Thank you to everyone who attended the live webinar with the 2015 Young Investigator Award (YIA) winner. Below are responses to the questions posed during the live event. We hope this is a useful resource and thank our webinar attendees and our speakers, Xiwei (Emmi) Zheng (winner of the award; University of Nebraska-Lincoln) and Kelly Doering (Waters Corporation) for their time.
1. How do you prepare the HSA affinity columns for these studies?
Emmi: In these studies, HPLC-grade silica was prepared in a diol-bonded form and then oxidized to form aldehyde groups on the surface of the silica. The amine groups on HSA were then reacted with the aldehyde-activated silica through the Schiff Base method.
2. Does the interaction between the injected drug or hormone and HSA that is on the column affect the measurement of dissociation rate constant and association equilibrium constant?
Emmi: The ultrafast affinity extraction method introduced in this webinar was used to measure the dissociation rate constant and association equilibrium constant for the interaction between a solute and protein in an injected sample (i.e., a solution-phase interaction) instead of the interaction between injected solute with the immobilized protein on the column (i.e., a solid-phase interaction). The binding of drug or hormone to the immobilized HSA on the column was simply utilized for the extraction of free drug/solute fraction to aid in this analysis.
3. For the multi-dimensional HPAC system, how did you to determine the optimum time to place the second affinity column on-line with the first?
Emmi: In the multi-dimensional HPAC system, the time for placing the second affinity column on-line was controlled by a valve switching event in the HPLC system. This event occurred over the time frame when the free solute peak eluted from the first column and was about to enter the detector. As the switching time was increased, the apparent free fraction decreased and approached a constant value because less contamination was passed onto the second column. However, a loss of precision in the measured free fraction can also be observed if the switching event was too long, causing only a small amount of the free solute to be delivered to the second column. The combination of these effects created a relatively well-defined window of time for placing the second column online with the first column.
4. Is there a requirement for the flow rate applied in a multi-dimensional HPAC system?
Emmi: The initial flow rate that is used in the multi-dimensional HPAC system needs to be high enough to minimize the dissociation of the solute from its protein-bound form as the sample passes through the first column. At the time when the second column is placed on-line, the flow rate needs to be decreased to a low or medium flow rate for better isolation and resolution of the retained peak on the second column.
The full webinar is available here – Webinar with the 2015 Young Investigator Award (YIA) winner.