Friday, February 1, 2013

Optimizing your nanoLC conditions part 4: The effects of a longer column

This is the 4th part of my somewhat convoluted monologue on optimizing your nanoLC gradient.
You can read
part 1,
part2, and
part 3 here.
Again, I'm not a huge chromatography expert,  I remember some things from college about theoretical plates and column loading, but not well enough to embarrass myself describing them here.  What I do know is that the image above, taken from this Dionex app note, very accurately reflects what I've seen when experimenting with column lengths.  In this experiment, they used three column lengths, the same flow rates, and the same injection volume.  The important part to me from the MS side of thing is the peak height.  When the column length increases, the peak height does as well.  You can't see it in the above screenshot, but we can assume this -- if we loaded the same amount onto the column and the height increased, that means that the peak width had to decrease.  Decreased peak width means increased chromatographic resolution, less coelution effect, and more results for your MS/MS, and this is just with more material.  The real way to jack up your results is to increase the chromatographic resolution AND your peak intensity AND your gradient length.  You can read about an old comparison of a 10cm 140 min gradient and a 30cm 240 min gradient here. In these limited experiments, I found that I could save time AND increase my coverage by using a longer column/gradient combination.  Again, you have to take a good hard look at the requirements of your lab and the study in question, but I hope this helps get you started.  As always, if I can answer further questions or if you have suggestions, don't hesitate to contact me or post questions here and I'll do what I can.


  1. I was just wondering regarding the use of TFA in mobile phase. I had problem of ion suppression when used it during Sep Pak (desalting step; washing and elution buffer). Ben, would you like to comment of this??

  2. Santosh,
    TFA ion suppression can be a pain in the butt. Now, I'm pulling straight from memories dulled from time and a surprising number of head impacts for someone my age, but I think that the end goal was to keep the TFA levels below 0.3%. This can be tricky when using a desalting step that prefers a bunch of TFA. You have this scary middle ground where you are drying your sample down; are you simply concentrating the TFA or is it evaporating at the same rate? I'm not sure I've used SepPak, but for other desalting columns I've simply substituted TFA for formic acid. Man, I need to know more chemistry; but what I found was that the pH was really what seemed important. If you were acidified then peptides stuck. If any readers (who actually know some chemistry and about the C-18 acidic binding dynamics) it would be awesome if you'd commment here!