High-performance liquid chromatography (sometimes referred to as high-pressure liquid chromatography), HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.
HPLC typically utilizes different types of stationary phases, a pump that moves the mobile phase(s) and analyte through the column, and a detector to provide a characteristic retention time for the analyte. The detector may also provide additional information related to the analyte, (i.e. UV/Vis spectroscopic data for analyte if so equipped). Analyte retention time varies depending on the strength of its interactions with the stationary phase, the ratio/composition of solvent(s) used, and the flow rate of the mobile phase. It is a form of liquid chromatography that utilizes smaller column size, smaller media inside the column, and higher mobile phase pressures.
With HPLC, a pump (rather than gravity) provides the higher pressure required to move the mobile phase and analyte through the densely packed column. The increased density arises from smaller particle sizes. This allows for a better separation on columns of shorter length when compared to ordinary column chromatography. (Adapted from Wikipedia)
The Varian HPLC we have has three major components: ProStar 230 Solvent Delivery Module, 410 Autosampler, and 325 Dual Wavelength UV-Vis Detector.
ProStar 230 Solvent Delivery Module
- Maximum operating pressure: 6000 psi (410 atmospheres)
- Flow rate range: 0.01 to 10.0 mL/min in 0.01 mL/min steps between 0.00 and 1.00 mL/min, and 0.1 mL/min steps between 1.0 and 10.0 mL/min
- Flow precision: 0.3% RSD at 1.0 mL/min MeOH/water
- Flow accuracy: ± 0.5% at 1.0 mL/min isopropyl alcohol
- Compositional accuracy: ± 0.5% absolute
- Compositional precision: ± 0.1% absolute
- Delay volume: 700 µL + 1 µL/ATM pressure
- Pressure pulsation: Less than 2% at 150 atmospheres (1 mL/min with 100% isopropyl alcohol)
- Proportioning modes: 1, 2, or 4-stroke (user selectable)
- Mixing: High pressure, static or dynamic mixing modes
- Display: 40 Characters, double-line, back-lit LCD
- Contact closure outputs: Six time-programmable external event relays, and three synchronization signals: READY, START, and FAULT
- Contact closure inputs: Three synchronization signals: READY, START, and FAULT
- Bus communication: The 230 solvent delivery systems communicate with the Star Workstation by means of the IEEE 488 Bus (GPIB)
410 Auto-sampler Features
- Sample trays in 3 configurations: 96 standard 1.5mL vials;84 standard 2 mL vials plus 3 10 mL vials for mix solvents; 24 10 mL vials for large injection volumes. Trays change in under a minute.
- Easy to automate. Internal standard addition, sample dilution, or derivatization steps are easily programmed to reduce sample preparation time and errors due to manual procedures.
- High-resolution syringe design for maximum reproducibility.
- Integrated column oven.
- Cooling and biocompatibility options available.
- Easy-access needle, injector valve and syringe for quick maintenance.
325 Dual Wavelength UV-Vis Detector
- Wavelength range: 190 to 900 nm
- Light sources: Deuterium for UV and quartz halogen for Vis
- Spectral bandwidth: 6 nm
- Wavelength precision: ±0.1 nm
- Wavelength accuracy: ±1 nm at 260 nm
- Noise, single wave length: ±5x10-6 AU peak-to-peak at 254/280 nm, 2 sec. response time, 9 mm flow cell
- Noise, dual wave length: ±10x10-6 AU peak-to-peak at 254/280 nm, 2 sec. response time, 9 mm flow cell
- Noise, dry cell: Typically, ±3x10-6 AU peak-to-peak at 254/280 nm, 2 sec. response time, 9 mm flow cell
- Drift: less than 1 mAU/hr
- Linearity: with acetone in water; analytical 1% to 2 AU; preparative 1% to 20 AU at 265 nm
- Absorbance range: up to 70 AU with preparative flow cell
- Response time: selectable 0.05, 0.5, 1, and 2 sec.
- Data rate: 20 Hz single channel
- Analog detector outputs: two with selectable absorbance range of 0.001 to 10 AU full scale at 1V
- Flow cell pressure rating: 1000 psi maximum for all cells
- Maximum flow rates, single pathlength flow cells: Analytical 9 mm, 15 ul; 100 mL/min