BioToF –USERMANUAL ver. 1 (Feb 2010) The BioToF machine can
Contents
1. 657 3668 657 6685 657 3676 1 243088 PEG_15_Na 701 3930 701 7147 701 3944 1 919761 PEG_16_Na 745 4192 745 7592 745 4195 0 330706 PEG_17_Na 7894454 789 8025 789 4433 es 101 613 6219 5757 657 6685 701 7147 437 4406 393 3932 349 3462 a 62 4948 384 i 076 g le Bistert Yshtmp Poit FieMaker ro 1 modifed Erma RO IID sm Prepared by Angel Ugrinov NDSU Fargo ND Page 72. BioloF USERMANUAL ver 1 Feb 2010 Electro Spray lonization Mass Spectrometry ESI MS Practice Training by Angel Ugrinov The BioloF machine can be used only by users who have already passed the training and have their own account to log in Call 701 231 5256 or e mail Angel Angel Ugrinov ndsu edu to schedule a practice training This short step by step illustrated user manual is made to help the new users to perform their experiment without additional help but only after they have passed the training More detailed user manual provided by Bruker Daltonics is located placed on the desk next to the computer in Dunbar 160 do not remove It from their Sample Preparation Polar solvents with high volatility are preferable The best solvents are CHOH HPLC CH3CN HPLC and Water HPLC ESI MS is a very sensitive method and the best concentration of the solution for small molecules is 4ppm 10 ppm for proteins Appendix A Collecting a spectrum 1 Log in with your username the first 3 200 letters of your first name and the first 3 letters of your last name and password and then click the blue run button to get paali to the main navigation screen Optional Type the name of the project sample in Sample ID note book Click the grey Bio TOF button to maximize the Bio TOF application 600 Mode Chromatogram Calibration ESI Source ENE Manual Stop 2 Check the status of the Instr
3. ess UpdateAnalyses How to Calculate the mass error Exp Mass Theor Mass Mass error Exp Mass Exp Mass experimental mass which you observed after internal calibration external only if internal is impossible Theor Mass the mass from the simulated pattern calculations Prepared by Angel Ugrinov NDSU Fargo ND Page 4 Appendix A Parts per million oom works as percent by mass but is more convenient when there is only a small amount of solute present PPM is defined as the mass of the component in solution divided by the total mass of the solution multiplied by 10 one million A solution with a concentration of 1 ppm has 1 gram of substance for every million grams of solution Because the density of water is 1 g per mL and we are adding sucha tiny amount of solute the density of a solution at such a low concentration is approximately 1 g per mL Therefore in general one ppm implies one milligram of solute per liter of solution Finally recognize that one percent 10 000 ppm Therefore something that has a concentration of 300ppm could also be said to have a concentration of 800 ppm 10 000 ppm percent 0 03 percent by mass Density of CHOH 0 791 g mL at 25 C Density of CH3CN 0 786 g mL at 25 C Prepared by Angel Ugrinov NDSU Fargo ND A Da eg 5 Appendix B Internal and External Calibration Internal calibration means that you run your sample and one standard together The inter
4. mini e mm are with Na Recalbiate Spectum Oniy Recalbrate a 5 After clicking OK Fig 6 you will return in to the main Calibration window 437 2357 I Quadratic calibration Search ranges m z 0 5 Edit Lists 6 Click Auto Search button The software is now searching for observed picks of Fig 6 the standard in the spectrum which you i oe ran The picks are listed in the window see aa aix together with the observed errors see Fig 8 All picks with error less than 5 i Coltxaion go ES ppm are good to use If one or more picks Sm rane lt not available gt Angiotensin CID have error bigger than 5 select the one JAPCI APPI Calbrant neg with the biggest error and remove it After that check the errors again Remove as many picks as necessary to make the Na TFA pos rest of the picks with errors less than 5 ri Nae MafemitePo ppm At the end you should have a minimum of 3 picks more is better with errors less than 5 ppm see Fig 8 I Quatatic cal gr T Find Mass List Charae Deconvolution Library Search Exclusion Masses Recalibration Masses Layouts Display Process Export 7 Click Recalibrate button Internal calibration is done Prepared by Angel Ugrinov NDSU Fargo ND E Page 6 a Internal Mass Spectrum Calibration 1 Er 1 0 784246 PEG_12 Na 569 3144 569 5757 569 3137 1 223746 PEG_13_Na 6133406 613 6219 613 3404 0 216180 PEG_14_Na
5. nal calibration is impossible if your compound reacts with all possible standards The external calibration is necessary only if the internal is impossible The internal calibration is much more accurate than the external 1 modified hrms m modified Bruker Daltonics DataAnalysis To make internal calibration aie Ba ASQ 1 In analysis list window click on thE se cst rod masus Decorvolite Identiv process Calbrate Anotato A tera A Method View Tools Windc sample which you want to calibrate to 4455 s as ott ee Bem Ay a select it PESO Free aot conmentFre farmat conmenisFres Y Parameters Mass Spectrum 1 M5 _ Analysis List HY ilk 1 1 2 Click Calibrate Internal see Fig 5 3 Click Select list button to select your Step 1 Calibration list The default list is for Select the sample positive spectra of any PEG standard Fig 5 therefore if you are using PEG you should skip step 3 5 The list of PEG ionize with Na 4 On the new window click the drop menu called Calibration List see Fig 6 and 2s D 7 and select the desired standard for 45nn ay o Find Mass List Charge Deconvolution Library Search Pravou CD fa02 567s ci fizsoris c2 Exclusion Masses Recabbration Masses Layouts Display Process Expat example PPG with Na all our standards Me a PEES c NAS a nin eT Quackatc calibration Search range m 2 0 5 Zoom Ci
6. seat the needle inside but when you feel a lot of resistance stop pushing dbu Oneration _ ae eS Lo Sich ia Hold down the Forward Arrow button and the Run Stop button on the syringe pump panel simultaneously see the picture above this makes the pump inject at a fast rate to clear the injection tubing and fill it with your sample Once you see the Spray current to stabilize you can let go off the two buttons of the syringe pump and press once the Run Stop You should see a flashing arrow on the syringe pump LCD screen In the Bio TOF software press Start to start acquiring a spectrum Page 2 Once you have collected at least 500 scans of your solvent this is your background click Save to save the background to disk Make sure that the data Path is set to C XMASS In the Sample Name field you may enter your name the name of the sample or any name which is convenient for you The Experiment No field will automatically increment to create a unique ID for every spectrum you save with a particular Sample Name You should always insert the characters d into the Experiment ID field to make it easier to open the files in the DataAnalysis Software Finally the Comment fields allow you to enter detailed information about the spectrum you are saving Repeat steps 4 8 with the sample and the sample mixed with internal standard Optional You can run a spectrum of the standard I
7. t can be used as an external standard The internal standard is better than external but some samples are very reactive and cannot be mixed with standards The Bruker Bio TOF is capable of extremely high mass accuracy lt 5 ppm mass error but peak positions must be calibrated against a set of known mass standards in order to achieve this resolution In this experiment you will use PEG or PPG PEG stays for polyethylene glycol H O CH2 CHz2 OH PPG stays for polypropylene ip it te Issel Sissi glycol H O CHMe CHz2 OH PEG 600 in MeOH The contain polymer molecules with range of well defined molecular masses and set of multiple peaks is observed w One example of PEG600 is shown on Fig 3 Based on your needs you will use glycols with different masses such as a PEG PPG samples PEG200 PEG400 PEG600 e 3 PPG 725 PPG1000 etc 10 After collecting all necessary spectra minimize Bio Tof application and click the gray DataAnalysis button to maximize this application Choose File Open to navigate to your folder within the CAXMASS Select the file you want to open and click the Open button 11 Once the file is open you can see the labeled peaks and the mass list For the sample by itself you can simply print the spectrum and make note of the masses observed 12 For the spectra where you mixed your sample with PEG PPG you will need Prepared by Angel Ugrinov NDSU Fargo ND to calibra
8. te See Appendix B each one to obtain the accurate mass of your sample Page 3 Unknown sample Make a note of the exact mass of your sample peak Open Tools Generate Molecular Formula and use the exact mass measured to calculate a list of possible molecular formulas that match the measured mass to within 10 ppm Remember to use all possible tools to narrow the list of the possibilities Known sample lf you know have assumption for the molecular formula of your sample Open Tools Simulate Pattern and type your formula to simulate the theoretical pattern do not forgot to add H or Na if you have ran neutral molecule which were ionized Use the exact mass of your sample peak and its theoretical pattern and calculate the mass error Mass error lt 5 ppm is publishable and confirms that your assumption for molecular formula is accurate Sp modified hrms m modified Bruker Daltonics DataAnalysis eH Se the Be Q Qa Q y File Edit Find MassList Deconvolute Identify Process Calibrate Annotation Method View Tools Window Help A Did a A a S aR ag A ag ag rar i DP a gt Tr BIF Control Tii Peso Free Format commentsFree format commentsFree format comments H LibraryEditor Mass Spectrum 1 MS El ReportDesigner Automation Analysis List Yi dh 1 Simulate Pattern MF Generate Molecular Formula y g Options Colors 65 0 8 ProcesswithMethod Reproc
9. ument Standby or Operation Make sure Spec Cant that the instrument is set to Standby Fig 1 Ae Fig 1 Instrumentation Mode lonizatic ms fe ESI A nESI f APCI se MS Only Chromatography a M Polarity Data Display Positive C Negative ya m z C Flight Time Gas Heater Control WV Dry Gas Mass Range m z V Neb Gas m z from 76 to V DG Heat 3 ONLY in Standby load the desired method for your experiment Method Load Method Please use only the methods which are started with the word user For most of your samples the most appropriate method will be user 1pass_pos tofpar Fig 2 Prepared by Angel Ugrinov NDSU Fargo ND E Pagel VERY IMPORTANT Do not change Polarity NEVER If you want to observe negative species you should change the method with user 1pass_neg tofpar and the computer will change the polarity automatically ito 810 T0F Last Good Method The methods Can De ees mr mo sas some u changed only on standby mode 4 6 Prepared by Angel Ugrinov NDSU Fargo ND tjer 1 ROL lA Oe ls Load the syringe with the desired solution you better start with pure solvent Reconnect the injection tubing to the syringe needle Do not touch the needle as doing it Push the connector until it feels like the needle is encountering a little resistance it is the Fig 2 Se needle hitting the connector j seat Push just a little harder to
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