Basline oscillation

[Amar]

Dear people,
I am trying to do reverse phase HPLC using Vydac C18 analytical column
(4.6 x 250 mm ) with guard column and gradient elution. Buffer A is 5%
Acetonitrile 0.18%TFA. Buffer B is 90% acetonitrile 0.15%TFA. However
there seems to be some problem in baseline. Baseline drifts towards
negative AU @ 4% buffer B and again returns to normal @ 11 % buffer B.
What might be the rason? is there air bubble in column? or any other reason?[/img]

[Mark Tracy]

This is probably normal for these conditions. TFA does this. Dirty TFA does it worse. If it does not interfere with any of your peaks of interest, you may just have to live with the baseline. The baselilne drift and noise are both worse with TFA compared to nice buffers like phosphate. From your description, it does not sound like a pump problem.

A few things you can do-- Get the best TFA you can. Don't keep mobile phases more than a few days; they go bad. Reduce the %TFA as much a possible without losing resolution or peak shape. Try a different column that does not need so much TFA to get good separation.

[Amar]

Hi Mark,
Thanks for reply. Well I see what you are saying. Such high TFA can do the things described by you and certainly it does give basline noise. But my problem is not exactly what u understood. Actualy its not baseline noise that is bothering me. But there is baseline oscillation. Baseline is 0.0001 AU at 3% B but it gradualy becomes -0.016 AU till 10%B and again recovers to 0.0001 at 12%B. I see this in almost each and every run . so whats happening?
Amar[/img]

[Mark Tracy]

Most likely it is your sample injection. Do you see it when you make no injection? The TFA has some significant equilibrium concentration in the stationary phase, and that concentration changes with the mobile phase composition. Sudden changes in the gradient or large samples with different ionic strength perturb the equilibrium, and that wave propagates through the column. All ion-pair agents do this, but TFA has the misfortune of also being UV detectable.

I have been making large injections on a similar system; my negative dip is -120mAU about 1 min after the void, and takes another 1.5 min to stabilize the baseline.

[Amar]

Hi Mark,
I realy appreciate your comments. Well I am also doing large injections and certaily my sample is not clean. When I do blank run however, sometimes I see such thing and sometimes I dont. So may be left over/ residual things are interefering during blank run??? Comments?
Amar

[Mark Tracy]

When you say blank run, I assume you mean no injection, as opposed to an injection with no analytes. Perhaps you are not allowing sufficient time for re-equilibration. Is the dip smaller or at a different time if you equilibrate longer?

[skunked_once]

When I purify peptides/proteins, I use acetonitrile/water at 5%/95% mobile phase A and acetonitrile/water 95%/5% mobile phase B. I add 0.1% TFA to A and about 0.08% TFA to B. In order to get a flat baseline, I measure the absorbance of each mobile phase at the wavelength that I will be using, usually 215 nm. The trick is to adjust the UV absorbance of mobile phase A so that it is between 0.03 and 0.04 absorbance units greater than the absorbance of mobile phase B. This usually gives a flat to slightly raising baseline during the gradient. Using good quality TFA is critical and always do a blank gradient run.

[Amar]

Well Mark, yes blank run means no injection. I have not tried diffferent equlibration time. I usualy do for 30 min @ 1ml/min. Is it ok or should be done longer?
Amar

[Mark Tracy]

30 minutes should be more than enough equilibration. Unless your pump has an unusually large mixing volume.