电喷雾四级杆飞行时间Qtof理论ppt课件

1、micrOTOF-Q 运用培训培训目的：了解ESI-Q-TOF的根本原理 掌握仪器的根本操作和维护 数据处置软件的运用潘晨松 博士布鲁克道尔顿公司运用技术支持工程师培训安排第一天根本实际简介及运用中应留意的要点microTOF Control软件引见(1)测样练习tunemix、小分子、多肽、蛋白质microTOF Control软件(2): Calibration; DataAnalysis 软件引见(1)练习DataAnalysis软件的运用学员练习校正及测样第二天 复习及操作调查 HyStar LC/MS 练习小分子混合样品和蛋白/多肽样品测定 DataAnalysis软件(2) 练习处

2、置LC/MS数据第三天 复习及操作调查软件引见与练习LibraryEditor, Isotope Pattern, Generate Molecular Formular, ReportDesigner.) 练习处置LC/MS数据样品测定样品测定答疑、讨论液质联用型高分辨串联质谱仪 电喷雾四级杆飞行时间 (ESI-Q-TOF)商品名：micrOTOF-QDry Gas HeaterOrthogonal AcceleratorGlass CapillaryCollision Gas SupplyHexapole电喷雾离子源去溶剂系统飞行管检测器反射镜四级杆Q碰撞池产生离子离子传输与聚焦分辨离子I

3、on GenerationIon Transmission and focusIon ResolutionmicrOTOF-QIon Generation: Atmospheric Pressure Ionization (API)Atmospheric Pressure Ionization- Electrospray Ionization (API-ESI)Ion generation for MS analysis Nebulization Desolvation Coulomb explosions DesorptionUnder proper source conditions, i

4、ndividual ions only will enter the capillary. An unstable signal or capillary current may indicate a need for adjustment of gases, flow rate, or spray needle. See the Users Manual for Troubleshooting tips. Nebulizer GasSampleDry GasCapillary ( 4 kV)Spray NeedlegroundedDry GasGeneration of Ions - Neb

5、ulizationDroplet formation in presence of electrical field at the needle tip within the spray chamberHVGeneration of Ions-DesolvationGeneration of Ions-Ion EvaporationTo obtain spectral information, ions need to be created and introduced to the instrument for interrogationMust consider flow rates of

6、 Sample:NebulizerDry gasGeneration of Ions - ESI sourceThe sample is introduced via the nebulizer. The ESI process is supported by nitrogen gas.The drying gas heats up the source and dries the spray.It acts as a counter current flow to the nebulized sample ensuring complete evaporation of the solven

7、t in order to prevent droplets from entering the glass capillary.The desolvation unit contains the dry gas heating block and capillary housing. The glass capillary interfaces the atmospheric pressure region of the spray chamber with the fist vacuum stage of the ion optics separates atmospheric press

8、ure from first vacuum stage parts: drying gas heater glass capillaryGeneration of Ions Desolvation unitElectrospray Factors Affecting IonizationNeedle set-up Inner Needle Position Nebulizer Pressure Needle ConditionHigh voltage electrodes Capillary Voltage settings Condition of Capillary and Chamber

9、 High Voltage Elements Condition of InsulatorsSolution Chemistry Flow Rate Solution pH Sample pKa Solution ConductivityElectrospray Solution ChemistryMobile phase pH has a major effect for analytes that are ions in solution. Basic pH (7.0; 9 preferred) for negative ionsAcid pH (7.0; 5 preferred) for

10、 positive ions*Manipulation of pH can enhance performance for analytes that are not normally ionized in solution.Electrospray Sample ChemistryPositive Ion Mode Negative Ion Mode Base + acid Sample Acid + base Sample+:-Electrospray BuffersChoose buffers carefully for TOF instrumentsElectrospray Buffe

11、r Selection: Volatile buffers are used to modify mobile phase pH. May be added in mobile phase as a post-column addition. Acidic solutions favor positive ion mode. Formic acid, 0.1-1.0% Acetic acid, 0.1-1.0% Ammonium salts favor production of single ammonium adducts. General buffers Ammonium acetate

12、 Ammonium formate Triethylamine Other volatile solvents (离子传输率大大提高，从而提高灵敏度。增宽传输离子质量范围 离子流方向传统的锥式离子传输DCACResonant ionNonresonant ionQuadrupole Mass Analyzer Collision Cell+Parent ion FragmentsArgon Gas EnergyMS : Ions were cooled by argon gasMS/MS: Fragments were produced by argon gas and energy Dry

13、Gas HeaterOrthogonal AcceleratorGlass CapillaryCollision Gas SupplyHexapole电喷雾离子源去溶剂系统飞行管检测器反射镜四级杆Q碰撞池产生离子离子传输与聚焦分辨离子Ion GenerationIon Transmission and focusIon ResolutionIon Resolution Contains last lens package (lens4 & 5) Accelerates the ions for measuring the time of flight Fill mode - pusher an

14、d puller are grounded, corrector is on “fill potential - the region between pusher and puller is filled with ions Extract mode - pusher and puller are set to “high voltages opposite to each other (about 400V) - corrector is on extract voltage - ions are accelerated into vertical direction (towards t

15、he reflector)Ion Optics Orthogonal AcceleratorIon beamIon beamIn the ideal case:mi = mass of analyte ionzi = charge of analyte ionE = extraction fieldti = flight time of ionls = length of source (orthogonal acceleration stage)ld = length of the field-free drift regione = electronic charge (1.06022 x

16、 10-19 C)TOF Theory22=disiilteElzmThe aim of an electrostatic reflector, also called reflectron is to improve mass resolution. It creates a retarding field that acts as an ion mirror by deflecting the ions and sending them back through the flight tube.The reflector corrects the energy dispersion of

17、ions with the same m/z ratio. Indeed, ions with more kinetic energy will penetrate the reflectron more deeply and will spend more time in the reflectron. Thus they reach the detector at the same time as slower ions of the same m/z.Ions receive kinetic energy from electric field. E = 1/2mv2Resolution

18、 of Ions TOF Assemblym1 = m2, but E1 E2Orthogonal Detector acceleratorThink of the TOF operation as a drag race between vehicles of different sizes, but all having identical engines: “Start line = orthogonal accelerator; “Finish line = TOF detector Just as all vehicles have the same engine (i.e., ho

19、rsepower), all ions are pulsed up the flight tube with the same kinetic energy. Since m = 2E/v2, the smaller vehicles/ions will reach the finish line/detector before the larger ones.STARTFINISHPrinciple of the TOF Mass AnalyzerA detector converts an ion signal into an electrical signal. Here, the de

20、tector is a micro-channel plate detector. It has millions of small pores which are coated inside with a semi-conductive layer. Each of these channels work as an independent electron multiplier.Micro-channel plates are effective for the detection of signals over a large dynamic range without saturati

21、on. In addition, the time response of this detection system is very rapid, therefore avoiding deterioration of peak resolution.Multiplication process in a channelMicrochannel Plate DetectorQ- SeparationCIDDry Gas HeaterDual Ion FunnelAnalyticalQuadrupoleCollision CellOrthogonal AcceleratorDetectorRe

22、flectronFlight TubeGlass CapillaryCollision Gas SupplyAPI Spray ChamberSprayerHexapolemicrOTOFQ MS2 PossibilitiesTOF-MSIn Source CIDMS3质谱根本术语： 分辨率 Mass Resolution同位素分布方式 Isotope Patterns单同位素质量 Monoisotopic Mass.It is a measure of a mass spectrometers ability to distinguish two compounds of nearly eq

23、ual mass.60080010001200140016001800200022002400m/z 100 200 300 400 500 600 700 800 900 1000 1100a.i.161816231628 m/z“easy“not so easyResolutionWhat is meant by Resolution?The narrower the FWHM, the higher the resolution. With the same FWHM, higher masses will have higher resolution than lower masses

24、.Resolution = (m/z) / FWHMFWHM Full Width at Half Maximumm/zHow do we measure mass resolution?Isotopic Distribution Patterns13121009080706050403020100C11221211201009080706050403020100C101,2061,2041,2021,2001009080706050403020100C10012,03012,02012,01012,0001009080706050403020100C1000Isotopes are atom

25、s with the same number of protons in the nuclei, but with different numbers of neutrons. Only 21 elements have only one stable isotope. All other elements are mixtures of at least 2 stable isotopes, and the proportions of these isotopes can vary greatly depending on the element. Carbon has 2 stable

26、isotopes, C-12 and C-13, with natural abundance of 98.892% and 1.108% respectively. As the number of carbons increase in a molecule, the isotopic distribution pattern will reflect the mass contribution of the isotopes with their extra neutrons. pQLYENKPRRPYIL MW 1672.9Res. 1,0001,6821,6801,6781,6761

27、,6741,672100908070605040302010 1673.9Average Mass401,6821,6801,6781,6761,6741,67210090807060503020100Res. 10,000M+H+M+H+1+M+H+2+M+H+3+M+H+4+ 1672.9Monoisotopic MassNeurotensinResolution = (m/z) / FWHMAverage and monoisotopic massesExact mass or accurate mass is the mass of the monoisotopic peak meas

28、ured accurately to within a few millimass units. Lower resolution results in an “average -inaccurate determination of peak center -calculated average mass is inaccurate at best multiple charged ion m/z =M + nHnH质荷比的计算：2+1+Determining Multiple Charge StatesExample: m1 = 1000, isotope m2 = 1001Charge

29、(z) = 1 1000/1, 1001/1 m/z = 1000, 1001Charge (z) = 2 1000/2, 1001/2 m/z = 500, 500.5Charge (z) = 3 1000/3, 1001/3 m/z = 333.33, 333.66The m/z difference is incremental to the charge. Charge 2 = 0.5Charge 3 = 0.333Charge 4 = 0.252+3+1+质量准确度计算Mass accuracy can be expressed as a percentage or ppm:e.g.

30、 % mass accuracy = Measurement errorTrue massx 100%Very small percentage errors (0.01%) are expressed as parts per million or ppm. 0.01% = 100ppme.g. ppm = Measurement errorTrue massx 106Measured mass: 1296.970 True mass: 1296.685 0.02% errorppm = 0.001 per thousandmicrOTOF Q TuningThe goal of tunin

31、g is to maximize the intensity and/or resolution of ions within an m/z range of interest.To achieve this, the parameters of the source and TOF portions of the instrument must be properly set.Source Parameters largely affect the intensity; Optics Parameters - determine the m/z range over which ions a

32、re transmitted.TOF Parameters largely affect the resolution.micrOTOF Q TuningTune ParametersIon OpticsThus we give the rule of thumb: the larger the mass, the higher the value for the tuning parameters.How the voltages affect the ions is largely mass dependent.and smaller mass ions require less ener

33、gy to control them.Larger mass ions need more energy to direct them into the trap, ScansSummationsRollingAvg. of 1A single spectrum is obtained each time the TOF is pulsed. A data point is the spectrum that is saved and/or displayed in the GUI. To make one data point, several spectra are summed toge

34、ther. When rolling average is turned on, each data point is an average of itself with the “X number of data points prior. Each averaged spectrum is weighted proportionally to its recency. RollingAvg. of 2less weightmore weightRolling Average and SummationSumming and averaging increases the statistic

35、al reliability of your data set.78910111213Time min0.00.51.01.52.02.58x10Intens.mAURolling 5: TIC AllRolling averages together is used for infusion of low level samples to increase the signal to noise (S/N) ratio for the low intensity ions of interest. Rolling provides a smoothing effect to the data

36、 and tailing can be observed in a chromatographic peak if there is too much rolling. This is because older scans with higher intensity make up part of the data point after the peak has eluted. 78910111213Time min0.00.51.01.52.02.58x10Intens.mAURolling off: TIC AllRolling offPeak tailing Loss of peak resolution Decreased peak intensityRolling 5To smooth chromatographic data, rolling of one to three is acceptable. Rolling values higher than this can cause considerable tailing and loss o