F LORIDA I NTERNATIONAL U NIVERSITY Advanced Mass Spectrometry Piero R. Gardinali/Yong Cai/ Bruce McCord Revised on August 23, 2009

First Steps Introduction to mass spectrometry Definition of common terms A generic mass spectrometer Formation of ions The mass spectrum Why doing mass spectrometry?

Survey: No previous knowledge of MS? Know basic operation but no theory? Comfortable user? I can do a manual tune! I took your MS class (did you pass?) I will be helping with the class!

Mass spectrometry is a powerful analytical technique that is used to identify unknown compounds, to quantify known materials, and to determine the structural and chemical properties of molecules. Detection of compounds can be accomplished with very minute quantities. (as little as grams).

Anode rays Goldstein 1886 Electron m/z Thomson 1897 (Nobel Prize in 1906) Magnetic deflection Positive Ions Wein 1898 Unit of charge Millikan & Fletcher 1909 First parabola Spectrograph Thomson 1912 Metastable ions Thomson Nier U Exact Mass Benyon Develop first MS with velocity focusing, Nobel Prize in 1922 Fenn developed ESI in 1988; Concept was proposed by Dole in Fenn received Nobel Prize in 2002

Progress in Experimental Methods and Improvements in Solution, Sensitivity, Mass Range, and Accuracy Resolution: m/Δm from 13 (Y1913) to 8,000,000 (Y1998) Sensitivity: as low as 800 yoctomoles (~480 molecules)( measure peptide in solution) Mass range: several tens of millions D (40.5 MD) Accuracy: isotope measurement

Did you know that mass spectrometry is used to… Detect and identify illegal steroids in athletes Monitor the breath of patients during surgery Determine the composition of molecular species found in space Determine whether honey is adulterated with corn syrup Locate oil deposits by measuring petroleum precursors in rock Monitor fermentation processes for the biotechnology industry Detect dioxins in contaminated fish, water and soil DNA Fingerprinting! Establish the elemental composition of semiconductor materials Speed the development of new drugs

Basic concepts The basis of mass spectrometry (MS) is the production of ions that can be separated or filtered according to their mass to charge ratios (m/z) and detected. The resulting mass spectrum is a plot of the abundance of the produced ions as a function of the m/z ratio.

Still spectroscopy?

What is a mass spectrometer? Illustration of the basic components of a mass spectrometry system. Ionization Source Mass Analzyer Detector Inlet all ions selected ions Data System

% Relative Abundance  m/z  The mass spectrum

What do I get from the instrument? Mass spectrum of methanol by EI, presented as a graph and a table

More definitions Base peak The most intense peak of a mass spectrum is called base peak Usually mass spectra are normalized to the most intense peak by setting its intensity to 100 % relative intensity. This offers the advantage of being independent from absolute intensities which may extremely vary between measurements.!!!!!! Thus, normalization to the base peak makes comparison of spectra much easier.

More about ions and fragments Molecular ion The molecular ion is directly created from the corresponding neutral molecule by addition or removal of one electron i.e. it still has the same molecular formula. The molecular ion is usually labeled as M +. or M -., respectively. Quasimolecular ions are different from molecular ions in that they are formed by addition or removal of some ionic particle.

Ways to define and calculate the mass of an atom, molecule or ion Average mass: calculated using the atomic weight, which is the weighted average of the atomic masses of the different isotopes of each element in the molecule. Often used in stoichiometric calculations. Nominal mass: calculated using the mass of the predominant isotopes of each element rounded to the nearest integer value that corresponds to the mass number. Monoisotopic mass: calculated using the extract mass of the most abundance isotope for each constituent element. Use monoisotopic mass if possible in MS

Differences between Masses C 20 H 42 C 100 H 202 Nominal: (20 x 12) + (42 x1) = 282 u(100x12) + (202x1) = 1402u Monoisotopic: (20 x12) + (42 x ) = (100x12) + (202x ) = Average: (20 x ) + (42 x ) = (100x12.011)+(202x ) =

HOW DO I CREATE AN ION? M + e -  M e -

HOW DO I CREATE AN ION? M + e -  M e -

HOW DO I CREATE AN ION? M + e -  M e - An then what?

HOW DO I CREATE AN ION? M + e -  M e - Radical cation

Ion free path ??? Free mean path ??? K: Boltzmann constant, T: temperature (in K) p: pressure (in Pa)  : collision cross-section (in m 2 ) P in pascals P in milli Torrs