Turning HTE Agilent LC/MS Data to Knowledge in 1hr

Transcription

1 With permission Reaction Analytics Inc Page 1 Turning HTE Agilent LC/MS Data to Knowledge in 1hr Neal Sach

2 With permission Reaction Analytics Inc Page 2 Contents Acquiring Data Fast demo method (0.8min) Batch Analysis peak labeling mass extraction Exporting Data Collating Data (ichemexplorer) Visualizing Data (Spotfire)

3 With permission Reaction Analytics Inc Page 3 Acquiring Data Fast 1 Agilent Online Chemstation > Sequence > Sequence Table > Insert/Fill Down Wizard Button Wizard Selections Select Append Location P1-A-01 to P1-H-12 (full plate) or P1-A-01 to P1-D-12 (half plate) Browse Method (length and mass range vs. products, reagents (i.e. DIPEA 130MW and potential sideproduct (i.e. SM-halide). Sample name (match CeN) with conditions hrs-100C Injection/Location 1 Press OK Sequence automatically filled (Figure 2) Press OK Link to 0.8min Method

4 With permission Reaction Analytics Inc Page 4 Acquiring Data Fast 2 Agilent Online Chemstation > Sequence > Sequence Parameters Set output sub-directory to CeN experiment number (Figure 1) Press OK Agilent Online Chemstation > Sequence > Save Sequence As Save as CeN experiment number These two things make tracking your data down later much easier

5 With permission Reaction Analytics Inc Page 5 Acquiring Data Fast 3 Turn everything (lamps, MS, DAD) ON with small ON button Press START Should start and green ready bar turn to blue running You can walk away now 96 reactions = approx. 2hrs

6 With permission Reaction Analytics Inc Page 6 Batch Analysis Finding and Agilent Offline Chemstation > Batch > Load Batch Load batch file (in directory specified on slide 4) Select only the required samples, no blanks or replicates (press Select All and then un-select others) Press Other Method Load 254.M (starting point wavelength method, this may change later) Loading Data

7 With permission Reaction Analytics Inc Page 7 Browsing your data Examine peaks by selecting Spectrum and click on peak in UV spectra to see DAD spectra or MS spectra to see MS data UV Spectra MS Spectra Use Batch Analysis up/down arrows to peruse data *DAD1, (2000 mau, - ) Ref=0.279 & of 2EB-0201.D mau * M S D 1 S P C, t im e = o f C : \ C H E M 3 2 \ 1 \ D A T A \ \ M a x : nm m / z

8 With permission Reaction Analytics Inc Page 8 Choose a Wavelength Using the Batch Analysis up/down arrows and Spectra buttons try and find a reaction with both your SM and Product. Use the UV DAD spectra to choose an appropriate wavelength in which both SM and P absorb well and are in correlation of MS peaks (assuming ionizable centre of molecule is not greatly changed). What would be an appropriate wavelength for this reaction mixture? mau SM *DAD1, (2000 mau, - ) Ref=0.279 & of 2EB-0201.D mau Product *DAD1, (282 mau, - ) Ref=0.380 & of 2EB-0201.D nm nm

9 With permission Reaction Analytics Inc Page 9 Choose a Wavelength 2 Keep in mind interfering factors and if you have the option to do so, avoid them. Toluene UV Spectra *DAD1, (1715 mau, - ) Ref=0.336 & of 2EB-0201.D mau DMF UV Spectra *DAD1, (1813 mau, - ) Ref=0.000 & of 2EG-0701.D mau nm nm For Toluene 225nm is a minima or go above 280nm For DMF go above 240nm

10 With permission Reaction Analytics Inc Page 10 Choose a Wavelength 3 There are a set of preprepared methods at these wavelengths 210nm 225nm 240nm 254nm 265nm 280nm 310nm 330nm Link to Methods Agilent Offline Chemstation > File > Load > Method Select appropriate method where the name corresponds to the wavelength i.e M = 225nm wavelength Use up/down arrows to refresh screen and have new wavelength take effect

11 With permission Reaction Analytics Inc Page 11 To simplify analysis we now need to label peaks of interest Agilent Offline Chemstation > Calibration > Calibration Table Press add peak button Press OK (not relevant) Select peak on UV spectra Enter peak name using nomenclature Name MWXX+ve i.e. SM MW158+ve Add all peaks by perusing data using Batch Analysis up/down arrows Labeling Peaks Important : No peaks should have the same name.

12 With permission Reaction Analytics Inc Page 12 Labeling Peaks 2 You should label all peaks of interest, even ones that you may want to remove from %Area calculations, such as ligands and Toluene In some cases you may wish to extract a particular mass ion, for a SM or product. The advantage of doing so is that a plot of % Area of Product vs. Mass Ion Count of Product removes false hits. Examples include -: Co-eluting UV peaks that cause over-representation of product Degraded reaction mixtures that suggest high % Areas of product but absolute yields are low

13 With permission Reaction Analytics Inc Page 13 Extracting Mass Ions 1 Exit calibration table. Press OK and select No do dialogue box (not relevant) Agilent Offline Chemstation > File > Extract Ions Enter ion range Press OK

14 With permission Reaction Analytics Inc Page 14 Extracting Mass Ions 2 A third spectrum will appear representing your mass ion. As on slide 11, add new peak Agilent Offline Chemstation > Calibration > Calibration Table Press add peak button Scroll down to new extracted ion MS spectra Select peak The peak will automatically be labeled with the UV name previously assigned Note : You can extract as many ion channels as you want and label peaks in the same way

15 With permission Reaction Analytics Inc Page 15 Save Method and Specify Report Analysis is Finished Save Method Agilent Offline Chemstation > File > Save As > Method Use nomenclature CeNNo- Wavelength i.e Agilent Offline Chemstation > File > Save Method As Use nomenclature CeNNo- Wavelength i.e M.B We need to specify output file names that ichemexplorer will read in to collate data Agilent Offline Chemstation > Report > Specify Report Enter File Prefix Use nomenclature CeNNoWavelength i.e M

16 With permission Reaction Analytics Inc Page 16 Save Batch and Run the Analysis FYI the specified reports are what ichemexplorer uses..csv is an excel file with tabulated %Area and Mass Ion Count Data..EMF is a picture of the LC/MS trace Save Batch Agilent Offline Chemstation > Batch > Save Batch As Use nomenclature CeNNo- LetterPrefix i.e. 825A.B Run Batch Analysis Press Start System will now analyze all your data using your new method

17 With permission Reaction Analytics Inc Page 17 ichemexplorer Loading Data Load ichemexplorer Press Load HPLC Data Browse Data C > Chem32 > 1 > Data > CeNno. > anyfolder > CeNNoWavelength01.csv Link to Data Select No

18 With permission Reaction Analytics Inc Page 18 Remove Undesired Peaks ichemexplorer > Edit > Peak Name Tick peaks to be hidden from %Area calculations Tick Re-calculate %Area on Hide Select Apply

19 With permission Reaction Analytics Inc Page 19 Export Collated Report (UV only) For just UV Data (no MS extraction ion channels) Press Save Collated Report Browse Project Folder and Save

20 With permission Reaction Analytics Inc Page 20 Export Collated Report (MS only) For just data with MS extraction ion channels ichemexplorer > Development > Export Multiple Report Select Channels 00 (method details, do not select) 01 (UV channel, do not select as exported by default) 02 (total MS channel, do not select) 03 (extracted mass ion channel 1, select) 04 (extracted mass ion channel 2, select) etc. Press Done. Browse Project Folder and Save

21 With permission Reaction Analytics Inc Page 21 Example Collated Report Link to Collated Data Report Report contains multiple tabs %Area UV Data with all peaks labeled Mass Ion Count Data for each mass extraction channel Bar-charts of UV profile for each reaction Pictures of LC/MS chromatograms for all reactions

22 With permission Reaction Analytics Inc Page 22 Final Steps Collating Design and Result Excel Spreadsheets Open both Design and collated Results excel spreadsheets Goto design spreadsheet. Select tabs by holding shift key and left clicking Right click. Select Move or Copy Select Create a copy Press Down Arrow Select Collated Results data Press OK

23 With permission Reaction Analytics Inc Page 23 Final Steps Collating Design and Result Excel Spreadsheets Single spreadsheet now contains Design and Collated Results Data Select Collated Data (Area%) Tab Select irrelevant columns Delete irrelevant columns Copy columns

24 With permission Reaction Analytics Inc Page 24 Final Steps 2 Collating Design and Result Excel Spreadsheets Select Reaction_Information Tab Paste data adjacent to final column (Right click, Paste Special, Formulas)

25 With permission Reaction Analytics Inc Page 25 Final Steps Collating Design and Result Excel Spreadsheets (MS only) For just data with MS extraction ion channels Select CSV Tab (not %Area, we want absolute ion count) Select relevant column Copy columns Paste onto final column in Reaction_Information Tab as before Select entire spreadsheet Copy

26 With permission Reaction Analytics Inc Page 26 Final Steps Visualizing Data Open Spotfire Spotfire > File > Import > Import Data Select Clipboard Press Finish Press Finish