When it comes to sample separation for target-purification, liquid chromatography is among the most reliable and widely-used methods. Based on various chemical/physical properties of molecules, nucleic acids, and proteins, resin-based chromatographyselectively skips or captures multiple targets of interest in sample mixtures.
Here are some tips from world leaders when it comes to resin-based technologies that will help you choose the ideal liquid chromatography resin for your needs.
Most Basic Principles
Liquid chromatography, for the most part, uses resins (small particles) packed into columns (stationary phase). Resins are prone to physical or chemical modification, towards the goal of having them bind/repel specific molecules inside complex mixtures.
To address the modern and ever-evolving needs of various researchers, a wide variety of different chromatography resins exist.
Resin-based chromatography is used for preparative and analytical purposes. For analyses, the end goalsare identification, discovery, and quantification (sometimes) of various components found in complex mixtures. When it comes to preparative chromatography, the objectives are purification and isolation of isolated molecules.
On occasion, particular targets might require more separation steps with different columns. Mixed resin methods often needto cover the needs that a researcher may have.
This is what selecting a liquid chromatography resin is all about.
Different Liquid Chromatography Resin Types
Ion Exchange Chromatography
This method (also known as IEX) focuses on molecule isolation based on the molecules’ total charge. Both preparative and analytic purification phases make use of this process.
IEX is based on covalent linking negatively – or positively-charged functional groups. A solid matrix is created (agarose, polystyrene, cellulose, etc).
The basics are that you load a protein sample onto a column that’s at low ionic strength. To increase the strength, the column is washed with buffers, to remove undesired impurities and proteins.
Hydrophobic Interaction Chromatography
This type of chromatography (also known as HIC) is based on separating the proteins on the basis of their hydrophobic properties.
The best part about HIC is that it uses matrices, parameters, and buffers that are much less denaturing than other methods. This method is often applied in combination with IEX and downstream size-exclusion (more about this below).
Size-exclusion chromatography is also known as SEC, for short.Their size dictates the protein arrangement here. Using a gel that consists of spherical beads with pores of specific sizes, molecules are excluded or included within the sample.
The proteins go through the column while being eluted. They are then ordered based on decreasing molecular weight.
SEC is often used to complement IEX or HIC, in cases where the involved proteins aren’t resolved through other methods. SEC is commonly the last purification step.
This resin-based chromatography type makes use of binding interactions between the binding partner and the immobilised ligand. The general applications that involve this method are enzyme/substrate, enzyme/inhibitor, as well as antibody/antigen interactions.
Affinity chromatography can purify both recombinantly-generated and native molecules. The best quality of this chromatography is the fact that it offers specific interaction, which provides quicker results.
Affinity is predominantly used as the first step in purification workflow. In some instances, this type of resin-based chromatography is the only step in the process.
This chromatography type is also known as mixed-mode chromatography. It is based on using resins that are capable of multiple interactions. In other words, it uses resins that utilise ligands that can withstand numerous interactions.
The multimodal chromatography is widely known for purification proteins with unknown specificity.The resin is used to purify, identify, and screen the sites that are a part of the target protein.
The best thing about multimodal chromatography is that it works so well with other resin-based chromatography types. It can even be used in combination with other types inside a single medium. So, what multimodal chromatography brings to the table is faster results that save the precious sample material. This chromatography type is particularly effective in cases of product impurities with similarities with the target molecule.
Selecting the Perfect Liquid Chromatography Resin
There is no magic formula that can tell you exactly which resin-based chromatography to use. In reality, it all boils down to what you’re working with and on you, as an expert. Hopefully, this guide will help you choose the perfect methods for liquid chromatography going forward.