Efficiency in HPLC Separations

The basic aim in any chromatographic separation is to achieve retention and separation of two or more components of a sample. Separation is best achieved when peaks are narrow thereby giving rise to better resolution from neighbouring peaks. All Thermo Electron columns are tested following manufacture to ensure that they are packed efficiently. The result of this test is the column test certificate, supplied with every column.

Column efficiency and particle size are inversely proportional; therefore a reduction in particle size from 5 to 3 µm will bring about an increase in efficiency of approximately 60%. Particles of 1.9 µm will generate theoretical plates approximately 160% greater than 5 µm media. Resolution gains of approximately 60% are theoretically possible when moving from 5 to 1.9 µm particles.


Theoretical models of peaks obtained on columns packed with three different particle sizes: retention times 1.9 & 2.1 min on a 50mm column (a) particle size, dp=5µm; theoretical plates, N=3500; resolution, R=1.48 (b) particle size, dp=3µm; theoretical plates, N=5800; resolution, R=1.90 (c) particle size, dp=1.9µm; theoretical plates, N=9200; resolution, R=2.40



During normal use, partial blockage and contamination of the column will lead to deterioration in its efficiency with the extent of this very much dependent on the sample and conditions with which the column is used. A reduction of approximately 50% in the theoretical plates with the first few hundred injections is considered normal. Contamination can be reduced and column performance extended by the use of guard cartridges – see the Extending Column Lifetime section of this guide. Column performance can be checked by running the method supplied in the individual column phase section of this guide and then comparing the resulting chromatogram with that on the test certificate.