安捷伦-液质联用技术(LCMS)及其应用.ppt

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,by Li Ping,*,安捷伦-液质联用技术(,LC/MS),及其应用,1,by Li Ping,液质(,LC/MS),联用技术基础,2,by Li Ping,质谱分析仪(,MS),需要高真空条件,质量分析系统,最好使用挥发性缓冲液,液相(,HPLC),高效分离混合物,流动相挥发后产生大量气体,出口压力为大气压,无质量限制,可以使用缓冲盐溶液,液相(,HPLC),到,质谱(,MS),的接口,HPLC,MS,Interface,(Key to Success!),3,by Li Ping,LC-MS,的接口的发展和种类,传送带(,moving belt),式的,LC-MS:,目前已经停用,热喷雾(,TSP)LC-MS,接口:已经很少使用,粒子束,(,PBI),大气压电离源(,API):,目前最常用的方法,电喷雾(,ESI),大气压化学电离源(,APcI,),4,by Li Ping,LC/MS,联用技术的相对适用范围,5,by Li Ping,大气压电喷雾质谱应用范围,大气压电喷雾源,大气压化学源,分子量,化合物极性,1000,100,000,10,000,非极性,极性化合物,气相色谱/质谱,6,by Li Ping,库仑爆炸,（,Coulomb Explosions,）,Rayleigh,Limit,Reached,+,+,+,+,+,+,+,+,+,+,-,-,-,-,-,+,+,+,+,+,+,+,+,+,+,-,-,-,-,-,+,+,+,+,+,+,-,-,+,+,+,+,+,+,-,-,溶剂蒸发,+,+,带电液滴,分析物离子,溶剂离子束,分析物离子,溶剂离子束,盐,/,离子对,中性化和物,电喷雾(,ESI),的电离过程,7,by Li Ping,Neutral Molecules,Analyte,Ions,Clusters,Salts,毛细管,液相的入口,雾化器,加热,N,2,雾化气入口,碎片碰撞电压,(,CID),锥孔,八极杆,四极杆,高能打拿极/电子倍增器,+,+,+,+,+,透镜,+,+,+,+,+,+,+,+,+,+,+,+,+,液质联用的电喷雾(,ESI),接口,8,by Li Ping,ES,电离原理,9,by Li Ping,蒸汽,通过电晕针放电形成带电荷的反应剂离子,溶剂在蒸发器中蒸发,+,含有气溶胶的分析物,分析物离子,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,+,电荷转移至 分析物分子,+,+,+,+,流动相,分析物,溶剂,-,溶剂,+,H,+,溶剂H,+,+M-,溶剂,+,M,+,大气压化学电离源(,APCI),的电离过程,10,by Li Ping,毛细管,碎片碰撞电压,（,CID）,Corona needle,+,+,+,+,+,+,+,+,+,+,+,+,锥孔,透镜,四极杆,+,+,+,+,八极杆,热氮气,液质联用的大气压化学电离源(,APCI),接口,高能打拿极/电子倍增器,11,by Li Ping,APCI,电离原理,12,by Li Ping,大气压源子化技术,电喷雾(,ESI),化合物无需具有挥发性,是分析热不稳定化和物的首选方法,离子在溶液中以已生成,除了生成单电荷离子之外还可以生成多电荷离子,大气压化学电离源(APCI),化和物需具有一定的挥发性,化和物必需是热稳定的,离子在气态条件中生成,只生成单电荷离子,13,by Li Ping,电喷雾(,ESI),和大气压化学离子化,(,APCI),农药及残留化合物,多肽及蛋白的分子量测定,确定氨基酸序列,多肽及蛋白的纯度分析,单糖和多糖的结构分析,中极性到高极性,包括离子化合物的分析,如表面活性剂分析,农药及残留化合物,抗生素,碱性药物,小分子量样品,类固醇,雌激素,单磺酸偶氮染料,ESI,APCI,14,by Li Ping,什麽是诱导解离碰撞?,(,Collision Induced Dissociation)?,在特定的区域,通过与氮气分子碰撞导致分子解离,从而产生特征碎片离子,可以得到结构信息用于化和物的定性分析,可以提供用于定量分析目地的确任碎片离子,只要改变单一参数,“,fragmentor,”,来控制,CID.,Capillary,Fragmentation,Zone(CID),Skimmers,Octopole,Lenses,Quadrupole,M,n,M,1,+,M,2,+,DETAIL,15,by Li Ping,碰撞电压值对质谱图的影响,350,400,450,500,550,600,650,700,2000,4000,6000,8000,m/z-,Abundance,439,467,502,684.65,350,400,450,500,550,600,650,700,2000,4000,6000,8000,m/z-,Abundance,411,439,467,495,684.65,碰撞电压,=175,volts,碰撞电压,=100,volts,loss of C14 fatty acid,loss of C12 fatty acid,loss of C16 fatty acid,16,by Li Ping,Excellence through Evolution,安捷伦液-质联用仪介绍,17,by Li Ping,液质(,LC/MS),联用系统的示意图,离子源,离子束,聚焦,M/Z,分析器,检测器,数据,处理,HPLC,LC,接口,质谱图,18,by Li Ping,安捷伦,LC/MS,产品家族,液相/离子井质谱,(,VL,和,SL),液相/单四极杆质谱,LC/MSD VL,液相/单四极杆质谱,LC/MSD SL,19,by Li Ping,四极杆质谱,+,+,+,+,+,+,Main RF,Low Amplitude Dipole Field,(1/3 Frequency of the Main RF),Conversion Dynode,Electron Multiplier,Shutter,Optimized Asymptote Angle,Octapole,Ring,多极离子阱质谱,20,by Li Ping,安捷伦的专利技术,直角喷雾接口,+,+,+,+,+,+,+,+,提高灵敏度,减少污染,降低毛细管被堵塞,对非挥发性缓冲液的忍耐能力强,21,by Li Ping,独一无二的交叉喷雾器,交叉喷雾器,不需,X,Y,Z,位置调整,而且避免了离子光学透镜的污染,倾斜排水口可以快速去除冷凝液,22,by Li Ping,完成整个分析后,毛细管的入口处还是干静的,!,在非常复杂的基质的条件下,-,汉克盐溶液(,Hanks),Componentg/L,Sodium chloride,8,Calcium chloride0.1,Potassium chloride0.4,Potassium phosphate,monobasic,0.06,Magnesium Sulfate0.1,Sodium bicarbonate0.35,Sodium phosphate dibasic0.048,Glucose1,Phenol red0.011,23,by Li Ping,清洁喷雾室,在,APCI,的条件下,流动相为,HBSS,连续进样,635,次以后,完成整个分析后,毛细管的入口处还是干静的,!,24,by Li Ping,内置式较正溶液,用于全自动调谐和质量较正,全自动调谐和质量较正,25,by Li Ping,安捷伦,1100,系列,LC/MSD:,易懂,易用的图型化的液相和质谱控制及分析软件,26,by Li Ping,哪一种离子化模式最好,?,不知道要分析的化合物的性质是酸性或碱性,要分析的化合物中同时包含酸性和碱性化合物,酸性和碱性化合物的浓度不同,27,by Li Ping,可同时采集多种质谱信号,:,正离子和负离子模式,采集50组，30个离子/组离子,同时采集正负离子,28,by Li Ping,我经常筛选未知物.,最好是同时获得分子量和一些碎片信息,我正在筛选未知物,但我不知道最优的碰撞电压,我希望一次能获得在不同碰撞电压条件下的谱图,可同时采集多种质谱信号,:,高和低,CID,能量,29,by Li Ping,低,CID,能量,高,CID,能量,可同时采集多种质谱信号,:,高和低,CID,能量,30,by Li Ping,0,200000,400000,600000,800000,min,1,2,3,4,5,6,7,0,100000,200000,300000,400000,500000,600000,700000,800000,High fragmentor:130 V,Low fragmentor:65 V,m/z,100,200,300,311.1,312.1,156.1,311.1,108.1,245.1,218.1,可同时采集多种质谱信号,:,高和低,CID,能量,磺胺药物演示混合样,31,by Li Ping,可同时采集多种质谱信号,:,SIM/Scan,SIM,来,定量所选的目标化合物离子,对未知的化合物,SCAN,32,by Li Ping,Scan,SIM,0,100000,200000,300000,400000,500000,600000,min,1,2,3,4,5,20000,40000,60000,80000,100000,120000,140000,160000,180000,200000,279.0,m/z,100,200,300,279.0,280.0,301.0,磺胺药物演示混合样,可同时采集多种质谱信号,:,SIM/Scan,33,by Li Ping,可同时采集多种质谱信号,:,时间编程信号1),开始五分钟不采集信号,5,分钟开始采集信号,信号关闭采集,Improve data quality and manage data file size:,34,by Li Ping,从每一项分析中获得最大的信息,一针注射样品可获得任意以下分析模式最多达四种,1.,高,/,低,CID,能量可获得分子量及碎片信息用于确认化 合物的结构,2.,正/负 离子化模式,3.,SIM/Full Scan,模式,35,by Li Ping,如何建立自己的,HPLC/ESI,和,HPLC/APCI,谱库,36,by Li Ping,质谱库检索特点,检索用户自建谱库,使用,NIST,程序和检索规则,没有应用独特的筛选,如,PBM,谱库可以包含结构式,交互式或自动检索,DAD/MSD,组合检索报告,37,by Li Ping,自动生成谱库检索报告,Library search mode:Automatic library search,Library file No.:1,Library file name :S:SPYDERMSLIBR1DOUGLIB.UVL,Match threshold :950 Purity threshold:Calculated,Time window left%:None Case sensitive:No,Time window right%:None Whole word:No,Wavelength shift :0.0 Compare spectrum:Yes,Absorbance threshold :0.0 Search logic:OR,Search range :All,Signal 1:DAD1 A,Sig,=254,8 Ref=450,80,Signal 2:MSD1 TIC,MS File,NIST MS library search mode:Automatic library search,NIST MS library file No.:1,NIST MS library file name:,douglib,RetTime Sig,MW Library Match Formula Name,min#Id,-|-|-|-|-|-|-|-,2.746 2 270 1 4 720,C9H10N4O2S2,Sulfamethizole,2.987 2 278 1 5 748,C12H14N4O2S,Sulfamethazine,3.571 2 284 1 6 583 C10H9ClN4O2S Sulfachloropyridazine,6.143 2 310 1 25 868 C12H14N4O4S Sulfadimethoxine,38,by Li Ping,SIM,灵敏度,API-,electrospray,at 400 l/min or,APCI,at 1000 l/min,Selected ion monitoring of,m/z,609.3,Positive ionization,VL,10 pg,reserpine,50:1 RMS(10:1 peak-to-peak),SL,1.0,pg,reserpine,50:1 RMS(,10:1 peak-to-peak,),Unsurpassed performance!,卓越的操作性能指标,39,by Li Ping,LC/MSD SL:,利血平(电喷雾,SIM,模式),Chromatographic Conditions,Column:30 x 2.1mm Zorbax Rapid Resolution SB-C8,3.5 m(p/n 873700-902),Mobile phase:5 mM each ammonium,formate,in 75%methanol in water,Flow rate:0.4 ml/min,Injection amount:1 or 2 pg on column,ESI MS Conditions,Source:ESI,Ion mode:positive,SIM ions:609,Fragmentor:200 V,2 pg reserpine,0,2000,4000,6000,min,2,4,6,810,12,14,1000,2000,3000,1 pg reserpine,40,by Li Ping,LC/MSD SL:,利血平(大气压化学离子化,SIM,模式),Chromatographic Conditions,Column:30 x 2.1mm Zorbax Rapid Resolution SB-C8,3.5 m(p/n 873700-902),Mobile phase:5 mM each ammonium,formate,in 75%methanol in water,Flow rate:1 ml/min,Injection amount:1 or 2 pg on column,APCI MS Conditions,Source:APCI,Ion mode:positive,SIM ions:609,Fragmentor:200 V,Vaporizer:450C,Drying gas:350C,5 l/min,Nebulizer,:50 psig,min,2,4,6,8,10,12,14,400,800,1200,9.475,10.992,12.506,14.022,15.536,400,800,1200,1600,2000,9.472,10.988,12.502,14.015,15.528,2 pg reserpine,1 pg reserpine,41,by Li Ping,安捷伦-液质联用技术,1.具有多年的台式质谱研究经验；,2.直角接口的设计，保证仪器耐缓冲盐，提高灵敏,度及易于清洗离子源和接口多重目的；,3.同一厂家的产品，工作站的一体化控制，同时,采集色谱和质谱信号；,4.具有自动调谐和手动调协两种方式；,5.,APCI,和,API-ES,源转换方便，5,min,即可完成；,6.在实验室条件下，0.4-1.5,ml/min,流速下达到最高的,灵敏度；,7.同时采集正负离子模式；,42,by Li Ping,Agilent 1100 Series LC/MSD:,应用分析举例,在农药合成及农药残留分析中的应用,在高分子添加剂分析中应用,在染料(如偶氮染料)分析中的应用,在双酚,A,壬基酚及表面活性剂分析中的应用,在激素及兴奋剂分析中的应用,在蛋白质及多肽分析中的应用,在糖类化合物分析中的应用,在天然产物分析中的应用,43,by Li Ping,氨基甲酸盐类化合物(,Carbamates,),磺酰脲类(,Sulfonyl Ureas,),苯脲类(,Phenyl,Ureas,),其它农药及其残留化合物,LC/MSD,在农药及农药残留分析中的应用,44,by Li Ping,Tomato extract spiked with 11,carbamates,200 pg/l;50 l injected;Scan mode,APCI-LC/MS,氨基甲酸盐类化合物的分析,mAU,min,5,10,15,20,25,20,40,60,80,UV 254 nm,Tomato extract spiked with 11,carbamates,Norm.,40000,80000,120000,EIC,m/z 222,MS TIC,carbofuran,45,by Li Ping,APCI-LC/MS,氨基甲酸盐类化合物的分析,对于1,ppb,氨基甲酸盐类化合,HPLC,的分析:,柱后衍生,然后用萤光检测器分析,LC/MS:,进样量:,50,ul X 200pg/,ul,柱子:,100,X4mm I.d,3um,Hypersil,ODS,溶剂,A:Water,溶剂,B:Methanol,梯度:,25,to 75%B in 20min,UV Detector=214nm Flow rate:0.8ml/min,MS:Mass Scan range:100 to 510,amu,APCI,Vap,=400C Drying gas temp=350C,46,by Li Ping,氨基甲酸盐化合物的总离子图及提取离子谱图,60000,120000,6000,12000,750,1500,6000,12000,1000,2000,2.5,5,7.5,10,12.5,15,17.5,1500,3000,MS TIC,m/z 163,m/z 116,m/z 222,m/z 202,m/z 226,oxamyl,aldicarb,aldicarb sulfone,aldicarb sulfoxide,carbofuran,carbaryl,methiocarb,promecarb,草氨酰,涕灭威,涕灭威砜,涕灭威亚砜,西维因,猛杀威,47,by Li Ping,41.5 pg/component,Increased Sensitivity for,Carbamates,Using SIM,min,20,40,150,300,20,40,2.5,5,7.5,10,12.5,15,17.5,20,40,min,0,2.5,5,75,10,12.5,15,17.5,0,100,200,300,400,200,400,MS TIC,m/z 132,m/z 226,m/z 202,m/z 222,aldicarb sulfoxide,carbofuran,carbaryl,promecarb,48,by Li Ping,*,Final sample concentration for carbamates equals 2 g/ml,*3,ul inj,on a 2.0 mm x 150 mm,Phenomenex,C-18,*10,ul inj,on a 4.6 mm x 250 mm Pickering C-18,检测的比较,florida,CDFA Methodology,克百威,敌灭威亚碸,草氨酰,甲氨叉威,3-羟基克百威,敌灭威,西维因,灭虫威,49,by Li Ping,50 l of 1 ppb injected,SIM,除草剂的,APCI-LC/MS,分析谱图的提取离子图,0,2000,Cyanazine,0,1000,Simazine,0,4000,8000,Hexazinone,min,5,10,15,20,25,Atrazine,0,4000,8000,m/z 241,m/z 202,m/z 253,m/z 216,氰草津,西玛津,莠草津,环嗪酮,50,by Li Ping,*50,l of 1 ppb injected(50 pg on column),2-OH-,atrazine,may shift,and is very dependant on column equilibration and pH,COMPOUND*,(M+H)+,S/N,2,6-,dichlorobenzamide,190,2,DIP-,atrazine,174,84,Metamitron,203,43,Dimethoate,230,18,DE-,atrazine,188,73,2,-,OH-,atrazine,198,detected,Cyanazine,241,24,Simazine,202,14,Hexazinone,253,62,Atrazine,216,86,Isoproturon,207,51,Diuron,233,24,Terbuthylazine,230,75,Linuron,249,2,Pendimethalin,282,26,利用,APCI-LC/MS,正离子/选择离子模式分析除草剂的结果,51,by Li Ping,Scan mode,20,ppm,/component,APCI-LC/MS,用于苯脲除草剂的分析,min,1,2,3,4,5,50000,100000,150000,200000,Metoxuron,Linuron,Diuron,Chlortoluron,Monolinuron,立谷隆,敌草隆,绿谷隆,甲谷隆,绿麦隆,52,by Li Ping,分析苯脲类除草剂的,LC/MS,条件,LC conditions:Column 200 X 2.1 mm C18,Mobile phase A:Water B:Methanol,Gradient:50-80%within 3 min,Flow Rate:0.4ml/min UV Detector=254 nm,MS:Mass Scan:100-400amu,Positive in APCI,Corona needle=6uA,Vaporize Temp=400C,Drying gas Temp=325C,4.0 L/min,Nebulizer,Gas,Presure,:30psi,53,by Li Ping,Electrospray,positive ion mode,SIM,Sulfonylureas,min,1,2,3,4,5,50000,100000,150000,200000,Chlorsulfuron,(Glean),MW=357,S,N,H,N,H,O,O,O,N,N,N,C,H,3,O,C,H,3,C,l,Thifensulfuron,methyl,(Harmony),MW=387,S,N,H,N,H,O,O,O,N,N,N,C,H,3,O,C,H,3,S,H,3,C,OO,Metsulfuron,methyl,(Ally),MW=381,S,N,H,N,H,H,3,C,O,O,O,O,O,N,N,N,C,H,3,O,C,H,3,Nicosulfuron,(Accent),MW=410,N,S,N,H,N,H,N,O,O,O,O,N,N,N,C,H,3,O,C,H,3,H,3,C,C,H,3,54,by Li Ping,液质联用(,LC/MS),技术在 聚合物添加剂分析中的应用,55,by Li Ping,聚合物润滑添加剂:,作用:,减少摩擦系数,Behenamide,(,山俞酸酰胺),CH,3,(CH,2,),18,CONH,2,MW 311.3,和,CH3,(CH,2,),20,CONH,2,MW 339.3,Erucamide,(,芥酸酰胺),CH,3,(CH,2,),7,CH=CH(CH,2,),11,CONH,2,MW 337.3,56,by Li Ping,聚合物润滑添加剂:,APCI,正离子模式-分析山俞酸酰胺(,Behenamide,),样品,0,2,4,6,8,10,0,500000,1000000,1500000,2000000,2500000,3000000,Time(min.),Ion Abundance,CH,3,(CH,2,),16,CONH,2,m/z,Ion Abundance,250,300,350,400,284.2,285.2,250,300,350,400,312.3,313.2,250,300,350,400,340.3,341.2,CH,3,(CH,2,),18,CONH,2,CH,3,(CH,2,),20,CONH,2,57,by Li Ping,0,50000,100000,150000,200000,250000,300000,350000,Time(min.),Ion Abundance,0,2,4,6,8,10,12,14,m/z,250,300,350,400,450,0,20000,40000,60000,80000,100000,120000,338.3,339.3,250300,350,400,450,0,2000,4000,6000,8000,10000,12000,338.3,436.4,339.2,437.5,411.2,312.1,356.3,284.3,Ion Abundance,Ion Abundance,聚合物润滑添加剂:,APCI,正离子模式-分析芥酸酰胺(,Erucamide,),样品,58,by Li Ping,抗氧添加剂,作用:,防止聚合物在挤出时,由于氧化而受到破坏,Irgafos,168,MW 646.6,Irganox,1076,MW 530.4,O,P,3,HO,CH,2,CH,2,C,=,O,OC,18,H,37,59,by Li Ping,抗氧添加剂,Irgafos,168 by Positive Ion APCI,Irgafos,168,MW 646.6,O,P,3,600,625,650,675,700,0,50000,100000,150000,200000,250000,300000,663.5,664.5,665.5,607.4,600,625,650,675,700,0,200000,400000,600000,800000,1E6,647.5,648.5,649.5,M+16,m/z,0,2,4,6,8,10,12,14,16,0,500000,1000000,1500000,2000000,2500000,3000000,Time(min.),Ion Abundance,Ion Abundance,Ion Abundance,60,by Li Ping,Irganox,1076 by Negative Ion APCI,0,2,4,6,8,10,12,14,-5,-2.5,0,2.5,5,7.5,10,0,2,4,6,8,10,12,14,0,500000,1000000,1500000,2000000,2500000,UV Signal 280 nm,Total Ion Chromatogram,Negative Ion APCI,Time(min.),mAU,Ion Abundance,Irganox,1076,MW 530.4,HO,CH,2,CH,2,C,=,O,OC,18,H,37,m/z,Ion Abundance,(M-H),-,520,540,0,200000,400000,600000,800000,1000000,529.3,530.2,527.2,531.3,61,by Li Ping,抗氧添加剂,Irganox,1010,MW 1176.7,Irganox,B225,50%,Irganox,1010,50%,Irgafos,168,62,by Li Ping,LC/MS Analysis of,Irganox,B225 by APCI,0,2000040000,60000,80000,100000,120000,140000,160000,600,800,0,20000,40000,60000,80000,731.5,675.3,619.3,785.3,600,800,0,100000,200000,300000,400000,500000,647.5,679.5,m/z,Ion Abundance,Peak at 10.6 min,Positive Ion Mode,Irganox,1010.h,1100,1200,1175.4,1177.5,1178.4,Peak at 10.5 min,Negative Ion Mode,Irganox,1010,Peak at 15.5 min,Positive Ion Mode,Irganox,168,0,10,20,30,40,50,60,0,250000,500000,750000,1000000,1250000,1500000,1750000,0,100000,200000,300000,400000,500000,600000,700000,mAU,Ion Abundance,0,2,4,6,8,10,12,14,16,0,2,4,6,8,10,12,14,16,0,2,4,6,8,10,12,14,16,Time(min.),UV Signal 280 nm,Positive Ion APCI TIC,Negative Ion APCI TIC,Irganox,1010,Irganox,1010,Irgafos,168,63,by Li Ping,液质联用(,LC/MS),技术在 酚类化合物分析中的应用,64,by Li Ping,化学结构式,C,HO,OH,CH,3,CH,3,CH,3,OH,HO,C,C,2,H,5,HO,C,n,H,2,n+1,65,by Li Ping,双酚,A(M.W 228),(STD 1ppm),Bisphenol,A,TIC,LC/MSD,66,by Li Ping,双酚,A,(,流动注射分析,),LC/MSD,67,by Li Ping,双酚,A,分析,-,TIC,vs,Concentration(10-0.1ppb)-,LC/MSD,68,by Li Ping,双酚,A,分析,-,检测限,(,UV/MSD)-,Conventional LC System:LC with Fluorescence/UV,Deector,(220nm),LC/MSD,69,by Li Ping,烷基酚的质谱图,(1ppm),LC/MSD,70,by Li Ping,烷基酚分析,-EIC mode,5ppb-,LC/MSD,71,by Li Ping,min,1,2,3,4,5,6,4000,5000,6000,7000,Bisphenol,A,Abundance,0.5,pg/,m,l,S/N=4.8,LC/MSD SL,Fig.SIM chromatogram of,Bisphenol,A,72,by Li Ping,安捷伦,LC/MS,产品家族,73,by Li Ping,