Chromatography Technique
Chromatography Technique - Related Videos
03:03
An Affinity Chromatography Technique for the Purification of Monoclonal Antibodies
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2025
This video demonstrates a technique for purifying monoclonal antibodies from the culture supernatant obtained from an antibody-producing cell culture. The process involves using a matrix of crosslinked agarose beads with conjugated Protein A, which immobilizes the antibodies in the supernatant on the beads, allowing for effective separation from contaminants. By applying an elution buffer with low pH, the protein A-antibody interaction is loosened, facilitating the collection of...
02:54
An Affinity Chromatography Technique for the Purification of a Recombinant Bacterial Protein
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2025
Source: Becker, B., et al. A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast. J. Vis. Exp. (2017).This video demonstrates the purification of a polyhistidine-tagged recombinant protein using nickel-based affinity chromatography. It outlines key steps, including bacterial lysis by sonication, clarification and filtration of the lysate, and selective binding and elution of the target protein using imidazole.
06:04
Thin Layer Chromatography Based Enzyme Activity Assay: A Technique to Determine In Vitro Pyrophosphokinase Activity Using Thin Layer Chromatography
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2025
This video demonstrates a thin-layer chromatography-based enzyme assay to determine pyrophosphokinase activity using radiolabeled nucleotides. The method provides insights into nucleotide synthesis and phosphotransfer reactions.
04:38
A Size Exclusion Chromatography Technique to Purify Mycobacterial Extracellular Vesicles
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2025
This video demonstrates the size exclusion chromatography (SEC) technique for purifying bacterial extracellular vesicles (EVs) from the culture filtrate of Mycobacterium tuberculosis (Mtb). The technique utilizes a porous resin-packed column to obtain purified fractions of EVs. The porous particles selectively allow smaller proteins to enter the pores, resulting in slower elution. At the same time, EVs are excluded from entering the pores due to their larger size and are eluted first.
03:59
High-Performance Thin Layer Chromatography: A Technique for Preliminary Separation and Quantification of Lipids From Neutrophils
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2025
This video demonstrates a technique for the preliminary separation and quantification of lipids from human peripheral blood-derived neutrophils. This technique helps in the initial evaluation of target analytes before proceeding with HPLC.
FAQs
Separation quality depends on how differently each component interacts with the mobile and stationary phases. Adsorption, partitioning, size, charge, and chemical affinity can cause substances to move at distinct rates. When these differences are sufficiently large, individual components become easier to identify, isolate, measure, or evaluate for purity.
Adsorption causes a component to interact with the surface of the stationary phase, while partitioning reflects how it distributes between the mobile and stationary phases. A component that spends relatively more time associated with the stationary phase moves more slowly than one that remains preferentially with the mobile phase, producing separation.
Paper, thin-layer, gas, and liquid chromatography provide different analytical formats in which components encounter phases and conditions that influence movement. Consequently, the same mixture may show migration behavior in one method or retention times in another. The selected format affects how researchers identify components, monitor reactions, or assess purity.
A researcher places the mixture into a chromatographic system, allows the sample to move with the mobile phase across or through the stationary phase, and observes the resulting separation. Individual migration behavior or retention times are then compared with standards. This comparison supports component identification and helps evaluate the mixture’s composition.
Chromatography can be applied during reaction monitoring to examine mixture composition as a reaction proceeds. By separating the substances present and comparing their migration behavior or retention times, researchers can assess which components are present and track changes in the mixture. This makes the technique useful for following chemical transformations in laboratory work.
Separated components can be isolated when the method provides sufficient distinction between substances, supporting purification and compound characterization. For measurement, advanced chromatographic instruments provide sensitive quantitative results, while comparisons with standards help assess composition and purity. These capabilities extend chromatography beyond visual separation to analytical evaluation of chemical samples.