Sample Purification

Graph illustrating the key stages of affinity chromatography separation involving bind, wash and elute steps.

Various chemical and physical purification methods can be applied to separate or concentrate a target analyte prior to analysis. Among these are absorption, adsorption, chromatography, distillation, extraction, ion exchange, filtration, complex formation, crystallization, drying, and many more. Sample preparation prior to analysis helps bring a sample to a format that is compatible with the analytical technique, reduces sample complexity, removes interfering impurities, and concentrates the analyte prior to analysis. Appropriate sample preparation will prevent clogging, may reduce the need for stringent washing procedures, and can extend the life of the chromatographic medium.


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Some of the following most common purification techniques are used in the laboratory to prepare samples for downstream analysis.

Dialysis

Dialysis is a purification technique used to remove salt or other low molecular weight molecules from a sample. It is also used to exchange a sample’s buffer composition. Dialysis is a passive technique requiring large volumes of buffer.

Buffer Exchange & Desalting

Desalting is a simple method to remove salts and other low molecular weight contaminants from a sample. It is also used for buffer exchange before or after different chromatographic steps and for the rapid removal of reagents to terminate a reaction.

Fractional Precipitation

Fractional precipitation is used to remove gross impurities from small sample volumes. Precipitation techniques separate fractions by the principle of differential solubility. Because protein species differ in their degree of hydrophobicity, increased salt concentrations can enhance hydrophobic interactions between the proteins and cause precipitation.

Extraction

Sample preparation by extraction involves the isolation of target analytes from a complex sample, or much larger sample volume. The process removes interfering sample components that may block HPLC and GC columns. It also increases analyte concentration by a factor of 100 to 5,000, thereby significantly improving detection sensitivity. As part of selective and specific sample preparation, extraction helps ensure more reliable chromatographic analysis. Types of extraction are liquid-liquid extraction, solid phase extraction (SPE), and solid phase microextraction (SPME).

Affinity Chromatography

Affinity chromatography separates proteins based on reversible interactions between a protein and a specific ligand that has been coupled to a chromatography matrix. The technique is highly selective and allows for high capacity protein purification. Affinity chromatography can be used whenever a suitable ligand is available for the protein of interest.

Purification by affinity chromatography involves capture and elution steps. In the capture phase, the target protein is specifically and reversibly bound to a complementary ligand that has been immobilized on a chromatography matrix. The objective is to isolate, concentrate, and stabilize the target product, conserving its potency and activity. After washing the matrix to remove unbound material, bound target protein is recovered by changing conditions to those favoring elution. Elution is performed specifically, using a competitive ligand, or nonspecifically, by changing the pH, ionic strength, or polarity. Elution allows the target protein to be collected in a purified and concentrated form.




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