Perfusion chromatography is an emergent separation technique, primarily introduced to improve the resolving power of liquid chromatography. Obtaining single, sharp, well-resolved chromatographic peaks is the foremost goal of any liquid column chromatography. Inefficient mobile phase flow through the chromatographic column can lead to peak broadening and poorly separated peaks. To overcome all these issues, perfusion chromatography is considered a viable choice. All the information related to perfusion chromatography is included in this article so let’s begin reading.
What is perfusion chromatography
The word ‘perfusion’’ refers to the passage of flow provided to a fluid. Therefore, fluid dynamics is the most important factor on which perfusion chromatography is based. Perfusive type of chromatography employs a porous stationary phase material with pore dimensions equal to or larger than 100 nm. It is called the perfusion media.
Large pore sizes support a mass transfer of mobile phase and analyte molecules through the stationary phase packing inside the column. Perfusion chromatography is specifically important for the large-scale separation of biomolecules.
Perfusion media as stationary phase in perfusion chromatography
A perfusion media is composed of two types of pores: through pores and diffusive pores. The through pores have dimensions in the range of 600-800 nm while diffusive pores lie between 80-150 nm. This intricately porous structure enables a better mobile phase flow as well as providing better access to macromolecules into the pores of the stationary phase. Porous polymers such as polystyrene or co-polymerized poly(styrene-divinylbenzene) can be used as a perfusible stationary phase packing material.
How is perfusion chromatography different from other chromatographies
The porous stationary phases traditionally used for liquid chromatography are developed such that their morphology restricts mass transport of solute and mobile phase within the chromatographic packing. The pore size is decreased to provide a large surface area for analyte adsorption in conventional liquid chromatography. But this simultaneously hinders mobile phase flow, leading to a stagnant pool of mobile phase inside the column.
Additionally, a slow diffusion of analyte molecules occurs into and out of the stationary phase pores. Small molecules get strongly retained while large molecules easily pass through. This slows down chromatographic separation; peak broadening occurs with a large dead volume in between the peaks.
However, in perfusion chromatography, the pore morphology is so designed that the analyte and mobile phase flow through the stationary phase pores by a combined effect of diffusion and convection. The presence of through pores allows a fast movement without compromising the high surface area provided by diffusion pores. At higher flow rates, convection flow dominates over diffusion and vice versa.
Working principle of perfusion chromatography
As already discussed there are two types of pores in perfusion chromatography. Through pores are large enough to allow an unhindered, convection flow of solute and the mobile phase. On the other hand, the smaller diffusive pores provide an adsorptive surface area for selective analyte retention thus facilitating the overall separation mechanism. Due to the convection flow, all the solute molecules can quickly reach the diffusive pores thus the entire chromatographic process is sped up.
How to perform perfusion chromatography
Here is a step-by-step guide on how perfusion chromatography is performed, mentioning all its important components:
Step I: Column packing
Commercially packed stainless-steel columns are used for perfusion chromatography, just like HPLC. The difference lies in the packing material. Physically and chemically stable porous polymers are used for column packing. Column diameters can range from 2.1-16 mm.
Step II: Mobile phase
Buffer solutions based on alcohols, acetonitrile, and water, are degassed, and used as mobile phase solvents in perfusion chromatography. Tetrahydrofuran can also be added in a small proportion as a mobile phase additive.
Step III: Pumps
Instead of high-pressure pumps, relatively simple, peristaltic pumps are used in perfusion chromatography to pump the mobile phase solvent through the column. A high mobile phase flow rate (10 mL/min) is usually maintained while performing perfusion chromatography.
Step IV: Sample injection
The sample mixtures are dissolved in the mobile phase, centrifuged, and filtered to remove any impurities present. It is then injected into the perfusion column through an auto-injector. A large injection volume can be used at one time.
Step V: Elution
Analytical separation takes place in the column depending upon the mode of interaction for example reverse-phase interaction. The large pore size of the stationary phase material and different types of pores present allows a mass transfer and thus fast analyte elution.
Step VI: Detection
Perfusion chromatography is mainly used for sample purification thus different product fractions are separately collected at the end. But, various types of detectors can be coupled with a perfusion column in case component detection and identification is needed. An ultra-violet detector and a fluorescence detector are common detector choices for perfusion chromatography.
Step VII: Recorder
The detector sends a signal to the recorder which then plots a chromatogram. Extremely well-resolved peaks with reduced dead volume are the forte of perfusion chromatography, indicating a good sample separation.
Step VIII: Column cleaning and rejuvenation
At the end of the chromatographic separation, the mobile phase flow direction is reversed to flush out any particulate or contaminants stuck onto the stationary phase. This refreshes the column for the next batch of separations.
Strengths of perfusion chromatography
- Perfusion chromatography overcomes the mass transfer bottleneck experienced in conventional liquid chromatographic columns.
- It supports a high mobile phase flow rate.
- Perfusion chromatography allows faster analyte separation, 10-30 times faster than conventional high-performance liquid chromatography (HPLC).
- A separation that requires 100 mins at a flow rate of 1 mL/min via conventional liquid chromatography, can be completed via perfusion chromatography in just 10 mins at a flow rate of 10 mL/min.
- It offers good selectivity, extreme flexibility, and gentle chromatographic purification.
Applications of perfusion chromatography
- Perfusion chromatography allows both analytical as well as preparative separation of biomolecules.
- Perfusion chromatography is an ideal choice for analyzing high viscosity, unstable macromolecules that otherwise experience a high mass transfer barrier in conventional liquid chromatography.
- It is specifically used for the analysis of food proteins. The protein composition of a foodstuff can be determined using perfusion chromatography at high speed and low cost as opposed to other relatively expensive chromatographic techniques such as FPLC.
- Perfusion chromatography finds applications in genetic engineering and biochemical research. Preparative analysis of new polypeptides and proteins in their development and production phase can be done on a low budget using perfusion chromatography.
In conclusion, we can say that perfusion chromatography is a large-scale chromatographic separation technique that allows routine analysis under economically restrained conditions. It speeds up a chromatographic separation process without scarifying the quality of results obtained.
Perfusion chromatography supports separation and analysis in all chromatographic modes except size-exclusion chromatography.
Size-exclusion chromatography selectively depends upon analyte separation on the basis of size. Therefore, it cannot allow an unrestricted mass transport of all analyte molecules through the stationary phase pores.
1. Garcı́a, M. C., M. L. Marina and M. Torre (2000). “Perfusion chromatography: an emergent technique for the analysis of food proteins.” Journal of Chromatography A 880(1): 169-187.
2. Gordon, N. F., D. H. Whitney, T. R. Londo and T. K. Nadler (2000). Affinity Perfusion Chromatography. Affinity Chromatography: Methods and Protocols. P. Bailon, G. K. Ehrlich, W.-J. Fung and W. Berthold. Totowa, NJ, Humana Press: 175-193.