High-Performance Liquid Chromatography Columns

HPLC Columns

An HPLC typically includes two columns: an analytical column responsible for the separation and a guard column. The guard column is placed before the analytical column, protecting it from contamination.

Analytical Columns 

The most commonly used columns for HPLC are constructed from stainless steel with internal diameters between 2.1 mm and 4.6 mm, and lengths ranging from approximately 30 mm to 300 mm. 

These columns are packed with 3–10 μm porous silica particles that may have an irregular or spherical shape. Typical column efficiencies are 40,000–60,000 theoretical plates/m. Assuming V_max/V_min is approximately 50, a 25-cm column with 50,000 plates/m has 12,500 theoretical plates and a peak capacity (equation 12.18) of 110.

Microcolumns use less solvent and, because the sample is diluted to a lesser extent, produce larger signals at the detector. These columns are made from fused silica capil- laries with internal diameters of 44–200 μm and lengths of up to several meters. Micro- columns packed with 3–5-μm particles have been prepared with column efficiencies of up to 250,000 theoretical plates.

Open tubular microcolumns also have been developed, with internal diameters of 1–50 μm and lengths of approximately 1 m. These columns, which contain no packing material, may be capable of obtaining column efficiencies of up to 1 million theoretical plates.11 The development of open tubular columns, however, has been limited by the difficulty of preparing columns with internal diameters less than 10 μm.

Guard Columns 

Two problems tend to shorten the lifetime of an analytical column. First, solutes binding irreversibly to the stationary phase degrade the column’s per- formance by decreasing the available stationary phase. Second, particulate material injected with the sample may clog the analytical column. To minimize these prob- lems, a guard column is placed before the analytical column. Guard columns usually contain the same particulate packing material and stationary phase as the analytical column, but are significantly shorter and less expensive; a length of 7.5 mm and a cost one-tenth of that for the corresponding analytical column is typical. Because they are intended to be sacrificial, guard columns are replaced regularly.