Chromatography Forum

hi all..
any ideas on the ways to decrease noise in RP-HPLC method.
this is a method using a gradient of acetonitrile and water - both with 0.1% TFA.
i'm using agilent 1200 series system and detector.

are there ways to reduce noise in the method. i need to stick to a shallow gradient of 0.14% acn change/min to get the desired resolution.

wat are the detection system changes that can make a difference?

thanks!

I assume you're detecting by UV, but all the same... What is your detection scheme? Can you post a chromatogram of your noise and your critical peaks?

yes it is UV 215 nm.

i'll try to send a chromogram.
thanks.

You mobile phase will have a considerable UV-absorbance, which will cause noise, no way around that.

But try as pure reagents as you can find - buy the best acetonitrile and TFA on the market.

I have found a couple of times with acetatebuffers that the S/N improves if you choose a shorter flow cell (e.g. 6 mm instead of 10 mm). Hard to explain, but the effect is clear.

All other things being equal, if you have access to a single pump, low pressure mixing version, that should give you a quieter baseline than a binary pump, high pressure mixing unit. If you have both types available, make sure you try the single pump type.

Use gradient grade ACN.

I don't know if you're already doing this, but instead of having MPA = waer and MPB = ACN, pre-mix your mobile phases as much as possible.

If you have a DAD, optimize your settings (wavelength, bandwidth, slit, responsetime). Here's my story: I was working on an oligonucleotide method (UV@260nm) a few weeks ago and noticed that the DAD settings were not set properly - so a few quick changes (changed BW from 4 to 16, open the slit to 16 and increased response time by a little) and my noise went down threefold. I doubt you'll see anything that dramatic because there's only so much you can do with your bandwidth down @ 215 but play around.