LC/MS WORKSHOP

IOWA STATE UNIVERSITY Kamel Harrata  Instrument Description  Data Acquisition  Data Processing

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

LC/MS and GC/MS Mass Spectrometers

GC/MS LC/MS Micromass GCT Inlet Direct Probe Flow Injection GC, LC Ion Source EI, CI APCI, ESI Agilent QTOF 6540 m/z Analyzer Q, QQQ EB, Ion Trap TOF, QTOF FT, Orbitrap Detector EM, PM Data System

Applications of Mass Spectrometry

• Pharmaceutical analysis • Biomolecule characterization Proteins and peptides Oligonucleotides • Environmental analysis Pesticides on foods Soil and groundwater contamination • Forensic analysis/clinical

TOF EQUATION

TIME OF FLIGHT SCAN

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

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

ELECTROSPRAY IONIZATION Desolvation + Coulomb Explosions

AGILENT ELECTROSPRAY ION SOURCE (POSITIVE MODE)

ELECTROSPRAY SPECTRUM OF MYOGLOBIN

RELATIVE APPLICABILITY OF LC/MS TECHNIQUES

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

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

QTOF 6540 ION PATH ELEMENTS

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

MASS ACCURACY-DEVIATION IN PPM

EXACT MASS CALCULATION AND DEVIATION

HOW MUCH MASS ACCURACY IS NEEDED?

Elemental composition implication

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)

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

LC/ESI pos of : L-Selenocysteine (Peak#1); MethSeCy (#2); SeMethionine(#3); Etionine(#4) Agilent XDB C18, 1.8 um, 4.6mm ID, 150 mm L

L-Selenocysteine MW 168 MethylSelenoCysteine MW 182 SelenoMethinine MW196 MW 163

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

POSITIVE ION MODE LC/ESI OF SEMIX

SELECTED IONS FROM SEMIX

ESI MASS SPECTRA OF SEMIX

LC/ESI OF SURFACTINE MIXTURES

14 DALTON DIFFERENCE BETWEEN COMPOUNDS

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