Dual Flow Chromatography - Protein Purification in a Pipette Tip

Dual flow chromatography (DFC) is the unique process of performing chromatography separations in a column bed loosely packed at the end of a pipette tip, retained by a minimally absorbing, proprietary mesh – a PhyTip®. Why? To enable automated sample purification on automated liquid handling systems while obtaining high sample recovery and purity.

The DFC technology was developed by Douglas Gjerde in 2004 with the objective of automating highly manual processes such as small scale protein purification on common liquid handling robotics such as Hamilton STAR or Tecan Freedom Evo. Nowadays, DFC can also be used for automated plasmid purification or other compound types.

How Does it Work?

The pipette head lowers the PhyTip® column into the sample and pumps the sample back and forth across the packed resin bed inside the tip. As the sample mixture is cycled back and forth more than once with a controlled flow across the resin bed, the biomolecules in the sample is given enough time to interact with the available binding sites despite the slow transfer kinetics associated with large biomolecules. As a result, all of the protein or plasmid can be captured during the capture step, leading to very high recoveries at the end of the purification process. And if this is not the case, this is easily adjusted by simply applying more DFC cycles to increase the contact time between the sample and the binding sites on the resin. In this sense, as DFC methods are developed, traditional chromatographic principles are applied. The same applies to the wash step and elution step – the number of cycles is an easy way of fine-tuning the purification process for a specific application. And when each step of the protein purification protocol is completed this way and driven to equilibrium, the purification will perform the same every time. That means the results are highly reproducible with the same sample purity and yield every time.

Gentle Purification Yields Biologically Active Biomolecules

DFC is a very gentle purification process which causes less aggregation than single-pass chromatography processes, which maintains the biomolecule in its native, biologically active state. For proteins this means less aggregation and denaturation and more biologically active protein in following assays. For plasmids, this means that the plasmid maintains its supercoiled state during the purification process –which is beneficial downstream if the sample is to be used for transient transfection.

Active Transport Leads to Concentrated Sample

Then, what’s the point with using a packed resin bed? Well, as the sample is pumped through the packed resin bed rather than loose resin, the biomolecule is actively transported to functional groups available on the resin. The result is that the biomolecules are captured on a smaller resin bed and can be eluted in a smaller volume, which leads to a higher final concentration of the purified, eluted sample. A higher concentration can be especially useful in following assays with limited detection thresholds.

Dual Flow Chromatography – for Plasmids Too!

Not only proteins can use purified by the use of PhyTip columns and dual flow chromatography. This purification process can also be applied to plasmid miniprep too.