2. LC/MS WORKSHOP IOWA STATE UNIVERSITY Kamel Harrata  Instrument Description  Data Acquisition  Data Processing

3. WHAT IS MASS SPECTROMETRY? Mass spectrometry is an analytical technique that involves the study in the gas phase of ionized molecules with the aim of one or more of the following: Structural characterization Gas phase reactivity study Qualitative and quantitative analysis of components in a mixture. Molecular weight determination

4. LC/MS and GC/MS Mass Spectrometers GC/MSLC/MS InletIon SourceAnalyzerDetector m/z Data System Direct Probe Flow Injection GC, LC EI, CI APCI, ESI Q, QQQ EB, Ion Trap TOF, QTOF FT, Orbitrap EM, PM Micromass GCT Agilent QTOF 6540

5. Pharmaceutical analysis Biomolecule characterization Proteins and peptides Oligonucleotides Environmental analysis Pesticides on foods Soil and groundwater contamination Forensic analysis/clinical Applications of Mass Spectrometry

6. TOF EQUATION

7. TIME OF FLIGHT SCAN

8. QTOF 6540 CAPABILITIES  MS and MS/MS  Mass Accuracy of better than 3ppm  Resolution (FWHM) 45,000 for mass 1000  Rapid switching between positive and negative ion modes

9. LC SYSTEM  4 reservoir bottles A1, A2, B1, B2  Binary Pump can handle up to 8000 psi  UV detector  LC Column housing  Autosampler

10. ELECTROSPRAY IONIZATION Desolvation + Coulomb Explosions

11. AGILENT ELECTROSPRAY ION SOURCE (POSITIVE MODE)

12. ELECTROSPRAY SPECTRUM OF MYOGLOBIN

13. RELATIVE APPLICABILITY OF LC/MS TECHNIQUES

14. ADVANTAGES OF ELECTROSPRAY Soft ionization LC/MS interface with best sensitivity Extends mass range for multiply charged analytes Works with a wide range of moderate to high polarity compounds Low maintenance

15. ELECTROSPRAY SOLUTION CHEMISTRY - Mobile phase pH has a major effect for analyte that are ions in solution - Basic pH (>7.0; 9 preferred) for negative ions - Acidic pH (<7.0; 5 preferred) for positive ions - Manipulation of pH can enhance performance for analytes that are not normally ionized in solution

16. QTOF 6540 ION PATH ELEMENTS

17. MASS SPECTROMETER TYPE: MASS ACCURACY Quadrupole 200-300 ppm Linear Time of Flight 200 ppm Reflectron Time of Flight 25-50 ppm OR-Time of Flight 1-3 ppm Fourier Transform MS 0.1-1 ppm

18. MASS ACCURACY-DEVIATION IN PPM

19. EXACT MASS CALCULATION AND DEVIATION

20. HOW MUCH MASS ACCURACY IS NEEDED? Elemental composition implication

21. TOF RESOLUTION Resolution is the ability to distinguish between two mass peaks that are extremely close together Resolution = M / Peak Width (FWHM: full width at half maximum)

22. LC/MS OF SEMIX (10 UG/ML) 10 PPM

23. QUALITATIVE DATA PROCESSING MASS HUNTER QUAL SOFTWARE Open File Use default layout: Chromatogram, Mass Specta Process and print data Display mass spectrum Average few scans Background substraction Peak integration

24. POSITIVE ION MODE LC/ESI OF SEMIX

25. SELECTED IONS FROM SEMIX

26. ESI MASS SPECTRA OF SEMIX

27. LC/ESI OF SURFACTINE MIXTURES

28. 14 DALTON DIFFERENCE BETWEEN COMPOUNDS

29. POSITIVE ION MODE APCI OF CAROTENOIDS m/z Positive ion mode LC/APCI is the method of choice for analyzing and quantifying Carotenoids