FPLC stands for fast protein liquid chromatography and HPLC stands for high performance liquid chromatography. While both techniques are similar, there are significant differences in their chromatographic methods. HPLC is an analytical technique used primarily for quantitative and qualitative analysis of liquid samples. On the other hand, FPLC is a purification technique which is used to separate protein mixtures and to collect the separated components of interest. While both HPLC and FPLC use pumps, detectors, valves and software, FPLC also uses pH and conductivity monitors as well as fraction collectors. Another way to differentiate HPLC from FPLC is that HPLC is considered an analytical technique while FPLC is a preparative technique.
In HPLC several methods are used for separations with reversed phase chromatography being the most prevalent. HPLC uses solvents for the mobile phase and FPLC uses salt buffers for the mobile phase. FPLC uses several purification methods including ion-exchange, gel filtration, and affinity as well as other lesser used methods. FPLC purification is usually a multi-step process including a combination of methods.
HPLC pump heads are made out of stainless steel and flow rate capability usually does not exceed 10 ml/min, whereas back pressure capability is typically 6000 psi and higher. FPLC pump heads are made out of either titanium or PEEK materials and bench top pumps can deliver flow rates as high as 150 mls/min but back pressures of approximately 725 psi at these high flow rates. Flow rates in FPLC are usually higher than HPLC, where the back pressures are typically lower.
The columns also differ as well as the column materials and particle sizes. The HPLC columns are made out of stainless steel. FPLC columns are made out of either glass bodies with high performance resin tops and bottoms or PEEK.
Most HPLC resins are made of silica beads with small particle sizes that can stand very high back pressures. FPLC uses agarose, polymer materials, or silica materials. The particle sizes for FPLC are larger and have large pore sizes. The FPLC resins are not as capable of handling very high back pressures due to the previously mentioned material and sizes.
HPLC typically uses stainless steel in the flow path. In an FPLC flow path, stainless steel is avoided and replaced with titanium, PEEK and other high performance composites.
HPLC is used to separate small molecular weight compounds. FPLC is used to purify large biomolecules such as proteins, nucleotides and peptides.
The software differs between the two techniques as well. HPLC chromatography software controls the instrumentation and analyzes the data. FPLC software controls the modules as well as integrating collection of the purified samples into the fraction collector. In addition to monitoring UV absorption, FPLC systems also monitor pH and conductivity.
The performance of both HPLC and FPLC have improved since their introductions. In 1969, the first HPLC was commercially introduced by Waters Corporation. The first FPLC system was developed by Pharmacia in 1982. Today, more companies offer HPLC solutions then FPLC solutions.
ICON Scientific, Inc
