 mL graduate. Release pres-sure when bottom of meniscus touches top of column and outer partof meniscus is still clearly visible. Pipet 1 mL solvent, (c), downsame side of tube onto column and drive into column. Repeat with2 additional 1 mL portions solvent. Fill tube to within 2 cm of topwith solvent. Drive solvent through column at rate of5 mL/22.5 min, collecting 5 mL eluate fractions, alternating two10 mL graduates during collection. Pour fractions into separate testtubesinrack,numberingfractionsconsecutively.Draingraduatebe-fore reusing by inverting on towel. Collect 10 fractions and deter-mine A at 270 and 325 nm against solvent, (c), as reference in 1 cmsilicacells.Draincellsbyinvertingontowelbeforerefilling;rinsingis not necessary. Permit column to elute by gravity while readingfirst 10 fractions, changing graduates for each 5 mL portion.Coumarin elutes first, ethyl vanillin second, and vanillin third. Inidealcolumncoumarinbeginstoeluteinfraction67,reachesmaxi-mum in 89, and fades considerably in 10. Earlier elution does notseparate compounds entirely; later elution takes more fractions andtime but does give good recoveries. Somewhat slower elution doesnot matter. Ethyl vanillin elutes in ca fraction 1118, and vanillin inca 1930.Ifcolumnissatisfactory,collect25additionalfractions(35inall)oruntilvanilliniscompletelyeluted.DetermineAofeachfractionat270 and 325 nm as above.Ifcoumarinbeginstoeluteatfraction5orearlier,discard,prepareanother column with less H2O in the silicic acid, and recalibrate. Ifcoumarin does not elute by fraction 910, prepare new column withmore H2O in the silicic acid.Use calibrated column for identification, G.F. Preparation of Test SolutionIfconcentrationofnoneofthecompoundsis0.4g/100mL,pipet25mLvanillaextract into250mLcentrifugebottle.Ifconcentrationof any compound is 0.4 g/100 mL, dilute 25 mL test sample withH2O to volume specified in Table 955.31A, and use 25 mL aliquot.Add 75 mL H2O, 20 mL H2SO4(1 + 4), and 50 mL CHCl3. Stopperwith rubber stopper and shake well 3 min. Centrifuge 5 min at1500 rpm. If emulsion persists, break with thin glass rod and 2000 AOAC INTERNATIONALTable 955.31A Dilutions and dilution factors for flavoringsConcentration ofmost abundantconstituent, g/100 mLDilute to: (mL)Dilution factor (F)3.2400200食品伙伴网recentrifuge. Pour contents slowly through large-bore, short-stemfunnel into 250 mL separator. Break emulsion with glass rod anddrain CHCl3into 100 mL volumetric flask. Pour aqueous phasethrough same funnel back into bottle.Rinse separator with 15 mL CHCl3, add to bottle through funnel,andrepeatextractionbymixingphasesthoroughlywithrockingmo-tion.Donotshakevigorouslyasinfirstextraction.Centrifuge,sepa-rate, and drain CHCl3into 100 mL volumetric flask. Repeatextraction with 15 mL portions CHCl3until flask is filled to mark.Pipet 2 mL aliquot into 25 mL volumetric flask, dilute to volumewith isooctane, and mix.G. IdentificationPipet 2 mL test solution (use 1 mL if concentration of most abun-dant compound is 3.2 g/100 mL) onto prepared column, letting itflow down one side of tube without disturbing column. Drive solu-tionintocolumnwithca2lb/sqin.(10.4cmHg)airpressureandcol-lect eluate in 10 mL graduate. Pipet 1 mL solvent, (c), down sameside of tube and drive into column. Repeat with 2 additional 1 mLportions of solvent. Fill tube with solvent and elute compounds bysame technique and conditions as for calibration. Collect 3 morefractionsthanindicatednecessarybycalibration.DetermineAofallfractions at 270 and 325 nm as in calibration.Positions of absorbing fractions compared to those obtained dur-ing column calibration reveal compounds present in sample.Coumarinisalsoidentifiedbyabsorbingat325nmslightly13itsAat270nm.Vanillinandethylvanillinabsorbverylittleat325nm.Ifdesired, confirm by obtaining UV spectrum of one high absorbingfractionofeachcompound.Comparewithspectrapreparedonsameinstrument.Spectraofcompoundsexhibitapproximatemaximumand minimum given in Table 955.31B. Approximate ratio of A atgiven wavelength to that at highest maximum for respective com-poundisgiveninparenthesesafterthatwavelength.Ratioof1.00in-dicates highest maximum.H. DeterminationAdd A at 270 nm of all fractions containing coumarin and calcu-late coumarin concentration in original sample:c = F A/awhereAissumofAat270nmoffractionscontainingcoum