Acetone as DNPH-aldehyde mobile phase?

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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Hello all,

as many of you know, the separation of acrolein, acetone, and propanal DNPH derivatives is both challenging and important for certain applications. The standard methods are a reversed phase HPLC separation using C18 or phenyl-hexyl columns using water and acetonitrile as the mobile phases with typically 10-20% THF added to mobile phase 1.

Still my application note search found separations between the three critical peaks to be marginal and prone to causing problems down the line. In addition, I am not eager to use THF in the lab or have to resort to MS detection on a routine basis.

I have found an Agilent application note using acetone as mobile phase B that demonstrates very good separation between acrolein, acetone, and propanal. You can drive a truck between those peaks!

However, I am concerned about the mobile phase being a CARBONYL. Won't leftover unreacted DNPH react with the mobile phase upon injection and give a potentially very large and smeared out acetone-DNPH peak? Or am I thinking about something incorrectly?

Here is the application note in question: "Analysis of DNPH-derivatized Aldehydes and Ketones using the Agilent 1220 Infinity LC System with Diode Array Detector" Agilent. Sonja Schneider. 2016.

Thank you for your help in advance.

Arne
I've got a couple of comments.

Here is the link for that application note, which I've never tried (we only assayed for formaldehyde and glutaraldehyde). https://www.agilent.com/cs/library/appl ... 1545EN.pdf

I wonder if acetone is intentionally used for this so it will react with any excess DNPH.

Look closely at the HPLC chromatogram in the Agilent application note; you'll see that it starts at 1 minute, not at zero. I wonder if there's a large early eluting peak that's intentionally not shown.

I'd also guess that the standard mix was made from individual DNPH hydrazones that had previously been purified.

My suggestion: buy the same standard as used there, and buy one of the columns from Agilent; if the application note procedure doesn't work for you, simply return the column, bet Agilent will honor that.
Thank You, Consumer Products Guy,

your assessment of the situation is like mine. I had been wondering if I had missed a key point.

The most important point is that the chromatograms shown are for standards of previously derivatized aldehydes. Thus, no free DNPH would have been present.

I am not concerned about the the chromatograms starting at 1 min. If anything, we'd see a large early-eluting DNPH peak, which is normal and ok.

Our concern is that we get a GIANT acetone-DNPH peak from real samples.

For now, I will brute-force the method with a phenyl-hexyl column and soem THF in MPA. If that does not work I will take up your Agilent suggestion.


Thanks again,

Arne
Not sure about the Acetone eluent wonder if that's a typo otherwise you would form Acetone-DNPH, but there are columns that will separate the 3 with just ACN;Water isocratically maybe not baseline but close

Restek Allure C18 which they might not make anymore
Restek Ultra C18 works ok 70:30 ACN H20

There are lots of other app notes for baseline separation of those 3 peaks etc
http://tools.thermofisher.com/content/s ... PN2984.pdf
Agilent application note used DNPH derivertized standards; Bet acetone was a typo, it should be ACN, does't it seem odd to use a solvent with strong uv absorption as mobile phase with a DAD? Aldehyde/ketone derivatization with DNPH requires buffer conditions generally.
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