Newbie HPLC help: Detecting Quaternary Amines

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Hi all,

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)!
For starters, step away from the chromatograph and think about the spectrophotometry. A quaternary ammonium group, per se, does not have any significant absorbance in the UV, so detectability boils down to what it's attached to: Any double bonds (e.g. carbonyls)? Any conjugated double bonds (even better!). Any aromatics (better still!). Then get on Google and look up UV spectral info for similar structures. While you're at it, look at the UV spectrum of acetic acid (acetate).

Unless you approach this sytematicallyh, you will waste a lot of time trying things at random.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
tom jupille wrote:
For starters, step away from the chromatograph and think about the spectrophotometry. A quaternary ammonium group, per se, does not have any significant absorbance in the UV, so detectability boils down to what it's attached to: Any double bonds (e.g. carbonyls)? Any conjugated double bonds (even better!). Any aromatics (better still!). Then get on Google and look up UV spectral info for similar structures. While you're at it, look at the UV spectrum of acetic acid (acetate).

Unless you approach this sytematicallyh, you will waste a lot of time trying things at random.


Thanks for the reply Tom. I'm working on getting some information on the exact structures of these compounds. The UV spectrum scan of the entire chemical is really confusing me right now though... The best responses I've got from the HPLC, by far, have been at 210/4 and 230/4... however, these wavelengths correspond with areas of zero absorbance when I scan the entire chemical! The peak absorbance corresponds to 280 nm according to

Is the ion-pairing reagent, or whatever reactions take place on board the column, potentially altering the absorbance?

Also, wondering how I can increase the spread of the peaks... pretty sure the large peak I'm getting at 210/4 and 230/4 contains a few co-elutions.
Yes, you can see "system peaks" just from injecting blanks (especially at or near t0).

And without knowing what you're looking for or what you're seeing, there's no way to tell you how to proceed. Don't take this the wrong way, but I suspect you don't even have the vocabulary to state the problem, much less interpret any advice.

Get hold of a copy of Mike Dong's book Modern HPLC for Practicing Scientists and read it. Here's a link to it on Amazon.com (http://tinyurl.com/7dnm8dw ); the price isn't too bad, but I don't know how much shipping to Australia will add. There are more detailed books on method development, but that one will give you the groundwork.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
Thanks Tom, I managed to track down a copy of the book... I'll have a scan over the weekend.
What compounds are you looking for?
Thanks for the help...

One of the mixtures I've been looking at, I've been able to get some pretty good results using an acetate buffer and acetonitrile (Isocratic). However, a second mixture containing quaternary amine salts is still giving me problems. I think the problem lies with the acetate buffer. I've run the buffer in a spectrophotometer and it shows some quite high absorbance below 280 nm. The first mixture contains a major component with a peak absorbance of 280 nm, so I've had no trouble there... a flat baseline and a well defined peak. However there are three compounds which all peak around 215 nm and I reckon the buffer is interfering.

Can anyone help here? Particularly in terms of suggesting a suitable buffer. I read that acetate shouldn't interfere above 205 nm, however the spectrophotometer results suggest otherwise...
Hi SOTR,

I think you will find the heptane sulphonic acid may be causing an issue. Go back to basics and try a simpler method before trialling any additives. . .
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