Hi RG community,
I run a Proteomics Core Facility with a few LC-MS/MS systems (Dionex U3000 nano LC system) running at 300nl/min flow rate on 0.1mm ID C18 monolithic columns with NewOjective non coated emitters (10um tips). I've read a few papers of people running at 200nl/min, just wondering (before I start the tedious work of comparing and adjusting methods) if anybody has any statistical evidence or experience to support the move from 300 to 200 nl/min?
Thanks!
There is van Deemter and the theoretical benefits, and then there is the reality of a core facility. The flow rate you are using also determines the dead time and re-equilibration time for your chromatography setup, as dead volume and column volume will not change. Dropping the flow rate from 300 nl/min to 200 nl/min will increase both by 50%, plus make your chromatography more susceptible to tailing caused by small mixing volumes in your connectors. As the sensitivity gains obtained by dropping the flow rate are minimal (at least in our hands), we stick with 300 nl/min as much as possible.
Hi Alejandro! Presuming the columns is associated with a relatively shallow van Deemter curve, it will be less likely that the chromatographic performance will be significantly different at these two flow rates. And at lower flow rates, the compounds will elute in smaller volumes, but this possible gain in sensitivity can be cancelled out by emitter position etc. and our experience is that the differences between these two flow rates are not that large. However, when going up to 500 and higher, differences become far more apparent.
There is van Deemter and the theoretical benefits, and then there is the reality of a core facility. The flow rate you are using also determines the dead time and re-equilibration time for your chromatography setup, as dead volume and column volume will not change. Dropping the flow rate from 300 nl/min to 200 nl/min will increase both by 50%, plus make your chromatography more susceptible to tailing caused by small mixing volumes in your connectors. As the sensitivity gains obtained by dropping the flow rate are minimal (at least in our hands), we stick with 300 nl/min as much as possible.
Good points above, but one should always keep chromatography basics in mind when changing LC parameters, to avoid unnecessary method weaknesses. PS. you don't have to equilibrate the column at the same flow as the LC method (can instead set this to a max pressure when washing with a set volume), as the separation hasn't started yet at this point.
Steven, Christof,
Thanks for your valuable contributions. I will keep both in mind and do a few tests when a have the time. One reason I have been inclined to reducing the flow rate is that I find the emitters start wearing out (electrospray turns into intermittent micro droplets) with time and the spray voltage needs to be slightly increased. I realized that reducing the flow rate from 300 to 200 reestablishes the nice taylor cone and plume ... so I thought it would be good to extend the lifetime of the emitters... but again, I should factor in the points you both brought up.
- Badges
- Science method