I am going to check the activity of an enzyme on a substrate which gives acetic acid as one of the products.
Hi Sushil, you should have better chances with PDA (near 220 nm) but I doubt that you can detect it below the mmolar concentration.
Hi,
we have done calibration curves for acetic acid using 20-500 ppm standards [Phenomenex Synergy 4u hydro-RP C18 4.6 x 250 mm column, 20mM KH2PO4, (pH=2.6) as eluent]. As detectors we use both DAD/PDA (at 210,214 and 220 nm) and RID. For actual samples, DAD/PDA at 210/214 nm tends to give a better response/larger peaks. However, this is sample dependent (e.g., if there's disturbing compounds).
For our analyses (water extracts) this works fine, but I haven't worked with enzymes so I don't know if it is applicable for your substrate. As Massimo Capobianco said, it might be tricky to detect low concentrations.
Regards,
JR
The choice of the detector will depend on the concentration of acetic acid that you are trying to analyze. For lower concentrations, PDA works better as suggested above.
Thanks a lot Mr. Massimo Capobianco, Mr. Jan-Erik Raitanen and
Mr. Keerthi Prasad Venkataramanan for your suggestions.
@Mr. Massimo & Mr. Keerthi : As you suggest PDA detectors can detect in lower concentrations of acetic acid, I should go for PDA detector than RID.
@Jan-Erik Raitanen: I am using an enzymatic reaction, which contains substrate, enzyme and multiple products. So there are chances of disturbing compounds. So according to your suggestion also I should go for PDA detector.
We use a DAD to detect acetic acid to sub 1mg/L levels accurately. RID would not give you as good lower limits. We use a BioRad Aminex column with 2.5 mM H2SO4 as eluent with column temp set at 60C and DAD set at 210nm. We do also use RID but do not use it for quantification of the signal
I have used DAD also with a C18 column around 210 nm. Methanol and some acid on water may work as mobile phase. My LOD was around 0.5 ppm
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