HPLC

Old HPLC systems had manual injectors between pump and detector for injecting samples. Modern systems have automated samplers and injectors which are programmed to inject samples and run the chromatograms for specified period of time. These systems are also equipped with mobile phase degassers as well as temperature controls for sample solutions and HPLC columns.

The fuction of the pump is to deliver the mobile phase through the system at a constant flow. The function of the injector is to deliver a specified amount of the sample solution into the mobile phase. The mobile phase then carries the sample into the HPLC column where the components of the sample are separated based on their affinity to interact with the stationary phase of the column. The separated components then pass out of the column at different time intervals into the detector which quantifies the components on the basis of their ability to absorb or reflect specified wavelength of light.

Mobile phases are of two types – Normal Phase and Reverse Phase

Normal mobile phase consists of organic liquid reagents which are non-polar and does not have any aqueous solutions like water. Reverse mobile phases are made up of mainly water based solutions (polar) of solid reagent buffers generally mixed with less polar liquid reagents. Normal mobile phases are used for separation of non-polar compounds and require pure silica columns. Reverse mobile phases are for separation of polar compounds and require silica based columns coated with a substance containing carbon chain atoms.

Some of the popular HPLC systems are as below,

1. Shimadzu
2. Waters
3. Agilent
4. Dionex