Chromatography Forum

I need to develop an HPLC method for analyzing lactic and C8, C10 carboxylic acids. I am using solvent A: water adjusted to pH 2.5 with Phosphoric acid, solvent B acetonitrile. Unfortunately my baseline shows a major "dip" during the gradient and again is fine at the end, when I have 100% AcCN. I am using HPLC water and acetonitrile from Alfa Aesar - could this be the issue. Suggestions?




Do have to appologise: this was originally posted in the GC section- my mistake.

How are you detecting (presumably UV, but what wavelength). You could be looking at a refractive index effect. Mixtures of water and polar organics such as ACN are non-ideal, and the RI of mixtures can be different from the average of the RIs of the components. Unfortunately, most UV detectors also respond somewhat to RI; depending on the optics and alignment of the flow cell, the baseline can shift up or down (this is sometimes referred to as the "mid-gradient hump". It can be aggravated if the flow cell is out of alignment. If you run the method on a different instrument and it looks OK, that would suggest the need for service on this one.

If you are detecting at short wavelength (say, in the 205 nm region) you may also be seeing some response from dissolved air, so you could try better degassing and see if that helps.

And yes, it could be due to contaminants in the ACN, the water, or the phosphoric acid, also those would tend to give sharp peaks rather than a broad dip. Getting water from a different source is relatively easy; a fresh bottle of ACN is not *that* expensive.

Of course, if it's not *too* bad, you could handle it the old-fashioned way (ignore it!)