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Flow splitting before mass spec

Posted: Wed Apr 01, 2015 11:37 am
by ger225
Hi everyone,

At our facility, we have a microTOF QII (Bruker) coupled to an Agilent 1100 HPLC. They are used for metabolomics and lipidomics research, and now, we want to expand their use into proteomics.

We have a really tight budget so we are able to purchase a nanoESI source (Captive Spray NanoBooster) but a nanoHPLC is not an option. So, I wanted to know if anyone has experience with flow splitting in order to connect the Agilent + capillary column to the mass spec equipped with the nanoESI source.

I would appreciate any info you can provide, especially materials that we should buy for this purpose.


Posted: Thu Apr 02, 2015 7:49 am
by Christopher
There are lots of good write ups online about doing flow splitting prior to the MS (e.g. and getting a good flow setup for nanospray ( A lot of companies sell splitters, but it is very easy to make your own.

Just be mindful of the pressure limits of your HPLC system when switching to a microbore column setup.

Posted: Thu Apr 02, 2015 8:11 am
by Christopher
Also...I will just is not necessary for you to switch to nano flow rates to get good proteomics results, especially on a qTOF. I mention this, in case you are having trouble getting your system down reliably to a lower flow rate at a reasonable pressure.

You can run wider ID columns (>200um) at higher flow rates (>2uL/min) on a conventional electrospray source as long as you are catering the spray tip to your flow rate (see the newobjective article above for info on that).

We frequently use longer columns (25cm) with wider IDs (3um particles) at higher flow rates (2-5uL/min) on some of our instruments. You can load a bit more material in this setup and get nice fast separations, without sacrificing sensitivity. Because we are loading a bit more material we can run our tripleTOF very fast (Top50, 50ms accumulation time) and retain the quality of our MS/MS spectra. This is about the same speed as the Fusion runs at. This is not a perfect setup on the TripleTOF because of the way its spray operates, but with the Bruker Captive Spray it would work quite well I think.

This can be especially advantageous for SRM experiments. Sciex actually has some nice posters on it (

Posted: Thu Apr 02, 2015 10:03 pm
by zougman
Reading this forum thread Secrets of flow splitting? may be also of help


Posted: Fri Apr 03, 2015 5:04 pm
by ger225
Thanks Christopher and zougman for your replies. Great info!

Posted: Sat Apr 04, 2015 6:57 am
by tsbatth
It depends the type of proteomics you want to do. If it's targeted proteomics you can get away without splitting and loading more. If it's for discovery proteomics ... you could probably get away even at higher flow rates (ul/min range) granted you run longer gradients and higher loading, but you will take a hit on the numbers and not recommended if looking for low stoichiometric PTMs. A demonstration for this was published in regards to targeted proteomics , where they show basically for targeted proteomics high flow with higher loading can prove to be advantageous over nano.

Also a shameless plug where at my previous lab we just took this to the extreme where I was able to do targeted proteomics on like 800+ peptides (~400 proteins) using a 5.5 minute gradient at standard flow ;) .

Additionally since you are using an Agilent 1100 system, you can try attaching different flow cells for optimal flow rates, which might be better then splitting downstream. I'm pretty sure the agilent 1100 system has a nano-flow rate module or flow cell but I could be wrong. Be aware you might need to change the tubing on the autosampler (or even the sampler switching valve) for different flow rates.



Posted: Mon Apr 06, 2015 4:10 am
by ger225
Thanks T

I completely agree with you. But this is one of the cases in which the researchers (over 20 different groups) with no background in mass spec want something that can "do everything". So, we might take a shot at both targeted and discovery proteomics. I feel that right now we are more interested in the latter, since many groups want to compare cell A vs mutant B, condition A vs condition B or eluates from affinity chromatography and pull down assays (interactomics).

Thanks for your tips!