Liquid chromatography systems perform relatively low-temperature analyses of bodily fluids.

Liquid chromatography systems perform relatively low-temperature analyses of bodily fluids.

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The word chromatography is a combination of two Greek words, chroma (meaning color) and graphein (meaning to write)—the resultant word literally means “color writing.” Chromatography systems are used to separate a complex mixture into its components for further examination or identification. A chromatograph separates components of a mixture by taking advantage of the fact that each component has a different affinity for a particular media.

All forms of chromatography use this principle and employ two different media—a sorbent-packed column and a solvent—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 each component to pass through the column with a unique transit time. This is the basic principle of chromatography.

Chromatography systems are used to separate a complex mixture into its components for further examination or identification.

The sorbent-packed column is referred to as the “stationary phase” probably because it does not move. Some of the unknown substance is first mixed with a solvent, called the “mobile phase,” to form a “sample.” This can lead to confusion with the sample of blood or other biological from a patient. In clinical and research chromatographs, the sample containing the liquid phase is introduced into one end of the column and forced, under pressure, to transit the stationary phase. When the sample comes into contact with the sorbent, the analytes have an attraction for the sorbent, but the level of attraction varies with the analyte. As a result, the mixture components cling to the surface of the sorbent, but are not absorbed by it. The different analytes begin to separate from the mixture as they pass through the column at different rates.

This process is analogous to runners in a marathon. All runners simultaneously start from the same point, but as the race progresses, they spread out because of their individual speeds. In much the same way, each analyte moves through the stationary phase at its own speed. As each analyte emerges from the column, the movement rate through the sorbent is calculated as a retention time. Since the retention time is unique to the component-solvent-sorbent combination, precisely determining the retention time of each analyte as it emerges from the column will identify individual components of the unknown mixture.

The liquid chromatograph performs relatively low-temperature analyses of bodily fluids and is best suited for separating proteins and peptides as well as measuring toxins.

The chromatograph most commonly associated with healthcare activities comes in one of two forms—the original liquid chromatograph and the newer gas chromatograph. Although the basic principles upon which they are founded and outlined above remain the same, each is better suited for a specific purpose. Liquid chromatography is most often used for serum analysis where sample preservation is important because this process exposes the sample to lower temperatures, below the boiling point of the sample mixture and its components. That's important for fragile substances such as amino acids. This is an important consideration since up to 85% of all chemical mixtures of interest fall into this category.

The liquid chromatograph performs relatively low-temperature analyses of bodily fluids and is best suited for separating proteins and peptides as well as measuring toxins. It is also used to measure drug levels in serum for both therapeutic monitoring and unknown drug identification. The gas chromatograph performs similar analyses on volatile samples and does an excellent job of separating mixtures with similar vapor pressures and chemical structures.

The most common high pressure liquid chromatograph, or HPLC, found in the modern healthcare facility consists of a solvent reservoir, pump, preprocessing components, sample injector, analytical column, and detector. The pump must produce an unvarying, stable, steady flow of the mobile phase throughout the system. To accomplish this, either a constant displacement or constant pressure pump is used. Regardless of which pump is used, it must pressurize the mobile phase between 500 and 5,000 psi. Due to the characteristics of the solvent used in the mobile phase, chemical-resistant materials such as jewels (typically sapphires and rubies), ceramics, Teflon, and stainless steel are typically used in its construction. When the solvent leaves the pump, it may enter one of two optional preprocessing components, the precolumn.

Figure 1.

A Typical Liquid Chromatography Unit

Figure 1.

A Typical Liquid Chromatography Unit

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ORIGIN AND EVOLUTION

The earliest chromatograph was literally a column of semisolid material through which was poured an unknown mixture to determine what comprised it. History is vague on the exact origin of chromatography and tends to give some of the credit to several western European scientists who developed precursors to chromatography. For example, in 1855, German chemist Friedlieb Ferdinand Runge described the use of treated filter paper to analyze dyes and their individual components. Other sources give some credit to Christian Friedrich Schönbein and Friedrich Goppelsroeder, his student, because of the 1860s publishing of their initial attempts to study the different rates at which substances move through filter paper. However, most sources agree that Russian botanist Mikhail Tsvet (or Tswett, or other variant spellings) was somewhat influenced by these other published techniques, and he coined the name “chromatography” in 1903. This simple and effective technique would have been lost to history had it not been for a German organic chemist named Richard Martin Willstatter.

Willstatter is cited as rediscovering the technique of chromatography a few years later and introducing it to Western scientists. With numerous scientists throughout Europe packing glass columns with all sorts of chemicals, advances in chromatography were rapid. This new technique was found to work on a plethora of mixtures, both colored and colorless. Colored mixtures were broken down into rainbows of unique patterns and colors. (Apparently, even colorless mixtures displayed rainbows of color with the right column contents.) A variant of this process, called ion exchange chromatography, allowed scientists to separate individual ions from mixtures. Further experimentation continued, with researchers employing both polar and non-polar solvents to separate plant pigments, chlorophyll, and carotene from various plants.

Eventually researchers realized this new method of chemical extraction could be useful for more than just extracting pigments from plants. In the 1930s, continued tinkering with the technique led to the development of synthetic resins for complex ion-exchange processes. Probably one of the most important uses of this state-of-the-art chemistry technique occurred during World War II. Sturdy chromatography columns packed with a synthetic resin were provided in survival kit-equipped life rafts to partially purify sea water. Although not tasty, it did provide the essential desalinized drinking water necessary for survival on the open sea.

In a 1941 series of scientific papers, chemists Archer Martin and Richard Synge developed a process called partition chromatography to separate amino acids and other organic chemicals. Although better known for their work in column and paper chromatography, which led to the development of gas chromatography, Martin and Synge developed techniques that formed the basis of high-performance liquid chromatography.

The preprocessing components are found in several locations throughout the solvent path and are unique to their task. The first consists of a tank of gas (usually helium), filters, and valves that are connected to the solvent reservoir. The purpose of these components is to bubble inert helium gas through the solvent for about 30 minutes to prevent oxygen from interacting with the solvent and affecting the results.

The next preprocessing component, referred to as the precolumn, is one of two protector columns. The silica filled precolumn is positioned between the pump and the injector valve, and prevents stripping of the packing material in the analytical column. The second protector column is located after the sample injector and is called the guard column. It collects any impurities or strongly retained components of the sample and thus prolongs the life of the analytical column. Typically it is 1/15th to 1/25th the volume of the analytical column and is packed with a material similar to that found in the analytical column. Some chromatograph designs allow the precolumn and guard columns to be used interchangeably.

Some chromatograph designs allow the precolumn and guard columns to be used interchangeably.

The sample injector is much more complex than the one found on a blood gas apparatus. Because an exact volume of the 0.5 to 50 microliter (μl) patient sample must be introduced into the mobile phase to form the sample volume at a precisely controlled rate, the sample is not injected directly into the mobile phase. Instead, it is placed in a sample injector which, based upon available patient sample volume and desired dilution factor in the mobile phase, then slowly meters the sample into the mobile phase. Therefore, the precision of the injection is determined by the instrument itself, not by the skill of the laboratory technician.

The most common sample injector, called the fixed-loop injection valve, is rotated after sample injection allowing the patient sample to be drawn into the column by the solvent stream passing through it. The volume of the loop determines the sample volume entering the mobile phase. Some chromatograph systems semi-automate the testing process and automatically clean the closed loop after each injection so that several patient samples can be injected into the sample injector in succession with minimal sample crossover. Typically, the mobile phase now containing the patient sample, becomes the test sample, and enters, if used, the guard column or the analytical column.

The analytical column is literally the centerpiece of the system. This column contains the stationary phase, which separates the components of a mixture from the mobile phase as it passes through it. Physically, this stainless steel tube can vary from 10 to about 150 centimeters (cm) long by 2 to 5 millimeters (mm) in diameter and can withstand as much as 10,000 pounds per square inch (psi). This column is densely packed with the chosen material in one of three sizes - 3, 5, or 10 micrometers (μm) in diameter. Normally the size of the packing material must be as uniform as possible for column efficiency and reasonable operating pressure, but special-purpose columns are available with both spherically and irregularly shaped particles. Like the mobile phase, the stationary phase is unique to the separation process. Silica and alumina are used for over 95% of the separations.

These systems perform their analyses with remarkable accuracy. Therefore, maintenance should be tracked uniquely, specific to the instrument by serial number or locally assigned maintenance management control number.

The detector continually samples the output from the analytical column and provides a quantification of its output. The most common detector types are photometers or spectrophotometers, fluorescence, and electrochemical.

Photometers and spectrophotometers employ either a fixed or varying wavelength of light, and provide conductance and/or absorption information about the column's output. For certain components, fluorescence detectors are more sensitive than photometers or spectrophotometers. They provide information on the ability of the column's output to emit light after it has been excited by the radiation produced by visible light. When the quantification is observed as a function of time, volume, or distance, a chromatogram is produced. The resultant chromatogram is evaluated either by hand or by the use of computer technology employing a signature library to determine what components, and in what amounts, comprise the unknown sample. Since each component of a mixture has a signature as unique as an individual's fingerprint, the signature library comparison results reveal the components that comprise the unknown mixture.

Liquid chromatographs are complex devices that have traditionally been found in the largest hospitals, medical centers, and research facilities, but are now finding their way into smaller facilities. Special purpose chromatography systems can be assembled from the various modules available to the biologist or laboratory manager. These systems are designed to optimize the detection of specific chemicals or to perform only a few specific tests. Typical special-purpose systems include those assembled to detect trace amounts of hazardous substances in urine samples from workers who handle such substances, and for large-scale testing of lead in adolescents living in certain geographic areas.

These systems perform their analyses with remarkable accuracy. Therefore, maintenance should be tracked uniquely, specific to the instrument by serial number or locally assigned maintenance management 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 should consider an annual maintenance contract. The contract should cover, at a minimum, software upgrades and new releases, 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 not recommended unless a sufficiently large density of similar systems exists to warrant personnel training and a “first call” contract is available or can be negotiated.

There are no specific federal regulations addressing liquid chromatography systems; however, certain federal and local fire regulations impact system operations. Numerous volatile solvents, many of which are flammable, are used for the mobile phase. The U.S. Occupational Safety and Health Administration (OSHA) has regulations covering worker exposure levels and duration for many of these solvents. The agency also has a general duty clause requiring employers to protect employees from workplace hazards, and some of the solvents used in liquid chromatography are considered hazardous chemicals. Local fire authorities have storage requirement for flammables, and often require notification of the nature and quantities of hazardous and flammable chemicals located on the premises.

Unlike chromatographs used in pure research, most clinical applications require the liquid chromatograph to be registered with the Food and Drug Administration (FDA) as an in vitro medical device. The Clinical Laboratory Improvement Amendments (CLIA) of 1988 have criteria for the training, competence, certification, and experience of personnel performing laboratory tests. Typically, the sample preparation required for liquid chromatography place this testing into at least the moderately complex category. Some procedures are classified as high-complexity testing, which requires additional experience and training to perform.

Accurate analyses depend on sample preparation, thus making improper sample preparation a risk management issue. Interfering agents, either in the patient sample or introduced during sample preparation and not stopped by filters, as well as altering the pH during preparation can alter the outcome of the analysis. Bubbles in the mobile phase, irregular particles in the stationary phase, or a leaking filter can create a noisy baseline adversely affecting the results. Cross-contamination between samples, overloading the column, using contaminated solvent, or a sluggish flow rate through the column can also adversely affect the results. As with most laboratory instruments, these are considered risk management issues because the flawed results can lead to misleading diagnosis and inappropriate and ineffective treatment.

Unlike chromatographs used in pure research, most clinical applications require the liquid chromatograph to be registered with the Food and Drug Administration (FDA) as an in vitro medical device.

Most common problems with liquid 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. Two potential trouble spots for the biomed are the power supply and the solvent pump. The power supply, like that found in other medical devices, is usually a modular design with no schematics or parts layout diagram available to the biomed. It is usually replaced as a single unit, although limited internal troubleshooting may be accomplished by the bench technician if a schematic is available. The solvent pump, having multiple internal moving parts, is subject to wear and can usually be repaired and/or rebuilt as necessary if parts are available from the pump or system manufacturer.

If the system is to be serviced partially or entirely in-house, manufacturer training of a seasoned and experienced biomed is virtually mandatory.

If the system is to be serviced partially or entirely in-house, manufacturer training of a seasoned and experienced biomed is virtually 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 particular chromatographic system being supported.

The overall design of a liquid chromatography system is considered mature technology. However, incremental improvements of the individual components are regularly introduced. More sensitive detectors are constantly being developed. Improvements in the consistency of particle size and density of column packing, pump capabilities, and flow consistency are being made to enhance throughput and reduce test time. Better instrument automation and easier-to-use instrumentation are the products of both improved computer technology and overall laboratory automation. Such incremental improvements are expected to continue in the future, with the instruments themselves designed in a more modular fashion to incorporate these changes more easily.

Glossary of Terms

Absorbent: a solid material which holds liquid in the spaces between its particles. The liquid can oftentimes be squeezed out by mechanical action.

Adsorbent: a solid material which holds liquid on its surface by chemical-type binding. The bond is usually 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.

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, from point of insertion to exit.

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 or 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.

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Healthcare Product Comparison System for Chromatography Systems, Liquid, High-Performance
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Congratulations to the following new Certified Biomedical Equipment Technicians, Certified Radiology Equipment Specialists, and Certified Laboratory Equipment Specialists who passed their exams in May and June 2012.

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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: robert.dondelinger@mepcom.army.mil