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The CHROMacademy Essential Guide:
The Secrets of Optimizing GCMS Methods

Thursday 13th December 2012, 11:00 AM EST, 16:00 GMT, 5:00 PM BST

The definitive guide to optimizing methods for GCMS analysis.  In this session we consider the major instrument and method decisions that allow you to get the very best from your analysis. 

Topics covered include:

  • GC column, flow rate and sample prep. considerations
  • Auotune - why and when
  • Improving your instrument tuning performance
  • Optimizing EI and CI source variables
  • Quadrupole – tuning for specific analytes
  • Spectral data acquisition – overlooked parameters
  • Optimizing methods for quantitative or qualitative analysis

Who Should Attend:

  • Anyone working with GCMS who wants to better understand the working principles of their instrument
  • Anyone wanting to achieve better qualitative or quantitative results with GCMS

Find out more about this Month's Essential Guide Webcast »

 

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The CHROMacademy Essential Guide Tutorial
The Secrets of Optimizing GCMS Methods

The difference between a useable and optimised GCMS method can often be differentiated by the optimisation of ‘secondary’ method parameters and attention to detail in areas perhaps not cosidered as of great importance.  This may make the difference between getting a result and getting the best result you can.

 

For example, whilst most users will choose to accept the autotune paremeters derived by the instruments tune algorithm, it is udoubted that some simple manual tuning will be able to improve the intrument quanitive and/or qualitative response.  In order to do this successfully,  you need to understand the basic operational principles of the instrument.

This tutorial demonstrates the working principle of a generic GCMS instrument and the highlights the parameters which, when optimised, have a dramatic effect on sensitivity, sepacificity or robustness of the data produced.

 

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Sample preparation

Sample preparation is of overriding importance to achieve optimum GC-MS results.  Sample cleanliness directly relates to lower detection limits and improved mass spectral data wih lower incidences of chromatographic, or worse, isobaric mass spectral interferences.

For complex samples, an ‘extraction’ technique might be used to isolate a particular analyte chemsitry and reduce the number of components within the sample. Common extraction technqiues in modern GCMS include:

 
  Solid Matrix Liquid Matrix
Volatile Sample
  • Gase Phase Extraction
    • Static Head Space
    • Dynamic Head Space
  • Solid Phase Microextraction
  • Supercritical Fluid Extraction
  • Gase Phase Extraction
    • Static Head Space
    • Dynamic Head Space
  • Solid Phase Microextraction
  • Supercritical Fluid Extraction
Non-Volatile Sample
  • Solid-Liquid Extraction
  • Soxhlet
  • Ultrasonic Extraction
  • Matrix Solid Phase Dispersion
  • Microwave Assisted Extraction
  • Supercritical Fluid Extraction
  • Liquid-Liquid Extraction
    • Separatory Funnel
    • Continuous Extraction
    • Purge and Trap
  • Solid Phase Extraction
  • Solid Phase Microextraction

Table 1.  Selected GC-MS Sample Extraction Procedures (GC Sampling)

 

Sample preparation not only increases the overall quality of a separation but reduces the incidence of contamination in the GC-MS system. 
The MS source becomes contaminated via two main routes:

  • Column bleed: background signal generated by thermal and chemical decomposition of the stationary phase
  • Dirty samples: the GC-MS ion source components become coated with involatile sample material, leading to loss of instrument sensitivity

A further important sample preparation consideration is sample solvent choice.  As well as the usual considerations of boiling point, expansion co-efficient etc., we have to know the elution time of the solvent peak into the mass spectrometer source.

Electron ionisation filament lifetime can be seriously compromised through the vacuum reduction associated with the solvent peak elution - effectively ‘burning out’ in the reduced vacuum.  As such the filament is usually switched off during the solvent elution time.  Without accurately knowing the start and end time of the solvent peak, one may obviously risk missing important analyte peaks during this time.  Consider the volatility of analytes relative to the sample solvent chosen.

 

 

Boiling Point Typical Solvents MS Analysis
Analyte of Interest GC Solvent
High (over 140oC) Low Dichloromethane Detect high-boiling analytes.  Set the MS instrument ON after the low boiling solvent has passed
Low (below 80oC) High Xylenes / Ethyl Acetate Detect low-boiling analytes.  Set the MS instrument OFF before the high boiling solvent begin to pass

Table 2.  GC-MS solvent selection.
 

If you are unsure of the elution time of your solvent, try putting a drop of solvent in the bottom of an autosampler vial and using the syringe to sample the headspace gas.  This usually introduces enough solvent vapour into the MS detector to allow estimation of the solvent elution time without compromising the filament lifetime.

For more on sample preparation click here »

For more information on GC sampling techniques click here »

 
 

Analytical Chemists

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  • I can troubleshoot effectively
  • I know where to go for help
  • I understand my analyses
  • I know where to get applications
  • I’m up to date
  • I’m more employable
  • My career is progressing
 

Laboratory Managers

  • Improved equipment utilization
  • Faster method development/problem solving
  • Flexible workforce with a common standard
  • Better quality data
  • Get up to speed quicker
  • Lower T&E
  • Less reliant on me
  • I spend less time on training
 

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Column Bleed

We considered the importance of carrier gas cleanliness in a previous Essential Guide, please follow the link below:

GC Troubleshooting Gass Supply and Pressure Issues »

Column bleed is a problem in GCMS for several reasons:

  • It creates a high background signal – reducing detector sensitivity
  • It adds ions into the background which will appear in all spectra for unknowns – reducing the quality of spectra generated
  • It will contaminate the source components – reducing sensitvity and altering the voltages required for optimum response

Clean carrier gas is essential to prolong the lifetime of GC columns, and is required to achieve less noisy baselines and good peak shape. 
Figure 1 shows a typical oxygen catalysed stationary phase degradation mechanism.

 

fig 01
« Figure 1.
Siloxane bleed – six membered ‘back-biting’ O2 catalysed degradation of polysiloxane based capillary GC stationary phases

 

 

 

 

 

 

 

 

 

 

 

 

 

One should take possible precautions to reduce the amount of column bleed enetering the detector.
This includes:

  • Using gas traps / filters to ensure the carrier is dry and free from oxygen
  • Use low bleed (MS certified) columns where possible
  • Use thinner stationary phase films (bleed increases with film thickness)
  • Use low polarity phases (bleed increases with column polarity)
 
 

Gas Traps / Filters

To reduce gas contamination, high purity gases are used in conjunction with gas purifiers (‘traps’). The traps are fitted as close as possible to the GC instrument to reduce contamination between the trap and the instrument. Table 1 shows which trap types are recommended for the various gas supplies and detectors typically used in G

Molecular sieve traps: are used to trap water vapour thus increasing column and oxygen trap lifetimes.  Molecular sieves are known to reduce baseline noise from sensitive detectors such as ECDs and mass spectrometers.

Moisture traps: usually contain molecular sieve that is heat treated and packed under vacuum. Removing moisture from the carrier and detector fuel gases prolongs column lifetime and ensures low noise baselines with good detector sensitivity. The moisture trap will also protect the oxygen filter further down the gas line.

The indicator for modern moisture traps have been altered from the traditional cobalt blue dye according to European Safety directives. Newer traps use a silica based indictor which changes from orange/brown to off-white when exhausted.

Modern moisture traps have capacities of around 7-10 grams (H2O) and will typically reduce the carrier hydrocarbon content to less than 1ppm.

fig 02Oxygen traps: Oxygen contamination in the carrier gas can produce excessive column bleed at elevated column temperatures caused by oxidative degradation of the stationary phase.  It may also cause oxidation reactions with the gas phase ionisation products in the ion source of the MS detector.

Oxygen traps usually contain aluminium oxide which has a high capacity for getting and binding (fixing) moisture.

Modern oxygen traps have capacities of around 1000 mL (O2) and will typically reduce the carrier hydrocarbon content to less than 50ppb.

 

Figure 2 . Combined gas trap unit.  Courtesy of Agilent Technologies »

Gas suppliers all have proprietary names for high quality gas – however in general they all carry products that can be categorized as ‘Certified’ and ‘High Purity’. Wherever possible the gas supplied to the GC should contain less than 1ppm of the relevant impurity and be designated as (‘five nines’) 99.999% pure or better

Table 3 shows typical gas purity when using commercial gas filter systems.  This can be achieved with an input gas of ‘four nines’ (99.99%) purity or above:

 

Trap Capacity (g) Efficiency (ppb) Dimensions (cm) Weight (kg
Oxygen (high capacity) 3.5 < 10 5.0 x 37 1.2
Oxygen indicating 0.05 < 0.1 3.2 x 26 0.2
Moisture 12 < 10 3.2 x 26 0.3

Table 3. Typical trap capacities

 

fig

Figure 3. Column bleed produces high background and poor sensitivity in GCMS - bleed product ions are predictable and are highlighted above

 
 
 

Low Bleed Columns

Most stationary phases can be used with GC–MS systems, however, it is a good idea to choose a phase for your application that has the lowest amount of column bleed possible to avoid spurious ions within the mass spectrometer that will confound elucidation and reduce detector sensitivity. Column bleed results from the breakdown of the siloxane backbone of the stationary phase polymer and is promoted by higher temperatures and the presence of oxygen within the carrier gas.

 

Many manufacturers produce stationary phases that are chemically modified to reduce the amount of ‘bleed’ suffered by the column. A typical example is shown here – j b8na 5% phenyl polydimethylsiloaxne column can be produced in two forms; one which has the phenyl groups pendant to the polymer backbone; the other (a so called ‘arylene’ phase), incorporates the phenyl moieties into the backbone of the polymer, so reducing column bleed. A further advantage of these phases, therefore, is their higher upper temperature limit .

Note that whilst bleed is reduced, the selectivity of these two phases will differ and one should take care when choosing a low-bleed ‘equivalent’ phase for an existing method. The following rules of thumb are useful when selecting a stationary phase for GC–MS analysis:

  • Polar stationary phases bleed more than their less polar counterparts
  • Columns with thicker stationary phase films bleed more
  • Longer columns bleed more
  • Be aware of both the gradient and isothermal maximum temperature limits for the stationary phase you are using and do not exceed these limits.

Some of the typical ‘fingerprint’ ions from column bleed products are shown in Table 4.

 

stationary phases

Figure 4. chemically modified stationary phases

 

Ions (m/z)

Compound

Possible Source

13,14,15,16

Methane

CI gas

18, 28, 32, 44 or 14, 16

H2O, N2, O2, CO2 or N, O

Residual air and water, air leaks

31, 51, 69, 100, 119, 131, 169, 181, 214, 219, 264, 376, 414, 426, 464, 502, 576, 614

PFTBA and related ions

PFTBA (tuning compound)

31

Methanol

Cleaning solvent

43, 58

Acetone

Cleaning solvent

78

Benzene

Cleaning solvent

91, 92

Toluene or xylene

Cleaning solvent

105, 106

Xylene

Cleaning solvent

151, 153

Trichloroethane

Cleaning solvent

69

Foreline pump fluid or PFTBA

Foreline pump oil vapor or calibration valve leak

73, 147, 207, 221, 281, 295, 355, 429

Dimethylpolysiloxane

Septum bleed or methyl silicone column coating

77, 94, 115, 141, 168, 170, 262, 354, 446

Diffusion pump fluid

Diffusion pump fluid and related ions

149

Plasticizer (phthalates)

Vacuum seals (O-rings) damaged by high temperatures, use of vinyl or plastic gloves

Peaks spaced 14 Da apart

Hydrocarbons

Fingerprints, foreline pump oil

Table 4:  Some common contaminant ion masses in GC-MS analysis.

 
 

Ferrules

Despite their importance, ferrules are sometimes underestimated; without ferrules the airtight sealing that is required to secure the column at the MS detector and injector of a GC system would be impossible to achieve.

The connections between a GC column injector and detector are points at which leaks can develop. Mass spectrometers are particularly prone to air leaks that can also draw contaminants from the atmosphere into the instrument via the vacuum system within the detector. While all unions are potential leak points, the most problematic is the seal at the transfer line interface of the mass spectrometer.

The ideal ferrule will provide a seal avoiding leaks, must not stick to the column and must tolerate wide temperature changes during programming.

Under the analysis conditions, ferrules should not fragment or allow oxygen to permeate into the system.  Typically,  Graphite/Vespel composite ferrules or metallised ferrules are used for GC-MS applications.


Figure 5. GC and GC-MS ferrules »

 

 

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Column Conditioning

GC columns have relatively small amounts of volatile contaminants originating from their handling, installation and storage; column conditioning involves the heating of the column to remove such contaminants.  So, before it is used, the GC-MS column must be thermally conditioned; otherwise, column residual volatile matter as well as low-boiling point species coming from the stationary phase will be released.  This column degradation process will affect the detector (usually by fouling the ion source).  The lack of column conditioning could result in noisy and drifting baselines.  From this, it follows the GC column should not be connected to the detector during conditioning - -however in practice this is not always possible.

When conditioning your capillary column, please bear in mind the following:

  1. Use a dry and oxygen free carrier gas
  2. A continuous flow of carrier gas must be ensure whenever the column temperature is above ambient temperature
  3. Eliminate any leaks
  4. Do not exceed the maximum temperature limit of the stationary phase

The first part of the GC column conditioning is to purge the column with carrier gas at room temperature.  This removes dissolved oxygen from the stationary phase and will prevent wasteful stationary phase oxidative degradation which might otherwise occur if the column is immediately heated without first purging the dissolved gases from within the bonded phase.

 

Column ID (mm)

Minimum Flow Rate (mL/min.)

Minimum Purge Time(min.)

0.53

5.0

10

0.32

1.5

20

0.25

1.0

25

0.18

0.8

30

0.10

0.5

40


Table 5.  Matching column purge time to column i.d

 

Remember that conditioning is intended to produce an acceptable background signal, free from system peaks, with the required signal to noise ratio for the intended application.  Any further conditioning will unnecessarily shorten the working lifetime of your column.  The practice of overnight column conditioning at elevated temperature should be avoided.  This is usually only necessary when using very thick film columns with highly sensitive detectors or when the column is intended to be operated at its upper temperature limit for extended periods of time.

To condition the column after purging:

  1. Program the column oven from ambient to 20oC above your intended maximum operating temperature or to the columns isothermal temperature maximum limit (whichever is lower) at 10oC per minute
  2. Program the inlet temperature from ambient to the intended operating temperature as the oven begins to heat
  3. Hold the column at the upper temperature according to the table below
  4. Cool the oven and, with the carrier still flowing, connect to the detector if required

Column Length (meters)

Column Film Thickness (µm)

Time for conditioning (mins.)

< 15

< 0.25
0.5 - 1.0
1.0 - 1.5
1.5 - 3.0

20
40
60
80

15 - 30

< 0.25
0.5 - 1.0
1.0 - 1.5
1.5

40
60
80
100

60

< 0.25
0.5 - 1.0
1.0 - 1.5
 1.5

80
120  
160  
200

Table 6.  Suggested column conditioning times according to column geometry

 

Please note that these times are suggestions and you should condition the column sufficiently so as to meet the background response and signal to noise criteria acceptable for your particular analysis.

 
 

Column Dimensions

In GCMS the column flow and sensitivity will depend upon the type of high vacuum pump attached to your detector.  More often than not, the column flow should be something between 0.5 and 2.0 mL/min, with some pumps capable of extended operation at up to 4.0 mL/min.  Therefore, the combinations of column length and diameter are restricted to those that can operate within this range:

  • Columns with internal diameters not exceeding 0.25 mm can be installed directly into the GC interface
  • Columns with internal diameters exceeding 0.45 mm should not be directly installed into the GC interface.  In these cases, the use of vapour concentration devices is recommended
  • In any other case consult your GCMS manual or system manufacturer

At higher flow rates in excess of the recommended operating range, the vacuum in the detector will be comprimised and sensitivity will be reduced, the possibility of analyte ion gas phase reactions increases and the

A large volumetric gas flow into the instrument when using a direct interface will mean a lower vacuum level is attainable.  This may increase the number of background molecules which can collide with the ions formed, leading to a potential reduction in sensitivity and a change in the relative abundance of ions within the mass spectrum.  Ion source filaments will also show reduced lifetimes.

The carrier gas flow rate through the column is influenced by the pressure difference between the inlet and outlet column ends.  This difference is usually larger in GC-MS than in any other forms of GC due to the vacum at the column outlet, so columns tend to deliver larger amounts of carrier gas to MS detectors than non-MS detection systems.

 

Figure 6. GC-MS couplings »

 

 

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Some column dimension / flow rate combinations may lead to very low inlet pressure requirements.  This problem is worst when performing split injection (>10:1 split) with shorter, wider bore columns and with these combinations issues with pressure stability and retention time reproducibility may result.

The figures below show typical pressure drops resulting from range of column dimensions with a vacuum outlet – the regions shaded in blue are those in which accurate and reproducible pressure control may be an issue.

 
inlet pressure and column length   inlet pressure and column length
     

Figure 7: Plots of inlet pressure and column length, with helium carrier gas and vacuum compensation on. Column inner diameters (mm): (a) 0.20, (b) 0.25, (c) 0.32, (d) 0.53; column temperature: 50 °C; average linear velocity: 40 cm/s. The blue shaded area designates negative inlet pressures.

 

Figure 8. Plots of inlet pressure vs. column length, with hydrogen carrier gas and vacuum compensation on. Column inner diameters (mm): (a) 0.20, (b) 0.25, (c) 0.32, (d) 0.53; column temperature: 50 °C; average linear velocity: 40 cm/s.

 

Alternatives to the direct interface that help alleviate the underwater effect include the open split interface and the use of restrictors.

By diverting only a portion of the column’s effluent into the detector, the open split interface uses a restrictor tube to transfer the column’s effluent and make-up gas to the MS source.  In this design, the column exit remains close to atmospheric pressure. As only a fraction of the solutes enter the source, sensitivity is reduced.

Figure 9. The open split interface (only a fraction of the columns effluent and make-up gas are delivered into the MS source) »

  inlet pressure and column length
 

It is also possible to connect a restrictor at the column outlet with a zero-dead-volume connector and then pass the restrictor through a direct interface into the MS source. The restrictor length and inner diameter need to be chosen according to the column flow rate, so that the column outlet pressure will be high enough to bring the inlet pressure above atmospheric levels. This arrangement is comparatively simple, but it will cause EPC display of the column flow and velocity to be in error.



Figure 10. The GC-MS Restrictor »

 

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Analytical Chemists

  • I feel empowered to fix things
  • I can troubleshoot effectively
  • I know where to go for help
  • I understand my analyses
  • I know where to get applications
  • I’m up to date
  • I’m more employable
  • My career is progressing
 

Laboratory Managers

  • Improved equipment utilization
  • Faster method development/problem solving
  • Flexible workforce with a common standard
  • Better quality data
  • Get up to speed quicker
  • Lower T&E
  • Less reliant on me
  • I spend less time on training
 

Subscribe for $399 per/year and access:

  • The entire e-Learning archive
  • All Essential Guide Webcasts and Tutorials
  • LCGCs archive of articles and webcasts
  • Expert troubleshooting advice when needed
subscribe now

 

Ionisation and the Ion Source

We are not going to fully develop the theory of ionisation in GC-MS as that is out of the scope of this Essential guide; however, rather, a short introduction to the theory is presented here.  For more information on this subject, please follow the links below:

The CHROMacademy Essential Guide Tutorial to Understanding GC-MS Analysis - available to Premier Members only

The CHROMacademy Essential Guide to Understanding Electron Ionization for GC-MS - available to Premier Members only

 

 
 

Electron Ionisation (EI)

A typical electron ionization source takes the gas phase analyte ion from the gas chromatography column and, under vacuum conditions, exposes the analyte to a stream of thermionic electrons produced from a resistively heated filament. These electrons cause analyte molecules to ionize (and subsequently fragment or re-arrange) producing positively charged molecules (cations). These cations can then be repelled from the source whilst being formed into a collimated ‘ion beam’, focused and accelerated into the mass analyzing device using electrostatic lenses

 

Figure 11. The EI ion source process »

 

 

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Most EI spectra are recorded at 70 eV, primarily because most species which are likely to be investigated using GC-MS will ionize as the electron de Broglie wavelength (matter wavelength) matches the length of common bonds in organic molecules (circa. 0.14nm) and at this energy the transfer of energy from the ionizing electron is maximized. Typically at 70eV the energy inparted to the analyte will resultin the abstraction of a single electron from the analyte resulting in a single charged radical cation as shown by the general schema below.

eq 1

(where M is the analyte molecule)

 

Under these conditions around 1 in every 1000 analyte molecules is ionized (0.1% ionization efficiency). Maximum energy transfer also leads to maximum fragmentation of most common organic compounds – which can be both a good or bad thing depending on the required outcomes of the analysis.

Further 70eV represents an ionization energy plateau on which small variations in electron energy will not adversely effect ionization efficiency and so library searching will be a practical reality.

At higher electron energy, the de Broglie wavelength of the electrons becomes smaller than the bond lengths in typical analytes and as such the analyte molecules become ‘transparent’ to the ionizing electrons and ionization efficiency decreases very quickly.

At lower electron energy, the efficiency of ionization also decreases, although some ionization will occur down to around 20eV. This can be usefully used when attempting to reduce the degree of analyte fragmentation in order to generate a more intense molecular ion (i.e. the intact radical cation of the parent molecule) within the spectrum, which can be of great use for spectral interpretation. As electron energy reduces, the degree of analyte fragmentation reduces and thus the chance of recording an intact molecular ion increases. The efficiency of ionization also decreases dramatically however, and a reduction in the absolute ion intensity must be balanced against any enhancement in the molecular ion intensity.  It should also be noted that library searching of the spetrum will not be poissible when using an electron energy of anything other than 70eV due to the effect on relative intensities of ions within the spectrum.

The useful ionization energy window for most bench top GC-MS instruments will be around 20-80eV.

The mass spectrum of a β-lactam (a cyclic amide) illustrates the effect of ionizing electron energy on ion abundance - use the slider to alter the ionization electron energy.

 

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« Figure 12:  Effect of ionizing electron energy on ion abundance and degree of ionization for a lactam compound under electron ionization conditions.

 

At 70eV the impacting electrons are usually energetic enough to break chemical bonds within the molecule and so molecular fragments are formed.  The fragmentation of ions formed under given conditions will always give the same quantity and pattern of ions.  This fragmentation pattern becomes a fingerprint that can be used to identify and quantitate the molecule being ionised.

 
 

The EI Source

An exploded diagram of an electron ionization source is below. Most EI sources have a cylindrical geometry to ensure the proper concentric alignment of the source elements, will typically have low thermal mass and be slotted in some way to improve the vacuum level within the source.

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Figure 13. The EI ion source components.

 

Source Body: The source has a cylindrical geometry to ensure the proper concentric alignment of the source elements.  Electrons are emitted by one of the filaments and enter the ion source volume that is at ground potential. A small permanent magnet serves to focus the electron beam.  The positive ions formed are expelled from the source volume through a series of electromagnetic lenses that focus the ions into a tight beam prior to entry into the mass analyser. The source body is slotted to allow the vacuum system to pump away carrier gas and unionised solute molecules. The source is usually independently heated to avoid condensation of sample components.

Filaments: In addition to the ac power source that heats the filaments, each filament carries a –70 V dc bias voltage. The emission current is variable to adjust the energy of the electrons being emitted.  A ‘trap’ may be fitted to the opposite side of the source to monitor the electron density which regulates the circuit to ensure this is kept constant.

Repeller: positively charged plate or ‘funnel’ used to repel the ions from the ion volume towards the mass analyser.

  • A positive charge on the repeller plate pushes positive fragments and parent ions out of the source via the lenses.  The ions only receive a fraction of the repeller energy
  • The repeller voltage can be varied to achieve tune target ion abundances
  • Too low a repeller voltage results in poor sensitivity and poor high mass response
  • Too high a repeller voltage results in poor mass filtering (precursor ions) and poor low mass resolution
  • The repeller voltage required to achieve specific ion abundances during tuning will increase as the source becomes dirty through deposits of neutral and less volatile sample materials – the repeller voltage can therefore be used as a diagnostic tool to evaluate source cleanliness

 

Ion Focus and Transport Lenses: The voltage on the ion focus lens or lenses can be varied to collimate the ion beam and may also contain a ‘beam former’ in the guise of a slot or hole within the lens or an associated plate element which is typically at ground potential.

  • In general increasing the ion focus voltage improves sensitivity at lower masses
  • Decreasing the ion focus voltage improve sensitivity at higher masses
  • Poor ion focus adjustments result in poor high mass resolution

 

Lens stacks typically have an increasing potential applied to them to accelerate the ions through this region of the ion source.

Entrance Lens: The entrance lens is situated at the entrance to the mass filter and passes ions into the tunnel radius of the mass analyser – thus minimising the effects of fringing fields at the rod ends that discriminate against high mass ions.  Accelerating and focussing voltages may be applied to this lens to ensure rapid transit of a collimated ion beam.

 
 

EI Source tuning
The voltages applied to the various ion source components must be ‘tuned’ to achieve target ion abundance, relative and isotopic abundances for various analyte ion masses across the likely mass range. This ensures optimum instrument sensitivity and a predictable response across a range of masses, which allows for subsequent library searching of spectra.

During instrument-led tuning (sometimes called ‘autotuning’), the relative and absolute abundances of fragments of a known tune compound are established and the mass assignment, resolution and spectral peak width generated by the mass analyser are also adjusted and set.

The tuning process involves adjusting a number of mass spectrometer parameters. Some are purely electronic and only affect the way the electronics process the signal. Other parameters affect the voltage settings or current to ion source components, the mass analyser and detector. In general terms, the tuning process will set:

  • voltages on the source elements
  • gain and offset of the mass analyser for correct spectral peak width (resolution)
  • the electron multiplier voltage
  • the mass axis for proper mass assignment

 

The importance of tuning cannot be overemphasized and is performed to check the mass spectrometer is working correctly and/or to ensure that spectra (mass assignment and relative abundance of spectral signals) resemble a previously determined standard. The process of tuning will:

  • check that spectrometer contamination or degraded electronic components have not changed assigned mass positions or the sensitivity of the instrument measured using the absolute abundance of specific ions in the tune compound
  • ensure repeatable analysis from instrument to instrument (or lab to lab)
  • check the spectrometer gives expected relative ratios of ion fragment intensities for a target compound
  • act as a diagnostic tool and system chronicle to indicate the service/cleaning requirements of the spectrometer
  • match fragments from a known calibration compound and adjust mass axis so it agrees with the expected mass assignments
 

Several compounds might be used to calibrate the mass spectrometer, one of the most common tuning standards is perfluorotributylamine, also known as PFTBA, FC-43 or heptacosafluorotributylamine (heptacosa).

 

Figure 14. Structure of PFTBA Tune compound (C12F27N, M.Wt. 671.09 Da) »

 

 

 
 

Perfluorotributylamine has several characteristics which make it amenable for use as a tuning standard including:

  • It is a liquid that is volatile at room temperature and under the vacuum conditions inside the spectrometer. The liquid is kept separate from the system by means of a valve. When the instrument requires calibration the valve is opened and the calibration gas allowed to volatilize into the spectrometer. The calibration gas is ionized in the mass spectrometer EI source and its fragments are mass filtered and detected
  • It fragments very reproducibly and predictably under EI conditions to yield ions across a wide mass range and with reproducible relative ion intensity
  • It does not contain protons and therefore there is no issues with mass deficit and the C13 isotope peak can be easily used to measure relative isotopic abundance

EI-MS spectrum of perfluorotributylamine

« Figure 15. EI-MS spectrum of
perfluorotributylamine (oversimplified).

 

 

Although PFTBA is the dominant calibration compound for EI there are other alternatives such as perfluorokerosene (PFK), perfluorotributylamine, perfluorophenanthrene, etc.  Tables ## to ## provide EI tuning spectral information for such compunds.

 

Table 7: EI Positive Ion Spectra for Perfluorotributylamine

m/z

Rel. Abundance

m/z

Rel. Abundance

69

100

176

1.5

70

1.1

181

1.8

76

0.7

214

1.2

81

0.6

219

50.7

93

1.3

220

2.3

95

0.6

226

1

100

17.7

264

13.4

101

0.7

265

0.7

112

0.8

314

1.8

113

0.8

326

0.3

114

7.9

352

0.7

119

13

376

0.4

131

47.9

414

3.4

132

1.7

415

0.4

145

1

426

0.6

150

2.8

448

0.2

164

1.4

464

1.5

169

4

502

2.6

 

Table 8: EI Positive Ion Spectra for perfluorokerosene

m/z

Rel. Abundance

m/z

Rel. Abundance

m/z

Rel. Abundance

18.01

30

330.98

3.7

666.96

0.5

28.01

50

342.98

1.8

680.96

0.2

31

3.8

354.98

0.8

692.96

0.25

39.96

1.7

368.98

0.9

704.96

0.4

51

6.7

380.98

2.3

716.96

0.25

69

100

392.98

1.1

730.95

0.2

81

0.5

404.98

1

742.95

0.25

84.97

1

416.98

0.55

754.95

0.5

93

3.3

430.97

1.85

766.95

0.2

99.99

5.6

442.97

1.2

780.95

0.25

118.99

26.4

454.97

0.8

792.95

0.3

130.99

24

466.97

0.5

804.95

0.15

142.99

1.9

480.97

1.4

816.95

0.05

154.99

1.4

492.97

1.1

830.95

0.1

168.99

17

504.97

0.65

842.95

0.1

180.99

8.75

516.97

0.5

854.95

0.1

192.99

8.3

530.97

0.7

866.95

0.05

204.99

1.5

542.97

0.6

880.94

0.1

218.99

8.6

554.97

0.5

892.94

0.1

230.99

8.8

566.97

0.6

904.94

0.05

242.99

3.8

580.96

0.7

916.94

0.05

254.99

1.2

592.96

0.65

930.94

0.05

268.98

4

604.96

0.6

942.94

0.05

280.98

6

616.96

0.5

954.94

0.05

292.98

2.7

630.96

0.5

966.94

0.05

304.98

1

642.96

0.5

980.94

0.05

318.98

2

654.96

0.55

992.94

0.05

 

 

Table 9: EI Positive Ion Spectra for perfluorophenanthrene

m/z

Rel. Abundance

m/z

Rel. Abundance

55

1.83

217

2.05

56

1.09

219

2.66

57

2.97

231

4.06

69

100

243

7.66

70

1.17

255

2.09

93

5.97

267

2.55

94

1.27

286

2.09

100

11.09

293

7.65

112

1.5

305

1.14

119

16.8

317

2.27

124

1.49

331

1.12

131

47.08

343

1.17

143

5.26

367

1.88

155

3.05

405

4.23

162

5.64

455

18.3

169

7.66

505

4.41

181

11.23

517

1.17

193

6.73

555

4.62

205

2.82

605

1.64

 

The following table contains information regarding the signals typically used by manufacturers to tune the mass spectrometer.
 

 

Table 10: Selected important signals and abundances in a typical EI-MS spectrum of perfluorotributylamine.

Mass

Exact Mass

Indicative Abundance

%Relative Abundance (approx.)
(m/z 69 as base peak)

%Relative Isotopic Abundance (approx.)

69

68.99521 (CF3)

1974784

100.00

 

70

 

 

 

1.0

219

218.985629

1161216

58.80

 

220

 

 

 

4.0

502

501.971161

56648

2.87

 

503

 

 

 

10.0

 

 

When tuning with perfluorotributylamine, the calibration (or ‘autotune’) process typically begins by matching a single ion mass-to-charge ratio (m/z) to the internal instrument mass scale; this is typically achieved using the base peak of the spectrum, which will typically be m/z 69 and this represents the first mass ‘calibration’ point. Calibration continues by adjusting the internal instrument mass-to-charge scale until all m/z ratios across the range 69 to 502 are accurately calibrated. Note — the accuracy of the mass-to-charge axis calibration will depend upon the type of mass analyser being used. A quadrupole instrument will typically be calibrated to unit mass resolution (integer value of mass), whereas as a high resolving power instrument such as a time-of-flight (TOF) analyser may require more accurate calibration (typical GC–TOF-MS mass accuracy will be in the range of 5 ppm or better).

The calibration spectrum (Figure ##) should show low levels of both air (m/z 18) and water (m/z 28) as well as low number of background peaks (less than 100 background peaks is considered to show a ‘clean’ MS instrument).

Signals from the main PFTBA fragments should have a Gaussian peak shape and intensities should reflect the relative intensities shown in Table 1, within manufacturers tolerance. The C13 isotopic peak at m/z +1 higher than the monoisotopic signal should be clearly separated and should also have the correct intensity relative to the monoisotopic signal.
See Figure 16.

 

Signals from the main PFTBA fragment ions

 

Figure 16. Signals from the main PFTBA fragment ions
typically used to calibrate GC–EI-MS instrument response.

 

It is important that the EI source and mass analyser combination is able to report the correct relative intensity of the fragment peaks as the relative abundance of ion signals is critical in both ab initio spectral interpretation (indicating the relative stability of the ions created) as well as being essential for library searching, where relative intensity is a key search criteria. The relative isotope peak area ratios are also important in analyte characterization where the cluster of isotope peaks (especially around the molecular ion) can be used to postulate an empirical molecular formula.

Typically, during the tune process, each key ion source component will have a ramped voltage applied whilst the abundance of key fragment ions are monitored for abundance. The resulting instrument output is shown in Figure 17.

 

Optimization of the repeller voltage

« Figure17. Optimization of the repeller voltage during an EI source tune. Ion abundance of fragment ions across a wide mass range are plotted against applied voltage to yield the optimum ‘average’ value.

 

Manufacturers use the repeller (‘ion extraction’) or other source component voltage to track source cleanliness. As the repeller surface becomes coated with uncharged sample component residues, the electrostatic voltages which are required to generate a target ion abundance will increase, hence a correlation can be drawn between source cleanliness and the applied voltage. As each source component becomes occluded with neutral sample component residues, its performance will deteriorate until the source needs to be cleaned in order to successfully meet tune targets. More information on source cleaning is given later in this topic.

Figure 17 highlights an interesting dilemma. It should be obvious that the ‘optimum’ value chosen for the repeller voltage is an average value and in fact the ion abundance for the fragment of m/z 69 is not optimized at this value. When one is using EI GC-MS in quantitative mode, where the mass of the major fragments produced by the analyte molecule are known, one may prefer to ‘manually’ tune the instrument response in order to optimize on a PFTBA fragment whose mass lies close to the analyte masses used for quantification.  This optimises the instrument settings, and hence it’s sensitivity, to masses more closely aligned to that of your analyte and somewhat helps to mitigate the fact the EI sources cannot be tuned using an infusion of the analyte of interest – unlike many LC-MS instruments where this is possible.

 
 

Tuning in Practice

The auto tune routine is outlined below and is driven by a computer algorithm that iteratively determines the optimum combination of parameters. This is the easiest way to tune the spectrometer and will produce a optimised tune which is widely applicable across the mass range:

  • Find mass peaks • Coarse adjustments of EM voltage and peak widths
  • Adjustment of ion source components to optimise tune mass 3
  • Fine adjustment of EM voltage and peak widths
  • Mass axis calibration
  • Save tune file

The results of a typical auto-tune report (PFTBA) are shown below, together with details of expected ranges and indications of possible degradation of system performance.

 

typical auto-tune report« Figure 18: typical auto-tune report

 

 

 

There are several parameters that need to be examined in this top section of the autotune report.

Peak Shapes should be smooth and Gaussian.  Do not be concerned with peak shoulders unless they become a major component of the spectral peak.  The shoulders should increase in area to reflect the isotopic abundance of 13C.

The following tables reflect some typical manufacturers specifications for an autotune routine and are shown here as ‘typical’ values and to highlight the specific information that can be gained from a tune report.

 

Table 11. Relative ratios for prominent masses.

m/z

% Relative Abundance

69

100 (base peak)

70/69

0.5 - 1.6

219/69

40 - 85

220/219

3.2 - 5.4

502/69

2.0 - 5

503/502

7.9 - 12.3

 

The absolute abundance of mass 69 should be something between 200,000 and 400,000

More on isotopic peaks later, however the isotopic mass should always be an (M+1 ion) i.e. 70, 220 and 503 amu. As PFTBA contains only C13 and N15, the relative isotopic ratios can be easily calculated.  These values should be approximately 69 (1.0) / 219 (4.0) / 502 (10.0).  Any drift in these ratios can indicate that the spectrometer mass axis has not been correctly calibrated or that the system resolution is grossly degraded.

 

Table 12. Target relative abundances.

m/z

% Relative Abundance

50

1.0

69

100.0

131

55.0

219

45.0

414

3.5

502

2.5

 

If there are peaks at 18, 28, and 32 amu there may be an air leak in the system. 

Mass peak widths (PW50) should be 0.55 ±0.1.

Mass assignments:
69.0 ±0.2
219.0 ±0.2
502.0 ±0.2

Note the electron multiplier voltage – a clean source with a relatively new EM horn should report a particular voltage on the detector which attains the absolute ion intensities specified in the manufacturers tune targets.  As the lifetime of the dynode increases and the source becomes contaminated this value will increase.  If the voltage consistently needs to be ramped to a higher value, this indicates that the dynode is reaching the end of it’s useful life, or that the source is contaminated.  Increasing the electron multiplier voltage will increase absolute signal intensity but will not increase instrument sensitvity (as measured by signal to noise ratio) as the noise intensity will also increase commensurately.

All of the other parameters within the autotune will differ from instrument to instrument, but general trends from subsequent autotune reports should be noted.  Any consistent drift in either the positive or negative direction should be noted and repeat autotuned or manually tuned or maintenance carried accordingly.

The lower part of this autotune report shows the mass spectrum obtained for the calibration gas (PFTBA).  Look for the major tune ions at 69, 219 and 502.  This section of the report will also give the relative abundances of each of the ions, and these can be compared with the table above.

 
PFTBA auto-tune report  

« Figure 19: PFTBA auto-tune report

Features to check in the PFTBA spectrum include:

Presence of large peaks at 18, 28 or 32 will indicate an air or water leak - The data system will allow the operator to perform an air and water check to quantify the amounts within the spectrometer at any given point in time.  This can be a useful tool to check the vacuum system and spectrometer operating temperatures are equilibrated, or to check if a suspected leak has been properly fixed. Remember that the quality of the vacuum within the GCMS will have a direct bearing on it’s sensitivity and regular vacuum pump maintenance, including ballasting of the roughing pump, is recommended.

Presence of a large number of peaks across the spectrum constitutes what is known as a ‘high background’.  This can arise from several sources of contamination such as column bleed, septum bleed, oil contamination and various other sources.  A reference table of masses is given below to help identify the source of several common contaminant ions.

 

Table 13: Some common contaminant ion masses in GC–MS analysis

Ions (m/z)

Compound

Possible Source

13,14,15,16

Methane

CI gas

18, 28, 32, 44 or 14, 16

H2O, N2, O2, CO2 or N, O

Residual air and water, air leaks

31, 51, 69, 100, 119, 131, 169, 181, 214, 219, 264, 376, 414, 426, 464, 502, 576, 614

PFTBA and related ions

PFTBA (tuning compound)

31

Methanol

Cleaning solvent

43, 58

Acetone

Cleaning solvent

78

Benzene

Cleaning solvent

91, 92

Toluene or xylene

Cleaning solvent

105, 106

Xylene

Cleaning solvent

151, 153

Trichloroethane

Cleaning solvent

69

Foreline pump fluid or PFTBA

Foreline pump oil vapour or calibration valve leak

73, 147, 207, 221, 281, 295, 355, 429

Dimethylpolysiloxane

Septum bleed or methyl silicone column coating

77, 94, 115, 141, 168, 170, 262, 354, 446

Diffusion pump fluid

Diffusion pump fluid and related ions

149

Plasticizer (phthalates)

Vacuum seals (O-rings) damaged by high temperatures, use of vinyl or plastic gloves

Peaks spaced 14 Da apart

Hydrocarbons

Fingerprints, foreline pump oil

 

 
 

Manual Tuning

Whilst it is useful to apply auto-tune values and parameter targets for EI tuning, there is no facility to tune on ‘YOUR COMPOUNDS’. This is often overcome by optimising the tuning parameters to optimise response of the mass spectrometer using a tune compound mass nearest to your compounds of interest – e.g. if your compounds are typically in the mass range 100 – 150 amu (Da) then the 131 peak within the PFTBA spectrum may be selected (in most instruments) as being ‘typical’ of your analyte species, and the tune centered around this mass. Note that autotune will not allow the change in mass – rather the instrument is autotuned first, and then each element manually tuned using the 131 peak to optimise at this mass. Some manufacturers however  will allow the operatuor to build specific autotune routines where new alternative ions and tune performance targets may be specified.  There are several strategies for developing manual (user) tune approaches but ultimately it is the experience of the operator in optimising the MS parameters that will govern whether or not this approach is valid.

These techniques can be very useful tools for quickly optimising certain parameters.  For example if maximum sensitivity is required at lower mass, the user tune will allow the operator to ramp the amu offset (a detector parameter – more details to follow in this Essential Guide) to its optimum value by providing a graphical output to parameters entered by the operator.

To examine each of the tuneable components in turn would take too long however a general principle applies to each of the voltages which may be ramped within the spectrometer.  In each case selected ion abundance is measured across the voltage range selected for each source component, the optimum value for the ion or ions of interest may then be selected.

When optimising lens voltages ensure that the curve produced is smooth in every case.  A noisy or non-sigmoidal curve will indicate that the component is dirty, damaged or very far from its optimum value.

In every case an optimum (or curve maxima), should be able to be located.  This indicates that the voltage (or current) applied to that particular component is the optimum with respect to the other system voltages at any given time.  Of course all of the system voltages are interactive and the process should be repeated iteratively to optimise each of the system components.

 
Repeller voltage optimization  

« Figure 20: Repeller voltage optimization.  Note that 25 V ensures good intensity for all masses, however the voltage chosen optimises on the response of the 131 amu (Da) signal

 

AMU Gain and Offset are Quadrupole related parameters that can also be ramped either manually or using the ‘Ramp’ function to enable spectral mass resolution and mass abundance to be optimised for a particular ‘Target’ mass.  These parameters will be explained further in the “Mass Analysers” section.

 
 

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Quadrupole Operation

Mass AnalyzersIn quadrupole mass analysing devices, electric fields are used to separate ions according to their mass-to-charge ratio (m/z) as they pass along the central axis of four parallel equidistant rods (or poles). Ion separation is performed by using controlled voltages applied to the mass analyser rods, which impart an electrostatic field inside the analysing device.

Figure 21: Schematic representation of a Quadrupole Mass Analyser – note that the internal radius has been extended for clarity »

 

 

 

 

 
 

As long as x and y, which determine the position of an ion from the centre of the rods, remains less than r0, the ion will be able to pass through the quadrupole without touching the rods.  This is known as a non-collisional or stable trajectory.

 

« Figure 22. Quadrupole

 
 

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. This is known as an unstable or collisional trajectory.

 

Where;
Ф is the applied hyperbolic field within the analyzer
x and y are the distances along the co-ordinates axis
ro is the distance from the z-axis to either quadrupole surfaces (Quadrupole Radius)
All other terms are as defined in the previous section
U is the DC voltage applied to each rod
V is the AC voltage applied to each rod
ω is the oscillation frequency
t is the time

 

« Figure 23. Stable and collisional trajectories within the quadrupole

 

 

When using a Mathieu Function to solve the equations of motion for an ion within a quadrupole, two factors a (directly related to the applied voltage U) and q (directly related to the applied voltage V) emerge as being important in defining regions of stable ion trajectory:

Where z is the number of electrostatic charges that the ion carries and the other parameters were defined above.

 

« Figure 24. Mathieu diagram for a quadrupole

 

 

 

Figure 25. Changing quadrupole gain and offset

 

The region denoted by the letter A is often referred to as the ‘first stability region’ is the typical operating region of quadrupole mass analyzers.  When you view the exploded diagram of this region, the shaded area denotes the ratios of a against q (U against V or DC against RF) where any particular m/z value will possess a stable trajectory in the quadrupole.  The symmetrical nature of the diagram around the q axis denotes that this region can be operated when looking for both positive and negative ions.   The DC (U or a) and RF (v or q) voltages are altered in a linear relationship and this is referred to as the scan line.  Each ions stability region is bounded by the scan line and the outline of the stability diagram, representing ratios of DC and RF voltages that will result in a stable trajectory within the mass analyser.  As ion stability diagrams often overlap, particularly for similar m/z values, the scan line is adjusted such that only the apex of ion stability region is taken.  This ensures that only one m/z is stable in the quadrupole mass analyzer at any one time. 

 

The slope of the scan line is often referred to as the quadrupole gain and the intercept on the y-axis is often referred to as the quadrupole offset.  Figure 9 demonstrates quadrupole gain, offset and the scan line along with how the scan line affects the observed peak for each of the ions, m1, m2 and m3.

  • The DC and RF voltage ratio (scan line) is adjusted such that all m/z ratios across the defined mass range possess equal peak widths.   The mass range of the mass analyzer is the difference between the highest and the lowest m/z ratios – typically from 2000 - 2 m/z
  • As the scan lines moves toward the apex of the ions stability region an increase in mass resolution (decrease in the width of the spectral signal) is observed, and vice versa
  • Quadrupoles are low resolution mass analyzers with resolving powers in region 500 – 2000.  They are capable of unit mass resolution whereby they can resolve adjacent m/z’s that differ by only 1Da
  • When the scan line moves towards the apex of the ions stability region a decrease in mass sensitivity is observed, and vice versa
  • Quadrupoles are highly sensitive instruments and are capable of detecting down to 50pg in scanning mode and 500fg in SIM mode
 
 

Quadrupole Resolution and Sensitivity

At this point it is important to study the effects of the slope and intercept on the SCAN line.
Increasing the mass GAIN (the slope of the scan line) by increasing the voltages U against V in a constant proportion, will lead to increased spectral resolution but decreased sensitivity (and vice versa). These concepts are explored further in the diagram opposite. It is very important to note that changes in mass gain affect higher mass ions TO A GREATER EXTENT than they affect lower mass ions due to the nature of the changing slope of the line and the larger stability regions of higher mass ions as shown on the examples opposite. Also, if the slope of the line is increased so that it misses the apex of the stability regions of any of the ions then these ions will not be seen in the mass spectrometer.

When increasing the mass quadrupole DC OFFSET voltage (or U intercept) the resolution will increase but sensitivity will decrease –these concepts are represented in the examples below. Again the converse is true when decreasing the DC Offset voltage. It should be noted that altering the DC offset voltage affects ions of all masses to the SAME EXTENT.

Figure ## explains how quadruple gain and offset can be adjusted and their resulting effect on observed spectral peak shapes and hence resolution and sensitivity.  Adjusting the quadrupole offset affects all peaks to the same extent.  Adjusting quadrupole gain affects larger m/z ratios to a greater degree than smaller m/z ratios.

 

Figure 26. Changing quadrupole gain and offset »

 

 
 

It is worth noting at this point that with a linear scan line through the origin the peak widths will increase geometrically with increasing m/z.  This is referred to as constant resolution mode and modern systems are scarcely operated in this way.   As you take a larger part of the ion stability region for the higher m/z ratios the peak widths increase and this will also alter the mass calibration.  Due to the characteristic shape of the ions stability region, with the leading edge increasing three times more slowly than the trailing edge decreases, the center point of the peak will move to a lower apparent mass.   Modern quadrupole instrument operate in unit mass resolution mode whereby a curved scan line will be approximated by either an electronic or software implementation.

However, for ease of understanding it is simpler for us to continue to think of scan lines as linear for the remainder of this article.

 
 

Data Acquisition Modes

Broadly speaking, MS selectivity and sensitivity are determined by the operational principle of the analyser and the optimisation of the applied voltages to ensure good performance. We are going to consider two operation modes, specifically with regard to quadrupole mass analysers in this instance:

  • Scan Mode: the mass spectrometer ‘scans’ over a range voltage settings  to sequentially allow the passage of ions over a selected mass range.  In this case, all ions which transit the mass analyser are recorded and a full mass spectrum is available at several points over the chromatographic peak – depending upon the ‘scan rate’ of the detector
  • Selected Ion Monitoring (SIM): the mass spectrometer voltages are set to allow the passage one (or a few) ion(s) of set molecular mass – all other masses are filtered out.  The selected ion(s) are plotted and only the compounds that produce ions of the selected mass fragments are represented by a peak in the ‘chromatogram’. Obtaining a full mass spectrum or information on non-selected ions is not possible post acqusition.
 
 

Scan Mode

In scanning mode complete mass spectra are repeatedly acquired over a specified mass range. One complete spectrum is obtained prior to the start of the subsequent acquisition.

The abundance of each ion within the spectrum are summed to provide a data point for a plot of total abundance against time — this plot is know as the Total Ion Chromatogram (TIC). The TIC is analogous to the ‘chromatogram’ obtained by LC with UV detection.

 

Figure 27. TIC sulphamethiazole »

 

 
 

In the scan mode of data acquisition, complete spectra are repeatedly measured between two set masses i.e. 50 – 550 Da.  Depending upon the scan range used, this operation can be carried out several times per second to obtain the corresponding number of spectra.  The rate at which whole spectra can be acquired is known as the scan rate and is measured in hertz (Hz)

 

Figure 28. Orthogonal nature of MS data »

 

  Orthogonal nature of MS data
 

As we have seen spectra contain peaks which represent ion fragments at differing intensity.  The intensity is directly proportional to the number of ions of that particular mass which reach the detector.  It follows therefore that if we sum the intensity of each spectral line it will give us an indication of the amount of that compound present within the system at any point in time. 

By plotting the results of these summations on a chart we can produce a plot which shows the amount of a solute present against time.  The sampling rate of this plot will be several hertz and will correspond to the scanning rate of the spectrometer.  This plot is known as a total ion current (TIC) and a representative example is shown in the diagram below.

 

Total Ion Chromatogram

Figure 29.
Total Ion Chromatogram

 

 
Integrated TIC peak  

 

 

The total ion current resembles a chromatogram that has been acquired on a data system that is sampling at the scan rate of the spectrometer.  If the TIC is to be used for quantitative determination it is essential that the scan rate be kept short so that each ‘peak’ within the TIC is modelled as well as possible. 

Individual spectra may by examined by choosing various points from a peak that has been enlarged using the data system.

 

« Figure30. Integrated TIC peak(top) and constituent spectra

 

The recommended number of data points across and chromatographic peak is 20 to ensure reproducibility (ICH Guidelines 1996).  If the scan range is wide it is often possible to attain as many points as this in the super efficient peaks produced by modern gas chromatographs.

Depending upon the ‘peak’ width in the TIC the scan rate must be adjusted so that enough data points (spectra) are collected to model the peak. The amount of data points across a peak to accurately represent the peak shape is estimated at 10-15. The scan rate depends upon the mass range measured and the dwell time at each m/z value.

If the mass range measured is 50-550Da i.e 500Da and the peak FWHM is 10sec., then a scan rate of 0.01sec. per Da is required to ensure that at least one spectrum lies entirely within the peak. From here it necessarily follows that if sensitivity is to be increased, then either the range of masses should be decrased (but some information will be lost) or the scan time should be increased (but even though, a good mass spectrum may not be obtained).

Scan rates for various mass analysers are shown in the figure below:

 

Figure 31. Scan rates for selected mass analysers »

  Scan rates for selected mass analysers
 

Spectral Tilting: when deciding upon the scan rate for data acquisition an important consideration is that of spectral tilting. This phenomenon gives rise to spectra that do not properly represent the ion ratios. If the concentration of analyte eluting into the mass analyser changes during the course of scanning a single spectrum, then the abundances of ions across the spectrum will reflect these changes.

The differences in spectra obtained from scans in various parts of a peak within the TIC are shown in the figure below:

 

differences in spectra

Figure 32. differences in spectra obtained from scans in various parts of a peak within the TIC

 

For qualitative purposes, average spectra are usually taken across the peak at half height. This approach helps to overcome the effects of spectral tilting. To further improve the quality of spectral data, a spectrum of the background - typically obtained from the baseline near the peak - may be subtracted from the analyte spectrum.

 
 
     

Critical MS SCAN Mode Parameters

Start Time: The time (in minutes after the start of the run) at which to activate the scan parameters defined by the entries in each row of the table. A maximum of three scan ranges can be active during a run. The first group is always defined and starts at the end of the solvent delay.

Mass Range: Low and high masses (amu) specify the range to be scanned by the MS. The larger the range, the lower the scans/second.

Threshold: Only ions with abundance equal to or greater than this value will be retained in the mass spectrum of each scan.

Sampling: This value is used to calculate the number of times the abundance of each mass is recorded before going on to the next mass. A value of 2 is suitable for most analyses. The resulting number is reported in Samples and is calculated as 2^N. Range is 0 to 7 although 0 is NOT recommended.

Scans/sec: An approximate value calculated from the mass range and sampling values you have entered. It does not take into account the overhead time needed to process the timed events.

 

Scan parameters

 

Figure 33. Scan parameters

 

mass spectrum

Figure 34. Only ions with abundance equal to or greater than this value will be retained in the mass spectrum of each scan

 

Factors affecting scan time:

  • Number of data points averaged per 0.1 amu increment (sampling rate)
  • Mass range scanned
  • Actual number of ions detected (scan threshold)

Recommendations:

  • Try to achieve 5-10 scans across the chromatographic peak for good qualitative data!
  • Try to achieve 10-20 scans across the chromatographic peak for good quantitative data!
 
 

SIM Mode

To improve sensitivity of the analysis two approaches may be taken:

Initially the scan rate may be increased by decreasing the mass range in order to increase the acquisition time per Da and increase the total number of ions counted. However this may lead to a decrease in the amount of analytical information obtained.

The second approach is used for the analysis of target compounds whose spectra have been previously obtained. In this mode of analysis the highest abundance ions may be chosen and the use of  2-3 ions per ‘group’ is usual - so that the scan speed becomes extremely rapid (cf. 500 ions in the previous spectra). Gains in sensitivity can be enormous and by choosing three ions instead of the 500 in the previous example - 1.66s per Da is available instead of the 0.01s in the previous example. This mode of analysis is known as Selected Ion Monitoring (SIM) or Selected Ion Recording (SIR),

In SIM data acquisition characteristic ions may be chosen to enable identification of the analyte and ion abundance ratios may be measured to confirm identification.

In the SIM mode at least 2 signals of the most abundant or unique ions for a given analyte are monitored.  Actually, the selection of the signals should be done by considering the signal to noise ratio for a given mass.

It is a good practise to use the SIM mode to screen your sample, so if your SIM results suggest the presence of certain compound, then a full scan mode experiment should be done in order to provide a more definitive means of compound identification (complete mass spectrum).

By choosing to set the quadrupole to certain values of a/q (or U/V) 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 mode), or selected ion recording (SIR mode), has certain advantages over scanning wide mass ranges. Because not all m/z values are recorded the mass analyser can carry out a SIM experiment very rapidly (102 - 104 increase over scanning experiment speeds) therefore acquiring more data points.

Further, if only the ‘useful’ m/z values are recorded, i.e. those that are compound specific or most intense, the intensity of the SELECTED ION CURRENT (SIC), that is recorded will be much larger than for the TIC. In this way the sensitivity of the quadrupole mass analyser may be improved for quantitative purposes.

In practice quadrupole instruments can be scanned over mass ranges in excess of 1000 Da in a few milliseconds, giving the advantage of real time spectral monitoring for tuning and diagnostic purposes. However, it is usual to slow the scanning speed (100 ms scans) or carry out SIM experiments when analysing samples in order to increase instrument sensitivity by increasing the ion count at each mass.

It should be noted that when scanning a wide range, a lot of information is gathered which is not used. Points are recorded within each spectrum where there are no ions of interest or where the ions being recorded are associated with the general background and are not solute specific. 

This wastes valuable scanning time and adds the size of the very large data files that are produced by the system. It therefore makes sense to limit the scan range to points that encompass the important  / abundant analyte ions of particular compounds (i.e. 50-300 Da rather than 50 – 550).  Of course, this is not possible when dealing with unknowns, but once a sample has been run and the spectra of different solutes acquired, this can be done with ease.

Once the spectra of solutes have been obtained the most prominent and specific of the ions can be identified.  This information can allow us to operate the spectrometer in a different acquisition mode, selected ion monitoring (SIM).  Instead of defining the range over which we wish to acquire data, specific ions, characteristic of the solutes which we are analysing, may be selected.  In this way the spectrometer is adjusted to looking at a few, individual ions rather than scanning a wide mass range.  This brings several benefits to the chromatographer

  • The number of data points which cover any chromatographic peak is increased (as the scan rate is much faster) which helps us to better model the selected ion current (SIC) peaks for quantitative reproducibility
  • The increase in the number of points and the faster scanning rate gives a large enhancement in sensitivity for any solutes which contain the selected ions (increase in sensitivity of 103 is possible)
  • The selectivity of the experiment is increased – only those analyte ions containing the specified SIM ions are seen in the Selected Ion Chromatogram (SIC)

It is possible to program changes in the groups of ions that are monitored to match the expected elution times of various solutes. In this way it is possible to gain the advantages outlined above, for several solutes within an analysis.

When using SIM the disadvantages are:

  • A reference spectrum for the compound of interest must be available to select appropriate ions
  • Spectral information is lost so any unexpected peaks within the SIC cannot be identified – which risks quantitive innaccuracies if isobaric interferences occur in chromtaographically unreolved peaks (see later on Qualifier ions)
 
 

Critical MS SIM Parameters

  • The dwell time parameter is applied to each ion within a SIM group. The Dwell time specifies the amount of time spent sampling a specific ion. The default of 100 ms is satisfactory for two to three ions in a typical capillary GC peak. For more than three ions, use a shorter dwell time (such as 30 or 50 ms) to ensure there will be enough data points to define the peaks
  • Resolution can be set at either high or low mass resolution. High specifies the mass peak width from the tune file (usually 0.5 amu). Low specifies a mass peak width of 0.7 - 0.9 amu; the increased peak width increases sensitivity with a resulting loss in specificity

 

SCAN vs’ SIM Data Acquisition Strategies

Figure 35. SCAN vs’ SIM Data Acquisition Strategies

 

Choose:

  • Number of ions/group
  • Dwell time/ion to obtain requisite number of cycles/peak for good quantitation
    • Goal:  15 - 25 cycles across a peak
    • Use equal dwell times for all ions
 
 

Choosing SIM ions

  • Use minimum number ions/group for maximum sensitivity and precision
  • Choose ions for maximum specificity
    • High mass
    • Abundant
    • Unique to compound (usually large mass losses)
  • Can choose ions characteristic of compound class for screening purposes
 
 

Calibration

Quantitation (calibration) is accomplished by setting up a quantitation database, which relates the area of an unknown but integrated peak area to a calibration curve that is constructed from Standards of known concentration.

It is recommended that one set up quantitation for each compound by using the response of one ion (usually the base peak) to build the calibration curve and using the responses of 2 or more additional ions to qualify the presence of that compound. These ions must all be present and in the correct ratios to each other. Also, the peak must elute at the proper retention time and all ions purported to be from the same compound must coelute within +/- specified number of scans. This process is known as Target Compound Analysis.  It should be noted that this is the process whether the data is acquired in Scan or SIM mode!

 

Figure 36. Principles of GC-MS calibration and peak recognition »

  Principles of GC-MS calibration
 
When choosing qualifier ions you should bear in mind that the most unique ions are those at highest mass (above 100 m/z if possible) and which are furthest away (in mass) from the molecular ion – larger mass losses tend to originate from more unique fragments. When choosing an ion for quantitation then the most intense ion (base peak) is usually chosen for sensitivity reasons.

Choosing qualifier ions in GC-MS calibration

Figure 37. Choosing qualifier ions in GC-MS calibration

 
 

The calibration curve is the key element in the quantitation process. For a particular compound injected into a particular instrument under static experimental conditions, a plot of response (area) versus the amount injected looks like the figure below. You create this calibration curve by injecting known amounts of a compound, determining the areas of the quantitation ion peaks (extracted ions) by integrating them, and plotting the areas versus the amounts. To quantitate an unknown, you determine the area of the quantitation ion peak for the unknown injection, then calculate the amount using the calibration curve.

NOTE: If the compound, instrument, or experimental conditions change, the calibration curve may no longer be valid. It should be verified by injection of an appropriate test mixture, or the calibration curve should be updated by reanalyzing valid standards.

 

Figure 38. The quantitation / calibration process in GC-MS »

  The quantitation / calibration process in GC-MS
 
 

Internal Standards

The internal standard method eliminates the disadvantages of the external standard method by using an added component in a known amount to serve as a normalising factor. This component, the internal standard, is added to both the calibration and unknown samples.

The compound used as an internal standard should be similar to the target compound, both chemically and in retention time. It must be distinguishable by retention time OR by m/z.

An isotopically labeled target compound makes an ideal internal standard that can be distinguished from the target compound by its ions (signals).

The internal standard and the analyte of interest must exhibit structural similarity, so equal extraction efficiency can be expected for the two of them.  Three types of internal standard are currently used with GC-MS:

  1. A compound that in spite of having different structure than the analyte, it will render the very same ion fragment (this compound will have a different retention time than the analyte of interest)
  2. A compound of the same chemical class of the analyte, so it renders ions with the same m/z values than the analyte (this compound may or may not have the same retention time than the analyte of interest)
  3. A stable isotope labelled derivative of the analyte (usually containing 2H, 13C, 15N, 18O).  This compound will have the same retention time than the analyte of interest

Isotopically labeled derivatives of the analyte are in general the best internal standard selection.

As a rule of thumb, the analyst should be aim for a difference of at least three Daltons between the molar masses of the analyte and internal standard.  This is, if using a deuterated derivative of the analyte as the internal standard, then a minimum of three deuterium atoms per molecule of internal standard are required.

In the following example, testosterone was analysed by using its three deuterated derivative as the internal standard.  Figure39 shows the structure of these compounds and their main EI fragments.

EI main fragments of testosterone

Figure 39. EI main fragments of testosterone (C19H28O2, 288 Da) and its deuterated derivative (C19H25D3O2, 291 Da)

 
Figure 40 shows the EI fragmentation pattern (recorded at 70 eV) for testosterone
 

the EI fragmentation pattern

Figure 40. Testosterone (288 Da) EI Mass Spectrum

 

The selection of the signals corresponding to the molecular ion (288 Da for testosterone and 291 for the internal standard) and the ones corresponding to the most intense fragment (124 Da for testosterone and 127 for the internal standard) seems to be convenient.  In fact, they are intense enough for quantification.  A closer analysis will reveal (data not shown) low signal to noise ratio for the aforementioned signals.

 
 

testosterone and three deuterated internal standard

Figure41.  Selected ion current chromatograms for testosterone and three deuterated internal standard

 
The ration of ion currents is then plotted. See figure 42
 

ration of ion currents

Figure42.  Calibration plot for indicated ratios of ion currents. 
Please note that in this case the amount of internal standard (WInternal Standard) should be a fixed amount, for example 500 ng.

 

This is because, for as long as the ratio of ion currents to the ratio of quantities is linear, quantification is possible.  In other words, different slopes will not necessarily mean that quantitative analysis is not possible.

 
 

TIC vs SIC

In the full scan mode, spectra are continuously recorded (typical scanning speed in the order of 0.2 -1 s).  The chromatographic signal is the sum of the MS signals due to all relevant ions in the selected mass range.

Remember that molecules of different structures exhibit different ionisation, so the number of ions, intensities and peak areas can be different for compounds present in equal molar ratio (even if they have the same molar mass).  The use of a calibration procedure is mandatory.

The data obtained by the TIC can be used either to confirm the presence of certain analyte or to perform quantification.  The TIC data permits to select one or more signals of interest (ions) and extract the information we required in the form of a different chromatogram (also known as the extracted chromatogram).  With this approach not only high specificity but an increase in chromatographic resolution can be achieved.  See figure 43

 
Increased chromatographic resolution by using SIC  

« Figure 43. Increased chromatographic resolution by using SIC

 

Signal selection has to be made by considering ion fragment stability (abundance) and signal to noise ratio.

 

 

 

 

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The Essential Guide from LCGC’s CHROMacademy presents the definitive guide to optimizing methods for GCMS analysis. In this session, Dr. John Hinshaw (Senior Scientist , BPL Global Ltd) and Tony Taylor (Technical Director, Crawford Scientific) consider the major instrument and method decisions that allow you to get the very best from your analysis. We consider sample preparation, GC column and analyser parameters that are critical to getting the best qualitative or quantitative results. Ever wondered how to better select ion source parameters or mass analyzer settings for your target compound? Ever wanted to get a better instrument performance than can be achieved by an autotune routine? Learn how the experts get the best performance from GCMS in this must see webcast for all those working with quadrupole GCMS instrumentation..

John

John V Hinshaw
Senior Scientist
BPL Global Ltd

Tony Taylor
Technical Director
Crawford Scientific

Key Learning Objectives:

  • Understand the implication of good sample preparation in GCMS
  • Selecting the best GC columns and GC separation conditions to maximize GCMS data quality
  • Appreciate the limitation of instrument autotuning
  • Explore which EI and CI source parameters can be optimized and the best approach to achieve instrument optimization for target analytes
  • Learn the principles of quadrupole tuning and discover how to tune for target compounds
  • Appreciate the key spectral data acquisition and analysis variables and learn how to optimize each of them