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why does low flow rate improve LC-MS sensitivity?

Posted: Thu Aug 01, 2019 10:01 pm
by prk018
I'm trying to understand how LC flow rates may affect sensitivity in LC-MS, and found lots of papers saying that nano-LCs were invented because low flow rate helps to conserve samples and increase sensitivity. I'm confused with this concept, however, in two ways.

1. Why would flow rate have an impact on conserving samples? In other words, if I put in the same amount of samples on the LC column, why would my result with a higher flow rate method vs. a lower flow rate method look different?

2. Why is the sensitivity higher with lower flow rate? I would have expected the sensitivity to be higher with higher flow rate, since that decreases the dead volume between column connections and will make the peak shape sharper.

Re: why does low flow rate improve LC-MS sensitivity?

Posted: Fri Aug 02, 2019 2:45 pm
by James_Ball
I believe with ESI ionization the greater sensitivity comes with higher concentration of analyte in mobile phase, not just higher mass of analyte hitting the source. If you have 2ng of analyte in 1ml/minute flow versus 2ng analyte in 0.2ml/minute flow, then the analyte will be more concentrated in the lower flow rate. In theory this means that each micro droplet of mobile phase has more analyte in it, and more charge is transferred to more analyte as the droplet evaporates.

If you have higher sensitivity, you can inject less sample, so you do not need to use as much sample, thus "conserving" sample that may already be in microliter quantities.

Sharp peaks to improve sensitivity, but you can achieve sharper peaks with less flow if you use a smaller diameter column as the linear velocity and not the volume of mobile phase is what matters. So nanoflow with narrow bore column with sub 3um particles will give more sensitivity than a 1ml/minute flow on a 4.6mm column with 5um particles.

Re: why does low flow rate improve LC-MS sensitivity?

Posted: Fri Aug 02, 2019 7:18 pm
by Rndirk
There might be a misunderstanding for your question 1. I think conserving samples in nano-LC refers to the fact that people inject a much lower amount of sample in nano-LC - resulting in a similar signal compared to HPLC/UPLC because the signal is concentration dependent. "Conserving sample" means that you don't need as much sample, which can be important if the sample is considered precious.

For question 2 I agree with James. Consider a situation where you have 2 significant different flow rates (for example 50µL/min versus 500µL/min) but still the same peak width (due to column choice) to isolate the effect of the flow rate. The signal should be higher for the lower flow rate - I believe this is even true in the situation where the concentration is the same. Why? As far as I know, it has to do with the fundamentals of the droplet formation, a process that is not fully understood yet.

Re: why does low flow rate improve LC-MS sensitivity?

Posted: Fri Aug 02, 2019 9:02 pm
by James_Ball
https://www.chromacademy.com/lms/sco31/ ... Theory.pdf

This is a good reference to read through.

Re: why does low flow rate improve LC-MS sensitivity?

Posted: Mon Aug 05, 2019 9:18 pm
by prk018
Rndirk wrote:
There might be a misunderstanding for your question 1. I think conserving samples in nano-LC refers to the fact that people inject a much lower amount of sample in nano-LC - resulting in a similar signal compared to HPLC/UPLC because the signal is concentration dependent.

I think you're right! Thank you for the explanation!

Also thank you James for the explanation and the reference material!

Re: why does low flow rate improve LC-MS sensitivity?

Posted: Mon Aug 05, 2019 11:15 pm
by Multidimensional
Yes, the answer has everything to do with nebulization (atomization) of the liquid over time. MS can not pick up or work with a liquid sample delivered by the HPLC system. The liquid needs to be removed (we refer to this as desolvation) so the ion particles are left in the inert gas flow. The more liquid you have, the more energy you need to evaporate it, per time. Higher flow rate equals more energy (gas and heat) needed in less time to remove. The lower the flow rate, the less overall energy needed per time to remove the liquid.

Re: why does low flow rate improve LC-MS sensitivity?

Posted: Thu Aug 08, 2019 8:45 am
by lmh
The question has been answered well by James and others, so these are just a few loose-ends and irrelevant side-lines that are sort of fun to know about!
James mentioned particle size. Here you may find something counter-intuitive going on in proteomic world: you will probably find people using very narrow, very long, 5u particle columns. Why aren't they embracing UHPLC and running small particles? Such sophisticated LC with such last-decade particle size! The reason is that they're running very complex samples, so they need the highest possible resolution, and therefore the highest possible plate number. Very roughly, if you halve the diameter of the particle, you double the plate number, but you quadruple the resistance to flow, which means you can only run a column a quarter the length (for the same back-pressure), so you've only got half as many plates... so for the very highest resolution with pump-pressure as the limiting factor, you need very loooong columns of fairly large particles.

The other thing relates to "conservation of sample": yes, proteomic people often have very small amounts of very precious sample for their nanoflow operations. As a completely irrelevant aside, this is why they tend to use loop injection autosamplers with low-pressure needles, which can be made of Peek, which can be lowered right to the very bottom of an insert so it just touches the bottom and bends slightly, allowing removal down to the last uL. Peek, of course, is also less inclined to bind things (bioinert and all that...). Those of us who worry more about carry-over and have plenty of sample tend to favour high-pressure needle systems (it's demanding on the autosampler to wash the inside and outside of a loop-injection needle thoroughly).