High-performance liquid chromatography, or HPLC, is an analytical technique that separates liquid samples under high pressures. An HPLC instrument consists of glass bottles for storing solvents called mobile phase reservoirs. HPLC-grade solvents are used to maintain high purity, and the dissolved gases are removed using a degasser, such as a vacuum pumping system or sparging with helium. The solvents are then pumped into the analytical column using a screw-driven syringe or reciprocating pumps. Most modern HPLC instruments use reciprocating piston pumps that create a pulsed mobile phase flow, which is smoothed out by connecting them to a pulse damper. In some analysis, gradient elution is employed. This technique involves gradually changing the composition of the mobile phase, which can enhance the separation of complex mixtures based on their polarity. Gradient controllers are used to maintain the flow rate of solvents during this process.
Before the mobile phase reaches the column, the sample is introduced at the loop injector or autoinjector. The mobile phase, along with the sample, enters the column, which is packed with a stationary phase. The analytical column is placed in a column oven to maintain a constant temperature. Sometimes, pre-columns such as scavenger and guard columns are used to protect the longevity of the column. Once the analyte components are separated, they travel to the detector, which identifies them and converts the results into a chromatogram the computer displays. Finally, the eluent- the solvent carrying the separated components- either goes into the waste or is collected at the fractional collector.
The major components of an HPLC instrument are a mobile phase reservoir, pump, injector, column, detector, and data system.
The mobile phase reservoir consists of one or more solvent-storage glass bottles from which the solvent is drawn through pumping systems.
In gradient elution, the solvents are mixed before they enter the column.
An injection system, manual or automatic, introduces the analyte mixture into the mobile phase before it enters the column.
In the column, the analyte components are separated based on their interaction with the mobile and stationary phases.
Next, the separated sample constituents travel to the detector, which generates characteristic signals.
These signals are translated into a chromatogram by a computer-aided program, and the eluent goes into the waste.
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