It is also used to collect separated compounds of interest and identify protein profiles within single Biomolecule mixtures can include proteins, peptides, oligonucleotides, DNA, and RNA. Because biomolecules are highly sensitive and can’t withstand the type of high pressure, temperature, or solvents used in high-performance liquid chromatography (HPLC), FPLC is employed. FPLC is also used to collect separated compounds of interest and identify protein profiles within single proteins.
You may hear FPLC referred to as medium-pressure chromatography, biochromatography, or biopurification, but the title doesn’t change the process. It’s an important method for many researchers in the life sciences and biotech.
Separating a sample within a mixture depends on each molecule’s specific interaction with the stationary and mobile phases used during chromatography. FPLC utilizes a wide range of resins, composed of many small coated beads, for its stationary phase, and a an aqueous solution or buffer for its mobile phase.
The targeted protein adheres to the resin as it flows through the separation column and the buffer carries any other molecules that are present out of the system. It’s typical to see a mixture of buffers used to accomplish this elution.
For example, having flushed the system, a secondary buffer, called an eluent, will separate the protein of interest from the resin and pull it into a detection system for measurement, recording the concentration levels of the target protein in the sample.
FPLC is a type of preparative technique, unlike HPLC which provides quantitative and qualitative analysis of liquid samples. It is used in all sorts of settings, from academia to research facilities to industrial manufacturing.
Its versatility is due in part to its simplicity, rapidity, and reliability as a technique, producing consistent results that end-users can count on.
Furthermore, its ability to support multiple columns simultaneously is based on its use of low pressure and offers a wide flow range when combined with multiple pumps, making the entire process efficient and highly scalable.
Some of the most common FPLC systems on the market include the AKTA FPLC system, offered by Cytiva—formerly GE Healthcare and Amersham Biosciences—and the AZURA® FPLC system, offered by KNAUER. (And guess what? We lease them.)
Fast protein liquid chromatography chromatographs operate using several key components and chromatographic modes.
FPLC systems consist of the following features:
This method separates molecules based on their specific charge, which depends on the pH or buffer. If a protein is more charged, it will be more likely to bind with an oppositely charged resin. Depending on salt concentration, a protein will take shorter or longer to elute from the resin as the salt ions compete with the molecule for adherence to the resin.
Using a substrate that is linked with the stationary phase, a sample is passed through and binds to the solid substrate according to its affinity for the substrate used. The compounds that haven’t bound are then washed out using an aqueous solution. The remaining compounds that are bound to the substrate are then eluted using a solvent with a certain pH or salt concentration.
This method is also referred to as gel filtration chromatography. A gel medium that consists of specifically sized beads that separate the sample mixture as it passes through. Molecules that are larger cannot pass through and are eluted from the column, while the smaller molecules diffuse into the gel medium and remain fixed in place. This results in separation based on size and molecular weight, as elution happens in order of linear weight.
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