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Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Click to edit Master title style,*,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,2008 Waters Corporation,66,单击此处编辑母版标题样式,四极杆系统的校正,校正,硬件,vs,软件,硬件的校正是由原厂和维修工程师进行的，可以保证名义质量数的准度。,对于某些应用，硬件校正是足够的。,对于需要最高的质量准确度的情况，用户可以通过,Masslynx,软件进行软件校正，并将系统的质量校正变得更为精确。,在识别未知物以及,MRM,和,SIR,分析时，确认选择了准确的质量数，这是非常有用的。,校正中的可变因素,环境的改变,温度或者湿度的改变,随着时间变化而发生的电源变化,改变了分辨率设置,在仪器的校正范围内工作是非常重要的,!,如同您不愿意在定量曲线之外进行定量计算一样，您也不希望在您的质量数校正曲线之外工作。,仪器的校正曲线（,Calibration Curves,）,一台三重四极杆的仪器需要有三种校正曲线：对于,MS1,和,MS2,有六条校正曲线,静态校正（,Static calibration,）可以使四极杆质量分析器准确地定位于感兴趣的特定质量数上。,(,例如 调谐，,SIR,和,MRM).,扫描校正（,Scanning calibration,） 保证在扫描采集中峰采集的质量数准确性。,(,例如 全扫描,Full Scan).,扫描速度补偿（,Scan speed compensation calibration,）：当仪器快速扫描是，补偿系统中的“滞后时间” 。,质谱仪的每一种校正都需要参考溶液。不过，,MassLynx,可以在一个步骤中执行所有必须的校正。,校正步骤,每个参考溶液的质谱图都是已经采集好的。采集过的质谱图中的峰与储存于参考文件表格中的预期理论质量数相匹配。,A mass spectrum of a reference solution is acquired. The peaks in the acquired spectrum are matched against a table of expected theoretical masses which are stored in a Reference File.,参考文件中的每一个峰与采集的质谱图中对应的峰相匹配。,Each peak in the Reference File is matched to a corresponding peak in the acquired spectrum file.,A calibration curve is created that adjusts the masses of the,experimantal,spectral peaks to the theoretical masses of peaks as listed in the reference file.,一般注射泵,Typically a Syringe Pump capable of delivering a slow, steady flow rate of 5 or 10 L/min is used with an Infusion Kit consisting of a fused silica capillary tubing with appropriate fittings.,参考溶液（,Reference Solutions,）,参考溶液用于在一个宽,mass,范围内产生多离子。,质谱仪可以在此范围内被校正。,常用的参考溶液,:,PEG, PPG,NaI/CsI,NaI/RbI,Horse,Myoglobin, Poly,Alanine,在此章节的最后有一些常用的参考溶液的配制方法。,参考文件,Reference File,参考文件中包括参考溶液产生的质谱峰的信息，,Masslynx,软件将在校正过程中对其进行搜索并匹配之。,该文件既有,mass,（,m/z,）信息，也有这些峰预期的强度。强度信息在校正,LC/MS,系统时，通常是不用的。,参考文件置于,Masslynx,软件的,Ref,文件夹下。,参比文件都是,text,文本文件，可以用,Notepad,写字板浏览之。,参比文件必须具备一个“,.ref”,的扩展名。,MS,校正的方式,从调谐页面进入，选择校正仪器校正,使用自动调谐中的校正功能,对于,SQD,、,TQD,，可以使用,IntelliStart, Instrument Setup,功能进行仪器校正,校正仪器,Calibrate Instrument,校正窗口,校正的参考文件,Examples of,Calibration,Reference,Files -,Myo.ref,Myo1500.ref,Myo1500n.ref,Myoneg.ref,Myotrp.ref,Pigb.ref,Naics.ref,Naics1.ref,Naics2.ref,Naics3.ref,Naics4.ref,Naineg.ref,Nairb.ref,Pegh.ref,Pegh1000.ref,Pegh2000.ref,peghna.ref,peghnam.ref,peghnh4.ref,ppghna.ref,ppghnh4.ref,Horse,Myoglobin,and Pig,NaI with,Cs or Rb,PEG or PPG,Calibration Reference Files Are Stored in: C:MASSLYNXREF,Note:,Na,+,= 22.9898,Cs,+,= 132.9054,I,-,= 126.9045,Na(NaI,),+,= 173,Na(NaI),2+,= 322,Na(NaI),3+,= 472,校正的参考文件,NaI,参考溶液的特性,NaI,基质的溶液有,Na,、,I,的优势，并有单一同位素。系统不必区分来自于不同同位素的各组峰。,solutions have the advantage of sodium and iodine being,monoisotopic,. There are no sets of multiple peaks from different isotopes that the system has to differentiate between.,使用,NaI/RbI,溶液校正低于,85,amu,，使用,NaI/CsI,溶液校正低于,133,amu,时，存在问题。,There may be a problem with calibrating below 85,amu,with,NaI/RbI,and 133,amu,with,NaI/CsI,.,完成了校正，在将所有的,NaI,清除出系统之前，可以看到,ESP-,的灵敏度降低，同时,ESP+,可以看到一系列,Na,的加合物,You may see a decrease of sensitivity in negative ESP and increased formation of sodium adducts in positive ESP right after you calibrate till all of the,NaI,washes out of your system.,NaI,溶液生成的离子可以高达,4000,amu,。,Magnified by 450X,Magnified by 5X,NaI,RbI,质谱图,PEG/PPG,参考溶液的特性,PEG/PPG,基质的溶液形成来源于不同同位素,(,主要是,C),的多个峰。,这些同位素峰组可以检查质谱仪的分辨率。,PEG/PPG,溶液非常容易电离。,PEG (,或者,PPG),的混合物可以在一个宽质量数范围内产生离子,(,高达,2500),。,PEG/PPG,溶液产生的碎片可以用于形成离子低至,50,amu,之下。,PEG/PPG,可能变得很“粘稠”，清洗出系统可能需要一些时间。,在确定的条件下，,PEG/PPG,溶液可以产生一系列的多电荷离子，也可以产生一系列的单电荷离子。 这些系列可以交叠并导致在一定质量数范围内的校正困难。,PEG 1000,质谱图实例,单电荷的铵加合物,双电荷的铵加合物,PEG 1000,和醋酸铵,马心肌红蛋白（,Myoglobin/PigB,）参考溶液的特性,当分析蛋白和多肽时，质谱仪常常测定多电荷离子的,m/z,.,对于一些化合物，电荷状态可以轻易地多达,50,。,当校正分析多电荷离子的四极杆系统时，校正通常是在能产生多电荷离子的校正溶液基础上进行的。,When calibrating,quadrupole,systems that are analyzing multiply charged species, the calibration is often performed on a reference solution that produces multiply charged ions.,这类应用中的参考溶液的代表是马心肌红蛋白以及猪和,/,或牛血清蛋白。,17,15,14,13,12,11,10,9,1885,18,16,19,20,21,22,23,24,多电荷化合物的质谱图,Mass,校正概述,选择并准备参考溶液（,reference solution,）。,设置针,/,泵（,syringe/pump,）与正确的源。,清除现有的任何软件校正。,对质谱仪进行调谐，找到参考溶液最佳灵敏度的参数。,检查,Mass,校正中用到的参数。,开始,mass,校正步骤。,必要时，查看并编辑校正。,The Calibration ,Uncal.cal, should be a blank calibration (no software calibration) which you can load in (use “File/Open” menu item).,未校正的参考文件,Uncalibrated,Reference File,清除当前的校正,选择一个参考文件,质量分析器的设置,质谱仪的调谐,设置峰显示的参数,从校正范围内选择四个代表性的峰并输入到,Mass,一栏中。确认选择了计划校正范围的顶端和底端的质量数。,Make sure you check a mass near the top and bottom of the mass range over which you plan to calibrate.,对于每一个峰，设置范围（,Span,）大约为,5,。,确认选择了质谱上信号最弱的峰,峰优化,针对四个峰，尽可能地优化毛细管和锥孔电压。,记住针对一个峰的绝对优化条件也许会完全消除另一个峰,此处的目标是找出符合四个峰的条件。,同时也要检查参考溶液中强度最大的峰，以此确认系统并未被饱和。,编辑参考文件,Editing the Reference File,如果您不能找到某一个峰，它们可以通过在峰前面添加一个“；”而加以注释。在校正步骤中，将不使用此标示出的峰。,确认保存了,(File, Save),参考文件。 如果标识出了特定的峰，保存尤其重要。,AutoCal,Check Parameters,校正参数,Calibration Parameters,Quad Mass Measure Parameters,开始校正采集,Start Calibration Acquisition,采集参数,Acquisition Parameters,针对静态校正,针对扫描速度补偿,针对,scan,校正,采集参数,Acquisition Parameters,Acquiring for Calibration,After all the parameters (mass range, run duration, etc.) are entered, Click,OK,and you will return to window shown to the right.,This brings you back to the Automatic Calibration window (shown to the right), Click,OK,to begin acquisition,Monitoring the Acquisition,一旦校正采集开始，可以从此窗口监测校正过程。,每当一个校正完成，每一阶段都会显示状态（,pass/fail,） 。,检查校正文件,如果你选择了“打印报告” （,Print Report,）选项，,Masslynx,将在校正完成后打印一份有所有校正的报告。,检查报告，查看校正是怎样进行的，报告包括：,采集的质谱图,Acquired Spectrum,参考峰的质量数,Reference Peak Masses,采集的峰与校正线之间的偏差。,Deviations of the acquired peaks from the calibration line.,采集的谱图,参比峰的质量数,采集的峰与校正线之间的偏差,打印出的校正报告的实例,Acquisition of Spectra for Calibration,针对每一个校正类型，质谱图都采集并存放在一个后缀是,.raw,的文件中。,质谱文件名,静态,Static:,Static MS1 = StatMS1.raw,Static MS2 = StatMS2.raw,扫描,Scanning:,Scanning MS1 = ScnMS1.raw,Scanning MS2 = ScnMS2.raw,扫描速度补偿,Scan Speed Compensation:,Scan Speed Compensation MS1 = FastMS1.raw,Scan Speed Compensation MS2 = FastMS2.raw,Scanning,Calibration,Scan Speed,Compensation,校正中采集的质谱图实例,对于静态校正，质谱图只采集每一个峰附近较小的质量范围。,对于,652,峰，质谱图只测定,648,至,657,Da,的范围,From a Static Calibration,校正质谱图,质量范围,分辨率 离子能量,慢和快的扫描速度,检查校正,回顾校正报告,回顾校正报告,查看曲线,:,选择校正类型,(Static, Scanning, Scan Speed Compensation),以及四极杆,(MS1 or MS2),2),可以用,Browse In,浏览选择相应的文件,:,3),点击 ,OK,随后出现校正报告。,回顾校正文件,编辑校正,有时选择峰会出错，如此例所示,.,鼠标右键点击并托拽到感兴趣的峰上，可以编辑并手工选择正确的峰。,编辑校正,选择了错误的峰,错误的峰取消选择,选择正确的峰,保存校正,校验校正,校正结果也可以被校验。,With the calibration that is to be verified in place on the mass spectrometer, use the same setup as that used for the calibration.,注意应该使用同样的分辨率和离子能量。,从采集对话框中，选择,Acquire & Verify,选项，并点击 ,OK,。,Verifying a Calibration,Spectra will be acquired and peak masses compared against reference file masses just like in the calibration procedure.,The mass differences will be reported to see how good the current calibration is. The spectra acquired for the verification process will be stored using slightly different filenames.,Static:,Static MS1 = StatMS1V.raw,Static MS2 = StatMS2V.raw,Scanning:,Scanning MS1 = ScnMS1V.raw,Scanning MS2 = ScnMS2V.raw,Scan Speed Compensation:,Scan Speed Compensation MS1 = FastMS1V.raw,Scan Speed Compensation MS2 = FastMS2V.raw,查看校正验证,Reviewing a Calibration Verification,需要时可以查看每种校正的验证，从,If,it is necessary to review the curves for each type of verification, then from the window menu, click on Process, Verification From File and browse in the appropriate file. (Similar to reviewing a calibration as shown in Step 16b.),Verification Report,A The top graph shows the peaks found in the infused reference solution.,The middle graph shows the Reference File peaks.,The lower graph shows the difference between the mass found (acquired using the calibrated system) and the mass in the Reference File. The mass differences should be less than 0.1,amu,.,保存校正质谱图,Saving Calibration Spectra,The calibration spectrum data files are overwritten every time a calibration is performed. Save or move the calibration data folders off into another folder for later use if needed. For example, you can load ,Uncal, and practice changing the mass measure parameters. Then choose the Recalibrate From File option and select one of your saved files.,AutoTune Wizard Calibration,AutoTune Wizard Calibration,AutoTune Wizard Calibration,AutoTune Wizard Calibration,AutoTune Wizard Calibration,AutoTune Wizard Calibration,AutoTune Wizard Calibration,Calibrate Check,PEG 1000 with Ammonium Acetate,Sodium Iodide Solution for Positive Ion,Electrospray,Prepare a solution of sodium iodide at a concentration of:,2 g/L (micrograms/microliter) (2mg/mL) in 50:50 propran2ol (IPA)/water with no additional acid or buffer.,Add cesium iodide to a concentration of 0.05g/L (micrograms/microliter) (0.05mg/mL).,Cesium iodide is to obtain a peak at,m/z,133 (Cs,+,).,Without this, there is a gap in the calibration file of 150amu from,m/z,23 (Na,+,) and the first cluster at,m/z,173, which leads to poor mass calibration in this mass range.,Do not add more CsI than suggested, as it can result in a more complex spectrum due to the formation of NaCsI clusters.,Use reference file: NAICS.REF,Sodium Iodide Solution for Positive Ion,Electrospray,(Alternative),Prepare a solution of sodium iodide, as described previously, but instead of adding cesium iodide, use rubidium iodide at a concentration of 0.05g/L (micrograms/microliter) (0.05mg/mL).,This gives a reference peak at,m/z,85 (85Rb) with an intensity of approximately 10% of the base peak at,m/z,173.,The advantage of Rubidium Iodide is that rubidium clusters are not formed, which can complicate the spectrum.,Use reference file: NAIRB.REF,Calibration Solutions,PEG solution for positive ion electrospray and APCI,Prepare a solution of the following polyethylene glycols in 50:50 acetonitrile/0.002 M ammonium acetate in water (50:50 ACN/2 mM NH4Acetate in Water).,PEG 200 at 25ng/L, PEG 400 at 50ng/L,PEG 600 at 75ng/L,PEG 1000 at 250ng/L,This produces a series of PEG+ ammonium ions allowing calibration up to approximately 1200amu.,To calibrate further up the mass scale, add PEG1500 or preferably use a sodium iodide solution.,Use reference file: PEGNH4.REF,Calibration Solutions,Horse heart myoglobin for positive ion electrospray,For use when measuring large multiply charged proteins, prepare a 5M solution of horse heart myoglobin in 0.1% formic acid in 50/50 water/acetonitrile.,Use reference file: MYO.REF,
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