Chromatography Forum

i am wondering if i can decrease the noise of my baseline( 1.3mAU).
Agilent 1290(new), M.Phase: 0.5%formic acid in H2O,PH8.5 by NH3/MeCN
DAD:230,4 ref:300,100
By the way, why when we use needle wash the vial must be uncapped?

That is a lot of noise!

The first question to ask is "long-term or short-term noise?"

Short-term noise (moving up and down in a second or less) is often electronic. Check the integrity of your data cables, grounding of the power system, and so on. If it persists, it can usually be filtered out algorithmically in the data system (settings like time constant, sampling rate, bunching rate, etc.)

Long-term noise (moving up and down in a few seconds to a few minutes) is more of a problem because it can't be filtered out without also degrading the peak shape. If that's what you're seeing, the first thing to do is to shut off the flow and measure the noise under static conditions. If the noise level is anywhere near what you're seeing now then you have either a dying lamp or an electronic problem. If the noise level is much lower (down around 10^-5 AU), then the optics and electronics are OK and you need to address the mobile phase:
- fluctuations in pump flow?
- purity of solvents and additives?
- clean glassware?

it is long -term noise and it's only for 230nm. In this analysis i have dual wavelength 230/320 and in 320 the noise is fine. This is a brand new LC, i wouldn't blame the lamp. The pressure is pretty steady.I don't blame the glassware either. i have done the same analysis 3 times in 30 days and i had the same problem.

Ok. You have exonerated the lamp. Does the noise go away under no-flow conditions? If it does, that points to an electronic problem, possibly something like a bad element on the diode array (analogous to a dead pixel on a display). In thatcase the noise should decrease as you invrease the bandpass.

Speculating about other posibilities: is there a chance you are seeing a bunch of late-eluting garbage from previous runs? In thatcase you would expect things to settle down after a series of blanks.

Hi
What's the noise and mAu readings when you pump 100% A. Now pump 100% B and again what are the readings.... If they are vastly different , you may have a mixing issue.

The idea behind not having a vial cap on the needle wash.

There is a possibility when the needle is removed from the sample vial that some sample is present on the outside of the needle. As the needle is pushed through the needle wash vial cap there is a possibility that this sample will be 'wiped off' the needle by the vial cap septa and deposited onto the septa. On removal of the needle from the wash vial there is a possibility that the sample ( that is on the septa ) is reintroduced back onto the needle.

That's my take anyway.

it must be the mobile phase. When i wash H2O/ACN: 50/50 the noise is fine. when i put M.phase A in water's channel and begin to equilibrate the noise is about 1mAU (only at 230nm). Formic acid maybe?

0.5% formic acid will have a considerable absorbance at 230 nm. Not sure how much though? 1 AU/cm?

This could be a case that there is almost no light passing through the cell. With 1 AU only 10% of the light passes. And that is with just baseline and no peaks.

With very little light, the UV noise looks "quadratic" if you zoom in. Almost as if the detector is counting single photons.

What column are you using at pH 8.5? At what temperature are you running your column?

i use prontosil c18 250x4.6 ,5μm but i tried one from phenomenex as well with the same results.I "play" with the temperature a bit but it's from 20 to 35C.

i use prontosil c18 250x4.6 ,5μm but i tried one from phenomenex as well with the same results.I "play" with the temperature a bit but it's from 20 to 35C.

Sorry to be a stickler, but *which* Prontosil C18, specifically? Which Phenomenex column? Do you know the pH limits of each?

Have you tried pumping 100% B? What does your baseline look like hen you pump 100% B? Have you switched the mobile phases (A on the B pump and vice-versa) and repeated the test to see if it follows the mobile phase or the pump?

it is long -term noise and it's only for 230nm. In this analysis i have dual wavelength 230/320 and in 320 the noise is fine. This is a brand new LC, i wouldn't blame the lamp. The pressure is pretty steady.I don't blame the glassware either. i have done the same analysis 3 times in 30 days and i had the same problem.

i changed channels several times. i don't believe PH 8.5 (+ %ACN) will be much trouble for these columns. the 1 of 2 prontosil i used was brand new.