Aldehyde detection with 2,4-Dinitrophenylhydrazine

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GerardL
GerardL's picture
Aldehyde detection with 2,4-Dinitrophenylhydrazine

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

mbicking
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You are correct that this is

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.

ghunter
ghunter's picture
It is wonderful to hear from

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

newbern1
newbern1's picture
I have recently looked into

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.

mbicking
mbicking's picture
The reaction can be very fast

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.

MarkK
MarkK's picture
I'm looking at DNP for

I'm looking at DNP for measuring aldehydes (e.g. acetaldehyde, glyceraldehyde, vanillin, syringaldehyde,  furfural, etc.) in wine.  My concern is with the matrix and using DNP. 

Preferably, my hope was/is to find a simple semi-quantitative colorimetric assay I could run via uv/vis.

Dr. Analytical
Dr. Analytical's picture
I am not sure what conerns

I am not sure what conerns you have about the matrix, can you be more specific?

Do you want to use a standard UV/VIS instrument, or are you just referring to a UV/VIS detector for HPLC?  A standalone instrument is not likely to be useful, because of interference from unreacted DNPH.  An HPLC method should be relatively easy to develop, based on the information in this thread, in standard methods, and in the literature.

MarkK
MarkK's picture
Other components in the

Other components in the matrix having similar absorbancies and non-aldehydic carbonyl cmpds.    HPLC I have.

Dr. Analytical
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The DNPH derivatives will

The DNPH derivatives will absorb at about 360 nm.  Any other substance with a yellow-orange color could interfere, but there are not that many compounds that do this.  They would also have to elute at the same time.  This seems unlikely, but simple analysis of an underivatized sample would answer the question quickly.

I would try the reaction in mildly acidic solution and see what happens.