Gas Chromatography-Mass Spectrometry (GC-MS)

Overview

GCMS involves the combination of two powerful techniques, gas chromatography (GC) and mass spectrometry (MS).  GC separates the volatile compound(s) of a sample while the MS portion of the tool can provide a mass spectrum from each separated component, helping to confirm its identification.  Combining these two systems help overcome their limitations when operating alone.  Conventional GC does an excellent job at separating mixtures of samples.  However, it is difficult to positively confirm the identity of unknown samples.  For this reason, it is most often used for the analysis of knowns.  MS, on the other hand, is very good at providing detailed spectral information of samples.  But MS operated alone is not a separatory technique.  As such, MS spectra of mixtures can be extremely complex making it difficult to know which signals within the spectra are associated with each compound.  Operating these two techniques together helps to overcome these limitations making the combined technique a valuable tool in the analysis of unknown samples in many areas (e.g. environmental, pharmaceutical, food and beverage, forensics, consumer products). 

 

Samples can be gases, liquids or solids (must be dissolved in a volatile solvent for the latter two).  For substances to be considered volatile, and thereby suitable for GC analysis, their vapor pressures should typically be <300C and their molecular weight <1000 Da.  The compounds should also NOT undergo decomposition when vaporized.

​

In GCMS, the sample (commonly a mixture) is first separated in the GC.  The individual components are then ionized as they leave the GC column prior to entering into a mass spectrometer to separate the ions by mass.  A detector follows to count the number of ions at each mass. 

 

Chromatography

The process of GC is relatively simple.  The sample, typically containing a mixture of compounds, is injected into the instrument through a heated injector (typically to ~200-300C) to vaporize the sample facilitating its entry into a flow of an inert carrier gas (i.e. the mobile phase) which transports it down a column containing an appropriate chemical (i.e. the stationary phase).

 

Sample Introduction

Samples can be injected into the instrument manually using a syringe or via an autosampler, which also uses a syringe, but is more controlled and reproducible.  Injectors can be operated in a Split or Splitless mode.  Other less common methods of introducing samples exist as well and are used for special applications.    

 

The Split method of injection is most common and involves introducing only a percentage (typically 0.2-20%) of the sample onto the column.  The remainder is vented out of the injector to waste.  This is a fast method resulting in narrow analyte bands on the column and subsequently narrow peaks in the chromatogram.  It also is easy to use and automate and helps protect the column from contaminants and involatile sample components that may be present.  However, it is not suitable for trace analysis since a portion of the sample is discarded.   

 

The Splitless method of injection transfers the entire sample volume onto the column.  This is primarily used for trace analysis applications.  However, this is a much slower process than the Split method resulting in broader analyte bands on the column.  Further, this method is more susceptible to column contamination since the entire sample enters the column. 

​

Columns

The sample flows from the injector into the chromatography column where the separation occurs.  Capillary columns are typically used for GCMS.  These are typically 0.1-0.75mm in diameter, 15-60m in length and wound in a circle.  The stationary phase can be simply coated onto the wall (referred to as 'non-bonded') or it can be immobilized and/or incorporated (via cross-linking) into the column wall (referred to as 'bonded').  The latter is preferable as less 'bleeding' of the stationary phase occurs during the analysis resulting in lower noise.  Columns are easily switched out as needed as specific types are often required for different types of analyses.  Further, columns degrade with use and are sometimes ‘poisoned’ by components within a sample which negatively affect their performance.  As such they must be periodically replaced. 

 

Stationary Phases

A wide variety of stationary phases are used in GC columns with the actual one chosen dependent on the compounds being analyzed with the general chemical principle of 'like dissolves like' being considered.  Understanding the interaction of the analytes of interest with the stationary phase is important because this is key to the separatory aspect of the technique. 

 

The analysis of Polar compounds such as alcohols, acids, ethers, esters, thiols and amines utilize Polar stationary phases (e.g. Polyethylene Glycol) while Non-Polar compounds such as alkanes require a Non-Polar Stationary phase (e.g. siloxane, hydrocarbon/siloxane mixture).  Polarizable compounds such as alkenes, alkynes and aromatics often require a very polar stationary phase (e.g. poly(biscyanopropyl siloxane)). 

 

A large variety of compounds exist within each of these 3 categories of stationary phase.  The actual compound chosen is one important factor in determining the maximum temperature for a particular GC analysis.  For example, the maximum temperature of the non-polar stationary phase polydimethylsiloxane is 350C while that of the polar polyethylene glycol is only 250C.

​

Column Heating

The GC column is located in an oven allowing the temperature to be increased during the analysis to values as high as 200-350C (or higher for some applications).  During a sample ‘run’ the oven is typically heated to a set temperature and held for a period of time before being ramped to a higher temperature (ramp rates are typically ~10-80C/min) and held again.  Multiple hold and ramp events can occur within a single run to accomplish the desired separation of analytes within the sample. 

 

The sample components interact with the stationary phase within the column.  Those compounds that have a strong interaction take longer to travel down the column (i.e. elute) compared to those which interact weakly.  Heating the column facilitates the desorption of species that are tightly held to the stationary phase.  This process ultimately results in the separation of the sample components.  As the individual components exit the column, they are detected by a suitable detector.  The time it takes for a compound to traverse the GC system is termed the 'Retention Time'.  Each component will have a suitably different retention time if analysis conditions have been chosen to allow for their appropriate separation within the column.  Ultimately, separation is based on differential partitioning of the components between the mobile and stationary phases.  Heating the column helps drive the partitioning towards the mobile phase.   

 

Ionization                                  

Once the analytes exit the GC column they are ionized for analysis in the mass spectrometer.  Two ionization modes are commonly used, electron ionization and chemical ionization with the latter being subdivided into two additional categories. 

​

Electron Ionization (EI)

In EI mode electrons emitted from a filament are accelerated into an ionization chamber through which the analytes (and carrier gas) are flowing.  The electrons impact with the molecules present.  This is typically considered a ‘hard’ ionization source resulting in fragmentation of the molecules.  The fragments can be useful in helping to identify the structure of a particular analyte.  However, not all molecules fragment.  Some are ionized simply by removal of an electron.  These are called ‘Molecular Ions’ (M+) and are useful in determining the molecular weight of a compound. 

 

EI is the most widely used ionization mode and spectra produced by this mode are easily compared against libraries of data acquired under similar conditions.  While the energy of the impacting electrons can be lowered (to minimize fragmentation) or raised (to maximize fragmentation), 70eV is most commonly used since that’s the condition used in many spectral databases.

 

Positive Chemical Ionization (PCI)

PCI is a soft ionization mode that results in considerably less fragmentation than EI.  PCI is an indirection ionization process involving ionization through a molecular reaction between the analyte eluting from the GC and reactant ions produced by ionization of a reagent gas.  This ionization process produces both protonated molecules whereby a proton atom from the reagent gas is transferred to the analyte molecule and adduct ions consisting of the analyte molecule combined with portions of the reagent gas.  The ability of a molecule to accept a proton (and thus form an ion) is dependent on its Proton Affinity.  If the analyte molecule has a larger proton affinity relative to the reagent gas, then proton transfer can occur.       

 

The most commonly used reagent gases are methane, iso-butane and ammonia.  Methane typically produces the protonated molecular ion [M+H]+ and [M+C2H5]+ ions.  Methane is a good relatively inert gas to work with and has a lower proton affinity (130.5 kcal/mol) relative to the other two reagent gases making it more likely to be able to protonate most, if not all, of the analytes as the majority have proton affinities higher than 180kcal/mol.  However, if the difference in proton affinity between the reagent gas and analyte is too large, the ionization process can be aggressive resulting in more fragmentation, which isn’t always desired.

 

Iso-butane has a proton affinity of 196.9kcal/mol making it a candidate if methane results in excessive fragmentation since it is an even softer ionization process.  However, Iso-butane can produce a myriad of adducts such as [M+C4H9]+, [M+C3H3]+, [M+C3H5]+ and [M+C3H7]+ in addition to the protonated molecular ion. 

 

Ammonia is also a soft ionization process as it has a proton affinity of 205kcal/mol.  But since ammonia is relatively corrosive, it requires more maintenance of the system.  The use of ammonia produces [M+H]+ and [M+NH4]+. 

​

Negative Chemical Ionization (NCI)

NCI operates similarly to PCI and the same reagent gases are commonly used.  However, the emphasis for this mode is the production of negative ions.  The process involves the transfer of an electron from the analyte molecule to the reagent gas producing a negative molecular ion [M]-.  Sometimes fragmentation can occur as well.  However, the spectra are almost always much simpler than that obtained via EI.  This type of ionization is best suited for the analysis of species such as chlorinated pesticides, organophosphorus pesticides and toxic chlorinated compounds such as PCBs, steroids and drugs, all of which tend to capture electrons.  This characteristic makes the ionization efficiency high for these types of compounds allowing for very good sensitivities.    

 

Mass Analyzers

Following ionization, the resulting ions are extracted into the mass spectrometer (MS).  It is here that the ions are separated based on their different mass-to-charge (m/z) ratios.  The most commonly used mass spectrometer is the quadrupole.  However, time-of-flight, ion traps and magnetic sectors are used as well.  Further, some instruments utilize two mass analyzers in series, and are referred to as MS/MS.  This combination helps to mitigate the poor mass resolution inherent of some mass analyzers and aids in structural determination.

​

Quadrupole

The Quadrupole mass analyzer has a number of advantages over other MS options.  Primarily, it is relatively inexpensive, compact and easy to operate.  Further, it has high sensitivities and fast scan rates.  These advantages outweigh its two primary disadvantages of limited mass resolution (only unit mass resolution is possible) and mass range (typically ~2-1200 m/z) relative to the other mass analyzers.  Unit mass resolution means that the system is unable to differentiate between two different ions with the same nominal mass. 

 

The Quadrupole mass analyzer consists of 4 rods (~20cm in length) which are aligned in parallel.  An RF and DC voltage are applied simultaneously to one pair of rods facing one another.  An RF and DC voltage of the same magnitude but opposite polarity is applied to the other pair of rods.  One set of RF and DC voltages allow a single mass of ions to pass down the center of the rods while all others collide with the rods or other parts of the spectrometer.  By scanning through a series of RF and DC voltages, the entire mass range of interest can be successfully scanned.     

 

Time-of-Flight

The Time-of-Flight (TOF) mass analyzer separates ions based on the time they take to pass through an evacuated tube.  All ions are pulsed into the tube at the same time.  But heavier ions are slower than lighter ions meaning that it takes them longer to travel through the tube.  Thus, separation occurs allowing a complete mass spectrum to be acquired from each pulse of ions entering the tube.  Repeatedly pulsing ions into the TOF throughout the run allows for mass spectra to be obtained for each analyte eluting from the chromatography column.  TOF mass analyzers have much better mass resolutions than quadrupoles which can be useful when analyzing unknown samples.  These mass analyzers have worse sensitivities than quadrupole when the latter is set to detect specific masses.  However, when scanning over a large range of masses, the TOF has better sensitivities. 

 

Ion Trap

Another mass analyzer used when higher mass resolution and mass accuracy are needed is the Ion Trap.  There are multiple configurations of ion traps but the one utilized most commonly on commercial instruments today is the electrostatic trap (i.e. Orbitrap).  Orbitraps can achieve mass resolutions (defined as M/ΔM where M is a particular mass and ΔM is the peak width at that particular mass) in the range of 100,000-140,000 at masses of ~400 m/z compared to only about 1000 for a quadrupole. 

​

An orbitrap mass analyzer consists of an inner spindle-like electrode surrounded by an outer barrel-like electrode.  It traps or ‘stores’ ions in a stable flight path orbiting around the inner spindle by balancing their electrostatic attraction by their inertia coming from an RF only trap.  The frequency of the axial motion around the inner electrode is related to the m/z of the ion.  An Ion Trap can also be utilized for MS/MS experiments without the need for a second mass analyzer. 

​

Magnetic Sector

The least common mass analyzer used in GCMS is the Magnetic Sector due to its expense and rather large size.  However, because of its extremely good sensitivities and mass resolution, its use is specified for a number of environmental applications.  In the magnetic sector mass analyzer, ions leaving the ion source are accelerated to a high velocity and then pass through a magnetic sector in which a magnetic field is applied in a direction perpendicular to the direction of ion motion.  Only a certain mass of ions can traverse the sector successfully for a given magnetic field.  Scanning the magnetic field allows a mass spectrum to be obtained. 

​

However, while a magnetic sector can separate ions according to their mass-to-charge ratio, the resolution is limited since the ions leaving the ion source do not all have exactly the same energy and therefore velocity. So to achieve better resolution, an electric sector that focuses ions according to their kinetic energy follows the magnetic sector.  Additionally, ‘Linked Scans’ in which the magnetic and electric fields are scanned together, can be used to perform MS/MS experiments without the need for a second mass analyzer, although it has some disadvantages over an MS/MS that uses two mass analyzers. 

​

MS/MS

Most commercial instruments capable of GC-MS/MS (also referred to as GC Tandem MS) utilize 2 quadrupoles and a collision cell and are often called ‘Triple Quads’.  The first quadrupole (Q1) is used to isolate ions of a particular mass.  Ions with the selected mass then travel into a collision cell (Q2) where they collide with an added gas and fragmentation occurs.  The resulting ions then travel into a second quadrupole (Q3) for analysis of the collision cell products.  GC-MS/MS instruments have extremely high selectivity and sensitivity due to the elimination of most background noise.  These instruments can also be utilized to provide additional structural information for unknowns.    

 

Detection

Once the ions exist the mass analyzer of the GCMS instrument, they travel into the detector.  The most common detector is the electron multiplier which converts the ions to electrons (i.e. electric current).  The electric current can then be processed via an electric circuit.  Electron multipliers, as the name implies, multiply the number of electrons emitted from the surface created by ions impinging on the detector surface using a series of dynodes.  This increases the signal intensity. 

 

Analysis Modes

GCMS instruments are typically operated in one of two modes, Full Scan Mode and Selective Ion Monitoring Mode.  The Full Scan Mode acquires data over a very broad mass range (e.g. m/z 50 to m/z 500).  Very low masses are often not collected to avoid contribution from potential interfering species such as nitrogen (m/z 28) from air and carbon dioxide (m/a 44).  The upper mass limit is set to include the highest mass expected in the sample although the larger the mass range, the worse the detection limits.  The mass spectral data are acquired in sequence at specific intervals (e.g. 0.25 sec) by changing the applied voltage to the rods.  This mode is typically used when analyzing unknown samples and is useful for qualitative analysis since mass spectra are available for the individual components of the sample.

 

When the species of interest and predominant ions that they produce are known, Selective Ion Monitoring (SIM) mode, can be used.  Here, data is only collected from specific ions of interest.  Thus, significantly more time is spent acquiring data from the important selected masses resulting in detection limits that are 10-100 times better than that achievable in the Full Scan Mode.  SIM mode is used for quantitative analysis. 

 

Data

Mass Chromatograms

The data produced in regular GC is called a chromatogram and consists of the monitored signal versus retention, the time in which an analyte elutes from the column and is detected.  The data produced in GCMS is similar but is typically referred to as a mass chromatogram since the signal being detected is from a mass spectrometer.  There are many different subsets of mass chromatograms. 

 

The Total Ion Current (TIC) chromatogram can be acquired from the Full Scan mode of operation.  It sums the intensity of all signals detected in the mass range being monitored from each mass spectrum acquired.  It is often used for the analysis of unknown samples, since the signals of interest produced from the analytes present aren’t known.  However, the TIC can be somewhat noisy since background signals are also included in the mass chromatogram. 

 

Other chromatograms that can be acquired from the Full Scan mode are the Base Peak chromatogram and the Extracted-Ion chromatogram (XIC).  The former plots the highest intensity signal from each mass spectrum acquired.  It is typically less noisy than the TIC.  The XIC, on the other hand, utilizes data from an initial Full Scan run to reconstruct a chromatogram using only one or more chosen masses that are characteristic of the analytes present.  This is also much less noisy than the TIC. 

 

The Selected Ion Monitoring (SIM) chromatogram is acquired from the SIM mode of operation.  It is similar to an XIC as only specific masses are monitored.  However, for a SIM chromatogram, only the masses of interest have actually been acquired (the data is NOT reconstructed from a Full Scan mode data) allowing for much better detection limits as discussed previously. 

 

A chromatogram often acquired in MS/MS experiments is the Selected-Reaction Monitoring (SRM) chromatogram also referred to as a Multiple Reaction Monitoring (MRM) chromatogram.  To acquire an SRM chromatogram, the first quadrupole of the instrument is set to pass the precursor ion of interest into the collision cell where it undergoes chemical induced dissociation (CID).  All other masses are filtered away.  The products from the collision cell are then sent to the third quadruple, Q3, and a specific fragment is monitored (i.e. plotted on the chromatogram).           

 

Mass Spectra

The real power of GCMS over conventional GC is its ability to provide a mass spectrum for each separated compound.  These spectra are indispensable in helping to determine the identity of unknown species present within a sample.  The mass spectra show specific, relatively reproducible patterns (when identical analysis conditions are used) for each separated analyte acting as its fingerprint.  Thus, the mass spectrum from an unknown analyte can be compared to a library containing mass spectra from a large volume of reference compounds.  Software included with the databases employ search routines to determine which references provide the closest match to the mass spectrum from any unknown in question.  Three types of data bases exist:

 

1. Database for General Compounds:  Examples of this is the NIST database which contains mass spectra from more than 265,000 unique compounds and that from Wiley which currently contains spectra from nearly 600,000 unique compounds.

 

2.  Specialized Databases:  These libraries contain compounds relevant to a particular field.  An example is the FFNSC Library that contains flavors and fragrances.  Pesticide databases exist for environmental applications. 

 

3.  Personal Databases:   These are databases constructed by individual users.  They often include references from materials specific to the processes from which samples are being pulled for analysis.     

 

Various ways exist to search through the spectral database being used.  The Forward Search utilizes most mass peaks found in the unknown spectrum.  Spectra from references which most closely match that of the unknown produce a higher Similarity Index Value.  This value determines how closely a reference spectrum fits an unknown spectrum.  The Reverse Search determines whether peaks found in the spectrum from the Reference compound exist in that of the unknown.  This type of search is useful when background peaks or other interferences may be present in the spectrum from the unknown.  Finally, an Index Search allows the library to be searched by compound name, CAS number, MW or some other variable.    

 

Most spectra included in databases were acquired using Electron Ionization with 70eV electrons and a quadrupole mass analyzer.  It should be stressed that while libraries are extremely valuable tools for aiding in peak assignments, an unknown mass spectrum will never exactly match that of any spectrum in the library, even when the unknown compound is one that is present in the library and the aforementioned conditions have been used.  This is because many variables affect the actual mass spectral pattern such as instrument age and operating parameters as well as statistical fluctuation.  Thus, the search routine provides a list of candidates whose spectra best resemble that of the unknown to which it is being compared.  Sometimes, the compound listed first is not the correct answer.  As such, analyst expertise is invaluable during data processing.        

 

Variations

While the vast majority of GCMS analyses utilize the conventional method of introducing samples into the system via injection using a syringe, a number of variations exist to aid in sampling of materials that may not be directly amenable to conventional GCMS analysis. 

 

Static Head Space GCMS

Static Head Space GCMS is a method of analyzing volatile compounds present in solids or liquids.  The sample is placed in a closed sampling vessel.  Volatile compounds that are present within the sample are released into the gas phase above the sample.  An equilibrium exists between the volatile species entering the gas phase and that portion returning to the sample.  The sample is then heated using a particular temperature profile which drives the equilibrium towards the gas phase resulting in higher concentrations of volatile species in the space above the sample, defined as the ‘head space’. 

 

At the end of the heating profile, the head space can be sampled by a number of methods.  The easiest utilizes a gas tight syringe that is typically heated to prevent condensation of any material within.  Other methods utilize a capillary transfer line or sample loop that is filled with the contents from the head space and then forced onto the GC column.     

 

If greater sensitivity is required, solid phase micro-extraction (SPME) is sometimes used to first concentrate the volatile compounds before GCMS analysis.  In SPME, a fused-silica fiber coated with appropriate material (can vary depending on application) is contained within a syringe needle.  The fiber is pushed from the needle into the headspace volume to extract the volatile materials before being retracted back into the needle to transport to the GCMS instrument.  The fiber is then extended again for desorption into the GCMS.

 

Static Head Space GCMS is well suited for a wide variety of analyses.  It is often used to look for residual solvents remaining on medical devices, pharmaceuticals and packaging materials for food and pharmaceuticals.  It’s also used to look for off-odor products as well as flavor components in beverages and food products.  It’s useful for looking at low molecular weight additives as well as monomers in polymers and plastics.     

 

Dynamic Head Space GCMS

Dynamic Head Space GCMS, also known as Thermal Desorption GCMS, varies from Static Head Space analysis in that a stream of inert gas (e.g. He) continuously flushes the head space region, carrying the volatile components to a trap where they are sorbed.  The trap can contain a variety of materials to facilitate sorption such as glass beads, Tenax and charcoal.  Additionally, traps are often cooled to temperatures as low as -100C to further enhance sorption.  Following the end of the heating profile of the sample, the trap is heated and the flow through it reversed and directed onto the GCMS instrument for analysis.  This method of collecting the volatile components of the sample serves to concentrate them providing much better detection limits compared to Static Head Space GCMS.

 

Purge and Trap

Purge and Trap is very similar to Dynamic Head Space GCMS.  However, this technique primarily involves the analysis of liquids (although solids are sometimes sampled as well) with the inert gas bubbling through it rather than passing only through the head space region.  The volatile components that are swept from the sample pass onto the cooled trap and analyzed like described above for Dynamic Head Space analysis.  This method is commonly used for the analysis of volatile organic compounds (VOCs) in drinking water as specified by certain EPA protocols. 

 

Pyrolysis GCMS

As has been stressed, GCMS is for the analysis of volatile compounds.  Nonvolatile species are not vaporized and thus are unable to be carried by the carrier gas down the column for separation.  Pyrolysis GCMS allows for the analysis of nonvolatile materials.  In this method the sample is placed in a quartz sampling tube and rapidly heated to extremely high temperatures such as 400-1200C with even higher temperatures used for special applications.  The high temperatures cause the sample to decompose into smaller, more volatile fragments, which are then amenable for GCMS analysis.  The entire process occurs in a fraction of a second and the products are carried into the GCMS instrument for analysis.    

 

Pyrolysis GCMS is used for the analysis of high molecular weight, insoluble, cross-linked and thermally stable polymers.  It can aid in their identification and look for the presence of additives and/or monomers or be used for quality assurance or deformulation.  It’s also useful for looking at rubbers, paints, papers, coal, petrochemicals and adhesives as well as forensic applications.    

 

Comparison

The key point of GCMS that helps set it apart from most other techniques is the analysis of mixtures of unknown volatile species allowing them to be separated and mass spectra obtained from individual species.  This greatly simplifies the process of identifying components of an unknown mixture.  Regular GC can separate components of mixtures as well, but it is not well-suited to identify unknowns that are present due to the lack of detector specificity.    FTIR and Raman, on the other hand, can provide valuable structural information on both volatile AND nonvolatile compounds, but when present within a mixture, the resulting spectra are often complex, sometimes inhibiting compound identification.  LCMS is one option for the analysis of mixtures containing nonvolatile species (volatiles can also be analyzed).  It has similar principles to GCMS except that the stationary phase is a liquid meaning that the sample components only have to be soluble in it allowing for the analysis of nonvolatile species.  However, LCMS instruments are more expensive to both purchase and operate and typically require more operator training/experience.

 

GC is used in a wide variety of industries.  It’s useful for looking for the presence of contaminants in environmental samples, industrial processes and food and beverages.  It’s also commonly used in quality control applications to ensure the purity of products.  Many products such as drugs or chemicals used in semiconductor processing must be extremely pure.  The superb detection limits of GC help insure their purity.