HPLC Station--Everything you need to know on HPLC
The following is a very simplified and general overview of HPLC theory.
For another excellent source of background reading, try the on-line HPLC textbook written by Prof. Yuri Kazakevich and Prof. H.M.McNair
of
The goal of any chemical analysis is to separate a sample (blood, urine, water from a well, etc.) into its individual components in order to evaluate each component free from interference from the other components. Chromatography is a general technique that separates a mixture into its individual components. Those components are referred to as analytes--the chemical compounds of interest to the analyst. Chromatography is then coupled with a detection system that can characterize each type of analyte appropriately. High performance liquid chromatography (HPLC) is one such method. It is used to analyze liquid samples or the liquid extract of a sample.
The fundamental basis for HPLC consists of passing a sample (analyte mixture) in a high pressure solvent (called the mobile phase) through a steel tube (called a column) packed with sorbents (called the stationary phase). As the analytes pass through the column they interact between the two phases--mobile and stationary--at different rates. The difference in rates is primarily due to different polarities for the analytes. The analytes that have the least amount of interaction with the stationary phase or the most amount of interaction with the mobile phase will exit the column faster. Repeated interactions along the length of the column effect a separation of the analytes. Various mixtures of analytes can be analyzed by changing the polarities of the stationary phase and the mobile phase.
The many types of columns on the market today can help refine your HPLC method. (Drop by SRIF to look at our catalogs!) Choosing the right column is essential in obtaining good HPLC results. Obviously, the polarity of the stationary phase can be altered significantly. The stationary phase is typically bonded to a support phase, usually consisting of porous beads. The pore sizes can be varied to allow certain sized analytes to pass through at different rates. Furthermore, the dimensions of the column can be varied to allow different sample sizes to be analyzed.
Changes in the polarity of the mobile phase is another variable that can effect the efficiency of your HPLC separation. The mobile phase polarity is generally the opposite of your stationary phase. Our multisolvent delivery system allows the polarity of the mobile phase to be changed during the course of the HPLC run. The rate at which the polarity is changed defines the "gradient." This gradient technique helps to further seperate mixtures of variously polar analytes.
As the analytes exit the column, they can be detected by various means. Refractive index, electrochemical, or ultraviolet-absorbance changes in the mobile phase can indicate the presence of an analyte. The amount of analyte leaving the column will determine the intensity of the signal produced in the detector.
The detector measures a signal peak as each analyte leaves the column. By comparing the time it takes for the peak to show up
(called the retention time) with the retention times for a mixture of known compounds, the components of unknown sample mixtures can
be identified. By measuring the signal intensity (response) and comparing it to the response of a known amount of that particular
analyte, the amount of analyte in the mixture.
1. Components of the HPLC system
2. HPLC Application guides and protocols
3.HPLC general protocol for new proteins
Web Guider
Ch 8.Immunohistoch / immunology
Ch 10.GC/MS, NMR and Proteomics