Sciencemadness Discussion Board

I Need HPLC Help

Kavion123 - 8-2-2015 at 09:55

For this HPLC -

I need help. First off, if I want to use acetonitrile as my mobile phase, I buy a separate jar and put it on top and put the tube inside, right? Then do I need another jar for something else? Like H2O? My biggest area of concern is purging this system and getting all the air bubbles out. I will know how to change the column/loop, but I just don't get the whole mobile phase situation with this machine. If anyone could give me directions on how to start, that would be great. I already have the method set up for running the samples and standards, I know how to use Chromeleon, just the mobile phase stuff and purging I need help with. Thank you for your time!

phlogiston - 8-2-2015 at 17:07

Generally you just pump some mobile phase through the system for long enough. Monitor the column pressure. It stabilises when the column is equilibrated and at constant temperature. Check when the solvent comes out of the waste line. Sometimes the software has a purge function somewhere.

acetonitrile and methanol are generally good for washing the column/system, but you will usually employ a second solvent for the mobile phase. You program the software/pump to mix the two components in various ratios during a run. The solvents are typically an organic solvent and an aqueous buffer. Straight unbuffered H<sub>2</sub>O can be used but is usually not as a good, because the pH is poorly defined which affects the chromatographic behaviour of many components.

Kavion123 - 8-2-2015 at 18:29

Thank you, phlogiston. So let's say my mobile phase is to be Acetonitrile:H2O (8:2 v/v). I just need one bottle of it, make sure I have the tube placed in the mixture. This bottled mixture or mobile phase is called the solvent reservoir, correct? Then I loosen some knob in the back, and then hit purge on chromeleon? Then I wait until I see the solvent drip into the waste bottle, and then I stop the purge, tighten the knob, and then I'm ready to run my samples? Is that it?

phlogiston - 9-2-2015 at 07:33

Yes, you could fill a bottle with 8/2 acetonitrile/H2O and use that. If your method requires isocratic elution (=same composition of mobile phase throughout the run), that will be fine. If you have a gradient method (composition of mobile phase is changed during the run), you need to have two separate solvent bottles, one with 100% acetonitrile, and one with 100% water, and setup the pump/software to handle the mixing for you.
That is the preffered method, because it is more flexible (you can change/optimise/experiment with the composition of the mobile phase in software, no need to prepare mixtures manually), and also offers the additional advantage that you can flush the column with 100% acetonitrile for a few minutes after each run to remove compounds from your sample that would otherwise remain behind (and cause gost peaks in later runs).

I suspect the purge function in chromeleon will purge the lines from your mobile phase bottle up to the sample valve but not the column and sample loop. So, after the purge has completed, start a constant flow for a while and allow the column to equilibrate with your mobile phase. Elute at least 3 times the dead volume of the column and allow the column pressure to stabilize. Then you are ready to start injecting samples.

[Edited on 9-2-2015 by phlogiston]