Converting UHPLC to Capillary HPLC?

[chris.singleton]

Greetings,

I currently have a Shimadzu UFLC XR system, and would like to use a 300uM capillary column with the system. Has anyone had success with 'converting' an HPLC to a capillary HPLC? I've seen a resource on the web:

http://www.ionsource.com/tutorial/capil ... uction.htm

And I have used the Acquity and other low-volume systems in the past, so I'm familiar with the need to reduce the volume in the system. That said, if I change the mixer to a ultra low volume mixer, minimize any extra tubing with capillary tubing, use a small sample loop etc. etc., and direct the effluent from the column straight into the MS source, does it sound feasible? Thanks for any help,



Chris Singleton
DMPK, BiogenIdec

[sam.pedraglio]

Depending on the flow rate you think to use, you could also find some problem in gradient reproducibility: on UPLC, i.e., if you don't use the electronic actuated check valves, at low flow rate (less then 100ul/min) the check valves will not work properly.

[Uwe Neue]

Chris: My first question would be, what the reason is for using a column diameter of 300 micron. What do you expect to gain?

[chris.singleton]

Hello Sam,


Similar to the web link I posted, I would probably use a higher flow rate and then split the flow, using a back pressure regulator on the waste line so that I could direct the flow to the capillary column.


Hello Uwe,

The main difficulty that I face is sensitivity. Since ESI is more sensitive at low flow rates, I was hoping to increase my sensitivity by running at lower flows. Although with today's heated ESI sources that can effectively desolvate at high flow rates, this may not be much of an issue. I don't know how much I'll gain, but I am willing to try.
I am aware of chromatographic dilution effects that will result in an increase in sensitivity, but I expect that any increase in sensitivity from this will be minimal, since I would have to scale all the other parameters like injection volume, etc. to avoid overloading the column and maintain good chromatography (Trace Quantitative Analysis by Mass Spectrometry, Boyd et al, 2008 p. 87).
Lastly, I am somewhat concerned about the packed bed being less dense with very narrow columns, from the wall disturbing the bed. I plan to use 3.5 or 5 micron particles. In your book (HPLC Columns: Theory and Practice, page 53) you mentioned this effect, but noted that this becomes important as the column diameter is less than ~30 particle diameters. Although I am well over this 30 fold limit, I am somewhat concerned about a bed not being as well packed, since I have an interference eluting close to my analyte that I need to separate. Resolution is good now, but I can't really afford to lose any.


Two things to note:
I am using MRM in ESI+ for detection. I wanted to try MS3 on an Qtrap (quadrupole/ion trap) to try to remove the interference, but my peaks are very narrow and I don't think I can get the proper number of points across the peak to quantitate.
I am extracting a compound from whole blood, the recovery is about 70% and the matrix effects are about 20% (matrix effects are enhancing signal). I am trying to improve recovery, but I can't gain too much from this.

[Uwe Neue]

Before I would try to set up a complete systems that is supposed to function in the micro-world, I would just check first with standards and flow splitting, how much signal-to-noise I can expect to gain by going to lower flow. Once I know that, I also know, how much work this is worth, or if there are not any better solutions to the problem.

[chris.singleton]

Good point, I'll give that a try first since going back and forth from a high-volume system to a low-volume system would be very labor intensive. Thanks,


Chris