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

LIQUID CHROMATOGRAPHY

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

LIQUID CHROMATOGRAPH

1) Samples are prepared and injected into vials. 2) Samples are injected through sample loop. 3) Solvent management system takes solvent and mobile phase to column (C18). 4) Gradient elusion starts. 5) Flow cell cables direct analytes to Mass Spec.

Sample Vials Flow Cell Injector Column Solvent Management System Sample Loop Mobile Phase

TANDEM MASS SPECTROSCOPY

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

ELECTROSPRAY IONIZATION (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.

 Molecules < 2000 Da- 1, 2,3 +  Molecules > 2000 Da- series of

multiple charges

Particle Sciences. (2009). Mass Spectrometry in

• Bioanalysis. Particle Sciences.

QUADRUPOLE

 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

COLLISION IONIZATION

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

MASS SPECTRUM

 Each bar represents an ion

having a mass-to-charge ratio (m/z).

 Height of the bar indicates

the relative abundance (%)

• f the ion.

 Small bars represent

fragments of compounds.

TANDEM MASS SPECTROMETER

1) Sample and mobile phase is directed from LC to

• probe. Both travel into

capillary to the probe tip where the solution is sprayed from nebulizer gas (N2). 2) Charged solution moves to quadrupoles and collision cell where fragments are filtered and segmented. 3) Desolvation gas (N2) evaporates analyte from sample throughout. 4) The chromatogram and mass spectra are created and analyzed.

Probe Nebulizer and Desolvation Gas Ion Source Capillary and probe tip Quadrupoles and Collision Cell

DEVELOPING A METHOD 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).