HPLC

Agilent Technologies (HP)
1100 Series HPLC Pump and Vacuum Degasser

Some Basic HPLC Theory

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 Seton Hall University. You can access it by the link in his homepage for Analytical Chemistry.

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.

Our most popular detector at SRIF is the photodiode array detector. The PDA can continuously scan various wavelengths of the UV spectrum. As an analyte peak is detected, the UV spectrum is recorded. This 3rd dimension is useful in identifying compounds and determining if the peak consists of an individual analyte or a mixture of analytes that wasn't effectively separated.

All aspects of the SRIF HPLC system are controlled by a PC using Millenium Windows-based software. This software controls an autosampler which injects samples at proper intervals. It controls the mobile phase gradient, the solvent flow rate, mobile phase pressure, and it measures the signals produced by the detector. The results of your sample run can then be interpreted and printed in a variety of report formats.

See our section on qualtitative analysis for more information!

How To Get Started with HPLC

First, let us know you are interested! Then take a look at the many HPLC supply catalogs we have at SRIF. The companies such as Phenomenex, Waters, Supelco, and Alltech all have an enormous wealth of information on different HPLC applications. Call them for help with your project, they love to have you call. Also, try the Waters Web site where there is some basic HPLC theory and application info from the makers of our instrument. Then join us at one of our SRIF instrument seminars and we will show you where to sign up!

E-mail List

We have formed an HPLC e-mail mailing list to keep interested persons informed about the recent developments with our HPLC systems. If you would like to receive updates on the HPLC or you would like to share your knowledge regarding this instrument or HPLC techniques in general, please contact the lab manager.

Hot Links!

HPLC is big business! The manufacturers of HPLC systems and supplies offer an abundant supply of information. They want you to use their products, and they will educate you so that you do! Try browsing their sites and their online catalogs. You may find a project title!

Here is a partial list of some of the suppliers of HPLC products.

Instrumentation

Our student instrument is an Agilent Technologies (formerly part of Hewlett Packard) 1100 Series HPLC system with quaternary pump, autosampler, and diode array, electrochemical and fluorescence detectors More information is available from the Agilent Technologies web site. Search their literature for project ideas.

Search the Waters Corporation site for further information about your experiment. Use their search engine in the Applications Library link for methods that others have used.

Follow the chromatography links at Varian for further HPLC stuff.

Supplies and Methods

Supelco--part of Sigma Aldrich--has a large site dedicated to supplies and techniques. See there online applications library.

Alltech has a great new web site with tech support, technical library and a ton of methods in their ChromAccess library.


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