試驗(yàn)2 高效液相色譜法測(cè)定梔子藥材中梔子苷的含量

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1.把握高效液相色譜法的基本原理及操作步驟；2.熟悉高效液相色譜法在中草藥有效成分測(cè)定中的應(yīng)用。試驗(yàn)原理

高效液相色譜法是用高壓輸液泵將具有不同極性的單一溶劑或不同比例的混合溶劑、緩沖液等滾動(dòng)相，泵入裝有固定相的色譜柱，經(jīng)進(jìn)樣閥注入供試品，由滾動(dòng)相帶入色譜柱，在柱內(nèi)各組分被分開后,依次進(jìn)入檢測(cè)器，色譜信號(hào)由記錄儀或積分儀記錄。

所用的儀器為高效液相色譜儀。色譜柱的填充劑和滾動(dòng)相的組分應(yīng)按待測(cè)組分的性質(zhì)而定，常用的色譜柱填充劑有硅膠和化學(xué)鍵合硅膠，后者以十八烷基鍵合硅膠最為常用，辛基硅烷鍵合硅膠次之，氰基或氨基鍵合硅膠也有使用。除另有規(guī)定外，柱溫為室溫，檢測(cè)器有紫外檢測(cè)器，二極管陣列檢測(cè)器，示差折光檢測(cè)器等。

影響色譜分開效率有兩大主要因素，其一為熱力學(xué)因素,如不同組分容量因子(K’)，其二是動(dòng)力學(xué)因素,如色譜峰的展寬。反映這些物理過程的指標(biāo)是保存時(shí)間和理論塔板數(shù)(N)。

其中K’=tR-to/to；n=5.54(tR/W1/2)2

密切凝眸和努力解決這兩個(gè)因素引起的矛盾，組分間的分開就成功了，分開的表現(xiàn)指標(biāo)是分開度“R〞。

R=2(t2-t1)/(Wb1+Wb2)試驗(yàn)條件

1.儀器：高效液相色譜儀，超聲波清洗器，ZorbaxC18柱（4.6×250mm）。2.試劑：梔子苷對(duì)照品，乙睛，甲醇，水。試驗(yàn)內(nèi)容

1.色譜條件：色譜柱：ZorbaxC18柱（4.6×250mm）；滾動(dòng)相：乙睛—水（15:85）；檢測(cè)波長(zhǎng)：238nm，流速：1mL/min。

2.對(duì)照品溶液的制備和標(biāo)準(zhǔn)曲線的繪制：縝密稱取梔子苷對(duì)照品適量，加甲

醇制成每1mL含100μg的溶液，搖勻后，分別取該液1，2，3，4，5mL，置10mL容量瓶中，加甲醇稀釋至刻度，搖勻后，分別進(jìn)樣20μl，按上述色譜條件測(cè)定，以峰面積為縱坐標(biāo)，濃度為橫坐標(biāo)繪制標(biāo)準(zhǔn)曲線，計(jì)算回歸方程和相關(guān)系數(shù)。

3.供試品溶液的制備：取本品粉末0.1g，縝密稱定，置25mL量瓶中，縝密參與甲醇20mL，超聲處理30mins。放冷，加甲醇至刻度，搖勻，濾過?？b密量取續(xù)濾液10mL，置25mL容量瓶中，加甲醇至刻度，搖勻，過微孔濾膜，取續(xù)濾液，作為供試品溶液。

4.測(cè)定法：吸取供試品溶液20μl注入液相色譜儀，測(cè)定。由標(biāo)準(zhǔn)曲線法或一點(diǎn)法計(jì)算樣品中梔子苷的含量（C17H24O10）。

五試驗(yàn)記錄

1.扼要說明操作步驟及試驗(yàn)結(jié)果；

2.繪制標(biāo)準(zhǔn)曲線圖，及回歸方程，相關(guān)系數(shù)；六思考題

1.在儀器操作過程中，應(yīng)重點(diǎn)注意哪些方面？

2.用HPLC法測(cè)定中藥中有效成分含量的主要優(yōu)勢(shì)是什么？

Experiment3DeterminationofGardenosideinFructusGardeniaebyHPLC

Classhour:3Objectives

1.MastertheprinciplesandoperationalmethodofHPLCthroughthisexperiment.2.BefamiliarwiththeuseofHPLCindeterminationofcomponentsintraditionalChinesemedicine.Principles

Inhighperformanceliquidchromatography,themobilephase,asolventorasolventmixtureofsuitablepolarity(orabuffersolutionofsuitableionstrength),ispumpedintoacolumncontainingthestationaryphase.Whilethesampleisinjectedintoaninjectorandcarriedintothecolumnbythemobilephase,thecomponentsareseparatedonthestationaryphaseandeachcomponentpassesthroughthedetectorinsuccessionandthusachromatogramisrecord.

Thestationaryphaseandthecomponentpartofthemobilephaseshouldbeinaccordancewiththequalitiesoftestsamples.Silicagelandchemicallybondedsilicagelarecommonlyusedasthebulkingagent.Forchemicallybondedsilicagel,octadecylsilanetypeismostfrequentlyused,followedbytheoctylsilanetype,andthecyanoandaminogroupbondedsilicagelareoccasionallyused.Thecolumnismaintainedatroomtemperature.ThetypeofdetectorincludesUV,DAD,SPDandsoon.

Therearetwoimportantfactorsresponsibleforchromatographicseparationefficiency.Oneisthethermodynamicfactor,suchasthecapacityfactorofdifferentcomponents(k’),theotheristhedynamicfactor,suchasthewidthofpeak.Theindexestoreflectthesephysicalprocessesareretentiontime(tR)andthenumberoftheoreticalplatesofcolumn(n).Calculatethevalueofk’andnasfollows:

k’=(tR－to)/to;n=5.54(tR/w1/2)2

Theseparationofdifferentcomponentswillbesuccessfulifcloseattentionispaidandgreateffortistakentosolvethecontradictioncausedbytheabovefactors.The

term“resolutionfactor〞isoftenusedasanindextoexpressseparationefficiency.CalculatethevalueofRasfollows:

R=2(t2－t1)/(wb1＋wb2)

Conditions

1.ApparatusHPLCinstrumentcoupledwithanultravioletdetector,Column:ZorbaxC18（4.6×250mm,ultrasoniccleaner.

2.ReagentsFructusgardeniae,gardenosideCRS,acetonitrile,methanol,water.Assay

1.Chromatographicsystem

Useoctadecylsilanebondedsilicagelasthestationaryphaseandacetonitrile–water(15:85)asthemobilephase;flowrate:1.0mL/min;thewavelengthofthedetectoris238nmandthecolumntemperatureissetinroomtemperature.2.Preparationofthestocksolutionandthestandardcurve

ThestocksolutionispreparedbydissolvingaquantityofgardenosideCRSinmethanoltoproduceasolutioncontaining100μgpermL.Thentransferthestocksolution1,2,3,4and5mLtofive10mLvolumetricflasks,anddilutewithmethanoltothevolumerespectively.20μLofeachofsolutionisanalyzedusingthechromatographicsystemshowedabove.Thestandardcurveisobtainedbyplottingconcentrationversuspeakareaandcorrespondingcoefficientarealsocalculated.3.Preparationoftestsolution

0.1gofthesampleisweighedandcontainedina25mLvolumetricflask.Addaccurately20mLofmethanoltotheflask,extractfor20minsinanultrasoniccleaner.Afterthat,takeitoutandallowittocool.Addmethanoltothemarkofthevolumetricflask.Shakeitwellandfilter.Afterfiltration,transferaccurately10mLofthesuccessivefiltratetoanother25mLvolumetricflaskanddilutetothevolumewithmethanol.Thenthesolutionaremixedwellandfilteredwithmilliporemembrane(particlesize0.45μm).Thus,thesuccessivefiltrateisusedasthetestsolution.4.Procedure

20μLoftestsolutionisinjectedaccuratelyintothecolumn,andthecontentofthe

sampleisdeterminedaccordingtothestandardcurveoronepointmethod.Experimentrecords

1.Describebrieflytheprocessofthisexperiment.

2.Plotthestandard