I'm runing a gradient. Components of mobile phases A and B are the same except for the content of acetonitrile, which is higher in the mobile phase B. The UV detection is set at 205 nm. The shift in baseline observed in not acceptable and can't be explained by the absorption of acetonitrile. Several run give the the same result. Does any one have any idea on how to solve this baseline problem?

providing detailed data would help. As it stands there could be dozens of suggestions as to what might be going on. You didn't even say if the baseline went up or down!

Bill is right, need more detail. What percentages of ACN are you using? What are all the components of your mobile phase?

You could be seeing absorption or effects of changes in refractive index. Different detector designs respond to changes in refractive index differently. If you can use a reference wavelength, setting this appropriately often helps.

If the shift is due to differences in absorption you may be able to use a spectator ion like nitrate and make both solutions absorb the same.

The change in ACN concentration could be disturbing an equilibrium between the MP and the SP of one of your mobile phase (or water) components.

Mobile phase A contain Acetonitrile - 2-Propanol -0.2M Ammonium acetate - Water (16 : 16.5 : 5 : 62.5, v/v/v/v). For mobile phase B,30% of acetonitrile is used and the percentage of water is reduced accordingly. Different gradient programs were tried, but all, including those with relatively slow increase in mobile B, result in subsequent increase in the baseline.
Tom,
What do you mean by spectator ion like nitrate to make both solutions absorb the same. I need more information so that I can try it.

Hi,

You are running at a high concentration of Ammonium Acetate. Look at your mobile phase in a UV spectrophotometer blanked against water 190 - 400.

If my foggy memory is correct, when I looked at a 10mM solution I had a uv cutoff of about 205nm.

You could also flush the hplc with water and autozero then switch to your mobile phase and see what the background absorption is.

Where does your baseline start and where does it end up. Good luck.

With UV detection, you will be better off with a phosphate buffer. No problems in the UV!

Thank u for ur comments. The method is intended to be further used with MS, and I can't use phosphate buffer. On the other hand, less good separation is obtained using lower concentration of ammonium acetate. The baseline goes from 0 mAU up to 200, 300 or even 400 mAU depending on the slope of the gradient.

Be aware that even ammonium acetate will suppress your MS signal at that high of a concentration, at least in ESI. Ideally 10 mM should be your maximum. Not sure about APCI.

Tom M.,
I'm still interested to know about the use of spectator ion like nitrate to make solutions absorb the same.

Unknown,

In your case I don't think that is going to help. As you go from 16% to 30% ACN I don't think the ACN is causing the change in absorbance at 205nm, assuming you are using HPLC grade ACN. Some of this could be refractive index change, but what I think you are mostly seeing is the result of a change in equilibrium concentration of acetate as the percent of ACN increases. There is probably not a lot you can do unless you can move to a longer wavelength and get away from the background absorption of your buffer or change buffers.

I am using ACN:water 90:10 and a abs wavelength of 200nm for my compounds. I get an initial sharp spike a negative peak and then the baseline remains steady at 100abs units. I am not able to bring the baseline to zero for my entire run.Does anybody have an idea how to solve this problem?