FPLC versus HPLC: Two divergent separation technologies with a common ancestor

Fast Protein Liquid Chromatography (FLPC) and High Performance Liquid Chromatography (HPLC) are both designed to separate biological molecules (although HPLC has extended usage in the small compound and synthetic chemistry worlds). That is where the similarity essentially ends. While they both arose from a common core technology, innovations coupled with expanded applications have led to functional diversity between the two. Key differences between FPLC and HPLC  include the target molecules, separation media, column types, methodology, which we explore below.

Target Molecules

FPLC chromatography of protein biomolecules is very demanding due to the inability of these analytes to withstand high temperature, high pressure, or the solvents typically used in HPLC. This has necessitated the development of hardware and techniques that will accommodate the wide range of biomolecules, each with specific behaviors and preferred conditions.

HPLC, conversely, excels in the separation of molecules or compounds that are solvent, pressure, and heat resistant. HPLC techniques have less to do with preserving complex structure and function and more to do with maintaining solubility.

FPLC is applied to purify large biomolecules several kilodaltons (kDa) in size including proteins, nucleotides, and large peptides, with the goal to obtain as much pure and native product as possible. A challenge in FPLC is purification of molecules from complex source materials (such as cell lysates) which are often rich with DNA, RNA, cell membranes and other matrix components. For this reason, sample loops much larger than those used for research scale HPLC are often used to effectively dilute the sample. Subsequently, higher flow rates can be employed.

HPLC, on the other hand, is primarily used for the identification, characterization, and quantitation of small compounds (biological or otherwise) ranging in size from a few atoms up to approximately 3000 Da. These compounds do not have the complex requirements for retention of native structure and function implicit in larger biomolecules. They are therefore often suspended in organic solution and are subjected to high pressure and sometimes heat during chromatographic separations. Furthermore, there is typically less of a concern of a complex heterogeneous background such as that of the typical cell lysate, and as a result smaller sample loops are typically used.

Separation Media 

This brings up another major difference — separation media. For HPLC, silica beads with very small particle sizes and with large resistance to high pressures are used as the column matrix. FPLC, conversely, requires agarose or polymer material with larger particle and pore size. The resins for FPLC are not nearly as pressure resistant and can lose integrity under conditions reserved for HPLC. They are also sensitive to air bubbles, a problem that must be monitored to prevent the separation bed and column performance from becoming compromised.

Columns

Not only are the resins different but the columns are distinct as well. In most applications, HPLC columns and sample loops are made of pressure-resistant stainless steel in order to handle the high pressure of the technique. Of course, the relatively low pressures used in FPLC mean that glass columns can be used. These are more compatible with biomolecules of interest, with the added bonus that the column bed can be readily checked for bubbles and integrity throughout a run.

Methodology

Separation methods between the platforms are both similar and distinct. Analytical HPLC can involve reversed phase chromatography with hydrophobic stationary phase and polar mobile phases. Alternative methods include: partition chromatography, normal-phase chromatography, displacement chromatography, size-exclusion, ion-exchange. FPLC encompasses a rather wide array of methods as well including: ion-exchange, affinity, size-exclusion, gel filtration, and others, all relevant to the complex nature of the background and the exclusive nature of the target molecule.

Other Differences 

Of course there are many other differences as well, including those centered around the organic and sometimes caustic nature of the solutions used in HPLC and the larger flow rates used in FPLC. The pumps, seals, fittings, injection ports, auto samplers, controllers, and others are unique to each platform. The differences are more distinct as the leap is made from analytical scale to preparative or process scale.

In the end, both platforms have a multitude of uses and advantages. The question comes down to the biochemical nature of the target molecule and the background, the desired yield, and other relevant objectives.

Major players in the HPLC field include: Waters, Agilent, Shimadzu, and a number of other high end analytical instrument manufacturers. Famous in the FPLC field is the AKTA brand from GE, who has had a past history as rich as the Agilent 1100/1200 series HPLC.

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Updated Dec 2021