23.5 What is High Pressure Liquid Chromatography?
Description
This article is from the Chemistry FAQ, by Bruce Hamilton B.Hamilton@irl.cri.nz with numerous contributions by others.
23.5 What is High Pressure Liquid Chromatography?
HPLC is a development of column chromatography. it was long realised that
using particles with a small particle size ( 3, 5, 10um ) with a very narrow
size distribution would greatly improve resolution, especially if the flow
rate and column dimensions could be adjusted to minimise band-broadening.
Pumps were developed that could handle both the chemicals and pressures
required. Traditional column chromatography ( nonpolar solvent and
polar surface ) is described as "normal" and, as well as silica, there are
columns with amino, diol, and cyano groups. If the system uses a polar
solvent ( water, methanol, acetonitrile etc. ) and a non-polar surface it
is described as "reverse-phase". Common surface treatments of silica include
octadecylsilane ( aka ODS or C18), and it has been the development of
reverse-phase HPLC that has experienced explosive growth. Reverse-phase HPLC
is the method of choice for larger non-volatile biomolecules, however it is
only recently that a replacement "universal" detector ( evaporative
light-scattering ) has emerged. The most popular detector (UV), places
constraints on the solvents that can be used, and the refractive index
detector can not easily be used with solvent gradients. There are several
excellent books introducing HPLC, including the classic "Introduction to
Modern Liquid Chromatography" [10]. HPLCs can be a pain to operate, and
novices should borrow "Troubleshooting LC Systems" by Dolan and Snyder [11].
There is also a handy basic primer on developing HPLC methods by Snyder and
Kirkland [12], however, unlike GC, you also need to search the journals
( Journal of Chromatography, Journal of Liquid Chromatography ) to find
relevant examples to assist with method development.
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