FPLC stands for fast protein liquid chromatography. Fast protein liquid chromatography (FPLC) is a form of high-performance liquid chromatography (HPLC). FPLC utilizes a small diameter stationary phase which enables higher resolution than achievable with traditional gravity flow chromatography.
In 1982, in Sweden, Pharmacia developed and marketed the first fast performance liquid chromatography system. Pharmacia developed this system to enable them to promote their new line of high-performance protein purification resins. Pharmacia trademarked the term FPLC. Pharmacia created the FPLC to take advantage of their small resin technology. Neither gravity flow nor peristaltic pumps could provide the necessary pressure to move the buffers through the resins to achieve a separation. Pharmacia promoted this as a revolutionary technique, proving that they could purify proteins faster and with higher resolution than was previously achievable.
Why FPLC Systems?
The primary focus of fast protein liquid chromatography is protein purification. It was developed to purify mixtures of proteins. FPLC incorporates high loading capacity, biocompatible buffer systems and fast flow rates.
A fast protein liquid chromatography (FPLC) system can offer a high level of automation by incorporating software, valves and a fraction collector.
Chromatography resins that can be used include ion exchange, gel filtration, hydrophobic interaction and affinity. FPLC uses aqueous salt buffer systems.
An FPLC system can consist of a combination of the following components:
FPLC pumps
UV detectors
conductivity monitors
PH monitors
fraction collectors
switching valves
injection valves
software
PEEK tubing
FPLC systems can be configured as simple as an isocratic system for gel filtration or as complex as a multi-step, multicolumn purification system using multiple modalities with automated fraction collection.
FPLC Systems can be used in teaching labs, biotech companies, University research labs, biopharma companies and any other lab interested in doing protein purifications. FPLC systems can scale up to preparative and process levels.
Fast protein liquid chromatography is a similar technique to HPLC but their chromatographic methods are significantly different. As we explained, FPLC is a purification technique used to separate protein mixtures and collect the separated components of interest. HPLC is an analytical technique mostly used for quantitative and qualitative analysis of liquid samples. Both methods incorporate detectors, pumps, valves and software. FPLC uses fraction collectors as well as conductivity and pH monitors. Another differentiating factor is that FPLC is considered a preparative technique while HPLC is an analytical technique.
Reversed phase chromatography is most often used in HPLC. HPLC uses solvents in the mobile phase while FPLC uses salt buffers. FPLC is a purification technique that is typically a multi-step process incorporating different purification modalities including gel filtration, ion exchange and affinity chromatography.
Fast protein liquid chromatography pump materials differ from that of HPLC pumps. FPLC pump heads are made of either titanium or PEEK materials and bench top pumps can deliver flow rates up to 150 mls/min. with back pressures of approximately 725 PSI at high flow rates. FPLC back pressures are usually lower than HPLC back pressures and flow rates in FPLC are usually higher. HPLC pump heads are made of stainless steel and flow rates usually do not exceed 10 ml/min. but back pressures can be as high as 6000 PSI or even higher.
FPLC and HPLC columns, column material and particle sizes differ as well. FPLC columns have either glass bodies with high performance resin tops and bottoms or are made out of PEEK. HPLC columns are made from stainless steel.
FPLC’s use agarose, polymer or silica materials. Most HPLC resins are made out of silica beads with tiny particle sizes able to stand very high back pressures. The particle sizes for FPLC are larger and have larger pore sizes. FPLC resins can’t handle very high back pressures due to their resin types and particle and pore sizes. HPLC uses stainless steel in its flow path. FPLC flow paths avoid stainless steel and replace it with PEEK, titanium or high-performance composites.
FPLC is used to purify larger molecules like proteins, peptides and nucleotides. HPLC is used to separate small molecular weight compounds.
As the two techniques have differences, so does their software. FPLC software controls the pumps, detectors and valves while integrating the functions of the fraction collector for sample collection. FPLC systems monitor UV absorption, pH and conductivity.
The first commercial HPLC system was introduced by the waters Corporation in 1969. In 1982, the first FPLC system was introduced by Pharmacia. Today many companies have HPLC systems but only offer a few FPLC systems. There have been dramatic improvements in both technologies since their introductions.
FPLC systems can incorporate the same detectors that are used in other forms of liquid chromatography. UV detectors used in liquid chromatography can be incorporated into FPLC systems, if they have inert flow cells.
ICON Scientific, Inc
