Chromatography Forum

Hi All,

I'm working on analysis of glutaraldehyde by HPLC following derivatization with DNPH (containing H3PO4). The problem I have is that multiple peaks with two major ones were observed to be related to glutaraldehyde after derivatization. LCMS analysis of the derivatized glutaraldehyde standard showed the two major peaks are isomers and both are the expected derivative. Searching online showed no report of two peaks for glutaraldehyde-DNPH derivatization by HPLC. Has anyone experienced the same problem?

Thanks.

We've assayed glutaraldehyde by HPLC after DNPH derivatization and only obtained a single peak.

Thanks CPG for your reply. I'm puzzled by the two peaks. The derivatization procedure is simple: 0.8 mL 5 mg/mL 2,4-DNPH (acidified with 1.5% H3PO4) was added to 2 mL of glutaraldehyde standard in ACN, incubate @40C for 1 hour, centrifuge and inject. Column XDB C8, 150X4.6, 3.5 um. mobile phase A 5% ACN in water mobile phase B 100% ACN
45/55 A%/B% for 3 minutes to 100 B% at 10 minutes, UV@360 nm.

Is your second peak DNPH?

Thanks, bunnahabhain. No, the second peak is not DNPH. DNPH elutes before the derivative peaks. My guess is that the glutaraldehyde reference standard is not pure. I've requested COA from the glutaraldehyde vendor. At the same time, I'm ordering a glutaraldehyde-DNPH standard in order to figure out the cause of the two peaks.

If you have 1,3-Cyclohexanedione in your lab, try this procedure:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3934741/
I tried for C13 and C17 and it worked.

It is normal. Depending on what chromatographic conditions you use. We just integrate two peaks together as one peak to get the area.

Off the top of my head, don't you expect two peaks ?

R1-CH=N-R2 with H and R2 syn

and

R1-CH=N-R2 with H and R2 anti

with the same at the other end of the molecule; because of symmetry you only have two different stereoisomers.

Yes, I forgot the one SYN one ANTI configuration.

cis and trans configuration isomers are the most likely reason for the double peaks. Thanks.

Agreed--you've got your answer already...this Uchiyama guy has written a ton about DNPH derivatization of asymmetric aldehydes:


"Isomerization of aldehyde-2,4-dinitrophenylhydrazone derivatives and validation of high-performance liquid chromatographic analysis", Journal of Chromatography A, Volume 996, Issues 1–2, 9 May 2003, Pages 95-102, by Shigehisa Uchiyama, Masanori Ando and Shohei Aoyagi.

this is one example. Not much you can do about it...EPA TO-11A is fraught with this kind of nonsense...controlling the pH of the derivatization of both standards and samples helps a wee bit (also including a lag time for the reaction to come to equilibrium), but always there are two isomers of the shorter-chain aldehydes.