Chromatography Forum

Hi,

I'm trying to develop a method for detecting acetone via derivatisation with DNPH using HPLC-DAD.

I have an issue with sample preparation and can't seem to fix it. I always get far more acetone-DNPH in my own derivatised standards than I do with a certified acetone-DNPH standard.

2mg/L acetone-DNPH standard peak area = 0.9938

My own derivatised blank peak area = 11.6328
2mg/L derivatised sample made with certified acetone standard = 15.5618
Blank corrected peak area = 3.9290

My DNPH is DNPH-Phosphoric acid (https://www.sigmaaldrich.com/GB/en/product/sial/42215) diluted 1:250 in acetonitrile and phosphoric acid.
1mL of standard mixed with 4mL of DNPH solution.

Results are consistent regardless of DNPH concentration (tried 1:2500 and 1:25 dilutions), derivatisation time (30 mins - 24 hours) or derivatisation temperature (room temp - 40C at 4 hours).

Method parameters:
60:40 Water:Acetonitrile mobile phase
C18 column (works well with formaldehyde-DNPH)
0.6mL/min
42C column temp

Now I know the blank peak area is big in and of itself, but I'm really struggling as to why even once blank corrected, my self made derivatisation standards are so much higher than the pre-derivatised acetone-DNPH standard and would really appreciate any help with what I'm doing wrong here.

Hello there!

i am sorry if this may sound silly but it is part of the troubleshooting process. How exactly did you prepare both samples? In other words, is there any chance that you prepared a 2 mg/L acetone mixture and then derivatized it?

Hello,

I have a similar problem. I have been performing HPLC analysis for the determination of formaldehyde in resin samples by derivatization with DNPH. I first recorded the chromatogram of DNPH and then made working solutions by adding a fixed amount of DNPH against increasing formaldehyde concentration. As the formaldehyde concentration increased, the peak area of the derivative compound formed (derivatization: 30 min at room temperature) also increased, while the peak area of DNPH decreased (which is what I expected). I obtained a calibration graph with a correlation coefficient of 0.9992. However, when I derivatized the formaldehyde in the resin under the same conditions, I could not obtain reproducible chromatograms, the peak area kept increasing continuously, and after 18 hours I obtained a constant peak area. This tells me that I need to do some optimization work for the derivatization process of the sample, I think.

However, when I derivatized the formaldehyde in the resin under the same conditions, I could not obtain reproducible chromatograms, the peak area kept increasing continuously

Could your resin be decomposing to release additional formaldehyde?