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The CHROMacademy Essential Guide to Understanding Electron Ionization for GC-MS

The Essential Guide from LCGC’s CHROMacademy presents the second in our series of webcasts on Gas Chromatography-Mass Spectrometry (GC-MS).  In this session, Dr. John Langley (Head of Mass Spectrometry, University of Southampton, UK) and Tony Taylor (Technical Director, Crawford Scientific), present the fundamentals of Electron Ionization (EI) for GC-MS, supported by Interactive Multi-Media from CHROMacademy.  The session will consider topics such as the anatomy of a typical EI source, the mechanisms of ionization, fragmentation and re-arrangement reactions and an introduction to EI mass spectral interpretation. A must see for everyone using or developing methods for GC-MS.

Tony Taylor
Technical Director
Crawford Scientific
Dr. G. John Langley
Head of Mass Spectrometry
School of Chemistry
University of Southampton

Topics include:

  • Why use Electron Ionization (EI)
  • Anatomy of a typical EI source
  • Vacuum and analyte introduction
  • Creation of radical cations
  • Energy of ionizing electrons
  • Transporting ions to the mass analyser
  • Analyte fragmentation and
    re-arrangement reactions
  • Tuning, Troubleshooting & Maintenance
  • Simple tools for EI interpretation

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Key Learning Objectives:

  • Understand when to use Electron Ionization GC-MS analysis and what type of information can the technique can provide
  • Understand the anatomy of a typical EI detector and how each component functions
  • Appreciate the effects of changing electron energy on the resulting mass spectrum
  • Explore the various mechanisms for radical cation formation – including simple fragmentation processes and re-arrangement reactions
  • Gain an insight into Auto-tuning, Tuning and Troubleshooting the EI source
  • Investigate some simple techniques for identifying the molecular ion and using the molecular ion cluster for fundamental interpretation
  • Explore the technique of Isotopic Normalization and other key skills in MS interpretation

The CHROMacademy Essential Guide to Understanding Electron Ionization for GC-MS

Tutorial 24 April 2011

In this session, Dr. John Langley (Head of Mass Spectrometry, University of Southampton, UK) and Tony Taylor (Technical Director, Crawford Scientific), present the fundamentals of Electron Ionization (EI) for GC-MS, supported by Interactive Multi-Media from The CHROMacademy.  The session will consider topics such as the anatomy of a typical EI source, the mechanisms of ionization, fragmentation and re-arrangement reactions and an introduction to EI mass spectral interpretation .  A must see for everyone using or developing methods for GC-MS.

Electron Ionization (EI) is used in mass spectrometry to ionize and fragment analyte molecules prior to mass analysis and detection. While there are a host of methods which can be used to introduce solid or liquid samples into an EI source in this tutorial we will concentrate on sources connected to a capillary column within a gas chromatograph (GC). This technique is known as gas chromatography-mass spectrometry (GC-MS) and typically involves a GC column connected via a transfer device to a mass spectrometer, the simplest and most popular of which will contain an electron or chemical ionization source with a quadrupole mass analyzer and a detector. More complex instruments with triple quadrupole and time of flight mass analyzers are also available.

Figure 1: Schematic of a typical bench top GC-MS system.
  Figure 2: Simplified diagram of a typical EI source.

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.1

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  Animation 1: Typical EI source and ion production mechanism.

Figure 3: Typical electron ionization spectrum of the cocaine molecule – note the large number of intense fragments created.



‘Standard’ electron ionization uses 70eV electrons to induce analyte ionization which is typically much higher than the energy required for ionization.2,3 
The excess energy imparted breaks bonds and leads to fragmentation and re-arrangement pathways which are well characterized and predictable according to some fairly rudimentary principles.  A typical mass spectrum produced from electron ionization is shown in Figure 3.  The high degree of fragementation induced by electron ionization often leads to it being known as a ‘harsh’ ionization technique. 

The fragments and re-arrangement products within the mass spectrum can be used to deduce a molecule's chemistry or identity and because ionization at 70eV is consistent between instruments, spectral libraries are available which makes compound identification a much more straightforward proposition.


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If an ionizing electron passes close to or ‘through’ a neutral gas phase analyte molecule, some of the energy will be transferred to the molecule in the form of translational or rotational energy. However the majority of the energy will be stored as internal modes, where vibration and electronic excitation are capable of the uptake of several electronvolts. The ionization energy of most molecules lies between 6 and 15eV and as such all gas phase neutral molecules which are ‘impacted’ by an ionizing electron will ionize via the removal of a single electron from the highest occupied molecular orbital (HOMO) of an atom within the molecule, forming a radical cation according to the general scheme.4

In general, the order of ease with which electrons are lost under electron ionization conditions is:

Lone pairs > Π-bonded pair > σ-bonded pair

The electron ionization of formic acid is represented in Animation 2.

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Animation 2:  Ionization of formic acid under EI conditions to form the radical cation which will be transferred to the mass analyzer using electrostatic fields and subsequently mass filtered and detected.

Having formed the radical cation from our analyte molecule we must consider what will occur to this highly energetic species.5

Energetics of Ionization
We have established that most organic compounds will have an ionization energy in the region 6 to 15eV.  We have also briefly seen that most of the excess energy imparted by the ionizing electron (55 – 64eV), will be internalized and dissipated via vibrational and electronic excitation modes.
It should also be stated here that radical species are highly excited states and of themselves, not energetically stable.

In electron ionization, the radical cation formed typically undergoes a single or a series of fragmentations OR a re-arrangement reaction, resulting in the elimination of a neutral species in order to achieve a more stable state. The degree of fragmentation or intermolecular re-arrangement will depend upon the analyte molecule (typically on the ionization cross-sectional area and the ability of the molecule to stabilize charge6) and the energy of the ionizing electrons (although this is typically 70eV as stated above).

While there are many complicated schema for the possible ionization and fragmentation/rearrangement pathways in electron ionization, for everyday purposes, we need only consider relatively few potential pathways which are demonstrated in Figure 4 and Animation 3.


Figure 4: Typical fragmentation pathways and the resulting spectrum for an excited radical cation molecule ABC formed during electron ionization.


There are many important considerations relating to the scheme above.7,8

  1. Only charged species are seen in the mass spectrum – neutrals and radical species are not influenced by the elecstrostatic fields within the instrument and therefore are lost to the vacuum within the instrument – i.e. if it doesn’t carry a charge, you won’t see it!
  2. All reactions are assumed to be unimolecular – under the high levels of vacuum within the instrument (10-6 Torr, 10-9 Atmospheres) there are no background species to react with our radical cations of their fragmentation/re-arrangement products.
  3. The intensity of the fragments gives an indication of the relative stability of each species.
  4. As will be discussed subsequently, re-arrangement reactions are restricted in the time domain and certain reactions will pre-dominate depending upon analyte chemistry.
  5. Each of the fragmentation products formed may undergo further fragmentation.
  6. Loss of a neutral typically results in a even mass fragment – something which is highly indicative in spectral elucidation as will see subsequently.
  7. The products formed under 70eV conditions will be predictable and the resulting spectrum can be used as a ‘fingerprint’ of the molecule which can be library searched or used for ab initio spectral interpretation/analyte characterization.

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Animation 3:  Homolytic bond fission within a radical cation


The fragmentation pattern of a branched alkane is shown below.  The intensity of each signal is related to the stability of the ion fragment.

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Animation 4:  Electron Ionization mass spectrum of
3-ethyl-2,3-dimethyl-Pentane (oversimplified)


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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. 01.4nm) and at this energy the transfer of energy from the ionizing electron is maximized. 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. 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.5
This is demonstrated further in Animation 5.

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. See animation 4 for more details.

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) is shown in Animation 5 - use the slider to alter the ionization electron energy.

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Animation 5:  Effect of ionizing electron energy on ion abundance and degree of ionization for a lactam compound under electron ionization conditions.

An exploded diagram of an electron ionization source is shown in Animation 5. 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. 10, 16

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Animation 5: Exploded diagram of the main components of a typical EI source.

  1. Ionization (ion) volume — manufactured from low thermal mass metal, an aperture permits the ingress of electrons to the ion source and a second aperture allows electrons to exit for collection and current monitoring. Most manufacturers include slots or openings in the ion source body to improve vacuum levels within the ion source.
  2. Filament — usually placed outside the ion source and heated by a regulated current to produce thermionic electrons of a constant energy.
  3. Collector — used to monitor electron current/density (some manufacturers only).
  4. Magnetic Field — produced by small permanent magnets used to constrain electron path and impart a ‘corkscrew’ motion to electron path to increase ionization efficiency.
  5. Repeller — positively charged plate of ‘funnel’ used to repel the ions from the ion volume towards the mass analyser.
  6. Accelerating potential — used to accelerate the repelled ions through the ion source.
  7. Draw-out plate — used to form the ion ‘beam’ and reduce noise from spurious free electrons and photons created in the ion volume.
  8. Electrostatic focusing lens — tube lens (will differ according to manufacturer) used to reduce the diameter of the ion beam for successfull transport into the mass analyser.
  9. Transport/Focusing lenses — further lens devices used to focus and accelerate the ions from the ion source, through any potential interfering fields (such as the ‘fringing fields’ at the end of quadrupole rods) and into the mass analyser.

In our generic source design shown in Figure 6, ionizing electrons are emitted by one of the filaments and enter the ion source volume that is at ground potential. The electrons are thermionically emitted from the surface of a resistively heated tungsten/rhenium filament at a constant voltage (typically 70eV as we have seen above). The filaments operate in a vacuum and hence are prevented from immediately burning out, which would occur if they were operating in an oxidative atmosphere. This is another reason for operating the mass spectrometer in a vacuum and why switching on the filament during the pressure surge created by the GC injection solvent passing into the MS isn’t a great idea!

  Figure 6: Straight and coiled examples of filaments used for electron ionization in GC–MS — note the guards behind to prevent back sputtering of electrons.

A small permanent magnet serves to focus the electron beam and provides some cyclical moment to the electron motion, which serves to increase the ionization efficiency by increasing the possibility of an ionizing electron encountering the ionization cross-sectional area of an analyte molecule.

The positive ions formed are expelled from the source volume through a series of electromagnetic lenses that focus the ions into a tight beam.

The source body is slotted to allow the vacuum system to pump away carrier gas and unionized solute molecules. For EI operation the source body temperature should be set to around 200–250 oC and this prevents condensation of the gas phase analyte molecules within the ion source, increasing sensitivity and minimizing ‘fouling’ of the source by analyte and other sample matrix components.

A series of electrostatic components are used to eject the ions formed from the ion volume, form them into a beam and accelerate them toward the mass analyser whilst focusing the beam into the required diameter.

The EI process is demonstrated in Animation 6.

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The voltages applied to the various ion source components must be ‘tuned’ to achieve target ion abundances for various analyte ion masses. This ensures optimal instrument sensitivity and a predictable response across a range of masses, which allows for subsequent library searching of spectra.

During 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
  • Set gain and offset of the mass analyser for correct spectral peak width (resolution)
  • Set the electron multiplier voltage
  • Set 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 to see that spectrometer contamination or degraded electronic components have not changed assigned mass positions (calibration of the mass axis)
  • 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 to indicate the service / cleaning requirements of the spectrometer
  • act as a chronicle of system performance
  • 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 7: Structure of PFTBA Tune compound
C12F27N, M.Wt. 671.09.

Perfluorotributylamine has several characteristics which make it amenable for use as an EI 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 (see later) and the C13 isotope peak can be easily used to measure relative isotopic abundance.
Figure 8: EI-MS spectrum of perfluorotributylamine (oversimplified).
The following table contains information regarding the signals typically used by manufacturers to tune the mass spectrometer.


Exact Mass

Indicative Abundance

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

%Relative Isotopic Abundance (approx.)


68.99521 (CF3)





























Table 1: Selected important signals and abundances in a typical EI-MS spectrum of perfluorotributylamine.

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 9) 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 9.

  Figure 9: 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 fragments are monitored for ion abundance. The resulting instrument output is shown in Figure 10.


Figure 10: 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 10 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.

When examining the spectrum generated by the tune, it is good practice to check for the presence of ions resulting from known sources of contamination. Table 2 lists some common contaminating ions.

Ions (m/z)


Possible Source



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)



Cleaning solvent

43, 58


Cleaning solvent



Cleaning solvent

91, 92

Toluene or xylene

Cleaning solvent

105, 106


Cleaning solvent

151, 153


Cleaning solvent


Foreline pump fluid or PFTBA

Foreline pump oil vapour or calibration valve leak

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


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


Plasticizer (phthalates)

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

Peaks spaced 14 Da apart


Fingerprints, foreline pump oil

Table 2: Some common contaminant ion masses in GC–MS analysis.


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Common ion source problems include:

  • Contaminated with sample/contaminant residues
  • Blown filament wire
  • Broken/shorted heater or sensor
  • Backstream of foreline pump oil

GC–MS ionization processes are typically 0.1% efficient. Non-ionized solute molecules will be adsorbed to the surface of the ion source or transported through the vacuum system to reside ultimately in the rough pump oil or vented to atmosphere.

The GC–MS ion source is therefore subject to contamination. The degree of this contamination is dependant largely on the concentration and nature of the sample. For example, analysis of samples that consist of a dirty/complex matrix will lead to rapid ion source contamination; here increased frequency of ion source cleaning becomes a necessity.


The system will become increasingly contaminated with each tune performed and each sample analysed, as neutral species are adsorbed onto the surface of the various electrostatic components. If possible, ‘screen’ unknowns using an FID or TCD detector before injection into the GC–MS. This will indicate if the sample needs a pre-cleaning step (typically solid phase extraction or similar) and will maximize the time between source cleans.

The GC–MS ion source contains no moving parts, so mechanical wear is minimal.

Broken or shorted filaments will result in the total loss of signal from the instrument. Filament lifetime can optimized by operating at high levels of vacuum and by switching the filament off during the pressure surge created by the elution of the sample solvent.

Backstreaming of pump oil will cause the spectrum to contain a very high number of prominent ions in the mass spectrum.
Ion Source Cleaning
  1. Obtain the necessary tools and supplies.
  2. Disassemble the ion source.
  3. Identify the parts of the ion source that need cleaning.
  4. Thoroughly scrub and clean the parts using fine emery paper (jewelers lap) or abrasive chrome cleaner.
  5. Wash the cleaned parts with solvent (typically washing with heptane/acetone/methanol) and over dry at 70oC.
  6. Reassemble the ion source whilst wearing lint free gloves.
  7. Replace the ion source into the mass analyser body.
  8. Reconnect the wires for the source heater and electrostatic components.
  9. Re-establish vacuum and tune to check source cleanliness.
  Figure 11: When cleaning the source, remember to use the right tools and wear gloves when re-building the cleaned components.

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Electron ionization sources produced ions that can be subsequently used for both quantitative and qualitative purposes. We will consider quantitative data analysis after we have studied mass analysers in the Essential Guide GC–MS Series.

The field of qualitative spectral analysis, or EI spectral interpretation is huge and well documented. However, as is the remit of the Essential Guide series, we will take this opportunity to present a selection of highly practical tips and tricks from the CHROMacademy to get you started on spectral interpretation. We will continue this series with a more comprehensive treatment of EI spectral interpretation in a future Essential Guide.

Of course – if you can’t wait to get access to over 100 topics on GC–MS spectral interpretation, sign up for CHROMacademy now at


EI Spectral Interpretation Stage 1 - Obtaining a Representative Spectrum

Begin by getting as many data points across each spectral peak as you can.

Quadrupole mass analyser scanning speed is increased by:

  1. Restricting mass range to only look for the compounds of interest - typically this will be in the range 40–700 Daltons.
    The narrower the mass range the faster the scan speed.
  2. Restricting the ‘oversampling’ of the mass analyser - i.e., how many measurements are taken for each mass (or 0.1Da).
    This will typically be 2 to 4 for most quadrupole mass analysers.

Whatever happens, try to get at least 7 data points across each peak which is to be structurally elucidated or library searched.

Take a ‘average’ spectrum across the peak at half peak height (which will average all spectra take from halfway up the peak upslope, across the peak apex, to half way down the downslope (See Figure 12).


Figure 12: Half height averaged spectrum taken from an EI total ion chromatogram.

This will avoid the problem of spectral ‘tilting’ which occurs because the analyte concentration is constantly changing across the elution profile. This issue is highlighted in Figure 13.

Figure 13: All of the spectra (634–636) are taken from the total ion current to the left— however due to the changing concentration profile, the relative ration of ions is varying markedly.

Tilting causes the relative intensities of spectral signals to vary according to the changes in analyte concentration during acquisition. Tilting phenomena are accentuated when the spectral acquisition time is ‘long’ in comparison to the peak elution time (i.e., there are a low number of scans across the peak). Spectral ‘averaging’ helps to avoid this problem.

Further, when generating spectra it is important that any ions arising from the ‘background’ signals are removed. This is generally achieved using ‘background subtraction’ data processing techniques, however one must take care not to remove ions from the analyte spectrum.


EI Spectral Interpretation Stage 2 — Library Searching

Some of the most useful tools in spectral interpretation are the many reference libraries which exist in digital form and which can be searched (using a variety of proprietary algorithms), primarily to match ‘unknown’ spectra to those within the library.

There are many comprehensive libraries available including those from:

  • Wiley
  • National Institute of Standards and Technology (NIST)

There are also hundreds of application specific libraries available from a host of providers.

Matching an unknown mass spectrum against a library file requires;17–19

  • The unknown and reference spectra were acquired under identical conditions (70 eV),
  • A database with sufficient information (for example, the NIST database contains spectra from almost 200,000 different compounds, however, smaller databases which are more application specific may also be very useful),
  • A well structured library searching algorithm.

Searching a database will result in a list of candidate compounds for our unknown, with a certain level of confidence based on the parameters of the search algorithm. These usually involve the uniqueness and abundance of ions within the unknown spectrum.


Case one. The spectrum of the unknown molecule we are trying to identify IS in the database

Modern matching algorithms implement an objective function that accounts for the “distance” between the unknown and the reference spectra. As expected, the smaller the value of the objective function (closeness), the more likely that both spectra correspond to the same molecule.

Most matching algorithms implement a pre-screen, where only a limited number of peaks (of certain intensity) are considered versus the full database. Once this step is done, a more refined matching procedure with the results of the pre-screen is performed.

In order to rank the “closeness” between two spectra, matching algorithms implement match factors. In general terms, you can find peaks in the unknown sample spectrum that are not reflected in the reference spectrum, as well as the opposite case (peaks in the reference spectra that are not reflected in the unknown sample spectrum). As a consequence, two different match factors are required:

  • Match factor: accounts for peaks in the unknown sample spectrum
  • Reverse match factor: accounts for peaks in the reference spectrum

The overall matching score parameter considers both the match and the reverse match factors. This parameter indicates how closely the sample spectrum matches the library spectrum on a peak by peak comparison of the two spectra.


Case two. The spectrum of the unknown molecule we are trying to identify IS NOT in the database

When there are good reasons to think that the spectrum of the unknown we are trying to identify is not in the database, one of the following options should be considered:

  • Similarity search: this mode compares the sample spectrum against the spectra of the database to see which spectra are similar, even if they do not match.
  • Neutral loss search: this mode looks for spectra in the database that exhibit the same neutral losses from the molecular ion.
  • Substructure identification: this mode helps to confirm the existence (or not) of substructures within the unknown molecule which give rise to certain characteristic ions.

Figure 14 shows a typical screen from the NIST library search. As can be seen there are many facilities available to compare spectra. Note the match factor in the lower left hand window, which is indicative of the confidence or quality of the match between the suggested spectrum and the unknown.

Figure 14: Typical library search results from the NIST EI GCMS 2008 library.

Many modern spectral libraries and specialist software offer the opportunity for spectral deconvolution - that is to extract spectra of individual compounds from overlapping or poorly resolved peaks. We will consider this topic in a subsequent webcast and essential guide.


EI Spectral Interpretation Stage 3 - Introduction to a Logical Strategy

So, what happens if the spectrum obtained from our unknown compound is not found within a spectral library, or the ‘match’ between the library spectrum and our unknown is poor.
Under these circumstances we must embark upon what is known as spectral interpretation. This type of interpretation falls into three broad categories:

  • We are required to ‘confirm’ the structure of a sample — for example from an organic synthesis.
  • Identify the differences between a suggested library match or our unknown spectrum — such as when the library has given us an indication of the analyte ‘type’ which we must refine.
  • We have no ‘clues’ as to the identity of the compound and must characterize the analyte structure from the spectrum (often known as ab initio interpretation).

The art and science of spectral interpretation is a vast subject, to which we may only hope to provide the reader with some fundamental tools and information. We shall be considering more advanced interpretation tools in a subsequent Essential Guide, but as a general introduction, a logical approach to the task of interpretation can be described as follows:20–21

  1. Obtain a “good” quality spectrum of the component.
  2. Identify the molecular ion.
  3. Consider the general appearance.
  4. Postulate a molecular formula using isotopic normalization/nitrogen rule/rings and double bonds formula.
  5. Identify significant structural fragment ions and elucidate/rationalize.
  6. Identify significant neutral losses and elucidate/rationalize.
  7. Combine information and propose a potential structure.

EI Spectral Interpretation Stage 4 — Identifying the Molecular Ion

The presence of a molecular ion (radical cation of the intact analyte) within a mass spectrum gives us a host of information and possibilities:

  1. It will give use the molecular weight of the species we are analysing.
  2. If we have a high mass accuracy analyser we will be able to postulate a molecular formula from the instrument data system (as mass accuracy decreases the number of possible formulae suggested will increase).
  3. If a low mass accuracy analyser is being used we can use the isotopic pattern around the molecular ion to help postulate an empirical formulae (using a technique known as isotopic normalization).
  4. The molecular weight may give us an indication of the presence of nitrogen within the molecule.
  5. We can look at the fragments at lower mass than the molecular ion and use the mass loss to look for major structural components and check for logical or illogical mass losses.
  6.  We can use the rings and double bonds formula to check the degree of unsaturation within the molecule and double check the fact that we are dealing with a molecular ion.

So how to check for the presence of a molecular ion?

To start, we should emphasize that the molecular ion will not necessarily be the highest molecular weight ion within the spectrum — especially if we have a noisy spectral background. Under these circumstances we should try to eliminate the source of the noise or increase the sampling threshold of the detector/data system combination in order to eliminate the noise (take care not to raise the threshold too high or you may eliminate important structural ions!).


Figure 15: We cannot assume that the ion at m/z 142 is the molecular ion (and therefore represents the molecular mass of the analyte assuming z = 1), several checks are available to help us understand the validity of the molecular ion.


We have previously seen that the molecular ion intensity will depend upon the energetic of the radical cation formed during the EI process and the ability of the analyte to stabilize this energy through various electronic effects (inductive stabilization, delocalization of charge, etc., etc.). Therefore the intensity of the molecular ion tends to be stronger for the following compound classes:

  • Aromatic
  • Highly unsaturated (conjugated)
  • Cyclic
  • Higher order of substitution (i.e., tertiary carbocations are more stable than their primary analogues)

Figure 16: Molecular ions can be more easily identified as the stability of the cation ion increases. Top: EI mass spectrum of benzene.

Bottom: EI mass spectrum of 3-Ethyl Octane.

As we saw previously, perhaps the quickest way to check for the presence of a molecular ion is to reduce the energy of the ionizing electrons below 70 eV in an attempt to reduce the degree of fragmentation induced. As always, this has to be balanced with the loss in absolute sensitivity and is not always achievable.

A second approach is to use a ‘softer’ ionization approach such as chemical ionization (CI), in which the degree of fragmentation of the radical cation is greatly reduced through the use of a ‘reagent gas’ which ionizes the analyte molecules via proton transfer and other related processes. This topic will be covered in more detail in a subsequent Essential Guide.

The final, simple check that can be carried out is to look for illogical losses from the molecular ion. Examination of the proposed molecular ion and the next highest mass fragments and the mass difference between the sets of signals can sometimes reveal that the proposed mass difference (mass loss) is not logical. Figures 17 and 18 contain more details.


Figure 17: The difference between the proposed molecular ion signal at m/z 84 and the fragment ion at m/z 69 is 15 Da — representing the fragmentation of the intact molecule to lose the terminal methyl group from the molecule. This ‘logical loss’ indicates the the ion at m/z 84 MAY be the molecular ion [other ‘losses’ from the molecular ion should be checked, however take care not to go too low in mass (half of the proposed molecular weight is a good guide), otherwise we may fall into the trap of considering second generation fragments (fragments of fragments), which is not a valid approach].


While Figure 17 considers a ‘logical’ mass loss from the propsed molecular ion, there are illogical losses which it is impossible to lose from a compound containing C, H, N or O. If you see ‘losses’ from a proposed molecular ion in these regions then the molecular ion probably isn’t the molecular ion!

    Figure 18: The use of illogical losses to test the validity of a proposed molecular ion in GC–EI-MS.
    • Losses of between 5 and 13 Daltons AND 21 and 25 Daltons CANNOT easily occur
    • This should be considered when attempting to identify a molecular ion
    • To illustrate the unlikely losses – some ‘outrageous’ losses are proposed opposite. NOTE: if your analytes contain Lithium or Berylliumthen these losses MAY occur!

This short introduction into EI mass spectral qualitative data handling represents a broad overview of the topics that can be found within the CHROMacademy General MS Interpretation Strategies module.

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  1. Raymond P. W. Scott, “Tandem Techniques” John Wiley & Sons. Pp 165-173. USA 1997
  2. E. De Hoffmann, J. Charette, and V. Stroobant. “Mass Spectrometry –Principles and Applications.” John Wiley and Sons 1996, 9-20.
  3. Zschornack Guenter, Ovsyannikov Vladimir Petrovich, Grossmann Frank, Koulthachev Oleg Konstantinovich. “Electron Ionization Ion Source” United States Patent No 6717155.
  4. Rosaleen J. Anderson, David J. Bendell and Paul W. Groundwater. “Organic Spectroscopic Analysis” The Royal Society of Chemistry. Pp 120-148. Cambridge UK, 2004.
  5. Robert L. Grob and Eugene F. Barry. “Moderm Practice of Gas Chromatography” Chapter 6. Fourth Edition. Published by John Wiley & Sons, Inc., Hoboken, New 2004.
  6. John McMurry. “Organic Chemsitry”. Brooks/Cole Publishing Company. Third Edition. Ch 5. California, USA 1992
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  8. Milton Orchin, Roger S. Macomber, Allan R. Pinhas, R. Marshall Wilson “The Vocabulary And Concepts of Organic Chemistry” Second Edition. Ch 17. A John Wiley & Sons, Inc., Publication. New Jersey 2005.
  9. James Barker. “Mass Spectrometry” John Wiley and Sons 1999, 19-35.
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  11. Johnson, Bruce S, Khosla, Mukul, Herron, John R, Brassil, John M, and Schoen, Alan E. “Filament assembly for mass spectrometer ion sources” United States Patent # 5,543,625. Aug 6, 1996
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  13. Norio Kameshima. “Electron Ionization ion source” United States Patent # 5196700. Mar 23, 1993.
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  15. Thomas Neil Horsky. “Dual Mode Ion Source for Ion Implantation” United States Patent # US 7800312 B2. Sept 21, 2010.
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  17. Stephen E. Stein, Donald R. Scott. “Optimization and Testing of Mass Spectral Library Search Algorithms for Compound Identification” American Society for Mass Spectrometry 1994, 5, 859-866
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  20. J. Throck Watson and O. David Sparkman. “Introduction to Mass Spectrometry -Instrumentation, Applications and Strategies for Data Interpretation” John Wiley & Sons Ltd, West Sussex PO19 8SQ, England. Chapters 3, 6 and 7. March 2008
  21. F. W. McLafferty. “Interpretation of Mass Spectra.” 3rd Edition. University Science Books: Mill Valley, (1980), 15.
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The following subjects are covered in

The Theory Of HPLC
Introduction (1.5hrs)
Chromatographic Parameters (3hrs)
Band Broadening (3hrs)
Column chemistry (4hrs)
Reverse phase (partition) chromatography (6hrs)
Ion-Pair Chromatography (3hrs)
Normal phase (absorption) chromatography (3hrs)
Gradient HPLC (3hrs)
Quantitative and Qualitative HPLC (3hrs)
FAST HPLC (4.5hrs)
HILIC (3hrs)
SFC (3hrs)
Ion Chromatography(3hrs)

Theory and Instrumentation of GC
Introduction (1.5hrs)
Chromatographic Parameters (3hrs)
Band Broadening (3hrs)
Gas Supply and Pressure Control (2hrs)
Sampling Techniques (4.5hrs)
Sample Introduction (5hrs)
GC Columns (5.5hrs)
GC Temperature Programming (3hrs)
GC Detectors (2.5hrs)
SFC (3hrs)

Instrumentation of HPLC
Mobile Phase Considerations (3.5hrs)
Solvent Pumping Systems (4hrs)
Autosamplers (4.5hrs)
Detectors (4.5hrs)

Solid Phase Extraction
Molecular Properties (4hrs)
SPE Overview (3.5hrs)
SPE Mechanisms (4.5hrs)
Method Development (5.0hrs)
Primary Sample Preparation Techniques (2hrs)
Liquid / Liquid Extraction Techniques (1.5hrs) Approaches to Automation for SPE (1.5hrs)

Fundamental GC-MS
Introduction (1.5hrs)
GC Considerations (4.5hrs)
GC -MS Interfaces (2.5hrs)

Fundamental LC-MS
Introduction (1.5hrs)
Electrospray Ionisation Theory (6hrs)
Electrospray Ionisation Instrumentation (4hrs)
Mass Analyzers (9.5hrs)
Atmospheric Pressure Chemical Ionisation (3.5hrs)
Atmospheric Pressure Photoionisation (3hrs)
Solvents, Buffers and Additives (3.5hrs)
Vacuum Systems (3hrs)
Flow Rates and Flow Splitting (3hrs)
Orbitrap Mass Analyzers (3hrs)

MS Interpretation
General Interpretation Strategies (11hrs)
Intro to MS Proteomics Research (3.5hrs)

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