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Chromatography systems, although similar in function, can have important variance in their standard equipment which decides their best use. The following information provides an overview to the instrumentation involved in chromatography systems of low-pressure liquid chromatography (LPLC), high-performance liquid chromatography (HPLC), and gas chromatography (GC).
Modern LPLC systems consist of a column, injector, low-pressure pump, detector, and often a fraction collector. The mobile phase is pulled into the chromatography system by the action of the pump. From there, it travels to the injector where the sample to be tested is added. The injector may also act as a controller, switching between many connected mobile phases throughout the assay. After the injector, the sample is carried into the column where separation occurs. From the column, analyte passes into a detector. The detector analyzes the effluent to identify different chemical species and to generate data as a result. From the detector, the mobile phase is pumped into the waste receptacle or collected for further study. (The machine which collects eluent from the column is called a fraction collector and organizes the collected samples based on their time of elution.)
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A peristaltic pump is commonly used when performing an LPLC assay. Peristaltic pumps use a rotor to pinch tubing and generate a vacuum. The vacuum pulls liquid from the reagent source, and the rollers move the reagent along the tubing pathway. The reagent only touches the tubing. The rollers move the fluid by collapsing the tubing from the outside surface and pushing the fluid captured between adjacent rollers over the roller head and away from the pump. After an assay is complete, the tubing can then be removed and cleaned, or replaced. Tubing is available in many formulations to achieve the right balance of chemical compatibility, tubing size, operating temperature, pressure, and tubing lifespan. The major advantage of peristaltic pumping for chromatography applications is the ability to create an entirely self-contained system that allows for easy changeover and maintenance.
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The column used for LPLC can be an empty glass container or prepacked. The stationary phase of a LPLC assay is either a solid or solid coated with liquid. The physical structure of the column has some effect on the distinguishable separation, known as resolution, of the end data.
The stationary phase within a column is decided by the type of separation which needs to occur. When paired with the correct mobile phase, analytes may be separated along the lines of polarity, affinity, chirality, size, and charge. Biological assays frequently make use of affinity columns, which use the preferential binding of active proteins or amino acids to separate biological components from a mixture. These columns may come packed with solid media that is uncoated or pretreated for an application. Immobile metal ion affinity chromatography (IMAC) columns are a common choice when creating an affinity assay. Protein A affinity uses a stationary phase coated in a bacterial protein that bonds with immunoglobulin A, allowing for its purification through LPLC. Desalting columns are used to separate macromolecules from smaller molecules in the same mixture, such as when removing salts from a protein mixture or phenol from nucleic acid preparations.
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Due to the need to transport the mobile phase throughout the process, LPLC often required various tubing fittings, adapters, and other accessories. When selecting components for a system, the chemical compatibility of the components should be considered. If working in sanitary conditions, components specifically designated for those conditions should be used. Syringe filters and membrane filters are also important to ensure the mobile phase remains contaminant-free and will not produce false readings at the end of the process. Generally, one solvent filter is placed as the mobile phase exits its storage and another before sample injection. Glass columns also need appropriately fitting O-rings and frits that should be changed with regular use.
While LPLC is characterized by modular components, most HPLC systems are usually made by a single manufacturer, but still spread into separate components: the reagent(s), pump, injector, column and detector, all connected by tubing. The pieces of an HPLC system which are regularly switched or replaced are the columns, filters, and other accessories that wear down with regular use. Other, more extensive maintenance is typically performed by the system manufacturer or by a trained technician.
HPLC columns are typically 25 cm long, stainless steel tubes of various inner diameters, the most common of which is 4.6 mm; however, significant variance from typical dimensions is often present. Columns available for HPLC are packed with silica beads with various surface chemistries allowing for subtle but useful effects on separation. The choice of column is often based upon the chemistry of the analyte as well as preferred mobile phase reagents, which combine to provide optional retention and selection.
In HPLC, the two most popular modes of separation are normal phase and reversed phase. In a normal phase assay, the stationary phase begins more polar than the mobile phase. This means the stationary phase consists of unmodified silica, which is naturally covered in polar, acidic silanol when exposed to water. To run a reverse-phase chromatography assay, the surface of the silica is coated by a nonpolar compound such octadecylsilane (ODS)—otherwise referred to as C18.
Chiral selection takes place where a racemic mixture is introduced to a column coated in a compound which interacts with each enantiomer to a different degree of preference. The molecule attached to the stationary phase is often a chiral molecule itself, but without its structural mate present. Because of this, the attached molecule can form an inclusion complex with one of the target analyte’s enantiomers and retain it. The other enantiomer is left in the mobile phase and will elute first. Selecting a chiral stationary phase relies on an understanding of the analyte’s structure as well as being able to predict its interaction to the stationary phase’s bound molecule.
To separate by ionic charge in ionic exchange chromatgraphy, prepacked columns typically make use of two resins. Anionic selection columns consist of DEAE-C, or diethylaminoethyl cellulose (a positively charged resin). This resin holds a single positive charge, providing an attraction to anions that is both strong and able to be out competed. Cationic selection columns hold a Q functional group bound to cellulose or sepharose. The Q group is a quaternary amino group that holds a negative charge.
Size exclusion columns are packed with many silica beads which create barriers to the analyte carried by the mobile phase. They leave few openings for large molecules to pass through, meaning they take fewer paths overall and elute first after a shorter journey through the column. Small molecules have more options and take more paths when moving through the column, causing them to elute later.
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HPLC systems rely on consumable parts to maintain their working order. Valve seals, frits, injection needles, and plungers used during the chromatography process will eventually need replacement. HPLC also requires a pure mobile-phase media, or damage to its column and detector can occur. To help maintain the purity of the mobile phase, using appropriate inlet solvent filters, bottle caps, and other HPLC accessories are useful.
A GC system is typically made by one manufacturer. However, each system shares similar components. There is a gas supply where the mobile phase originates. This supply may be a single-use tank or a container filled by a gas generator. The mobile phase leaves the supply to pass into the GC column while under pressure. The column itself is placed inside an interior compartment, or oven, of the chromatography machine where it is heated during the analytical process. Samples are introduced to the GC by an injector, which is frequently automated so multiple runs can be completed sequentially without intervention. Liquid samples are provided to the injector and injected into a superheated environment to induce vaporization. Extreme heat is maintained in the GC’s interior oven to prevent condensation in the GC tube. Once passed through the GC tube, analytes enter a detector generating a signal in the presence of the analyte, which is used for data analysis. After leaving the detector, the mobile phase travels to a waste receptacle, often a pressurized container.
The mobile phase used in gas chromatography is gaseous . However, the gas used needs to be undetectable by the GC system so that it does not interfere with data generation. To this end GC mobile phases or carrier gases are small and often inert. Oxygen can often damage the stationary phase of a GC column, so normal air should never be passed through a gas chromatography device. While the most common carrier gases are nitrogen, hydrogen, and helium, nitrogen is the most economical and stable of the gases, able to be generated by a gas generator and kept in a standard pressurized tank.
Gas chromatography columns are either glass or stainless steel and can be split into two major categories: packed columns and capillary columns. Packed columns contain spheres of diatomaceous earth particles coated with the desired stationary phase. These spheres are loose in the column. Packed columns are used for fixed gas separation and when sample concentration is very high. Capillary columns are a more recent technology and differ from packed columns in that the entirety of the interior column wall is coated in the stationary phase. Capillary columns are thinner and longer in length than packed columns. This enables the column to separate low concentrations of sample into defined peaks. Most GC methods take place within capillary columns due to their superior sensitivity and low sample requirements.
The stationary phase of a GC column is responsible for its retaining action. Unlike in liquid chromatography, the mobile phase during a GC application plays no role in the separation chemistry. As a result, GC columns have a wide variety of stationary phase formulations to cover a spectrum of possible analytes. These phases often have proprietary formulations that provide even more specialization, such as temperature resistance or separations ideal for specific detectors.
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GC columns require routine replacement parts to stay in optimal condition. The ends of the column which attach to the sample injection site often become burnt and damaged due to the introduction of freshly vaporized compounds. These ends can be protected with column guards, but regular trimming of burnt column ends with a ceramic razor must still occur. The column is held flush with the injection port via a tube called a ferrule. Ferrules become worn with heat and prolonged use and should be replaced when the column is trimmed. The injection site also requires a frit to filter incoming analyte of impurities that may damage the GC column. This frit should be replaced every few runs to prevent cross contamination of samples. The injection syringe becomes worn with use and requires periodic replacement of its needle and plunger. Failing to replace any of these components regularly can lead to a loss in sensitivity for the GC machine or damage to the GC column.
GC systems also make use of filters to purify the mobile phase before it enters the system, and clean the effluent waste at the end. Waste is often stored in pressurized containers that need to be monitored for leaks and stress points to prevent dangerous exposures. Other GC accessories help with the readability of certain compounds. One such system is an analytical thermal desorption unit, which concentrates trace volatile organic compounds into a readable range.
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When needing instrumentation involved in chromatography systems of low-pressure liquid chromatography (LPLC), high-performance liquid chromatography (HPLC), or gas chromatography (GC), there is much to consider from selecting a complete system to choosing accessories. The options presented are by no means comprehensive but provide a starting point to constructing a functional chromatography solution.