Absolute measurement of internal volume changes inside the pulse damper: Discrepancy between delay and dwell volumes

We have developed a novel technique for the absolute determination of the mobile phase volume stored inside of a variable volume pulse damper at different pressures. Using an Agilent HPLC pulse damper we found a linear volume increase of ∼1 μL/bar. We found that pump pressurization is a relatively slow process and takes ∼1 min to reach 90% and takes ∼2 min to reach 99% equilibration at flow rates below 1 mL/min. During pump pressurization, column flow rate will be less than the pre-set, since part of the mobile phase is retained inside of the pulse damper. During our experiments we observed a discrepancy between data obtained by UV techniques and direct absolute measurements. This difference can be explained by a fundamental difference between the gradient delay volume and dead (dwell) volume.