The CHROMacademy Essential Guide Understanding GC-MS Analysis Part 1

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1 The CHROMacademy Essential Guide Understanding GC-MS Analysis Part 1

2 Speakers John Hinshaw GC Dept. Dean CHROMacademy Tony Taylor Technical Director Crawford Scientific Moderator M ( g ) e M ( g ) 2e Peter Houston Editorial Director LCGC Magazine

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4 Aims & Objectives When to use GC-MS Overview of a typical GC-MS System GC Considerations Interfacing the GC and MS Components Ionisation Electron Impact Ionisation Chemical Ionisation Introduction to Mass Analysers Ion Detection Systems Vacuum Systems Introduction to GC-MS data analysis

5 When to Use GC-MS GC-MS combines the separating power of Gas Chromatography (GC), with the detection power of mass spectrometry (MS) GC-MS is primarily used for: - Identification or characterisation of analytes within the sample - Increased analytical sensitivity in the absence of a compound or element specific detector such as ECD, NPD, FPD or chemiluminesence Typical Electron Impact Ionisation Spectrum of the Cocaine molecule GC-MS Total Ion Chromatogram used to quantify an environmental pollutant

6 A Typical GC-MS System 1. Pneumatic controls 2. Injector 3. Oven 4. Column 5. Interface 6. Ion Source 7. Mass Analyser 8. Detector 9. Vacuum System 10. Control Electronics

7 The GC-MS Process 1. Sample Introduction 2. Sample components separation 3. Transfer from the GC Column into the high vacuum of the mass analyser 4. Ionisation of sample components 5. Separation and detection of gas phase ions

8 GC Carrier Gas Considerations Gas Purity of % recommended Oxygen, Moisture and Hydrocarbons should be scrubbed from the gas supply Carrier gas flow rates limited to <2ml/min. due to detector vacuum system which limits column choice to some extent Typical Column Flow Rates for a number of Carrier Gas / Column Geometry Combinations

9 GC Ferrules & Septa Ferrules form a seal between the column and the inlet and detector connections GC-MS has a special requirement for ferrules to be impermeable to air / oxygen (reduces air background signal in the mass spectrometer) Preferred ferrule materials for GC-MS are graphite / vespel composites or special metal ferrules Septum bleed products must also be reduced Septa Material & GC-MS Compatibility

10 Stationary Phase Selection for GC-MS Due to the nature of the detector low bleed phases are preferred for GC-MS This reduces the background signal and contributes spurious ions to the spectra of compounds of interest Column Bleed increases with Temperature Column Length, Film Thickness & Polarity Special low bleed phases exist with Silphenylene chemistry and many manufacturers offer MS designated phases Specially immobilised PLOT columns should be used to prevent evolution of particulate material into the ion-source Silphenylene low-bleed Phenyl phase chemistry Traditional Phenyl phase chemistry

11 Common Contaminant Ions in GC-MS

12 Interfacing GC with MS Detection The column outlet needs to be connected to the ion source of the mass spectrometer and fulfill the following conditions: - Analyte must not condense in the interface - Analyte must not decompose before entering the mass spectrometer ion source - The gas load (dictated by the mobile phase gas flow rate) entering the ion source must be within the pumping capacity of the mass spectrometer Most capillary GC-MS interfaces directly couple the column exit with MS ion source. The advent of capillary columns brought about a significant reduction in the volumetric gas flow exiting the column (typically 1ml/min or below for columns of 0.32mm id and less), and the need to split the analyte away from the carrier gas to reduce gas load into the ion source was eliminated.

13 Direct GC-MS Interfaces Column is inserted directly into the mass spectrometer ionisation chamber This interface gives the highest sensitivity Changing the GC column may be a time consuming process unless curtain gas devices are fitted Usually heated >10 o C above the final oven temperature program and at a lower temperature than the ion source Analyte degradation should be avoided Temperature Zones: Column Oven > Interface > Ion Source > Quadrupole

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15 GC Interface Connection The connection must be gas tight Traditional nut and ferrule couplings into the transfer device are common Several devices are available to facilitate vent-free columnchange including: - Curtain gas connectors - Couplings with restrictors - Couplings using deactivated silica tubing pig-tails

16 Analyte Ionisation Electron Impact (EI) 1. Sample introduced directly into the ion source 2. Electrons are emitted from a heated filament 3. Accelerated using an appropriate potential (5-100V) to achieve the required electron energy 4. The industry standard for the electron energy is 70ev (electron volts) which 5. Resulting ions are accelerated out of the source using an electrostatic potential 6. Ion beam is focused using electrostatic lenses

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18 Electron Impact Ionisation Process M ( e M 2e g ) ( g ) 1. The bombarding electron at 70eV abstracts an electron from the analyte molecule (M) 2. All 70eV is transferred to the analyte molecule which becomes a Radical Cation 3. Depending upon the magnitude of the analyte 1 st Ionisation Potential (energy required to remove 1 electron from an atom within the molecule) there may be excess energy remaining 4. This remaining energy may cause the analyte to fragment through further bond cleavage 5. The molecule may also undergo simple intermolecular rearrangement reactions 6. Any charged fragments and re-arrangement products will appear as signals within the mass spectrum

19 Typical EI Spectra 1. EI is a relatively harsh technique inducing fragmentation in many cases 2. The intensity of the molecular ion varies depending on analyte radical cation stability 3. The fragmentation pattern can be used to identify the analyte molecule and acts as a fingerprint of the molecule 4. Commercial (70eV) or in-house libraries may be searched by the instrument data system to provide a tentative match for the unknown spectrum 5. Experienced users may undertake ab. initio. interpretation to elucidate the molecular structure from the mass spectrum

20 Analyte Ionisation Chemical Ionisation (CI) 1. Chemical ionisation involves the ionisation of a reagent gas, such as methane or ammonia at relatively high pressure (~1 mbar) in an electron impact source 2. Once produced, the reagent gas ions collide with the analyte molecules producing ions through gas phase reaction processes such as proton transfer and adduction 3. Reagent gas, gas pressure and stability of gas pressure are critical

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22 Chemical Ionisation Process NH4 e NH4 2e NH 4 M NH3 [ M H] NH [ ] 4 M M NH4 (Production of the reagent gas ion) (Analyte ionised by Proton Transfer) (Analyte ionised by Adduct Formation) 1. CI is a soft ionisation technique compared to EI 2. All 70eV is transferred to the analyte molecule which becomes a Radical Cation 3. Appearance of the spectra heavily influence buy the choice of reagent gas methane / isobutane / ammonia / combinations 4. Tuning the mass spectrometer to calibrate the mass axis / resolution and relative response across the mass range is more difficult in CI

23 Typical CI Spectra 1. Energy of reagent gas rarely exceeds 5eV 2. Softer ionisation technique gives rise to more intense molecular ions and fewer fragments (library searching more difficult) Afalatoxin B1 3. Useful for obtaining the molecular weight of the analyte 4. Operates in two modes negative ion and positive mode 5. Negative ion mode is highly sensitive used for ultra-trace analysis of halogenated compounds (capable of electron capture) 6. Positive ion mode less sensitive as reagent gas is also detected

24 Mass Analysers The Quadrupole 1. Ions are separated according to their mass-to-charge ratio (m/z) as they pass along the central axis of four parallel equidistant rods (or poles) 2. Ion separation is performed using controlled voltages applied to the mass analyser rods which impart an electrostatic field inside the analysing device. 3. As long as x and y, which determine the position of an ion from the centre of the rods, remains less than r 0, the ion will be able to pass through the quadrupole without touching the rods. This is known as a non-collisional or stable trajectory.

25 Mass Analysers The Quadrupole (II) 4. Where the ion is caused to oscillate with a trajectory whose amplitude exceeds r0 it will collide with a rod, and become discharged and subsequently pumped to waste 5. This is known as an unstable or collisional trajectory.

26 Quadrupole Advantages & Limitations Advantages Disadvantages Reproducibility Low cost Low resolution Mass discrimination. (Peak height vs. mass response must be 'tuned ) Limited scanning speeds compared to other analyser types (Not suiable for very fast GC analysis with very narrow peaks)

27 Mass Analysers - Time of Flight (ToF) 1. Ions are extracted (or produced) in short bursts or packets within the ion source and subjected to an accelerating voltage. 2. The ions then drift or fly down an evacuated tube of a set length ( d ). Once free from the region of accelerating voltage the speed at which the ions travel down the tube is dependant upon their mass (m) and charge (z). 3. All ions are detected (almost) simultaneously. 4. Scanning the mass range of all ions is very rapid and as such the inherent sensitivity of the instrument is increased.

28 Mass Analysers - Time of Flight (ToF) (II) 5. Most modern ToF instruments use orthogonal acceleration ( oa ) pusher for improved instrument performance 6. Increasing the length of the flight tube using an ion mirror or reflectron leads to an increase in the resolution of the instrument

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30 ToF Advantages and Limitations Advantages High ion transmission Highest practical mass range of all MS analyzers Very Low Detection limits Disadvantages Fast digitizers used in TOF can have limited dynamic range Cost (but this is becoming less of a barrier) High mass accuracy and resolution (Will give elemental composition possibilities)

31 Mass Analysers Ion Traps 1. Ion trap mass spectrometers work on the basis of storing ions in a trap, and manipulating the ions by using applied DC and RF fields. 2. The amplitude of the applied voltages enables the analyser to trap ions of specified mass to charge ratio within the analysing device. 3. Non-selected ions are given a trajectory by the electrostatic field that causes them to exit the trap.

32 Mass Analysers Ion Traps (II) 4. By filling the trap with an inert gas fragmentation of selected ions is possible - useful when structural information is required. 5. Can perform multiple product ion scans with very good sensitivity (MS n ) 6. Spectra acquired with an ion trap mass analyser may be significantly different to those acquired from a triple quadrupole system due to the different collision regimes within the systems (collision energy/gas).

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34 Ion Traps Advantages & Limitations Advantages Disadvantages High sensitivity Multiple Product Ion scan capability (MS) n High resolution Good specificity and capable of data dependant scanning (i.e. Automated MS/MS) Produces very unusual spectra if the ions are stored in the trap too long. Easily saturated Poor for low mass work (below 100 Da) Poor dynamic range (except the most modern devices) and hence may have limited quantitative use

35 GC-MS Detectors Once the ions have passed the mass analyser they have to be detected and transformed into a usable signal. The detector is an important element of the mass spectrometer that generates a signal from incident ions by either generating secondary electrons, which are further amplified, or by inducing a current (generated by moving charges)

36 GC-MS Detectors Point detectors: ions are not spatially resolved and sequentially impinge upon a detector situated at a single point within the spectrometer geometry. Array detectors: ions are spatially resolved and all ions arrive simultaneously (or near simultaneously) and are recorded along a plane using a bank of detectors.

37 GC-MS Vacuum Systems The entire MS process must be carried out at very low pressures (~10-8 atm) A high level of vacuum within the instrument assists the processes of ion movement and separation in the following ways: By providing an adequate mean free path for the analyte ions By providing collision free ion trajectories By reducing ion-molecular reactions By reducing background interference Vacuum systems consist of a differentially pumped arrangement: a foreline pump establishing a rough vacuum and a high vacuum pump or pumps situated on the analyser body to establish the high levels of vacuum required for effective mass to charge ratio measurement.

38 GC-MS Vacuum Systems High Vacuum Turbomolecular Pump: 10-6 to 10-8 Torr Oil Filled Rotary Foreline (Rough) Pump: 10-2 Torr

39 GC-MS Data Quadrupole GC-MS systems operate in two distinct modes Scanning and Selected Ion Recording In SCAN mode, the quadrupole settings are ramped through a range of values which allow successively lower mass to charge ratio ions to pass through the analyser scaning the mass range of ions emerging from the quadrupole device The scanning operation takes a finite time to complete (although scan rates of 5-20Hz are typical) and each individual m/z value is measured for only a fraction of the time that they elute into the mass analyser The Total Ion Chromtogram (Current) (TIC) is constructed by summing the abundance of all ions within a spectrum at a particular time point and plotting total abundance against the time at which the spectrum was acquired

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41 GC-MS Data By choosing to set the quadrupole to certain voltage values, we are able to choose only certain masses for transmission through the mass analyser This type of spectral experiment, called selected or specific ion monitoring (SIM), or selected ion recording (SIR), has certain advantages over scanning wide mass ranges The advantage here is gained because the analyser concentrates on useful ion signals from the analyte of interest and not on noise gaining significantly on signal to noise ratio Because not all m/z values are recorded the mass analyser can carry out a SIM experiment very rapidly ( increase over scanning experiment speeds) therefore acquiring more data points to accurately model the peak shape

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43 CHROMacademy & CHROMmunity Today's Webcast was brought to you by CHROMacademy: Join CHROMacademy for $295 per year includes all CHROMacademy classes, labs, library and webcasts All questions will be answered within CHROMmunity:

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