By D. Ling, Ion Signature Technology, Inc.

Gas Chromatography/Mass Spectrometry (GC/MS) is widely used to identify and quantify organic constituents that are thermally stable and have low boiling points. Liquid Chromatography/Mass Spectrometry (LC/MS) is used for those molecules that are thermally unstable and have high boiling points. Both techniques are used in a broad range of applications. MS is the only analytical technique that can provide unambiguous identification of organic compounds; it is the method of choice when the data generated must be able to withstand scrutiny in a court of law. MS can only provide this level of analysis when sample mixtures are simple or when sample components have been separated from each other. GC/MS is used more often than LC/MS since it provides much better separation efficiency and it is simpler to operate and maintain. GC/MS is commonly used in combinatorial chemistry analysis, petroleum analysis, forensic analysis, detection of illicit drugs, environmental risk assessments, etc. LC/MS is most often used to separate polar compounds such as pesticides, polymers, and proteins. Both instruments are used extensively as a research tools in industry, government, and academia, and for routine analysis in a broad range of applications.

Both instruments are so-called "hyphenated" techniques. They consist of two analytical procedures in sequence, namely a Gas Chromatography (GC) separation or Liquid Chromatography separation followed by Mass Spectroscopy (MS) detection.

Separating Multiple Compounds

The purpose of the GC or LC step is to separate multiple compounds in a sample so that they can be presented to the MS detector, one by one. The GC uses a high-resolution fused silica capillary column, ranging from a few meters to a hundred meters in length, that is housed in a temperature-controlled oven. The inner wall of the tubing is coated with an adsorbed liquid (the stationary phase). The sample is introduced into one end of the column and is conveyed through the length of the tubing by a carrier gas. As the components of the sample travel through the column, they interact to varying degrees with the stationary phase depending on their affinity for the selected liquid coating. The LC operates in an analogous manner, except much smaller stainless steel tubes are used. In this case, the stationary phase is coated onto an inert support, packed into the column, and used to separate organic and bioorganic compounds. Different than GC, the oven is not heated (although it can be), with increased separation efficiency obtained by changing the mobile phase composition. Typically, the percent water/solvent ratio is changed for organic compounds, while the buffer mixture is changed for biomolecules, to increase compound separation efficiencies. Although separation efficiencies are improved so too is the time it takes to coeluting compounds from the sample.

As a result, different compounds will travel with different speeds through the capillary tubing and will exit from the column at different points in time (see Figure 1). The time that elapses from when the sample is injected into the column until a particular compound exits the column is known as the "retention time" for that compound. The temperature of the oven containing the capillary column can be controlled and programmed to optimize the separation.

Figure 1 - Typical Gas or Liquid Chromatogram

When the mobile phase, containing sample components, passes through the detector, a signal is produced related to the concentration of the compound present at any given moment. If this signal is plotted as a function of time, a series of symmetrical peaks is obtained, as shown in Figure 1. Such a plot, called a chromatogram, provides a certain amount of information about the composition of the sample. The retention time of the peaks may help identify the constituents by comparing them to the retention time of peaks from known compounds, while the heights of the peaks or the area under the peaks provide a quantitative measure of the amount of each component.

Ideally, the constituents of a sample exit the column one by one as shown in
the figure, where each peak represents a specific compound. In reality, however, compounds often travel with similar speeds, thereby producing overlapping or "coeluting" peaks.

Tracking Output with Detectors

A number of different types of detectors can be used to track the output from the GC or LC column. Flame Ionization Detectors (FID) and Electron Capture Detectors (ECD) are used in GC separations, with the ECD providing selective GC detection of organics.  Both detectors simply indicate that a compound is present but provide little information concerning the identity of the compound other than what can be derived from the retention time. Such detectors are known as "non-specific" detectors. Similarly, UV or fluorescence detectors are most often used for LC. Although UV provides somewhat more information than FID or ECD, the signal produced is so broad that it offers little help in identifying or quantifying complex mixtures. On the other hand, fluorescence detectors are selective. They respond only to those compounds that can fluorescence; in this sense, they are selective. On the other hand, compounds in the same family generally fluorescence at the same or similar wavelengths, thereby, limiting its usefulness.

MS detectors, in contrast are "compound-specific." They can provide unambiguous identification of the constituents in the sample. As an organic compound enters the MS detector from the GC column, it is bombarded with high energy electrons. The energy absorbed by the compound usually causes the break-up or "fragmentation" of the compound, leading to the creation of "fragment ions." The resulting mixture of ions is subsequently sorted by an ion filter according to their molecular weight (or rather their mass/charge ratio), producing a characteristic multiple narrow-band mass spectrum (see Figure 2).


Figure 2 - Mass Spectrum of Methylene Chloride mass/charge

Determining Weight and Abundance

In a mass spectrum, the location of a given spectral band on the x-axis corresponds to the molecular weight or mass-to-charge ratio of the ions that contribute to that band, while the height of each spectral line is an indication of the relative "abundance" of the ions in question. In most modern GC/MS or LC/MS instrument, this sequential process of ionization and fragmentation, followed by the sorting of the ion fragments to produce a mass spectrum, is repeated 1 to 3 times per second. However, some newer instruments are capable of repeating this entire process (known as a "scan") up to 500 times per second, thereby greatly increasing the amount of information obtained.

When the electron bombardment is carried out at standard conditions (usually using electrons with an energy of 70 electronVolts), a given compound will always fragment in the same manner and produce the same mass spectrum. This spectrum, consequently, becomes a unique fingerprint for the compound and can be used to unambiguously determine the compound's identity by comparing it to a standard library of mass spectra of known compounds.

Figure 3

Unlike the selective detectors mentioned above, where only a single dimension of data is produced, MS detectors yield two-dimensional information (see Figure 3). The time-band trace (dimension 1) is obtained by summing the signals produced from mass fragments (dimension 2) at some specified time-scan. Positive identification is made when sample constituents are separated during GC or LC and have sufficiently different fragmentation patterns. Results indicate that the most often used mass spectral search routines, e.g., NIST and Wiley data analysis software, produce good agreements between sample and library spectra (50-75% accuracy) when sample spectra are relatively clean (see Figures 2 and 3). Poorer results (25-50% match) are obtained for two-component or more mixed spectra where peak purity is less than 85%. Published findings indicate that pattern recognition algorithms have difficulty unambiguously identifying sample constituents when more than one compound results in the same GC peak. In addition to long GC or LC run-times, most laboratories spend 15 min confirming the MS data analysis for every 30 min of analysis time.

Figure 4

The resulting TIC chromatogram, Figure 4, shows several ill defined peaks superimposed on a "humptygram," while the mass spectrum reveals fragmentation ions from all organics that hit the MS at the same time, t. Untangling this signal at a specified scan(or time) is analogous to re-constructing 10-50 puzzles (i.e., compounds), where each puzzle (i.e., compound) may contain 10 to 50 puzzle pieces (i.e., fragment ions), and where each puzzle piece may be suppressed in color (i.e., fragment signal) relative to the more vibrant puzzle pieces (i.e., organic fragments in the mixture), see Figure 5.

Ionizing Compounds

LC/MS instruments use electrospray or atmospheric pressure ionization sources to ionize organic compounds. These techniques are much "softer" in nature, and intentionally produce less fragmentation of the molecule. This is a hardware solution to simplifying the MS spectra. Nonetheless, multiple ions are typically produced by the compound and compound-adducts produced through compound-solvent interactions. For IFD purposes, increased ionization can be obtained by slightly increasing the source voltage. In this case, the characteristic ions produced can easily be used to identify the compound.

GC/MS and LC/MS instruments display the result of the analysis in the Total Ion Current ("TIC") mode in which the sum of all ions detected in each scan is plotted as a function of time. This produces a chromatogram similar to that obtained by the non-specific FID, ECD, UV, or selective fluorescence detectors. However, in the case of MS detectors, the detailed ion fragmentation patterns for each scan of the detector are stored in a data file that can be accessed and used to provide unambiguous identification.

Computer-based Instrumentation

All modern MS instruments contain a microprocessor, usually a desktop PC, that controls the operation of the GC/MS or LC/MS combination, including temperature control, mobile phase, ionization voltage, and other instrument parameters. The PC also contains software provided by the instrument manufacturer that acquires and stores the spectral information generated in each of the several hundred individual MS scans performed during a single analysis, and that can manipulate and display this data in a variety of ways. The PC software includes one or more reference libraries of mass spectra that are used during the interpretation of the spectral data to help identify the constituents of the sample. Finally, the PC can be used to report the result of the analysis in any of a number of standard or custom formats.

Identifying Compounds

The identification of unknown compounds by MS is most accurate and dependable when only one compound at a time is presented to the detector. If two or more compounds are detected simultaneously, the ion fragmentation patterns may overlap, making positive identification difficult or impossible. In such cases, traditional GC/MS software will often use cross-correlation or other probabilistic statistical methods to generate one or more possible matches for the unknown compounds, with an indication of the likelihood of each match being correct. This problem becomes even more complex if the sample contains a large number of chemicals. A tissue sample containing fats and lipids, or a soil sample contaminated with oil may contain dozens of individual compounds that, upon electron bombardment, will fragment into hundreds of different ions. These, in turn, may interfere with or mask the spectral patterns of the targeted compounds and can thwart any attempt at identification.

Figure 6 depicts an example of a direct measuring MS for explosives detection. A 300 ft sampling probe was attached to an Agilent MS. The purpose was to demonstrate the power of the technology for detecting TNT (and other nitrotoluenes) around landmines. A total of 8 consecutive measurements were made, with TNT and its staring materials detected in < 20 sec/analysis. Unlike other early warning instruments used at airports today, which can only suspect the presence of a hazard, the MS provides unambiguous identification of a wide range of target compounds (see figures). This same approach can be used to detect chemical and biological warfare agents and drugs entering the country illegally.