Sunday, February 18, 2018

More details on our optimized gradients for C-18 PepMap

Thanks for the emails!  I legitimately love to receive them. Even if it starts with "you're an idiot!" Which, honestly, is reasonably rare. 

This is a follow-up to a recent post where I talked about how I'd been totally messing up my LC separations by treating C-18 PepMap like other resins.  I know I didn't provide enough details, but this has been a work in progress as we fine tune our instruments to be able to best handle the intimidating number of projects we've got going on.

After messing around with 6 EasyNLCs (1 Easy2, 2 Easy1200 and 3 Easy1000s) this is what appears to be the best separation I can get in 120 minutes of time on the 15cm 2um columns in 2 hours.

Please note: Recent versions of the EasyNLC user manual recommend using no more than 80% acetonitrile in Buffer B to protect your pump and valve seals. We switched all of our systems to 80% just last week. With 6 EasyNLCs running around the clock --- even a 1% increase in pump seal life will result in at least a 40% decrease in the number of loud profanities coming from this one weird bald guy in the lab.  Also worth noting -- these LCs use viscosity and temperature and some other stuff to determine flow rates. Don't change your solvents without consulting your manual (get the newest one online. there have been significant revisions over the years!), and don't trust what weird people post online on Sunday mornings.

Buffer A is 0.1% formic acid in 100% LC-MS grade water
Buffer B is 80% MS-grade acetonitrile, 20% LC-MS grade water, 0.1% formic acid.

This is with a 2cm C-18 PepMap trap column in line.One with a surprising amount of dead volume. More on that when I have more data.

I'm actually using 500nL/min when I get to the high organics just to flush things out, but I don't like how the graphic represents it.  Translation: I am displaying a method image above that is actually incorrect, purely for my own personal sense of aesthetics...

What happens if I don't ramp it up the organic pressure/flow rate? I get some trailing of some extremely hydrophobic peptides.

I know that is hard to see, but if you click on it - it should expand it. This is a human cell digest from Thermo with the PRTC peptides spiked in. The bottom is the most hydrophilic peptide of the 15 and the middle frame is peptide number 13. You can see that there is still some signal when the method cuts out at 130 min. There really are some peptides coming off at the end there.

If I up the flow rate on high organic, it trails off a good bit better and it sharpens the peak shape on my latest eluting standards.

I really dislike the boring 0-10 minutes at the front. We're running some tests in this weekend's queue, but on one run we've been able to negate it almost completely by using an alternative NanoViper trap column with significantly lower dead volume. I'll share those details when I get them.

For less complex samples we're getting the best separation by mimicking this gradient and ramping to 24% buffer B in 40 minutes and 36 in an additional 15. I brought some RAW files home, but I can't find the funny cord for my portable hard drive. I'll post later if I can find the stupid cord.

Why aren't all USB cords standardized yet?!? It's 2017, for crying out loud.

EDIT: *2018


  1. Dear Ben,
    thank you for all of your posts. We (the Garcia lab, UPenn) are all big fan of the blog. One question: did you think about removing the first 5 minutes of isocratic flow? You said that yourself: not much happens in the first 10 min of gradient. If the gradient starts right away you should have less delay.

  2. Simone,
    Nice to here from you! I'm also a big fan of your lab's work! Yeah...I should definitely do that! I'm trying to remember now why i even had that isocratic thing at the beginning...