Fast protein liquid chromatography (FPLC)

Instrument details:
Type: Fast protein liquid chromatography
Make: Akta purifier 10
Model No: GE Healthcare Bio sciences
Essential specification:
Measurement Range: 190-700 nm in step of 1 nm
Temperature Range: +4 to +60 °C
Flow Rate: 0–100 ml/min
Absorbance range: 0.001–5.0 AU (full scale)
Software Name: UNICORN
Sample type: Liquid
Minimum sample volume: 1ml
Accuracy: ± 2 nm
Precision / Repeatability: ± 0.01 nm
Result Type: Graph/ Chromatogram
Flow rate range; Isocratic and gradient mode: 0.05–20 ml/min in steps of 10 µl/min 0.1–20 ml/min in steps of 10 µl/min
Pressure range: 0–5 MPa (50 bar, 725 psi)
Absorbance range: 0.01–5.0 AU (full scale)
Wavelengths Hg lamp, fixed by changing filter Zn lamp: 254 and 280 nm 313, 365, 405, 436 and 546 nm 214 nm
Max. pressure: 4.0 MPa (40 bar, 580 psi)
Cell volume: 2 µl (30 µl detector volume)
Conductivity range: 1 µS/cm to 999.9 mS/cm
pH range: 0 to 14
Working principle:
Fast protein liquid chromatography (FPLC) is a type of liquid chromatography that provides high resolution by small-diameter stationary phases for protein characterization and separation. In fast protein liquid chromatography, the solvent velocity is controlled by a microprocessor through a software interface to maintain the constant flow rate of the solvents. The mobile phase is an aqueous buffer, and the flow rate is kept constant by a positive-displacement pump while the buffer composition can be varied by drawing fluids from external reservoirs. The stationary phase is composed of beads, usually of cross-linked agarose, packed into a cylindrical column. The eluent is passed through the detectors to measure the salt concentration (by conductivity) and protein concentration (by absorbing the ultraviolet light at a wavelength of 280nm)