All forms of chromatography separate components of a mixture by taking advantage of the fact that each component has a different affinity for a particular media. Both liquid and gas chromatographs use this principle and employ two different media—a sorbent-packed column and a solvent (termed the stationary phase and the mobile phase)—to separate a mixture into its components. The relative affinity of each component of a mixture, to both the stationary phase and the mobile phase, causes them to pass through the column with a unique transit time. This is the basic principle of chromatography.
In “Fundamentals of Liquid Chromatography” (BI&T, July/August 2012), we learned that chromatography systems are used to separate a complex mixture into its components for further examination or identification. The liquid chromatograph performs relatively low-temperature analyses of bodily fluids and is best suited for separating proteins and peptides, measuring toxins, and measuring drug levels in serum for both therapeutic monitoring and unknown drug identification.
The gas chromatograph (or GC for short) operates in a comparable manner, performs similar analyses on volatile samples, and does an excellent job of separating mixtures with similar vapor pressures and chemical structures. Gas chromatographs are used to provide fatty acid profiles and blood alcohol determinations. A GC is the only instrument sensitive enough to detect low concentrations of volatile organic mixtures, such as the aromatics (benzene, toluene, xylene, etc.). A GC connected to a mass spectrometer is often referred to as a gas chromatograph-mass spectrometer or GCMS, and is used extensively for performing confirmatory tests for both therapeutic and street drugs. As a general rule, if the sample is not compromised or degraded by the processing temperature, it is suitable for analysis by a GC.
Like its cousin the liquid chromatograph, the GC uses both a sorbent-packed column and a solvent. However, since the mobile phase is a gas, the stationary phase can be one of two types. The first is a solid sorbent packed into a tube, similar to that used in liquid chromatography. The other stationary phase employs a solid support with a nonvolatile liquid coating. As is the case in a liquid chromatograph, the relative affinity of each component of a mixture to both the sorbent and the solvent causes each component to exit the column at a particular time after sample injection. Using Kovat's Retention Index, the retention time of the unknown is compared to retention times stored in a sample library to determine the component(s) of the sample.
Although it is of comparable technology, the GC shares very few components with its liquid counterpart. The basic GC consists of a carrier gas supply with its associated controls, the sample injection port, the column, an oven enclosing the column, and detector electronics (Figure 1).
Gas chromatographs are used to provide fatty acid profiles and blood alcohol determinations.
Depending on the type of detector used, the carrier gas would be air, helium, nitrogen, hydrogen, or an argon-methane mixture. The two most common are helium and hydrogen. Helium allows a wider range of flow rates and is nonflammable, but it is expensive and may be impossible to obtain in some parts of the world. Hydrogen is more efficient, provides the best separation, and is much cheaper than helium, but it is highly flammable so disposing of the eluate can be problematic. The carrier gas is supplied through two controlling mechanisms. One is the regulator affixed to the gas supply tank, similar to the regulator on an oxygen tank. The second is a regulating mechanism that is part of the GC. On older chromatographs, this is a bubble or electronic flow meter at the outlet of the column. The flow rate is changed by adjusting the inlet (or column “head”) pressure. Correctly setting the required flow rate was a difficult process. Modern instruments use an electronic flow meter and employ a servo mechanism to control the flow rate. This allows the flow rate to be changed during the run, which was impossible with older units. Typically, one of two methods is used to check the flow rate prior to sample insertion. The first is through the use of a flow meter connected in the gas stream. The second is by injecting a specific volume of butane gas into the system. The retention time of the butane confirms the carrier gas flow rate.
Next, the carrier gas passes the sample injection port. Here the sample, usually a liquid substance, is injected into the gas stream and is almost immediately vaporized at the injection port. The sample port itself is an airtight assembly. This prevents both the escape of the carrier gas and the inadvertent addition of room air to the sample. Frequently the injection port is encased in or surrounded by a heating block that quickly vaporizes the sample. Some designs allow the column itself to vaporize the sample and is feasible where extremely small sample volumes are required. While other designs are used in applications, such as blood alcohol testing, they require a special-purpose heated injection port.
The flowing carrier gas, now containing the sample, next encounters the column. The column separates components of a mixture by taking advantage of the fact that each component has a different affinity for a particular media. The column contains the stationary phase, which separates the components of a mixture from the mobile phase as the mixture passes through it. However, since the GC uses the third state of matter as the mobile phase, both solid and liquid materials may be used for the stationary phase. Therefore, the column is usually found in one of three varieties. The first, and most obvious, is the packed column, similar to that used in a liquid chromatograph. Packed columns usually have a 1/8″ to 1/4″ outer diameter and are made of glass or stainless steel. They are used in older instruments and are often packed with silicate or another inert agent. The second is a replacement for the packed column used in older instruments, the large-bore capillary column. This column consists of a thin-fused silica (a high-quality glass) tube with a thick interior film forming the liquid phase. Large-bore capillary columns, in particular, can be as long as 90 feet and are often coiled up inside the oven. Capillary columns, the third and most common variety, are used on modern GCs which are designed to use smaller sample sizes. These are similar in length to their large-bore cousins, but are only about 0.01 to 0.03 inches in diameter and can analyze samples as small as 0.1 μL.
To maintain a constant temperature as the sample transits the stationary phase, the column is enclosed by an oven. The oven is usually very simple—a metal or plastic shroud, a small electric heating element, and a circulation fan—but sufficient to maintain a constant temperature during the separation process. Some instruments employ multiple ovens containing two or more columns, while others may enclose all columns inside the same shroud, and still others put two columns in one oven. Column-and-oven configurations are determined by the intended purpose of the GC since multiple samples can be processed nearly simultaneously. Multiple ovens housing one or more columns usually indicate an instrument that features high sample throughput. Modern microprocessor controls even allow the temperature to be changed during a test.
A number of detectors can be used on a GC. Those most commonly encountered include:
Thermal Conductivity Detectors (TCDs) and Flame Ionization Detectors (FIDs) which detect changes in carrier gas conductivity caused by sample components. While TCDs are suitable for general analyses, FIDs are better for detecting easily ionized organic components, such as amphetamines, barbiturates, and steroids.
Electron Capture Detectors (ECDs), Nitrogen- Phosphorus Detectors (NPDs or N/Ps) and Photoionization Detectors (PIDs) also detect changes in gas conductivity, but are specific to a particular substance. For example ECDs, with their radioactive source, are best suited for measuring halogenated gases, such as fluorine and chlorine. NPDs are better for compounds containing phosphorus or nitrogen, and PIDs are best for chemical structures, such as aromatics.
Mass Spectrometer Detectors (MSDs) separate the ions in the eluate according to their mass to charge ratio. This results in a characteristic spectrum unique to the analyte.
If the detector does not damage or change the sample, different detectors can be connected in series to provide different information about the eluate. The last detector in the chain could destroy or alter the sample since the sample will be disposed of next. In all cases, the detector provides its output to an electronics package that interfaces to some form of hardcopy printer, data integrator, PC, or other device that performs calculations and prints or displays the results.
The GCMS is an incredibly sensitive, accurate, and valuable analytical tool in the laboratory. Viewers of one of the popular CSI television programs might recognize the GCMS as that magical device into which a trace sample is inserted and moments later the technologist offers a wealth of incredible information about the sample, including when and where it was purchased. This, of course, supplies the missing lead that investigators need to solve the crime. The reality of both the GC and the GCMS is quite different. On the front end, some analyses can take hours, with separations as long as a half hour, and usually require complex sample and instrument preparation before the unknown material is ready for analysis. On the back end, both a comprehensive database of retention indices and data interpretation software must be loaded on the integrated personal computer and constantly updated to provide the plain text results shown on the show. But that is the magic of television—compressing hours of work into minutes of tension-building entertainment.
After the development of the liquid chromatograph in the 1940s, developers theorized that gas chromatography was also possible. Archer Martin and Anthony James began development of gas chromatography, and in 1952 Martin announced the successful separations of a number of compounds by gas chromatography. Although others, such as the German chemist Fritz Prior, were attempting to develop gas chromatographs, because of his 1952 announcement, Martin is given credit for its development.
Further development of the gas chromatograph spawned the parallel development of new detection methods for evaluating the output. In 1954, Neil H. Ray developed the thermal conductivity detector, which served as the foundation for developing the flame ionization detector by J. Harley and his colleagues in 1958. During that same year, James Lovelock developed the electron capture detector. Still others coupled the mass spectrometer to the gas chromatograph in the late 1950s, creating the GCMS.
In the years since their commercial introduction in 1961, gas chromatographs have undergone the same incremental improvements as other laboratory instruments. In 1969, at least two major laboratory instrumentation manufacturers were selling gas chromatographs in the USA. In 1973, industry giant Hewlett-Packard introduced its first gas chromatograph, making it the first microprocessor-controlled analytical instrument ever made. The introduction of electronic controls managed and monitored the instruments functions and adjusted instrument parameters in accordance with specific test protocols. As these controls and their programs became more sophisticated, the gas chromatograph went from a highly sensitive, complex, delicate analytical instrument found only in research laboratories to a modular device that could be custom tailored for various clinical applications. Over the next two decades, column manufacturers developed materials that are more inert, have improved thermal stability, and have more consistent particle size and chemical characteristics. Concurrently, new types of detectors were developed which greatly increased both their versatility and sensitivity.
How to Manage Gas Chromatography Systems
Gas chromatographs are complex devices that have traditionally been found only in the largest hospitals, medical centers, and research facilities. However, they are now making their way into smaller facilities. Since gas chromatography systems are assembled from the various available gas sources, columns, detectors, and other basic components, one can be as simple or complex as is required for the particular application. Therefore, maintenance should be tracked by instrument serial number or locally assigned control number. Due to the esoteric nature of the technology, the low density of liquid chromatography systems in the facility, and the importance of the column in the process, the maintenance manager may want to consider an annual maintenance contract. The contract should cover, at a minimum, software upgrades and new releases (if applicable), preventive maintenance visits, discounted or guaranteed labor rates, and a repair part discount schedule for billable repairs. In-house maintenance of the overall system is rarely cost effective.
No specific federal regulation addresses gas chromatography systems. However, local fire regulations, along with those from the federal Occupational Safety and Health Administration, may impact general system operations, especially systems using a flammable mobile phase such as hydrogen or methane mixtures. Therefore, where flammable mixtures are used, the waste eluate must be safely exhausted, not just vented into the room or into the facilities vacuum system.
Those systems employing an electron capture detector (ECD) contain a radioactive source within the detector. This source is what gives them their high sensitivity level. To achieve this, the source is more powerful than that found in most smoke detectors and because of this, the source falls under the licensure requirements of the Nuclear Regulatory Commission.
As it does with the liquid chromatograph, the FDA views the gas chromatograph as an in vitro medical device. The Clinical Laboratory Improvement Amendments (CLIA) of 1988 criteria for the training, competence, certification, and experience of personnel performing laboratory tests typically place tests performed by gas chromatographs into at least the moderately complex category, with some procedures classified as high complexity.
Risk Management Issues
Accurate analysis depends on sample preparation, thus making improper sample preparation a risk management issue. Interfering agents can alter the outcome of the analysis and cross-contamination between samples, overloading the column. A sluggish flow rate through the column can also adversely affect the results. These are considered risk management issues because the flawed results can lead to an incorrect diagnosis and inappropriate and ineffective treatment.
One additional risk associated with gas chromatographs lies in the gas chosen for the liquid phase. Helium and nitrogen are relatively safe gasses, although the user must take the same general precautions associated with compressed gasses and gas cylinders. Hydrogen, argon-methane, and other flammable gases warrant an extra level of precaution in handling both the cylinders and their associated fittings to find and eliminate leaks as well as the other connections inside the chromatograph. Disposal of the eluate also has its unique hazards and requires special handling.
Due to the esoteric nature of the technology, the low density of liquid chromatography systems in the facility, and the importance of the column in the process, the maintenance manager may want to consider an annual maintenance contract.
Most common problems with gas chromatography systems are well within the hands of the lab technician to resolve. Improper sample preparation, leaky columns, contaminated solvent, and cross-contamination are all operator issues that can be corrected through better attention to detail or procedural changes. The sample injection port can be a particularly troublesome source of a leak if the septum is not replaced on schedule. Replacing it before it begins to leak is good preventive maintenance. Symptoms of a leaky septum (also known as a “septum bleed”) include a rise in the baseline and extraneous peaks not associated with the sample. Additionally, as the septum deteriorates, it will emit volatile compounds and additives used during its manufacture. These will enter the mobile phase and show up in the substance analysis, thus compromising the results.
Training and Equipment Necessary
A service contract is recommended, especially if the system requires periodic software upgrades, such as those for a data integrator or compound database. However, if the system is to be serviced partially (on a “first-call” basis, for example) or entirely in-house, manufacturer training of an experienced biomed is mandatory. A well-equipped biomed toolkit is required, as well as any special tools and test equipment unique to the system being maintained. Guidance on accessories and repair parts sourcing information is best obtained from the manufacturer of the system being supported.
Unlike its liquid cousin, gas chromatography technology is evolving as these instruments become more commonplace in the clinical laboratory. Units are becoming more modular in design, which encourages continual improvements in the individual components. Column manufacturers are developing materials that are more inert with improved stability to compliment more sensitive detectors. Inexpensive, noninvasive screenings for diabetes, pneumonia, and cancer utilizing gas chromatography are currently on the marketing horizon.
Absorbent: a solid material which holds liquid in the spaces between its particles. The liquid can frequently be squeezed out by mechanical action.
Adsorbent: a solid material which holds liquid on its surface by chemical-type binding. Usually the bond is often quite firm and hard to separate.
Analyte: the substance of interest to be separated from a mixture during chromatography.
Eluate: the mobile phase leaving the column.
Kovat's Retention Index: a concept used in gas chromatography to convert analyte retention times into system-independent constants.
Mixture: two or more substances that have been combined, but each substance retains its own chemical identity. (This must not be confused with a compound where the atoms comprising it chemically join together to form a new substance. Salt dissolved in water is an example of a mixture of two compounds.)
Mobile phase: the substance which moves in a definite direction, usually downward.
Retention time: the characteristic time it takes for a particular analyte to pass through the system.
Solubilize: to make a substance, such as a lipid fat, soluble or more soluble, especially in water, by the addition of an agent such as a detergent.
Solvent: a substance capable of solubilizing another substance. In the confines of this article, it refers to the liquid used as the mobile phase.
Sorbent: a thin coat of adsorbent or absorbent material on a surface.
Stationary phase: the substance that is fixed in place.
Volatile: a substance which easily vaporizes at a relative low temperature.
About the Author
Robert Dondelinger, CBET-E, MS, is the senior medical logistician at the U.S. Military Entrance Processing Command in North Chicago, IL. E-mail: email@example.com