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).
LPLC system instrumentation and components
Modern low-pressure liquid chromatography (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.)
A peristaltic pump for chromatography
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.
The LPLC column
The column used for LPLC can be an empty glass container or prepacked. The stationary phase of an 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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