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- Posts: 4
- Joined: Wed Jun 18, 2014 8:02 am
I'm a total HPLC newbie and I'm looking for some help on detecting Quaternary Ammonium compounds (Quat Amines) in a chemical diluted in a distilled water mixture. Currently trialling at 50 ppm in H20 (components containing Quat Amines make up roughly 20% of chemical mixture).
The equipment I have at my disposal includes an Agilent 1220 infinity LC, with a thermo aquasil C18 column. Unfortunately I don't have literature access, all I have to work with is a conference paper from about 15 years ago, which contains a few methodology points. We are using a mobile phase of ACN (polar organic solvent) and an aqueous phase (acetate buffered to pH 5) containing 4mM heptane sulphonic acid (been trialing at 5/95 ACN/Buffer solution). All this stuff, I've just picked out of the conference paper... I know nothing about ion pairing! Flow rate I've been trialing at around 1 mL/min. injecting the maximum vol of 100 ul. Isocratic.
When I ran the chemical that we're targeting through a spectrophotometer (diluted to 50 ppm), I was getting a fairly broad peak absorbance around 285 nm (Peak between 282-289). Bizarrely though, when I've been fiddling around with the HPLC, the greatest response I've been getting has been around 210 to 230nm (on the spectrophotometer this region had nil absorbance)!
Basically I'm looking at some basic pointers on where to start and how to optimise, whether those relate to the flow rate, injection vol, mobile phase make-up and conc... or wavelength monitoring. At the moment I've been trying a number for wavelengths between 280 and 290 and also a few other random wavelengths, with a bandwidth of 4 nm (should I experiment here?). No reference wavelength is being used.
At the moment I've been getting one peak eluting within the first few minutes (depending on flow rate). I've altered the sample concentration and tested at various different ppm concs and quantitatively things stack up pretty well, however, I'm looking for better peak separation (there should be a number of peaks) and response.
Any comments, regardless of how trivial they may seem, would be helpful. I have no idea about HPLC (As you can probably guess)!