Fast protein liquid chromatography (FPLC) is a modern liquid chromatography developed by introducing the theory of gas chromatography from classical liquid column chromatography and reforming the phase body. It is equipped with high-pressure infusion pumps, high-sensitivity detectors, gradient elution devices, automatic collection devices and microcomputer. The FPLC is designed for the separation and purification of a wide range of biomolecules, including natural proteins, recombinant and fusion proteins, peptides, oligonucleotides, plasmids, viruses, antibiotics, alkaloids, etc. It is characterised by rapid, high resolution, high column capacity, high recovery efficiency and low deactivation of biomolecules.
FPLC Components
Fast protein liquid chromatography systems consist of a programmable controller, one or two high precision pumps (usually two, one each for buffers A and B), mixers, pre-filters, port M-7 valves, various sample rings (0.025-10 mL), columns, UV-1 ultraviolet monitors and fraction collectors.
Fig. 1 The components of FPLC chromatography (Sheehan, D.; Siobhan O'S. 2004).
The Difference Between FPLC and HPLC
FPLC is a liquid chromatography method for the purification of large biomolecules such as proteins, nucleotides, and peptides. HPLC is a liquid chromatography method used to separate small molecular weight compounds. The following is the difference between FPLC and HPLC.
|
HPLC |
FPLC |
| Definition |
HPLC is a type of liquid chromatography used to separate small molecular weight compounds. |
FPLC is a type of liquid chromatography used to purify large biomolecules like proteins, nucleotides and peptides. |
| Collector |
Does not use pH and conductivity monitors and fraction collectors. |
Uses pH and conductivity monitors as well as fraction collectors. |
| Technique |
An analytical technique. |
A preparative technique. |
| Motility |
Mobile phase is a solvent. |
Mobile phase is a salt buffer. |
| Pump heads |
Pump heads are made out of either titanium or PEEK. |
Pump heads are made out of stainless steel. |
| Helium or nitrogen gas |
Helium or nitrogen gas is used to move the sample. |
Helium or nitrogen gas is not used to move the sample. |
| Column |
HPLC columns are made of stainless steel. |
FLPC columns are made of glass bodies or peek. |
| Resin |
HPLC resins are made with small particle sizes. |
FPLC resins are made with large particle sizes. |
| Monitor |
HPLC chromatography software controls the instrumentation and analyses the data. |
FPLC software controls the modules. Regulates the integrating collection of the purified samples into the fraction collector and monitors pH and conductivity. |
The Advantages of FPLC
FPLC is a simple, reproducible separation technique with high separation efficiency.
Resistant to extremely high salt concentrations and corrosive liquids, the columns have a longer service life.
FPLC supports a wide range of columns.
The wide flow range makes it a suitable technique for analytical and preparative chromatography.
The Application of FPLC
Diagnosis of β-thalassemia
β-thalassemia is an inherited blood disorder in which hemoglobin production is reduced. Quantitative hemoglobin A2 (HbA2) levels are used for diagnosis. FPLC has 100% sensitivity and specificity for the diagnosis of β-thalassemia. Furthermore, the technique is simple, fast and inexpensive.
Separation of plasma proteins from urine and cerebrospinal fluid
FPLC has been used to rapidly identify protein profiles and microscopic variability within individual proteins. FPLC in anion-exchange column mode enables rapid separation and reproducible characterization of plasma proteins in urine and cerebrospinal fluid.
Assessment of protein unfolding
Fast protein size-exclusion liquid chromatography (SEC-FPLC) does not alter the equilibrium between unfolded protein states and can be used for qualitative and quantitative assessment of protein denaturation, including sperm whale myoglobin and hen white lysozyme, bovine and human alpha-Whey protein, bovine carbonic anhydrase B and 8-lactamase from Staphylococcus aureus.
Reference
- Dettmer-Wilde, K.; Engewald, W. Practical gas chromatography. A Comprehensive Reference. Springer, 2014: 902.
.
