Question about UV-cutoff with respect to an NH4Fm gradient

[Mike H.]

The UV cutoff of the formate buffer system is 200nm according to a method development chart on our lab wall. I'm trying to create a gradient for a compound that has 2 UV maxima. One at 230nm and one at 252nm. I'd prefer to use 230 because the sensitivity is twice as much as 252nm, but at 230nm the slope of the chromatography is close to 1(actually I guess it's -1 since it slopes downwards. My simple question is why?

My conditions are a simple gradient. 80:20 pH3.8 100mmol NH4Fm:ACN at 0min to 20:80 at 8mins.

Thank you.

[tom jupille]

Agilent's website lists the UV cutoff (A > 0.5) for 10 mM potassium formate as 210 nm (http://www.chem.agilent.com/CAG/cabu/buffersel.htm ).

0.5 is a *lot* of absorbance for HPLC and you're 10x more concentrated, so I wouldn't be surprised to see some residual absorbance at 230 nm. That said, check the purity of your ammonium formate and/or formic acid; that can make a *big* difference.

[HPLCaddict]

As a quick fix I'd try the following:
Use premixed mobile phases - mobile phase A: 100mmol NH4Fm/Water/ACN 20:60:20; mobile phase B: 100mmol NH4Fm/ACN 20:80. Run the gradient from 100%A to 100%B. With respect to the organic modifier, this gives you exactly the same gradient as before. But the Formiate content is the same in both eluents, so you won't see a baseline drift anymore.
Still, with so much formiate at 230nm, you may loose quite a bit of sensitivity, since the background absorption is rather high.

[Mike H.]

@Tom: I'm using 99.995% metal trace analysis from Aldrich. The formic acid is 98% from EMD and I'm using ammonium formate not potassium formate.

@HPLCaddict Mixing the mobile phases in the manner you suggested sounds like a great idea. I mean I've used mixed MPs before where the goal was to have ACN in both to stop algae growth, but never considered using it to eliminate gradient slope. Thanks for the idea. I know it will work.