LC-MS Julie Bliss CEE 772 11/24/2014 L IQUID C HROMATOGRAPHY 1) - PowerPoint PPT Presentation

LC-MS Julie Bliss CEE 772 11/24/2014

L IQUID C HROMATOGRAPHY 1) Mobile phase is pumped by the solvent management system to the column. 2) Simultaneously, the injector injects the sample to the mobile phase stream into the HPLC column. 3) The sample and mobile phase pass through the column into the mass spec. Waters. (2014). How Does High Performance Liquid Chromatography Work? Retrieved from Waters: http://www.waters.com/waters/en_US/How-Does-High-Performance-Liquid-Chromatography- Work%3F/nav.htm?cid=10049055&locale=en_US

L IQUID C HROMATOGRAPH 1) Samples are prepared and injected into vials. Flow Cell 2) Samples are injected Sample Mobile Phase Loop through sample loop. 3) Solvent management system takes solvent and mobile phase to column (C18). Sample Vials 4) Gradient elusion starts. Injector 5) Flow cell cables direct Column analytes to Mass Spec. Solvent Management System

T ANDEM M ASS S PECTROSCOPY 1) Sample enters ion source and is fragmented by electrospray ionization. 2) Charged fragments are filtered by mass (parent mass is identified) and transferred to collision cell. 3) Specific ions are further fragmented into specific masses by collision ionization with Argon. 4) New fragments are segregated into individual masses (daughter mass is identified) which are detected by the sensor. 5) The detector produces the mass spectrum (m/z) from ions of selected mass and chromatogram. Scott, R. P. The Quadrupole Mass Spectrometer . Retrieved from Analytical Spectroscopy: http://www.analyticalspectroscopy.net/ap8-23.htm

E LECTROSPRAY I ONIZATION (ESI)  Spray of charged particles  Nebulizer gas creates aerosols  Desolvation gas decreases droplet size by repulsion between charged droplets  fission.  Charge is proportional to size.  Factors: chemical composition, structure, solvent, instrument parameters, etc. Particle Sciences. (2009). Mass Spectrometry in  Molecules < 2000 Da- 1, 2,3 + Bioanalysis. Particle Sciences .  Molecules > 2000 Da- series of multiple charges

Q UADRUPOLE  Selects specific ions based on charge to mass ratios.  Charged molecules accelerate and move away from center line. Non-resonant ions are filtered by absorption. Swiss Laboratory for Doping Analyses. (2008). Mass Spectrometry . Retrieved from Swiss Laboratory for Doping Analyses: http://www.doping.chuv.ch/en/lad_home/lad-prestations-laboratoire/lad-prestations-laboratoire-appareils/lad-prestations- laboratoire-appareils-ms.htm

C OLLISION I ONIZATION Geändert, Z. (2013). Mass Spectrometry . Retrieved from Humboldt-Universitat Zu Berlin: https://www.biologie.hu- berlin.de/gruppenseiten/oekologie/meth/massspec/mass_sp

M ASS S PECTRUM  Each bar represents an ion having a mass-to-charge ratio (m/z).  Height of the bar indicates the relative abundance (%) of the ion.  Small bars represent fragments of compounds.

T ANDEM M ASS S PECTROMETER 1) Sample and mobile phase is directed from LC to probe. Both travel into Nebulizer and Desolvation Gas capillary to the probe tip where the solution is sprayed from nebulizer gas Probe (N 2 ). Ion Source 2) Charged solution moves to Quadrupoles and Collision Cell quadrupoles and collision cell where fragments are Capillary and probe tip filtered and segmented. 3) Desolvation gas (N 2 ) evaporates analyte from sample throughout. 4) The chromatogram and mass spectra are created and analyzed.

D EVELOPING A M ETHOD FOR LC-MS  Work backwards:  Disconnect cable from LC and add to MS probe.  Enter masses for analytes.  Turn on nitrogen gas to get peaks and optimize voltages to obtain highest peak.  Note optimized parameters.  Find daughter masses by scanning range of masses and optimizing collision energy.  Set up MRM (detection specification in software).