High Performance Liquid Chromatography – How Does It Work?

High Performance Liquid Chromatography HPLC is a widely used analytical technique that lots of industries and research areas rely heavily on. In this article you will discover how HPLC systems operate and the various kinds of HPLC machines which are generally used. If You are looking for more in depth advice, do not worry, There will be many more posts in the near future beginning with our next article which will insure HPLC equipment and methods for chemical applications, followed by our post on HPLC equipment and methods for biological or life science programs. HPLC is used to measure, isolate and identify the components of Non-volatile liquid mixtures volatile chemicals are separated by GC- Gas Chromatography. Solvent is pumped from a solvent reservoir and blended with the liquid sample. The solvent sample mixture passes through a HPLC column and into a detector, where a digital output signal is given as a chromatograph. The waste collects in a boat out of the machine.

what is hplc The HPLC column is the separation part of the system. HPLC Columns include packing material with different pore sizes, which is referred to as the ‘stationary phase’. The liquid sample which passes through the pores is referred to as the ‘mobile phase’. Since the mobile phase passes through the stationary phase, the bigger molecules become trapped by the smaller pores, allowing the smaller molecules to elute faster. This is the isolation and separation procedure. This causes the smaller molecules to have a faster retention period and larger molecules to have slower retention times. The retention elution period is the time it takes for the molecules to reach the sensor. The retention times are measured against known criteria, thus, the molecules in the sample could be identified. The more the molecules spend from the column, the broader the peaks become on the chromatogram.

Normal phase HPLC is not the most common method of HPLC, despite its name. Normal phase HPLC uses non-polar solvents as the mobile phase and silica particles as the stationary phase. In ordinary HPLC, polar compounds will stick with the polar silicone longer at the stationary phase in contrast to non-polar compounds. Therefore, non-polar compounds elute faster in normal HPLC. The pore sizes are usually around 3 microns. Reverse-phase HPLC is the most common type of HPLC. In reverse Phase HPLC, the stationary phase is altered by hydrocarbon chains typically 8-18 carbons long, causing the column to become non-polar. A polar solvent can also be utilized. In reverse HPLC, the polar molecules in the sample create powerful attractions with the solvent causing them to elute faster. They will also not socialize with non-polar stationary phase. When using an HPLC-MS machine, the molecules could be identified by the m or z ratio, with no need for understanding the retention times.