waters公司的uplctqd培訓(xùn)資料 ms_7_校正

1、四極桿系統(tǒng)的校正校正 硬件 vs 軟件硬件的校正是由原廠和維修工程師進(jìn)行的，可以保證名義質(zhì)量數(shù)的準(zhǔn)度。對(duì)于某些應(yīng)用，硬件校正是足夠的。對(duì)于需要最高的質(zhì)量準(zhǔn)確度的情況，用戶可以通過Masslynx 軟件進(jìn)行軟件校正，并將系統(tǒng)的質(zhì)量校正變得更為精確。在識(shí)別未知物以及MRM 和 SIR 分析時(shí)，確認(rèn)選擇了準(zhǔn)確的質(zhì)量數(shù)，這是非常有用的。校正中的可變因素環(huán)境的改變溫度或者濕度的改變 隨著時(shí)間變化而發(fā)生的電源變化 改變了分辨率設(shè)置 在儀器的校正范圍內(nèi)工作是非常重要的!如同您不愿意在定量曲線之外進(jìn)行定量計(jì)算一樣，您也不希望在您的質(zhì)量數(shù)校正曲線之外工作。儀器的校正曲線（Calibration Curves）一

2、臺(tái)三重四極桿的儀器需要有三種校正曲線：對(duì)于MS1和 MS2, 有六條校正曲線靜態(tài)校正（Static calibration）可以使四極桿質(zhì)量分析器準(zhǔn)確地定位于感興趣的特定質(zhì)量數(shù)上。 (例如 調(diào)諧，SIR和MRM).掃描校正（Scanning calibration） 保證在掃描采集中峰采集的質(zhì)量數(shù)準(zhǔn)確性。 (例如 全掃描 Full Scan). 掃描速度補(bǔ)償（Scan speed compensation calibration）：當(dāng)儀器快速掃描是，補(bǔ)償系統(tǒng)中的“滯后時(shí)間” 。質(zhì)譜儀的每一種校正都需要參考溶液。不過，MassLynx 可以在一個(gè)步驟中執(zhí)行所有必須的校正。校正步驟每個(gè)參考溶液的質(zhì)

3、譜圖都是已經(jīng)采集好的。采集過的質(zhì)譜圖中的峰與儲(chǔ)存于參考文件表格中的預(yù)期理論質(zhì)量數(shù)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. 參考文件中的每一個(gè)峰與采集的質(zhì)譜圖中對(duì)應(yīng)的峰相匹配。Each peak in the Reference File is matched to

4、 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/

5、min is used with an Infusion Kit consisting of a fused silica capillary tubing with appropriate fittings.參考溶液（Reference Solutions）參考溶液用于在一個(gè)寬mass 范圍內(nèi)產(chǎn)生多離子。質(zhì)譜儀可以在此范圍內(nèi)被校正。 常用的參考溶液:PEG, PPGNaI/CsI, NaI/RbIHorse Myoglobin, Poly Alanine在此章節(jié)的最后有一些常用的參考溶液的配制方法。參考文件Reference File參考文件中包括參考溶液產(chǎn)生的質(zhì)譜峰的信息，Masslynx

6、軟件將在校正過程中對(duì)其進(jìn)行搜索并匹配之。該文件既有mass（ m/z ）信息，也有這些峰預(yù)期的強(qiáng)度。強(qiáng)度信息在校正 LC/MS 系統(tǒng)時(shí)，通常是不用的。參考文件置于Masslynx軟件的Ref 文件夾下。參比文件都是 text 文本文件，可以用Notepad 寫字板瀏覽之。參比文件必須具備一個(gè)“.ref” 的擴(kuò)展名。MS 校正的方式從調(diào)諧頁面進(jìn)入，選擇校正儀器校正使用自動(dòng)調(diào)諧中的校正功能對(duì)于SQD、TQD，可以使用IntelliStart Instrument Setup功能進(jìn)行儀器校正校正儀器Calibrate Instrument校正窗口 校正的參考文件Examples ofCalibrat

7、ion Reference Files -Myo.refMyo1500.refMyo1500n.refMyoneg.refMyotrp.refPigb.refNaics.refNaics1.refNaics2.refNaics3.refNaics4.refNaineg.refNairb.refPegh.refPegh1000.refPegh2000.refpeghna.refpeghnam.refpeghnh4.refppghna.refppghnh4.refHorseMyoglobinand PigNaI withCs or RbPEG or PPGCalibration Reference

8、 Files Are Stored in: C:MASSLYNXREFNote:Na+ = 22.9898Cs+ = 132.9054 I- = 126.9045Na(NaI)+ = 173Na(NaI)2+= 322Na(NaI)3+= 472校正的參考文件NaI 參考溶液的特性 NaI 基質(zhì)的溶液有Na、I的優(yōu)勢(shì)，并有單一同位素。系統(tǒng)不必區(qū)分來自于不同同位素的各組峰。 solutions have the advantage of sodium and iodine being monoisotopic. There are no sets of multiple peaks from d

9、ifferent 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時(shí)，存在問題。There may be a problem with calibrating below 85 amu with NaI/RbI and 133 amu with NaI/CsI. 完成了校正，在將所有的NaI 清除出系統(tǒng)之前，可以看到ESP- 的靈敏度降低，同時(shí)ESP+ 可以看到一系列Na 的加合物You may see a decrease of sensi

10、tivity 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 溶液生成的離子可以高達(dá) 4000 amu。Magnified by 450XMagnified by 5XNaI RbI 質(zhì)譜圖PEG/PPG 參考溶液的特性PEG/PPG 基質(zhì)的溶液形成來源于不同同位素(主要是C)的多個(gè)峰。 這些同位素峰組可以檢查質(zhì)譜儀的分辨率。 PEG/PPG

11、溶液非常容易電離。 PEG (或者 PPG) 的混合物可以在一個(gè)寬質(zhì)量數(shù)范圍內(nèi)產(chǎn)生離子 (高達(dá) 2500)。PEG/PPG 溶液產(chǎn)生的碎片可以用于形成離子低至50 amu 之下。 PEG/PPG 可能變得很“粘稠”，清洗出系統(tǒng)可能需要一些時(shí)間。 在確定的條件下，PEG/PPG 溶液可以產(chǎn)生一系列的多電荷離子，也可以產(chǎn)生一系列的單電荷離子。 這些系列可以交疊并導(dǎo)致在一定質(zhì)量數(shù)范圍內(nèi)的校正困難。PEG 1000 質(zhì)譜圖實(shí)例單電荷的銨加合物雙電荷的銨加合物PEG 1000 和醋酸銨馬心肌紅蛋白（Myoglobin/PigB）參考溶液的特性當(dāng)分析蛋白和多肽時(shí)，質(zhì)譜儀常常測(cè)定多電荷離子的 m/z .對(duì)于

12、一些化合物，電荷狀態(tài)可以輕易地多達(dá)50。當(dāng)校正分析多電荷離子的四極桿系統(tǒng)時(shí)，校正通常是在能產(chǎn)生多電荷離子的校正溶液基礎(chǔ)上進(jìn)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.這類應(yīng)用中的參考溶液的代表是馬心肌紅蛋白以及豬和/或牛血清蛋白。1715141312111091885181619202

13、1222324多電荷化合物的質(zhì)譜圖Mass 校正概述選擇并準(zhǔn)備參考溶液（reference solution）。設(shè)置針/泵（syringe/pump）與正確的源。清除現(xiàn)有的任何軟件校正。對(duì)質(zhì)譜儀進(jìn)行調(diào)諧，找到參考溶液最佳靈敏度的參數(shù)。檢查Mass校正中用到的參數(shù)。開始mass 校正步驟。必要時(shí)，查看并編輯校正。The Calibration Uncal.cal should be a blank calibration (no software calibration) which you can load in (use “File/Open” menu item).未校正的參考文件Unca

14、librated Reference File清除當(dāng)前的校正 選擇一個(gè)參考文件 質(zhì)量分析器的設(shè)置質(zhì)譜儀的調(diào)諧設(shè)置峰顯示的參數(shù)從校正范圍內(nèi)選擇四個(gè)代表性的峰并輸入到Mass 一欄中。確認(rèn)選擇了計(jì)劃校正范圍的頂端和底端的質(zhì)量數(shù)。Make sure you check a mass near the top and bottom of the mass range over which you plan to calibrate. 對(duì)于每一個(gè)峰，設(shè)置范圍（Span）大約為5。確認(rèn)選擇了質(zhì)譜上信號(hào)最弱的峰峰優(yōu)化針對(duì)四個(gè)峰，盡可能地優(yōu)化毛細(xì)管和錐孔電壓。記住針對(duì)一個(gè)峰的絕對(duì)優(yōu)化條件也許會(huì)完全消除另一個(gè)峰

15、此處的目標(biāo)是找出符合四個(gè)峰的條件。同時(shí)也要檢查參考溶液中強(qiáng)度最大的峰，以此確認(rèn)系統(tǒng)并未被飽和。編輯參考文件Editing the Reference File如果您不能找到某一個(gè)峰，它們可以通過在峰前面添加一個(gè)“；”而加以注釋。在校正步驟中，將不使用此標(biāo)示出的峰。確認(rèn)保存了(File, Save)參考文件。 如果標(biāo)識(shí)出了特定的峰，保存尤其重要。AutoCal Check Parameters校正參數(shù)Calibration ParametersQuad Mass Measure Parameters開始校正采集Start Calibration Acquisition采集參數(shù)Acquisitio

16、n Parameters針對(duì)靜態(tài)校正針對(duì)掃描速度補(bǔ)償針對(duì)scan校正采集參數(shù)Acquisition ParametersAcquiring for CalibrationAfter 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 O

17、K to begin acquisitionMonitoring the Acquisition一旦校正采集開始，可以從此窗口監(jiān)測(cè)校正過程。每當(dāng)一個(gè)校正完成，每一階段都會(huì)顯示狀態(tài)（pass/fail ） 。檢查校正文件如果你選擇了“打印報(bào)告” （Print Report）選項(xiàng)，Masslynx將在校正完成后打印一份有所有校正的報(bào)告。檢查報(bào)告，查看校正是怎樣進(jìn)行的，報(bào)告包括：采集的質(zhì)譜圖Acquired Spectrum參考峰的質(zhì)量數(shù)Reference Peak Masses采集的峰與校正線之間的偏差。Deviations of the acquired peaks from the calib

18、ration line.采集的譜圖參比峰的質(zhì)量數(shù)采集的峰與校正線之間的偏差打印出的校正報(bào)告的實(shí)例Acquisition of Spectra for Calibration針對(duì)每一個(gè)校正類型，質(zhì)譜圖都采集并存放在一個(gè)后綴是.raw 的文件中。 質(zhì)譜文件名 靜態(tài) Static:Static MS1 = StatMS1.rawStatic MS2 = StatMS2.raw掃描 Scanning:Scanning MS1 = ScnMS1.rawScanning MS2 = ScnMS2.raw掃描速度補(bǔ)償 Scan Speed Compensation:Scan Speed Compensati

19、on MS1 = FastMS1.rawScan Speed Compensation MS2 = FastMS2.rawScanningCalibrationScan SpeedCompensation校正中采集的質(zhì)譜圖實(shí)例 對(duì)于靜態(tài)校正，質(zhì)譜圖只采集每一個(gè)峰附近較小的質(zhì)量范圍。對(duì)于 652 峰，質(zhì)譜圖只測(cè)定 648至657 Da 的范圍From a Static Calibration校正質(zhì)譜圖質(zhì)量范圍 分辨率 離子能量慢和快的掃描速度檢查校正回顧校正報(bào)告 回顧校正報(bào)告查看曲線:選擇校正類型 (Static, Scanning, Scan Speed Compensation) 以及四極桿

20、 (MS1 or MS2)2) 可以用Browse In 瀏覽選擇相應(yīng)的文件:3) 點(diǎn)擊 OK 隨后出現(xiàn)校正報(bào)告?；仡櫺Ｕ募?編輯校正有時(shí)選擇峰會(huì)出錯(cuò)，如此例所示.鼠標(biāo)右鍵點(diǎn)擊并托拽到感興趣的峰上，可以編輯并手工選擇正確的峰。編輯校正選擇了錯(cuò)誤的峰錯(cuò)誤的峰取消選擇選擇正確的峰保存校正校驗(yàn)校正校正結(jié)果也可以被校驗(yàn)。With the calibration that is to be verified in place on the mass spectrometer, use the same setup as that used for the calibration. 注意應(yīng)該使用同樣的分

21、辨率和離子能量。 從采集對(duì)話框中，選擇 Acquire & Verify 選項(xiàng)，并點(diǎn)擊 OK。Verifying a CalibrationSpectra 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

22、 verification process will be stored using slightly different filenames.Static:Static MS1 = StatMS1V.rawStatic MS2 = StatMS2V.rawScanning:Scanning MS1 = ScnMS1V.rawScanning MS2 = ScnMS2V.rawScan Speed Compensation:Scan Speed Compensation MS1 = FastMS1V.rawScan Speed Compensation MS2 = FastMS2V.raw查看

23、校正驗(yàn)證Reviewing a Calibration Verification需要時(shí)可以查看每種校正的驗(yàn)證，從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.)Verificatio

24、n ReportA 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 t

25、han 0.1 amu.保存校正質(zhì)譜圖Saving Calibration SpectraThe 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 pa

26、rameters. Then choose the Recalibrate From File option and select one of your saved files.AutoTune Wizard CalibrationAutoTune Wizard CalibrationAutoTune Wizard CalibrationAutoTune Wizard CalibrationAutoTune Wizard CalibrationAutoTune Wizard CalibrationAutoTune Wizard CalibrationCalibrate CheckPEG 10

27、00 with Ammonium AcetateSodium Iodide Solution for Positive Ion ElectrosprayPrepare 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/

28、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/z23 (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

29、 a more complex spectrum due to the formation of NaCsI clusters.Use reference file: NAICS.REFSodium 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/z85 (85