Gas Chromatography Mass Spectrometry Environmental Sciences Essay

Gas chromatography-mass spectrometry (GC-MS) is a method that combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample. [6] Gas chromatography (GC) and mass spectrometry (MS) make an effective combination for chemical analysis. [5, 10]

The use of a mass spectrometer as the detector in gas chromatography was developed during the 1950s by Roland Gohlke and Fred McLafferty. These sensitive devices were bulky, fragile, and originally limited to laboratory settings. The development of affordable and miniaturized computers has helped in the simplification of the use of this instrument, as well as allowed great improvements in the amount of time it takes to analyze a sample. In 1996 the top-of-the-line high-speed GC-MS units completed analysis of fire accelerants in less than 90 seconds, whereas first-generation GC/MS would have required at least 16 minutes. This has led to their widespread adoption in a number of fields. [6]

GC-MS theory and principle

The Gas Chromatography/Mass Spectrometry (GC/MS) instrument separates chemical mixtures (the GC component) and identifies the components at a molecular level (the MS component). It is one of the most accurate tools for analyzing environmental samples. The GC works on the principle that a mixture will separate into individual substances when heated. The heated gases are carried through a column with an inert gas (such as helium). As the separated substances emerge from the column opening, they flow into the MS. [3]

The GC separates the constituents of a sample as previously described, but as the gaseous sample exits the column and enters the Mass Spectrometer, it is bombarded with electrons that cause the molecules to become unstable and break down into charged fragments. The positive ions are collected and separated on the basis of their mass / charge ratio.  Various analyser types are available depending on what is being studied.  We have both a quadrupole type MS and an ion trap type MS available. The resulting mass spectra permit the identification of the analytes.  A typical detection limit would be 10 picograms which make it much more sensitive than the flame ionising detector on a GC. [2]

To effectively use GC/MS evidence one must understand the process.  First, the GC process will be considered, and then the MS instrument will be presented. [5]

Gas chromatography

In general, chromatography is used to separate mixtures of chemicals into individual components. Once isolated, the components can be evaluated individually. In gas chromatography (GC), the mobile phase is an inert gas such as helium. The mobile phase carries the sample mixture through what is referred to as a stationary phase. The stationary phase is a usually chemical that can selectively attract components in a sample mixture. The stationary phase is usually contained in a tube of some sort. This tube is referred to as a column. Columns can be glass or stainless steel of various dimensions.

The mixture of compounds in the mobile phase interacts with the stationary phase. Each compound in the mixture interacts at a different rate. Those that interact the fastest will exit (elute from) the column first. Those that interact slowest will exit the column last. By changing characteristics of the mobile phase and the stationary phase, different mixtures of chemicals can be separated. Further refinements to this separation process can be made by changing the temperature of the stationary phase or the pressure of the mobile phase. The capillary column is held in an oven that can be programmed to increase the temperature gradually (or in GC terms, ramped). This helps our separation. As the compounds are separated, they elute from the column and enter a detector. The detector is capable of creating an electronic signal whenever the presence of a compound is detected. The greater the concentration in the sample, the bigger the signal. The signal is then processed by a computer. The time from when the injection is made (time zero) to when elution occurs is referred to as the retention time (RT).

While the instrument runs, the computer generates a graph from the signal. This graph is called a chromatogram. Each of the peaks in the chromatogram represents the signal created when a compound elutes from the GC column into the detector. The x-axis shows the RT, and the y-axis shows the intensity (abundance) of the signal. [1]

Figure: schematic diagram of gas chromatography

3.1 Mass spectrometry

Mass spectrometry (MS) is a technique used for characterizing molecules according to the manner in which they fragment when bombarded with high-energy electrons, and for elemental analysis at trace levels. Therefore, it is used as a means of structural identification and analysis. Its widest application by far, is for the structural elucidation of organic compound. MS involves the ionization (conversion of molecules into positively charged ions) and fragmentation of molecules. Various methods are available to effect such a process: e.g.

(i) Electron impact ionization, by far the most common mode used,

(ii) Chemical ionization,

(iii) Field ionization or

(iv) Fast atom bombardment.

In the more commonly used electron impact (EI) mode, the sample molecules are bombarded in the vapour phase with a high-energy electron beam in the instrument known as a mass spectrometer. This process generates a series of positive ions having both mass and charge, which are subsequently separated by deflection in a variable magnetic field according to their mass to charge (m/z) ratio. This results in the generation of a current (ion current) at the detector in proportion to their relative abundance. The resulting mass spectrum is recorded as a series of lines or peaks of relative abundance (vertical peak intensity) versus m/z ratio. The sample is introduced into the inlet system, where it is heated and vaporized under vacuum, and then bled into the ionization chamber (ion source) through a small orifice. Sample sizes for liquids and solids range from milligrams to less than a nanogram, depending on the detection limits of the instrument. Once the gas stream from the inlet system enters the ionization chamber, it is bombarded at right angles by an electron beam (70 eV) emitted from a hot filament. Only ˜20eV is needed to remove one electron from the molecule, to create M+, the remainder is used to fragment the molecular ion into a mixture of radical cations, cations and free radicals.

The positively charged ion fragments are then forced through a series of negatively charged accelerating slits towards the mass analyser, where separation of these ion fragments takes place. This analyser tube is an evacuated curved metal tube through which the ion beam passes from the ion source to the ion collector.

In early instruments, the fragment ions were deflected in a curved path by a magnetic field only. Mass separation depended on the magnetic field strength, the radius of curvature of the magnetic field and the magnitude of the acceleration voltage. The introduction of an electrostatic field after the magnetic field in later instruments permitted higher resolution so that the mass readings could be obtained to four decimal places. In present day instruments, this double focusing system has been further modified to optimize resolution and most instruments now use a quadrupole mass analyser to effect separation of the ion fragments. The ions are collected one set at a time, with the aid of collimating slits, in the ion collector, where they are also detected and amplified by an electron multiplier. Mass spectral data is recorded on computer. Most mass spectrometers are computer controlled nowadays, and scans from mass ranges 12 to > 700 amu, Can be performed in seconds. [4]

Figure: schematic diagram of mass spectrometry

4. Instrumentation of GC-MS

The insides of the GC-MS, with the column of the gas chromatograph in the oven on the right.

The GC-MS is composed of two major building blocks: the gas chromatograph and the mass spectrometer. The gas chromatograph utilizes a capillary column which depends on the column’s dimensions (length, diameter, film thickness) as well as the phase properties (e.g. 5% phenyl polysiloxane). The difference in the chemical properties between different molecules in a mixture will separate the molecules as the sample travels the length of the column. The molecules take different amounts of time (called the retention time) to come out of (elute from) the gas chromatograph, and this allows the mass spectrometer downstream to capture, ionize, accelerate, deflect, and detect the ionized molecules separately. The mass spectrometer does this by breaking each molecule into ionized fragments and detecting these fragments using their mass to charge ratio.

Figure: GC-MS schematic

These two components, used together, allow a much finer degree of substance identification than either unit used separately. It is not possible to make an accurate identification of a particular molecule by gas chromatography or mass spectrometry alone. The mass spectrometry process normally requires a very pure sample while gas chromatography using a traditional detector (e.g. Flame Ionization Detector) detects multiple molecules that happen to take the same amount of time to travel through the column (i.e. have the same retention time) which results in two or more molecules to co-elute. Sometimes two different molecules can also have a similar pattern of ionized fragments in a mass spectrometer (mass spectrum). Combining the two processes makes it extremely unlikely that two different molecules will behave in the same way in both a gas chromatograph and a mass spectrometer. Therefore when an identifying mass spectrum appears at a characteristic retention time in a GC-MS analysis, it typically lends to increased certainty that the analyte of interest is in the sample. [6]

Figure: schematic of GC/MS

4.1 Inlet system

Samples are introduced to the column via an inlet. This inlet is typically injection through a septum. Once in the inlet, the heated chamber acts to volatilize the sample. [6]

4.1.1 GC-MS interface

In this GC-MS system, the link between the two instruments is called an interface; it is like a jet separator, whose purpose is to (1) enrich the sample and (2) adjust the vacuum to the high vacuum conditions needed for MS analysis of the column eluent. [11, 9] After separation of our components by the GC, we need away to introduce this sample into MS- interface.

An ideal interface should be

Qualitatively transfer all analyte

Reduce pressure flow/from chromatograph to level MS can handle

Not cost an arm (or a leg)

No interface meets all requirements

The major goal of the interface is to remove all of the carrier gas from- the majority of the effluents. Interface should cover

Molecular separator

Permeation separator

Open split

Capillary direct [7]

4.1.2 Molecular separator

It is the most popular approach when packed columns are used and based on the relative rate of diffusion. In this the smaller molecules will diffuse more rapidly and most will miss the MS entry jet. The larger molecules will diffuse more slowly will tend to lead the MS entry jet. [7, 11]

Figure: molecular separator

Advantages of molecular separator

It is relatively simple and inexpensive approaches

Disadvantages

Rate of diffusion is molecular weight dependent

If jet becomes partially plugged, you can end up with an excellent carrier gas detector [7]

4.1.3 Permeation interface

A semi permeation membrane is placed between the GC effluents and the MS

The major problem with this approach is

Membrane selectively based on polarity and the molecular weight slow to respond. Only a small fraction analyte actually permeates through the membrane. [7]

Figure: permeation membrane

4.1.4 Open or split interface

In a split system, a constant flow of carrier gas moves through the inlet. A portion of the carrier gas flow acts to transport the sample into the column. [6] The chromatographic column leads to a T-shaped that contains a smaller diameter tube. A platinum or deactivated fused silica capillary also leads to this tube and goes into the mass spectrometer source. The capillary is kept into the vacuum sealed device and is heated to avoid condensation. The T-shaped tube is closed at both ends but is not sealed, so that pressure remained equal to the atmospheric pressure. A helium gas is continuously passed to avoid any reaction of the gas. [9] The MS pulls the analyte in about 1mL/min through a flow restrictor. If flow is above that the excess is vented. If it is below the He from the external source is pulled in. it is the best source for that have flows close to1mL/min like capillary columns.[7]

Figure: open or split interface

Advantages

Any gas producing source will be used.

Relatively low cost and easy to use.

Disadvantages

Sample leaves columns in split.

Split changes as flow change.

Split system is preferred when the detector is sensitive to trace amounts of analyte and there is concern about overloading the column [7]

4.1.5 Capillary direct interface

This coupling consists of having the capillary column directly entering the spectrometer source by a set of vacuum- sealed joints. Here the pumping is not the problem because the capillary is very long. A length of at least 1.5m is necessary for the column with inside diameter of 0.25mm. [9] If we limit the GC to the capillary column only, the MS can actually use all column effluents. [7] The carrier gas flow gets directed to purge the inlet of any sample following injection (septum purge). Yet another portion of the flow is directed through the split vent in a set ratio known as the split ratio. [6]

Figure: capillary direct interface

Advantages

Low cost simple device

No dead volume

No selectivity

Disadvantages

Limits flow range that column can be used

Limits the column ID that can use

Part of column lost which serve as a flow restrictor[7]

4.2. Vacuum system

In order to the MS process to work, it must be conducted under vacuum condition. The major reason for this is to increase the mean free path.

“The average distance that ions or molecules will travel before colliding with another ion or molecule”.

A high mean free path is to ensure predictable and reproducible high sensitivity and reliable mass analysis. [7]

Since a vacuum is required to work a detector;

Detectors are design to use the vacuum as an insulator

Large voltage are used in the MS

Operation of the detector in the absence of the vacuum that can cause severe damage

Most instrument prevent operation if the vacuum is not high enough

A vacuum is produced by using a combination of the two pumps- two stage vacuum pumps. The rotary pumps produced vacuum 102-104torr. These are the turbomolecular or diffusion pumps work in the range of 105torr. These are actually like the compressor.

4.2.1 Turbomolecular pump

It relies on the series of blades or the air foils that tend to deflect the gas. It able to produce the clean vacuum in few hours and reliable

Disadvantage

It is expensive, short life time, can become noisy

Figure: turbomolecular pump

4.2.2 Oil diffusion pumps

It is another important type of the pump that produce high vacuum. These are reliable, maintenance free and quite but take much time and due to poor design oil enters into the vacuum. [7]

Figure: oil diffusion pumps

4.3. Ionization

A number of ionization techniques available

Figure: types of ionization

4.3.1 Types of ionization

After the molecules travel the length of the column, pass through the transfer line and enter into the mass spectrometer they are ionized by various methods with typically only one method being used at any given time. Once the sample is fragmented it will then be detected, usually by an electron multiplier diode, which essentially turns the ionized mass fragment into an electrical signal that is then detected. The ionization technique chosen is independent of using Full Scan or SIM. [6]

4.3.1.1 Electron Ionization

By far the most common and perhaps standard form of ionization is electron ionization (EI). The molecules enter into the MS (the source is a quadrupole or the ion trap itself in an ion trap MS) where they are bombarded with free electrons emitted from a filament, not much unlike the filament one would find in a standard light bulb. The electrons bombard the molecules, causing the molecule to fragment in a characteristic and reproducible way. This hard ionization technique results in the creation of more fragments of low mass to charge ratio (m/z) and few, if any, molecules approaching the molecular mass unit. Hard ionization is considered by mass spectroscopists as the employ of molecular electron bombardment, whereas soft ionization is charge by molecular collision with an introduced gas. The molecular fragmentation pattern is dependant upon the electron energy applied to the system, typically 70eV (electron Volts). The use of 70eV facilitates comparison of generated spectra with National Institute of Standard (NIST-USA) library of spectra applying algorithmic matching programs and the use of methods of analysis written by much method standardization Chemical Ionization. [6, 10]

Figure: EI graph

Figure: EI source

4.3.1.2 Chemical Ionization

In chemical ionization a reagent gas, typically methane or ammonia is introduced into the mass spectrometer. Depending on the technique (positive CI or negative CI) chosen, this reagent gas will interact with the electrons and analyte and cause a ‘soft’ ionization of the molecule of interest. A softer ionization fragments the molecule to a lower degree than the hard ionization of EI. One of the main benefits of using chemical ionization is that a mass fragment closely corresponding to the molecular weight of the analyte of interest is produced.

Figure: CI source

Positive chemical Ionization

In Positive Chemical Ionization (PCI) the reagent gas interacts with the target molecule, most often with a proton exchange. This produces the species in relatively high amounts.

Negative Chemical Ionization

In Negative Chemical Ionization (NCI) the reagent gas decreases the impact of the free electrons on the target analyte. This decreased energy typically leaves the fragment in great supply. [6, 7]

Figure: comparison of graph obtain from EI and CI

4.4 Mass analyzer

A mass analyzer or filter is the portion of the mass spectrometer that is responsible for resolving different mass fragments. Typically all ions will move with same kinetic energy (1/2mv2). Some aspects of these accelerated ions are exploited as the basis for resolving them.

4.4.1 Types of mass analyzers

There are following types of mass analyzers

Magnetic

Electrostatic

Time of flight

Quadrupole mass filter

Quadrupole ion storage(ion trap)

The last two types are most commonly used in GC/MS systems although time of flight making a come back [7, 6, 10]

4.4.1.1 Quadrupole mass filter

It consists of four rods.

Figure: rods of quadrupole

Rods operate in pairs (x or y) and each carries a voltage. Only ions of proper m/z value can successfully traverses the entire filter (z axis). The high pass rods filter out ions with too low of an m/z. the low pass filter outs the ions with too high of an m/z value. [7]

Figure: schematic of quadrupole

4.5 Detector

Ion detection

Once the ions are separated, we need a way to convert them to a response that can be used. An electron multiplier is the most common type of detector used. It is a continuous dynode type of detector. The inner surface of the detector is electroemassive material. When struck by ion electrons are ejected. Due to increasing potential, the electrons are accelerated and when they strike another surface, even more electrons are ejected. This significantly amplifies our signals. [7]

Figure: detector

4.6 Data system

Data system is the heart of our GC/MS system. Without it we would have no way to deal with the vast amount of information that even a single GC/MS analysis produce. Inexpensive fast desktop are the single most important advance in GC/MS. [7]

Figure: data system

4.7 Method of analysis

The primary goal of instrument analysis is to quantify an amount of substance. This is done by comparing the relative concentrations among the atomic masses in the generated spectrum. Two kinds of analysis are possible, comparative and original. Comparative analysis essentially compares the given spectrum to a spectrum library to see if its characteristics are present for some sample in the library. This is best performed by a computer because there are a myriad of visual distortions that can take place due to variations in scale. Computers can also simultaneously correlate more data (such as the retention times identified by GC), to more accurately relate certain data.

Another method of analysis measures the peaks in relation to one another. In this method, the tallest peak is assigned 100% of the value, and the other peaks being assigned proportionate values. All values above 3% are assigned. The total mass of the unknown compound is normally indicated by the parent peak. The value of this parent peak can be used to fit with a chemical formula containing the various elements which are believed to be in the compound. The isotope pattern in the spectrum, which is unique for elements that have many isotopes, can also be used to identify the various elements present. Once a chemical formula has been matched to the spectrum, the molecular structure and bonding can be identified, and must be consistent with the characteristics recorded by GC/MS. Typically, this identification done automatically by programs which come with the instrument, given a list of the elements which could be present in the sample.

A “full spectrum” analysis considers all the “peaks” within a spectrum. Conversely, selective ion monitoring (SIM) only monitors selected peaks associated with a specific substance. This is done on the assumption that at a given retention time, a set of ions is characteristic of a certain compound. This is a fast and efficient analysis, especially if the analyst has previous information about a sample or is only looking for a few specific substances. When the amount of information collected about the ions in a given gas chromatographic peak decreases, the sensitivity of the analysis increases. So, SIM analysis allows for a smaller quantity of a compound to be detected and measured, but the degree of certainty about the identity of that compound is reduced. [6]

5. Applications

5.1. Environmental Monitoring and Cleanup

GC-MS is becoming the tool of choice for tracking organic pollutants in the environment. The cost of GC-MS equipment has decreased significantly, and the reliability has increased at the same time, which has contributed to its increased adoption in environmental studies. There are some compounds for which GC-MS is not sufficiently sensitive, including certain pesticides and herbicides, but for most organic analysis of environmental samples, including many major classes of pesticides, it is very sensitive and effective.

5.2. Criminal Forensics

GC-MS can analyze the particles from a human body in order to help link a criminal to a crime. The analysis of fire debris using GC-MS is well established, and there is even an established American Society for Testing Materials (ASTM) standard for fire debris analysis. GCMS/MS is especially useful here as samples often contain very complex matrices and results, used in court, need to be highly accurate.

5.3. Law Enforcement

GC-MS is increasingly used for detection of illegal narcotics, and may eventually supplant drug-sniffing dogs. It is also commonly used in forensic toxicology to find drugs and/or poisons in biological specimens of suspects, victims, or the deceased.

5.4. Security

A post-September 11 development, explosive detection systems have become a part of all US airports. These systems run on a host of technologies, many of them based on GC-MS. There are only three manufacturers certified by the FAA to provide these systems, one of which is Thermo Detection (formerly Thermedics), which produces the EGIS, a GC-MS-based line of explosives detectors. The other two manufacturers are Barringer Technologies, now owned by Smith’s Detection Systems and Ion Track Instruments, part of General Electric Infrastructure Security Systems.

5.5. Food, Beverage and Perfume Analysis

Foods and beverages contain numerous aromatic compounds, some naturally present in the raw materials and some forming during processing. GC-MS is extensively used for the analysis of these compounds which include esters, fatty acids, alcohols, aldehydes, terpenes etc. It is also used to detect and measure contaminant from spoilage or adulteration which may be harmful and which is often controlled by governmental agencies, for example pesticides.

5.6. Astrochemistry

Several GC-MS have left earth. Two were brought to Mars by the Viking program. Venera 11 and 12 and Pioneer Venus analysed the atmosphere of Venus with GC-MS. The Huygens probe of the Cassini-Huygens mission landed one GC-MS on SaturnHYPERLINK http://en.wikipedia.org/wiki/Saturn’s’HYPERLINK http://en.wikipedia.org/wiki/Saturn’ss largest moon, Titan. The material in the comet 67P/Churyumov-Gerasimenko will be analysed by the Rosetta mission with a chiral GC-MS in 2014.

5.7. Medicine

In combination with isotopic labeling of metabolic compounds, the GC-MS is used for determining metabolic activity. Most applications are based on the use of 13C as the labeling and the measurement of 13C/12C ratios with an isotope ratio mass spectrometer (IRMS); an MS with a detector designed to measure a few select ions and return values as ratios. [6]