System High Pressure with respect to gradient change

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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Hello,

I am currently looking to optimize the run time for one of the main methods used where I work. So far I have gotten it down from 20-25 minutes to 14 minutes, with a chromatogram that looks like below.

[img](https://imgur.com/a/Eis7lMF)[/img]

The question I have is about the high pressure. Right now I use 2 mobile phases, A=H20 w/ 0.1%FA and B=MeOH w/ 0.1%FA. The gradient changes from 85%B - 100%B from 1min to 3min and then back down from 100%B - 85%B from 5min to 5.5min. Because of this change, the high pressure dips from about 4 min to restabilize around 12 min. This consumes the majority of my runtime, and everything elutes by 8 minutes. Is it normal for the system high pressure to have this much of a delay in restabilization after a gradient change? Is there a way to shorten this system high pressure change?
Reequilibration time will depend on the flow rate and the dead volume of the system.

What size column are you using and what is your flow rate?
The past is there to guide us into the future, not to dwell in.
James_Ball wrote:
Reequilibration time will depend on the flow rate and the dead volume of the system.

What size column are you using and what is your flow rate?


Flow rate = 0.4 mL/min

Column size = 2.1mm x 50mm + a guard column, (2.1mm x 5mm)
You could potentially save a little time by increasing the flow rate as soon as your last relevant peak has eluted, since the pressure at this stage is very low. In order to achieve the same re-equilibration you want to pass the same total volume of solvent through the column, but if you can increase the flow rate, you can correspondingly reduce the equilibration time. The difficulty is that as the column re-equilibrates, the pressure rises again, so you will need to drop the flow rate again, or maybe run with a gradually-changing flow-rate to keep the pressure near its maximum (but with a sensible margin of safety! You don't want to end up with the optimal super-fast assay that goes wrong the moment you run a single dirty sample and the column back-pressure rises by a few percent!).
I dream of manufacturer software that, during method development, can carry out a run at constant pressure rather than constant flow, but meanwhile monitor the actual flow, and then translate this to set up a method that uses predetermined flows for the final, developed method, so that the method remains constant thereafter, even if the column gets a bit clogged. That way I could take my column rated to 1000 bar, develop a method designed to give constant 700 bar, and have the flow-rate profile consistent with the fastest possible separation with known margin of safety. Get writing it, someone?? Agilent, Waters, Thermo, Shimadzu, you could all do this. It's not hard.

Alternatively, if you can get the separation with acetonitrile instead of methanol, the pressure fluctuation will be a lot less drastic.
The pressure change between 85% Methanol and 100% methanol is significant due to the viscosity of the mobile phase you're working with. Unfortunately, 80-100% Methanol in the mobile phase should result in the lowest possible pressure for the mixture, and reducing it to something like 40-60% would not help. As for shortening the pressure, I'm not very sure what could be done from this standpoint. However, some areas that you can look to reduce the pressure overall could impact this phenomena positively. You mentioned that you're optimizing this method for your job, have you tried the possibility of switching solvents, like to ACN for example? At 85-100% ACN you would not see that great of a difference, but if you have not tried it yet you might want to give it a go as it could improve your retention times. I assume that you have already tried this however. My other suggestion would be to look at your guard column, specifically the cartridge, and check if it is fouled and needs replacing. Usually when I change my cartridge I see an immediate drop of about ~300 psi, and if it's really clogged then this could potentially shorten your re-stabilization times if not at least provide you with less pressure overall.
What HPLC-system are you using?
From the chromatogram and the pressure trace, I'd suspect it has too much dwell volume to be used at such a low flow-rate with such a fast gradient.
HPLCaddict wrote:
What HPLC-system are you using?
From the chromatogram and the pressure trace, I'd suspect it has too much dwell volume to be used at such a low flow-rate with such a fast gradient.

Absolutely second that.
The pressure lags about 4-5 min the gradient, so your dwell-volume may be in the range of 1.5 ml, which is not that uncommon for a classical HPLC.

It would be good to determine the dwell volume of your system(s). Needed also if you want to translate gradients between systems.
Have a look at the procedure here:
http://www.lcresources.com/resources/TSWiz/hs410.htm
... but your first peak is at 0.6min, so if that's your unretained injection peak, your dwell volume is nothing like as bad as that. Dwell volume is not the same as re-equilibration volume.
If you really need a shorter method, yes, consider acetonitrile so you don't have the pressure issues, but also try out whether you truly need to equilibrate the column fully. Your first peak is eluting at 3min, which is way after the 0.6min blip, so if the 0.6min blip is your injection peak, you have good retention and good resolution of the first peak. This means that if you start off with a slightly under-equilibrated column, you'll probably still get away with it. This is a slightly dangerous approach because it relies strongly on your system behaving exactly the same every injection (but modern systems do) and you're creating a method that is less robust (if your column one day decides it needs a little bit more equilibration, it's not going to get it). Also if you move the method to a different instrument, it may suddenly fail, because a different instrument will have a different time between runs (this depends on the speed of the autosampler), which starts to become an important part of your column equilibration time (and, a different system will have a different pump dead volume, which is an important influence on how long it takes to start reequilibrating the column, from the point when you tell the pump to start pumping the initial conditions).
I know a number of people who have methods with ridiculously short equilibration times, which I find super-risky, and yet they get away with it, getting perfectly adequate results year after year, much to my disgust!
...but the injection peak doesn't care about dwell volume. And while dwell volume is not the same as reequillibration volume, the reequillibration time will heavily depend on the dwell volume.
Also what is the column temperature? If you can increase the temperature 10-15C then the pressure will be lower and you can run a higher flow rate, which will help with equilibration time, run time, and dead volume.
The past is there to guide us into the future, not to dwell in.
HPLCaddict, you're quite right, I made a totally unwarranted assumption that because the autosampler looks modern and low-volume, the pump would have been chosen to match. I should know better! I'm still wary of estimating the dwell volume from the pressure characteristics with a column fitted; columns just seem to delay things far more than their volume would suggest. I wouldn't like to make guesses without seeing data from a gradient test using a back-pressure regulator, but hey, that's another thing I never seem to get time to do.
It'd be really nice to know whether the OP tried any of these various suggestions. The resolution in that method isn't looking too bad, and the peak-widths are decent; it's just got some long bits where nothing much is happening. That's usually not a bad starting-point for some optimisation. I'd love to know how the story continued...
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