The LCGC Blog: HPLC Troubleshooting Tips–Poor Equilibration in Gradient HPLC

Our technical support center deals with many issues regarding irreproducibility of retention and selectivity in reversed phase HPLC. Very often, the problem lies in poor equilibration of the HPLC column between injection, which in gradient HPLC can affect the separation selectivity as well as analyte retention.

We have a rule of thumb when developing methods that 10 column volumes of mobile phase, at the starting composition of the gradient, should pass through the column for proper re-equilibration of the whole packed bed. Two important numbers are required to achieve this: the internal volume of your column (sometimes called the “interstitial volume”), and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column).

Column volume (V_M) is straightforward to estimate using the formula;

r = column radius (i.e. half of the column diameter) (mm)
L = column length (mm)

So, for a 5 cm x 2.1 mm HPLC column this would represent a volume of:

So, at a flow rate of 0.5ml/min., an equilibration of ten column volumes would mean allowing 2 min equilibration.

Now for that second important number-the gradient dwell volume. We will show you how to calculate this in a future newsletter-however a well plumbed LC system will have a dwell volume below 0.5 ml and some UPLC systems will be significantly lower. However, if we err on the side of caution and assume 0.5 ml-this will add a further 1 min to our equilibration time at an eluent flow rate of 0.5 ml/min.

Therefore, a total of 3 min will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity.

For method development advice, HPLC or GC column recommendations, or advice on troublesome methods, please feel free to get in touch with me directly.

Tony Taylor is the technical director of Crawford Scientific and ChromAcademy. He comes from a pharmaceutical background and has many years of research and development experience in small molecule analysis and bioanalysis using LC, GC, and hyphenated MS techniques. Taylor is actively involved in method development within the analytical services laboratory at Crawford Scientific and continues to conduct research in LC-MS and GC-MS methods for structural characterization. As the technical director of the ChromAcademy, Taylor has spent the past 12 years as a trainer and developing online education materials in analytical chemistry techniques.