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VAM Programme Training Guides on Mass Spectrometry

Vicki BarwickLGC

Overview

• The VAM programme• Knowledge transfer activities• Best Practice Guide to Generating Mass Spectra• Methodology for Accurate Mass Measurement of Small

Molecules

Valid Analytical Measurement (VAM) Programme

• A DTI funded programme which aims to:• Improve the quality of analytical measurements made in

the UK• Facilitate mutual recognition of analytical data across

international boundaries• Develop a robust and transparent infrastructure aimed at

achieving international comparability and traceability of chemical and biochemical measurements

• Measured anywhere accepted everywhere

VAM - Supporting chemical measurement

• Knowledge transfer to raise awareness and encourage the adoption of ‘best practice’

• Development of tools that enable laboratories to implement best practice

• Research aimed at long term improvements to the reliability and comparability of analytical measurements

Technical projects

• National Measurement Institute for chemical measurements

• Chemical calibration and standards– maintenance of supply of reference materials– production of pure substance and matrix reference materials– improving procedures for development and certification of pure

substance and matrix reference materials – UK chemical calibration facility

• Validation of measurement methods– development of high accuracy methods of analysis needed for

RM production/calibration services

Knowledge transfer

• VAM website• VAM bulletin and VAM brochure

– case studies of business benefits of adopting VAM principles

• Seminars and workshops• Development of resources

– books, guides, software…….

• Support for schools, colleges and universities

DTI VAM Programme Training Guides on Mass Spectrometry for Beginners and Making Accmass Measurements

Vicki Barwick - LGC, Teddington

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www.vam.org.uk

• Information on VAM activities– education and training– reference materials– proficiency testing– current projects

• Register to receive VAM bulletin

• Access to VAM publications– download publications– shop on-line for VAM

products• VAM advisory services

Best Practice Guide for Generating Mass Spectra

The need for guidance:• Quality of mass spectra used for identification/

confirmation of compounds highlighted as an area of concern

• Poor quality spectra can lead to incorrect conclusions• Novice users of mass spectrometry need guidance

– selection of appropriate analytical technique– recognition of good/poor quality spectra

Scope and aims

• Scope– qualitative analysis (identification of unknowns/confirmation of

identity)– ‘small’ molecules

• Aims to help novice users in universities and industry– select an appropriate technique and generate mass spectra that

are fit for purpose– recognise poor quality spectra and understand some of the

common causes– understand some of the limitations of mass spectrometry

Format

• Not a textbook!• ‘Short’ and snappy guide which can be used easily at the

bench– short sections of text (mainly as bullet points)– diagrams/example spectra to illustrate key points– sections structured to make key information easy to locate– bibliography to direct users to more detailed information

Key content

• Choice of ionisation mode– factors influencing selection, mode selection chart, summary of

ionisation modes (types of molecule, ionising mechanisms, sample introduction method, etc)

• Instrument set-up before acquisition– importance of peak shape and resolution, definition of resolution,

other instrument checks, common problems and possible causes• Evaluating spectral quality

– what to look for in a good quality spectrum, examples of acceptable and poor quality spectra, validity of the molecular ion

• Molecular ion recognition– definitions of molecular mass, how to find the molecular ion, use

of stable ion information

Selection of ionisation mode

TYPICAL SAMPLE

SOLVENT 0 100 200 300 400 600 800 1000 >1200

hexane hydrocarbons, PAH's; PCB's…toluene EI (note 1) EI EI EI EI EI EI EI EI

CI (note 1+2) CI CI CI CI CI CI CI CI common organics, alcohols, amines,organometallics, functionalised species...

dichloromethane EI EI EI EI EI EI EI EI chloroform CI CI CI CI CI CI CI CI

APCI APCI APCI APCI APCI APCI APCI APCI methanol ESI ESI ESI ESI ESI ESI ESI ESI

acetonitrile MALDI (note 3) MALDI MALDI MALDI MALDI MALDI water sugars, peptides, nucleotides, salts, multiply-charged species…

APCI APCI APCI APCI APCI APCI APCI APCI acid/base ESI ESI ESI ESI ESI ESI ESI ESI ESI

MALDI MALDI MALDI MALDI MALDI MALDI

Insoluble: Use EI / CIAcidic: Use negative ion mode

KEY: Examples in italicsPrimary technique ( => most likely to work well)Secondary technique ( => should work and will give complementary information to the primary technique)Tertiary technique ( => try it if you have no choice)

NOTES:1 Suitable inlet will also be required to match sample volatility (e.g. GC or solids probe or desorption probe)2 CI suitability will depend on gas selection (e.g. ammonia is unsuitable for hydrocarbons which will need e.g. methane)3 MALDI matrix ions cause interference at lower m/z [so MALDI without matrix ("SALDI") can be used more readily at lower mass]4 Mass range also depends on mass analyser and m/z of ion produced (e.g. ESI)

SPECIAL CASES:

POLA

RITY IN

CR

EASIN

G…

..

MOLECULAR MASS (note 4)

SELECTION OF IONISATION MODE for MASS SPECTROMETRY ANALYSIS of COMMON MOLECULES

semi-polar

polarnon-polar

DTI VAM Programme Training Guides on Mass Spectrometry for Beginners and Making Accmass Measurements

Vicki Barwick - LGC, Teddington

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Instrument set-up

• Peak shapeLorentzian

Gaussian

IDEAL PEAK SHAPES POOR PEAK SHAPES

m1 m2

10% intensity

a) 10% intensity definition

RP=m1/(m2-m1)

m1

50% intensity

∆m/z

b) FWHM definition

RP=m1/∆m/z

• Resolution

Methodology for Accurate Mass Measurement of Small Molecules

• Accurate mass measurement used to determine elemental formulae

• Accurate and precise mass measurement increases the certainty of identification of an elemental formula

• If the uncertainty in the measurement is known it can be used to reduce the number of candidate formulae to be considered

• Different techniques available– interlaboratory study indicated the

need for guidance on best practice

Achieving high accuracy mass measurements

• Key considerations– tuning and peak shape– ion abundance– resolving power– calibration– sample introduction– data manipulation– validation and quality control checks– selection of elemental formulae

• The guide describes best practice for these key areas• Also provides instrument specific guidance

Further information

• Best Practice Guide for Generating Mass Spectra– Authors: Vicki Barwick, John Langley, Tony Mallet, Bridget Stein,

Ken Webb– Available from the VAM website in September

• Methodology for Accurate Mass Measurement of Small Molecules– Co-ordinating editors: Tony Bristow, Mike Sargent, Bridget Stein,

Ken Webb– Available from the VAM website

vicki.barwick@lgc.co.uk

www.vam.org.uk

LGC is the lead contractor for the chemical VAM programme

DTI VAM Programme Training Guides on Mass Spectrometry for Beginners and Making Accmass Measurements

Vicki Barwick - LGC, Teddington