Principles of Chromatography

2.3.1.1 Column Chromatography

Chromatography is a purification technique by which sample components are separated according to their ability (or inability) to interact with a mobile phase or a stationary phase through which the sample is passed. Although there are many types of chromatography available today, the underlying core separation principle is essentially the same for all of them. One of the most basic and most common chromatographic techniques is column chromatography, in which a glass or plastic tube is filled with a mixture or slurry of a liquid mobile phase (typically a buffer) and a solid stationary phase. The sample, or solute, is applied to the top of the mixture (called the bed) in the vertically-supported tube and is allowed to pass through the two phases either by gravity flow or with pressure applied from an attached pump. The components of the sample distribute themselves between the mobile and stationary phases depending on their affinity for either (which is determined by the inherent chemical properties of each component). Molecules that have little or no affinity for the stationary phase spend more time in the mobile phase, pass quickly through the chromatography system, and are removed, or eluted. Molecules that have affinity for the stationary phase spend more time in the stationary, move more slowly through the system, and are eluted more slowly. The mobile phase that is collected after passing through the chromatography system is called the effluent and is or contains the sample molecules. Usually, the effluent is collected at different times or in fractions (such as for 2 Min periods or in 1-2 mL portions). Typically, fractions which are found to contain the desired components will be combined, or pooled, for further analysis.

In column chromatography, the solid phase is an adsorbing material which contains openings of various sizes (known as mesh sizes) through which the solute may pass. Low mesh sizes (20-50) have a lower number of openings per square inch of packed material and, thus, have a faster flow rate. Higher mesh sizes (200-400) have a higher number of openings per square inch and, thus, have a slower flow rate and are used mainly during high-resolution separations of nearly pure solutes.

2.3.1.2 Other Types of Chromatography

2.3.1.2.1 Ion-Exchange Chromatography Ion-exchange is a form of adsorption chromatography set up similarly to column chromatography in which solute molecules are separated on the basis of their affinity for a charged stationary phase. The column is packed with a stationary phase consisting of a synthetic resin that is tagged with ionic functional groups which electrostatically interact (reversibly) with ionic solutes. Depending on the experimental conditions, the solutes may have a negative or positive charge, or none at all (neutral). Solutes that are neutral, or that have a charge similar to that of the stationary phase, will elute with the buffer. Bound solutes can be released by increasing the ionic strength of the buffer (displacement) or increasing the pH of the buffer (weakening the interaction by charge reduction of the solute or resin).

2.3.1.2.2 Gel Exclusion Chromatography Gel exclusion chromatography is a form of column chromatography in which solute molecules are separated based on their molecular size. The column is packed with a stationary phase consisting of synthetic beads that contain small pores of a controlled size. Solute molecules that are too large to enter the pores will flow quickly through the column. Solute molecules that are small enough in size to enter the pores will remain in the column longer and thus are retarded in their travel through the column. The exclusion limit of the stationary phase defines the molecular mass of the smallest molecule that cannot enter the bead pore. Solute molecules with a molecular size greater than the exclusion limit will pass directly through the column. Gel matrices with an exclusion limit of approximately 6000 Daltons are routinely used for desalting of a protein sample following a salt precipitation procedure.

2.3.1.2.3 Thin-Layer Chromatography (TLC) TLC is performed by (1) coating a glass plate (similar in size to a microscope slide) with a thin layer of silica gel, alumina, or cellulose; (2) spotting a small drop of sample onto the coating and allowing it to dry; (3) and then standing the plate vertically in a shallow dish containing an appropriate solvent. As the solvent front moves vertically through the gel coating by capillary action, the solute components are separated. Samples that are not colored are sometimes detected by radioactive or fluorescent methods, or through treatment with chemicals that develop colors. Unknown sample components are identified by comparing their distances traveled to those of known standards.

2.3.1.2.4 High-Performance Liquid Chromatography (HPLC) HPLC, one of the most common chromatographic techniques performed by food scientists, is an analytical technique well suited to the separation and identification of biological molecules such as proteins, nucleic acids, phenolic compounds, carbohydrates, and lipids. With the development of stationary phases with very small particles sizes and large surface areas, HPLC improves elution rates by applying high pressure to the solvent flow. The result is high resolution of a solute into its individual components in a relatively short period of time (typically between 10-60 Min). Unknown compounds are identified by comparing their retention times (that is, the amount of time required for the compound to elute from the column under specific experimental conditions) to those of known standards, either alone or in combination. The computerized systems in use today are relatively simple to use as they are largely automated and can be controlled by accompanying software.

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