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

Tony Taylor
Tony Taylor

Tony Taylor is Group Technical Director of Crawford Scientific Group and CHROMacademy. His background is in pharmaceutical R&D and polymer chemistry, but he has spent the past 20 years in training and consulting, working with Crawford Scientific Group clients to ensure they attain the very best analytical science possible. He has trained and consulted with thousands of analytical chemists globally and is passionate about professional development in separation science, developing CHROMacademy as a means to provide high-quality online education to analytical chemists. His current research interests include HPLC column selectivity codification, advanced automated sample preparation, and LC–MS and GC–MS for materials characterization, especially in the field of extractables and leachables analysis.

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.

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 (VM) 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.