An Introduction to Mass Spectrometry
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1 An Introduction to Mass Spectrometry Guy Wilson UPC 2 & Purification Business Development, Northern Europe, Waters Ltd 2013 Waters Corporation 1
2 What is a Mass Spectrometer? Data System Sample Introduction Ion Source Mass Analyser Detector LC, GC etc. Mass Spectrometer 2013 Waters Corporation 2
3 Atmospheric Pressure Ionisation Atmospheric Pressure Ionisation (API) A term for any ionisation process that is carried out at atmospheric pressure The most common modes of API are Electrospray Ionisation (ESI) o Most commonly used Atmospheric Pressure Chemical Ionisation (APCI) o Less commonly used than ESI but still significant usage Atmospheric Pressure Photo Ionisation (APPI) o A more niche ionisation mode for certain compounds that will not ionise by either ESI or APCI Waters Corporation 3
4 Electrospray Ionisation (ESI) 2013 Waters Corporation 4
5 Electrospray Ionisation Overview 2013 Waters Corporation 5
6 Electrospray Ionisation Producing Charged Droplets More Negative Ions than Positive Ions More Positive Ions than Negative Ions Liquid Flow Taylor Cone Positively Charged Droplets Electrospray Probe Tip at high voltage 2013 Waters Corporation 6
7 Electrospray Ionisation Fission of Charged Droplets Droplets produced from the spray have a surface charge Surface charged droplets undergo solvent evaporation and droplet fission to produce smaller droplets Like charge repulsion becomes greater than droplet surface tension and fission occurs to produce smaller charged droplets Solvent Evaporation Coulombic Fission 2013 Waters Corporation 7
8 Electrospray Ionisation Formation of Gas Phase Ions Ion Desorption Mechanism Charged Residue Mechanism 2013 Waters Corporation 8
9 Electrospray Ionisation Z-spray Ion Source Atmospheric Pressure Sample ESI Probe Nebulizer Gas Sample Cone Isolation Valve RF Lens (Hexapole) Moderate Vacuum Full Vacuum Source Exhaust Cone Gas Quadrupole Desolvation Gas Extraction Cone Roughing Pump Turbomolecular Pump 2013 Waters Corporation 9
10 Atmospheric Pressure Chemical Ionisation (APCI) 2013 Waters Corporation 10
11 APCI Overview Liquid flow is forced through a narrow capillary to give it a high linear velocity The APCI Probe heater combined with nebulizer gas then vaporises the liquid flow The solvent and analyte vapour passes though the corona discharge region to produce gas phase ions Sample Solution Corona Discharge APCI Probe Heater ( C) Flash Vaporisation Corona Discharge Needle Solvent and Analyte Vapour Sample Cone Nebulizer Gas 2013 Waters Corporation 11
12 Atmospheric Pressure Photo Ionisation (APPI) 2013 Waters Corporation 12
13 APPI Overview APPI uses the same probe as APCI with a Vacuum UV light source instead of the corona discharge needle As with APCI: Liquid flow is forced through a capillary to give it a high linear velocity The probe heater and nebulizer gas vaporise the liquid flow Ionisation occurs by either direct or chemical ionisation type processes in the region of the light source Sample Solution Probe Heater Flash Vaporisation h Vacuum UV Source Repeller Electrode Solvent and Analyte Vapour Sample Cone Nebulizer Gas 2013 Waters Corporation 13
14 ESI / APCI / APPI Comparison ESI APPI APCI 2013 Waters Corporation 14
15 Universal Source platform ESI APCI ESCi APPI APCI APGC TRIZAIC UPLC nanoflow ESI ASAP Also compatible with 3 rd party source options: Phytronix LDTD IonSense DART Prosolia DESI Advion NanoMate Protea LAESI 2013 Waters Corporation 15
16 Quadrupole Theory Pre-filter Quadrupole Mass Filter Post-filter Rejected Ions Stable (Resonant) Ions 2013 Waters Corporation 16
17 Single Quadrupole MS 2013 Waters Corporation 17
18 Tandem Quadrupole MS 2013 Waters Corporation 18
19 Electric Field Transfer Region Ion Optics Diffuse Ion Cloud Maximising signal Maximising robustness 2013 Waters Corporation 19
20 Traveling Wave Ion Transport RF RF 2013 Waters Corporation 20
21 Traveling Wave Ion Transport Ions Time Traveling Wave Pulse 2013 Waters Corporation 21
22 Different Types of Experiments MS1 Collision Cell MS Waters Corporation 22
23 MS2 Scan MS1 Ion Guide (RF only) T-wave Collision Cell MS2 Scanning Works in the same way as MS Scan except: MS1 and Collision Cell act only as Ion guides MS2 is the scanning quadrupole Minimises distance and amount of ion optics to interact with between mass resolved ions and the detector Better performance for fast scanning (> 2,000 Da/sec) 2013 Waters Corporation 23
24 Extracted Mass Chrom Mass Spectrum MS Scan - Example TIC 2013 Waters Corporation 24
25 SIR Single Ion Recording MS1 Static Single Ion T-wave Collision Cell No Gas MS2 Ion Guide (RF only) Only selected ions are transmitted through the instrument and are monitored constantly Due to the instrument not acquiring other ions considerably more time is spent on each selected ion making this a much more sensitive acquisition mode than scanning acquisition modes No mass spectra are produced for this type of acquisition 2013 Waters Corporation 25
26 10ng/mL of each component SIR - Example S/N = 109 S/N = 38 S/N = 101 S/N = Waters Corporation 26
27 MRM Multiple Reaction Monitoring MS1 Static Single Ion T-wave Collision Cell MS2 Gas on Static Single Fragment Ion A selected ion is transmitted through the first quadrupole (precursor ion), fragmented in the collision cell, and a specified fragment ion is then transmitted through the second quadrupole (product ion). More selective and sensitive than SIR due to Specific transition needed for response to be seen Less interference by background ions of the same mass No mass spectra are generated by MRM experiments 2013 Waters Corporation 27
28 10ng/mL of each component MRM - Example S/N = 357 S/N = 772 S/N = 6600 S/N = Waters Corporation 28
29 Product Ion Scanning MS1 Static Single Ion T-wave Collision Cell Gas on MS2 Scanning Selected ions are transmitted through the first quadrupole (precursor ions) and fragmented in the collision cell MS2 is then scanned over a user defined mass range A mass spectrum of the product ions generated by fragmentation is acquired at each time point throughout the acquisition Waters Corporation 29
30 Product Ion Scanning - Example 2013 Waters Corporation 30
31 Precursor Ion Scanning MS1 Scanning T-wave Collision Cell MS2 Gas on Static Single Fragment Ion MS1 is scanned over a specified mass range and all ions are sequentially passed through to the collision cell where they are fragmented MS2 is set to transmit only the mass of a specific fragment ion and does not scan Any ions that fragment to give the specified product ion will generate a result Waters Corporation 31
32 Precursor Ion Scanning - Example Precurso r Ion Mass Spectra TIC Precursors of 156 Da Precursors of 85 Da 2013 Waters Corporation 32
33 Constant Neutral Loss MS1 Scanning T-wave Collision Cell Gas on MS2 Scanning with mass offset MS1 is scanned over a specified mass range and all ions are sequentially passed through to the collision cell where they are fragmented MS2 is scanned in sync with MS1 over the same mass range minus an offset A response in only seen if a precursor ion loses a neutral fragment in the collision cell of the same mass to charge ratio as the offset between MS1 and MS Waters Corporation 33
34 Waters Quadrupole Mass Spectrometers Xevo TQ- S micro SQD 2 Xevo TQ-S Xevo TQD 2013 Waters Corporation 34
35 Time Of Flight MS Time-of-flight mass spectrometry (Tof-MS) is probably the simplest method of mass measurement to conceptualise, although there are hidden complexities when it comes to higher resolution instruments. The inherent characteristics of Tof MS are extreme sensitivity (all ions are detected), almost unlimited mass range, speed of analysis (modern instruments can obtain >10 full spectra per second) and sub 5ppm mass accuracy Waters Corporation 35 35
36 Time Of Flight Theory A voltage pulse applied to a pusher plate (typically, V) initiates an orthogonal acceleration TOF (oa-tof) m/z measurement The energy imparted by this pulse in combination with an electric field is converted into kinetic energy (KE) The time it takes an ion to travel (TOF) a given distance (d) is proportional to its mass-to-charge ratio or m/z 2013 Waters Corporation 36
37 Time Of Flight Theory Pusher Detector Time from Push 2013 Waters Corporation 37
38 Maximising Resolution 2013 Waters Corporation 38
39 Accelerating the Ions P u s h e r P u s h e r P u s h e r Ideal Case: Spatial distribution: Energy distribution: perfectly straight line no orthogonal velocity component some ions are closer to the plate than others small orthogonal velocity component 2013 Waters Corporation 39
40 Waters TOF Mass Spectrometers Synapt G2-Si HDMS Xevo G2-XS QTof 2013 Waters Corporation 40
41 Xevo G2-XS 2013 Waters Corporation 41
42 LC/MS E Data Acquisition No selection of peptide ions Collision cell cycles between low (MS of peptides, 4V) and elevated (MS E of fragments, V) energy MS UPLC Global analysis: acquire data on all the ions Maximize the LC/MS duty cycle Minimize bias/selection of ions ( Reproducibility) Obtain qualitative and quantitative information from the same analysis run MS E 2013 Waters Corporation 42
43 SYNAPT G2-Si HDMS 2013 Waters Corporation 43
44 Ion mobility 2013 Waters Corporation 44
45 Ion Mobility Separation (IMS) The mobility of an ionised molecule is dependant on its Size Shape Charge 2013 Waters Corporation 45
46 Classic Ion Mobility Spectrometry The Bowers Group Website [Ion Mobility Theory]: Waters Corporation 46
47 TriWave Device Trap He Cell IMS Transfer 2013 Waters Corporation 47
48 information Selectivity (and Specificity) Retention CCS Mass Peak capacity QuanTof UPLC/HDMS 2013 Waters Corporation 48
49 Mass Detection At Its Most Advanced 2013 Waters Corporation 49
50 Know your peaks 2013 Waters Corporation 50
51 Complementary & Compatible DA PRE-OPTIMIZED ES± 20 HZ FS 100 HZ SIR 4 ORDERS INTUITIVE Information-rich mass spectral data Targeted for small molecule applications Complementary to and compatible with PDA UPLC, UPC 2, purification Optional ISM module to split and dilute Intuitive system for chromatographers 2013 Waters Corporation 51
52 6.5 mins 22 mins 2013 Waters Corporation 52
53 Thank You Questions? 2013 Waters Corporation 53
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