Chromatography Forum

Hi,

I have a method for aldehydes and ketones using DNPH with HPLC-DAD. Our formaldehyde method works great, but I can't seem to get Acetone to work.

I have tried 1-20ppm curves and also 20-50ppm curves. The peak area that shows up for Acetone increases slightly with increasing concentration, but it's nowhere near linear (1ppm gave 7 area and 20ppm gave 22).

My mobile phase is 60% Water : 40% Acetonitrile using a C18 column.

Reaction conditions: 1ml DNPH in 50ml acetontrile to give stock solution. 2.5ml of stock solution + 2.5ml acetone standard reacted for 30 minutes at room temperature.

Does anybody have any experience with Acetone detection with HPLC? All of the literature I can find says acetone reacts readily with DNPH.

Thanks

Can you just monitor acetone directly? It absorbs more than water/acn?

Could the acetone be reacting and just not able to be separated from the excess DNPH ???

The larger aldehydes and ketones often react much more slowly with DNPH than formaldehyde does. Try the same derivatisation but extend the reaction time out to a few hours, or try heating it up to around 40-50 degrees C.

Acidic conditions are required for derivatization of ketones and aldehydes if I remember correctly.

Acidic conditions are required for derivatization of ketones and aldehydes if I remember correctly.

Indeed, IIRC we used trichloroacetic acid with DNPH.