Baseline problems Waters UPLC

[Victor]

Mattias,

I do not understand your enthusiasm for this solution. I am not an expert on detector noise, but I would not think there would be a huge difference between a 10mm path length cell and a 25 mm path length. Has someone verefied that the detector noise will go away with the shorter path length cell? Do you know what the cell volumes of these two detector cells are? Will the change to a smaller path length cell improve the peak sharpness by reducing extra column band broadening?

[Bruce Hamilton]

I'm a little surprised at your "solution".

My understanding is the problem appears to be that the mobile phase absorbs at 220 nm, in which case the best solution would appear be to change the mobile phase so that it doesn't absorb, and that will improve the sensitivity. Alternatively, you may be able to change the detection wavelength.

Please keep having fun,

Bruce Hamilton

[Mattias]

I haven't tested the other flow cell yet, so I was maybe a bit too enthusiastic too early..

We have a 50 mm flowcell also (other instrument), and if I use that I cannot detect any peaks at all (just a very strange quadratic noise). I assume that if the amount of light is too small, the electronics go berserk. With 10 mm vs. 25 mm I will increase the amount of light 2.5 times, which hopefully will be enough. The signal will of course also be reduced, but the noise will hopefully be even more reduced.

I would be glad to change the buffer, but are there any non-UV absorbing buffers at pH 4? I could increase the wavelength about 10 nm, but not more since then I will not see the peptide bonds.

Thanks for all help by the way!

[Victor]

Mattias,

I believe the high sensitivity flow cell has a volume of 2400 nl whereas the analytical flow cell has a volume of 500 nl. Thus, if you are working with peaks of small retention factor, the cell you are using at the moment will broaden your peaks and worsen your signal to noise ratio. This is in addition to worsening of the noise that might result from the larger path length. So you are definitely going in the right direction....

However, I do not believe that there is some threshold value of beam energy that exists above which your noise will go away. So I think your noise will be reduced by some factor, but it will not go away. It just depends if it goes away enough to be usable for you.

Bruce-some compounds have vastly reduced molar absorptivity above 220 nm. Some people even use 210 nm for the detection of peptides, which would apparently generate even worse results for Mattias. It seems the problem Mattias is having is not a problem with his method because other people are using similar detection conditions very successfully.

[AA]

If your baseline noise is caused by compositional ripple (ie mixing issues) the noise generated by the HS flow cell will increase by about 2.5 times. The HS flow cell itself will not generate 2.5X noise if the noise is the regular type of chemical/electronic noise that all detectors generate. It will however, always give a larger signal for a peak, usually on the order of 2.2-2.3 times taller, it can never reach the theoretical maximum of 2.5 times due to band broadinging with the larger volume of the flow cell itself.

[zokitano]

Dear colleagues,

I have recently encountered similar problem during assaying benzalkonium chloride in eye preparation.
Namely, the method uses mobile phase ACN:THF:acidified water with HClO4 (pH=2.2) = 35:15:50 (v%/v%/v%)
Column: C8, 150x4.6mm, 5um
Flow:1.5 mL/min
Column temperature:35C
Detection: UV, 214nm

I tried to equilibrate the column for 2 hours and more, but still I was getting very noisy baseline (the drift was up to 10mAU). That's why I had problem quantifying the analyte, because analyte's peak was 25mAU high. Signal to noise ratio was very low beyond the S/N quantification limit.
I tried to transfer the method on other HPLC system (Waters, prevoiusly I was using Agilent 1100 series) but I didn't get any improvement in the baseline, so again I didn't meet the system suitability criteria.

When I carefully read this thread, I figure it out that my problem is in my mobile phase. THF absorbs in that UV range (214nm). I measured the absorbance of my mobile phase: 2.8 AU. So, I presume that only slight variation in mobile phase flow or pressure can lead to such noisy baseline, I was getting.

That's why, Mattias, I think that Bruce is right when he proposed changing of your mobile phase (to less absorbing adequate mobile phase) or changing the wavelength toward higher values, to eliminate the effect of absorbing mobile phase.

Best regards

[danko]

Hi Mattias,

I hope the smaller flow cell will reduce the problem, but I doubt it a bit. Please get back with the result, so I/we can be inspired
Unfortunately there are no good buffer “candidatesâ€

[Bruce Hamilton]

Without want to emulate a cracked record, the problem appears to be that your mobile phase absorbs too strongly at the wavelengths you want to use.

I understand you may not want to change buffers because the formic works well for the MS, but effort in reducing mobile phase absorbance will help both sensitivity and noise.

First option, ensure that you are using the lowest absorbing grades of solvents and formic acid available. Check manufacturers' catalogues ( noting that they measure absorbance differently - pure, 1M, 10% etc ). Use a UV spectrophotometer, or DAD to understand the absorbance curves of your chosen analyes and mobile phases.

Second option, reduce the concentrations, 0.1% may not be necessary, try 0.05% or 0.035%, and also simply the buffers where possible - less components the better.

Third option, play with detection parameters, moving to 225 and widening the spectral bandwidth, etc. etc.

Fourth Option, consider manufacturers' instrument specifications at the region you want to work, and talk to them. It may be they offer options that will help, or perhaps even a fixed wavelength high energy detector may be a better option...

Please keep having fun,

Bruce Hamilton

[JM]

Please correct me if my unrstanding is wrong. The issue is, Mattias could not run the method on UPLC due to noise, whereas it is working Ok on normal LCs.

If the mobile phase is working fine on normal LC with same wevlength, there is no question of MP absorbtion.It got to do with the difference in two systems, whether it is mixing chamber or flow cell.

JM

[Mattias]

JM> You are correct. The noise on a standard Agilent 1100 or Alliance is around 0.2 mAu (amplitude). On the Acquity it is above 2 mAu. Any peak below 1 area% is more or less hidden in the noise.

The first 10 minutes of my run is isocratic (100% bottle A). The noise does not increase when the gradient start. That's why I don't suspect the mixing chamber anymore.

The lamp is new, we run only on HPLC grade solvents (LabScan), the other chemicals are of pro analysi quality (Merck). The pressure ripple is extremely low (below 10 psi).

I did one puzzling observation yesterday, I collected the noise at 280 nm and it was even higher than at 220 nm. I have no idea of how to explain that. That is contradictory to the theory of the absorbing mobile phase.

[Victor]

Mattias-can you let us know whether the UV detector you are using is the variable wavelength UV or the photodiode array system? And what data gathering rate are you using, and what filter? I think the Acquity detector works up to 80Hz, at which speed the noise will be worst. Slowing down the data gathering rate will reduce the noise, but it will not remove it if is is as substantial as you have measured. It may also make your peaks broader too.....

I agree with JM. Bruce-Mattias is using standard conditions that have been employed in peptide analysis for years without apparently any problems.

[JM]

OK, So HPLC method is not an issue. Mattias!! could you check following things,

1. As Victor indicated : UPLC is with PDAD or normal UV detector? are you comparing PDA noise to UV detector noise?

2. If it is PDA please check what is the reference wevelength ?

3. what happen to noise , at no-flow ?

JM

[Peter Milland]

Hi Mattias,

The solution to the noise problem is better mixing, we tried it and solved it with to 50µl mixers in series. Just install the second mixer as would with the first one, and block one of the outlets. Is works beautifully when working with these buffers at low UV.

Best regards

Peter Milland
Waters Denmark

[Mattias]

JM> The UPLC has a PDA detector, as well as the "normal" instruments.

Here are the parameters that I could find:

PDA range 210-300 nm (220 nm is extracted afterwards)
Resolution 1.2 nm
Sampling: 20 Hz
Filter time constant: normal (I have no idea what normal means here)
Exposure time: auto

It is not possible too choose a reference wavelength. The instrument is occupied right now, so I cannot measure the noise without flow.

The mixer we have now is a 50 µl mixer, we have tried a 425 µl mixer without any improvement. I believe the mixer is put in the wrong direction (after recommendation from Waters).

[danko]

The solution to the noise problem is better mixing, we tried it and solved it with to 50µl mixers in series. Just install the second mixer as would with the first one, and block one of the outlets. Is works beautifully when working with these buffers at low UV.

Mattias has conducted an experiment with a premixed mobile phase!