Hello. Is there someone out there who has used 2,4-dinitrophenylhydrazine (DNP) for the quantification of low molecular weight aldehydes in aqueous solution? What concentration of reagent is required in the assay, and does the conjugate absorb at a different wavelength as the unreacted DNPH? or is separation of conjugate from unreacted DNPH required. This is an "old" assay but I cannot find a recent reference to the actual protocol. many thanks, Gerard

You are correct that this is an old reagent, but it is still used. EPA method 8315 uses this reagent, and it is generally useful for all aldehydes and ketones (I developed the method orginally).

An excess of reagent is required, and generally some separation method is going to be best. DNPH is easily separated from the derivatives by reversed phase LC, with absorbance detection near 360 nm.

I can provide more information if needed.

It is wonderful to hear from the original inventor for this method. I have seen many people used this method. Thank you for your contribution.

How fast does the reaction go? Would pH be critical for this reaction? If you have a protocol/description for this method handy, I will be honored to have one.

Best,

ghunter

I have recently looked into this myself. If you contact Supelco's apps lab - they have a method they use with reverse phase HPLC for this type of analysis.

The reaction can be very fast (one minute) but this usually requires high acid concentrations. Sometimes the use of high acid concentrations can result in generation of formaldehdye by decomposition of other compounds in the sample (a false positive result). For these samples a pH of 5 may be necessary.

As a suggestion, start with 0.1 % phosphoric acid, and add a 100 X excess of DNPH. Allow to react about 5 minutes and then analyze by HPLC. If you need low detection limits (< 100 ug/L) you may have to extract and concentrate the sample.

I am developing an automated method, but the results are not yet available.