Fast Protein Liquid Chromatography

Low pressure liquid chromatography is a rapid and robust method for purification of molecules from a mixture. The sample is applied to a column composed of functionalised beads to which the molecules in the sample can bind. Liquid is pumped through the column and, depending on the properties of the liquid relative to the beads, the sample molecules and any contaminants are sequentially removed.

Fast protein liquid chromatography is just liquid chromatography in which the beads are functionalised specifically to bind particular types of protein, such as those that carry a tag that binds metal ions. One of the most common types of protein chromatography is ion exchange, where positively charged proteins will bind to to negatively charged beads, or vice versa, and proteins are removed from the column by altering pH or ionic strength of the liquid. The output from the column is typically monitored by UV absorbance and collected in fractions.

Liquid chromatography is extremely widely used for preparative and semi-preparative separations and purifications of molecules ranging from small chemicals to large macromolecules.

Analyte molecules bind to LC columns based on hydrophobicity, hydrophilicity, charge or because they have an affinity for the beads of the column. Elution is by variations in ionic strength, pH or by inclusion of molecules that have a stronger affinity for the beads than the analyte molecules.

We have access to columns ranging from analytical to large laboratory preparative scales. We can pack these columns with a range of materials, including hydrophobic interaction resin, ion exchange resins, nickel-ion affinity or gel filtration resins. We also have a range of pre-packed columns, particularly for size exclusion separations, and have several FPLC instruments to choose from with flow rates ranging from 1-2 ml/min up to 40-50 ml/min.

Low pressure LC and/or FPLC work well for initial phases of purification of chemicals or biomolecules. Rapid enrichment can be achieved with minimal method development. Examples include:

• Purification of antibodies from culture supernatant
• Isolation of recombinant proteins from bacterial lysates
• Initial isolation of chemical reaction products from reagents
• Purification of components of a simple mixture of chemicals.

LC or FPLC are typically preparative or semipreparative scale processes, hence larger amounts of sample are required than, for example, HPLC or UPLC separations which tend to be performed at analytical scale.

Analytes need to be in solution or easily dissolved- solids and particulates must be removed prior to chromatography.