Fast protein liquid chromatography (FPLC) formerly known as fast performance liquid chromatography is a protein-friendly chromatographic technique. It is known for purifying proteins with a high resolution and reproducibility. FPLC is a medium pressure technique. Based on its infrastructure and operation conditions, FPLC can be considered an intermediate between column chromatography and high-performance liquid chromatography (HPLC).
This article will not only introduce you to fast protein liquid chromatography; its working principle and versatile applications but at the same time we will discuss how is FPLC different from HPLC.
What is fast protein liquid chromatography
Fast protein liquid chromatography is a type of liquid column chromatography. The stationary phase is packed into a column while a liquid mobile phase moves past it. It is primarily used for the chromatographic separation and purification of bulky protein molecules. However, it can additionally be applied to related biomolecules such as nucleic acids, oligonucleotides, plasmids, etc.
FPLC supports a wide variety of analytical separations from a few milligrams of proteins to the production of several kilograms of purified protein samples. This technique was introduced and used for the first time by Pharmacia, Biotech Sweden in 1982.
Stationary phase in fast protein liquid chromatography
Porous gel resins of varying particle sizes, bead volumes, and ligand surfaces are usually used as stationary phases in FPLC. The most common stationary phase material for FPLC is a highly crosslinked agarose gel. Chromatographic separation on the stationary phase may follow different retention mechanisms like a reverse-phase interaction, ion exchange, size exclusion, hydrophobic interaction and/or affinity-based interactions, etc.
Mobile phase in fast protein liquid chromatography
A wide range of aqueous buffers is employed as mobile phases in fast protein liquid chromatography. These buffers are aqueous solutions of a weak acid or base with its highly ionizable salt. A phosphate buffer (pH 6.4-7.2) is by far the popular mobile phase choice for FPLC.
How to perform fast protein liquid chromatography
Fast protein liquid chromatography (FPLC) is performed as follows:
Step I: A suitable buffer composition is set by mixing two or more solutions stored in external reservoirs.
Step II: The buffer solution is then pumped into the column. A constant flow rate is maintained using constant flow, positive displacement pumps. The flow rate is maintained at 125 mL/min. FPLC is a type of medium pressure chromatography so about 1-5 bar pressure is applied through these pumps.
Step III: The sample is then injected into the column. It can be injected manually or through an automatic injection facilitated via a sample pump.
Step IV: Titanium, glass, or plastic (polyether ether ketone, PEEK) based cylindrical columns are used for performing fast protein liquid chromatography. These biocompatible, polymeric materials are used to avoid corrosion in the presence of saline buffers.
Columns supporting a 5 mL sample capacity are used for small-scale analysis while columns with a sample loading capacity of several liters are used for preparative purifications. Sample separation occurs inside the column which is packed with the stationary phase material.
Step V: The buffer composition is changed such that the proteins retained onto the stationary phase get attracted to the buffer and are eluted.
Step VI: The components eluted out of the column with the running buffer then reach the detector. An FPLC detector is usually a multi-wavelength detector such as an ultraviolet detector. Proteins are detected around 280 nm. Other than that, a mass spectrometric detector can also be coupled with an FPLC column. The purified products and the unwanted ones from a protein sample can be separately collected.
Step VII: An electric signal is finally sent to the recorder which records it in the form of a chromatographic peak.
Where do we need fast protein liquid chromatography
Fast protein liquid chromatography (FPLC) aids in a diversity of applications. Some of these are as discussed below:
- Protein purification: Valuable proteins can be purified and collected from a protein sample using fast protein liquid chromatography. Special peptide sequences called protein tags can be added. The tagged protein then sticks to the FPLC resin while all the other unwanted components pass out of the column. In this way, FPLC supports a desired protein enrichment.
- Disease diagnosis: Small changes in proteins hold clinical importance. Hemoglobin A2 (HbA2) for instance, is a protein, vital to human bodily functions. DNA mutations in the cells that make hemoglobin can lead to a genetic blood disorder called thalassemia. Recording HbA2 levels in a blood sample can help diagnose thalassemia. FPLC in an ion-exchange mode allows this blood sample analysis with high sensitivity and reproducibility.
FPLC is also useful for studying other peptide markers in urine and cerebrospinal fluid for diagnostic purposes.
- Protein folding assessment: Fast protein liquid chromatography in size exclusion mode can help assess protein folding. Changes in temperature, pH, and, denaturants may lead to changes in the tertiary structure of proteins. Compactly packed proteins have a smaller size than an unfolded protein. Thus, the former is more strongly retained on a column than the latter. This allows their separation on the basis of size in an FPLC column.
How is FPLC different from HPLC
| Fast protein liquid chromatography (FPLC) |
High-performance liquid chromatography (HPLC) |
| Preparative purification technique for macromolecules | Analytical technique for micromolecules |
| Specific for biomolecules such as proteins | Used for the analysis of many different compounds |
| Medium pressure required (72 Psi or 5 bars ) |
Extremely high pressure required (6000 Psi or 40 bars) |
| Lower risk of back pressure in the column | High risk of back pressure in the column |
| Crosslinked polymeric resins such as agarose are used as the stationary phase | Silica is used as the stationary phase |
| Larger particle size | Small particle size with a large resistance to high pressure |
| Polymeric resins are very sensitive to pressure changes and air bubbles | Silica beads are more stable |
| Glass or plastic column | Stainless steel column |
| Columns with high sample loading capacity | Columns with a moderate to low sample loading capacity |
| Aqueous buffers preferred as a mobile phase | Buffers are rarely used |
| Higher mobile flow rate (125 mL/min) | Lower mobile flow rate (5 mL/min) |
| A qualitative purification technique | Deals with both qualitative and quantitative analysis |
You may like our article high-performance liquid chromatography (HPLC) for better learning.
FPLC limitations
- An FPLC column cannot withstand high pressures.
- It is difficult to purify heat-sensitive/thermolabile proteins through fast protein liquid chromatography.
- Extreme pH changes must be avoided to mitigate any risk of protein denaturation inside an FPLC column.
FPLC strengths
- Due to a medium pressure application, an FPLC column is less prone to back pressure development. Thus, FPLC columns have a longer lifetime.
- A reusable column ensures a one-time investment, so the technique is less expensive than the other chromatographic methods.
- Fast protein liquid chromatography is specific and extremely valuable for large-scale protein purifications.
Conclusion
Fast protein liquid chromatography is a biocompatible, efficient protein separation and purification technique . It offers high protein resolution and reproducibility. It comes with most of the beneficial features of HPLC while having a unique specificity for macromolecules such as proteins. The ultimate goal of an FPLC is to obtain as pure and native a product as possible.
Interesting fact!
The working temperature of most of the proteins is around 4°C. Therefore, an FPLC machine is usually installed in a cold room to avoid protein denaturation.
A video tutorial on how to perform FPLC is here.
References
1. Madadlou, A., S. O’Sullivan and D. Sheehan (2011). “Fast protein liquid chromatography.” Methods Mol Biol 681: 439-447.
2. Pontis, H. G. (2017). Fast protein liquid chromatography Elsevier.
3. Sheehan, D. and S. O’Sullivan (2004). Fast Protein Liquid Chromatography. Protein Purification Protocols. P. Cutler. Totowa, NJ, Humana Press: 253-258.
