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 approximately 1 microL/bar. We found that pump pressurization is a relatively slow process and takes approximately 1 min to reach 90% and takes approximately 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.
Rogatsky
Eduard
E
Analytical Core Laboratory, Institute for Clinical and Translational Research, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, USA. eduard.rogatsky@einstein.yu.edu
Zheng
Zheng
Z
Stein
Daniel
D
D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D011312:Pressure; D014466:Ultraviolet Rays