Chromatography Forum

I made an ion pairing method using 1-Octane sulfonic acid. The method is a gradient.
I have 2<k'<12, good separation and 1.1 tailing. I thought I was good until I noticed that 3 of the 4 compounds have max wavelength of 280nm but the other compound has a max of 229nm. If I understand correctly all compounds need to be measured at the max wavelength or the purity (area%) data will be skewed.

I tried a test run at 229nm and discovered that the baseline slope is way too high. It goes form 0mAu to 200mAu from start to finish. I'm assuming this is due to refractive index?

What options do I have? thx

Two runs at different wavelength or a PDA detector can help.

Skewed? It might be less robust to use a wavelength at the sides of a peak, but skewed?? Of course, the ideal would be to switch wavelength to the max abs. of the peaks during the run, but one can not always do things only under ideal conditions.
I wonder about the high noise (I assume that is what you mean to say) at 229 nm, though, strange gradient components?

At 229 the baseline goes from 0mAu to over 200mAu. Can this be from refractive index alone? The 1-octane sulfonic acid does absorb at this wavelength, but it is constant concentration throughout the gradient 20 mmol.

All methods will have to be validated so it's necessary to create a good one from the start if possible.
I have a couple more questions. Obviously I'm a novice at method development. I've had a class from Tom and that's it.

How do you proceed at determining a wavelength for your method if the compounds have different max absorbance wavelengths?

If you use a method where this is the case how does it affect the area% results? Will the area% data be valid at all without determining individual RRFs? thx

Skewed?

I think he's referring to the relationship between area% and mass%. However, in order for those to be equivalent, the compounds have the same extinction coefficients.

Skewed?

I think he's referring to the relationship between area% and mass%. However, in order for those to be equivalent, the compounds have the same extinction coefficients.

This is what I meant. I was under the impression that area% data would not be valid unless all compounds had the same max absorbance wavelength, but from your statement I'm thinking this isn't the case because they will still have different extention coefficients.
Technically what relevence does area% have unless you determine the RRFs?

The 1-octane sulfonic acid does absorb at this wavelength, but it is constant concentration throughout the gradient 20 mmol.

Yes, but how much sticks to the stationary phase will change! That's the reason that the classical ion-pair reagents are not recommended for use in gradients. I suspect that what you are seeing is adsorbed octane sulfonate beng stripped off the column.

I think our last two posts crossed paths, but at the risk of being repetitive, area% = mass% will be true only if all compounds have the same extinction coefficients. If you have standards, then you can correct for differences by using relative response factors (in that situation, each compound could be run at its own wavelength).

In general, I prefer to work at an absorbance maximum rather than on a slope; the response is much more reproducible. Small errors in wavelength can cascade out to big differences in absorbance on a slope. This slide from our Advanced Method Development course illustrates what can happen for a 1 nm error in each situation:

What do you do when developing a method for the separation of 4 compounds that don't have the same abs max? For the method I'm developing, 2 compounds have abs max of 280nm, one compound has abs max of 300 nm and the last one has abs max of 229 nm. We have PDA, but I've never seen a method yet that using more than 1 wavelength.

You mentioned traditional ion pair reagents. Are there non traditional ones, analogous to 1-octane sulfonic acid, that have lower UV cutoffs?

Pretty much anything that's a reasonably hydrophobic acid can function as an ion-pairing reagent. Heptafluorobutyric (HFBA) is fairly common for use in LC/MS and should be OK at 229 nm.

Thx Tom.