Glucosamine Rapid Assay
来自 : 蚂蚁淘
GlucosamineRapidAssayMRTHOD:- Placesample(containing0.5-10µgGlcN)inaPyrexscrewcappedtube.
- AddHCltoafinalconcentrationof2Nandafinalvolumeof0.6ml.Flushwithnitrogen.
- Stoppertightlyandhydrolyze16hat100oC.
- Preparestandardscontaining0-20µgGlcNfromastocksolutionof1.5mg/ml.(Use0,3,5,8,10,12mlofstocksolutionandaddwatertomakeupto300µl.Thenadd300µl4NHCltoafinalvolumeof0.6mlandfinalconcentrationof2N).Standardsneednotbeheated.
- Neutralizesamplesandstandardswith0.4ml2MNa2CO3(10.6gin50ml).(pH-7withtheslightexcessofNa2CO3)
- Shakegentlyandadd0.5ml(freshlyprepared)2%acetylacetonein1.5MNa2CO3.(15.9gSodiumCarbonate+2mlacetylacetonemadeupto100ml).
- Stoppertightlyandheatinboilingwaterbathfor20min.
- Cool.Add1mlEtOH.
- Add0.5mlEhrlichsreagent.(1gpdimethylaminobenzaldehydein15mlEtOHand15mlc/HCl)
- ShaketubesvigorouslytoexpelexcessCO2.
- Maximumcolourdevelopmentisreachedin5min.Chromophorestablefor1to2hours.
- Readabsorbanceat530nm.
NOTE:NaClaffectscolour.Thereforestandardsandsampleshouldcontainthesameamountofsalttoavoidcolourdepressionanderroneousresults.DialysishelpstoremoveNaCl.
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