Chromatography Forum

Hi everyone,

I am currently setting up an assay for paracetamol, paracetamol glucuronide and paracetamol sulphate however the paracetamol glucuronide is eluting at ~6.1 min which is too close to the solvent front - para sulphate elutes ~18 min and paracetamol at ~22 min. The assay conditions are as follows - Flow rate 1mL/min, run time 40 min, MP is 0.1M KH2PO4:isopropanol:THF 100:1.5:1. Column - Grace 5um 4.6x250mm column.

Does anyone have any suggestions for increasing the retention time of the PG peak?

Cheers,

Merrin

That mobile phase is somewhat out of the ordinary. What column are you using (Grace makes a wide range)?

Hi Merrin,

With the described column dimensions and flow rate, I would expect un- retained peaks (solvent front etc.) to elute at approx. 2.8 min – which is not that bad. It all depends on the un- retained peaks’ width of course, but if you have, say, Rs = 2 or higher which is not at all unrealistic, given the mentioned retention times, then you have nothing to worry about.
Actually I would try to optimize the method a bit if I were you (provided you’re allowed to do so).

You can try one of the following options or both.

1. Increase the flow rate to 1.2 or even 1.4 mL/min.
2. Install a shorter column, say, 150 mm.

Both options will shorten all retention times, but mostly the solvent front’s time. You will need to reduce the percentage of one or both of the organic components in your mobile phase. But it will only add to your savings in addition to shortening the run time.

By the way, I agree with Tom’s observation: It is very unusual mobile phase. I would fear the possibility of buffer precipitation, if I were you.

Best Regards

Hi again,

A few people have commented on the mobile phase - it came from a paper given to me by my boss however I think it was chosen because of the polar nature of the metabolites. I am certainly open to other suggestions for mobile phase for polar metabolites though.

As for the column it is a Grace Apollo c18 5um column. I originally used a 15cm phenomenex column however when I analysed plasma samples I wasn't achieving sufficient resolution so I started with the longer column - and so far it has been good.

The problem I'm having is that plasma proteins eluting just after the solvent front are affecting my earliest eluting metabolite. I am trying the different flow rates now - thanks for the suggestions - keep 'em coming ! I'm relatively new at small molecule work so all help/suggestions are warmly received.

Thanks again.

Merrin

How are you extracting your plasma samples? A different procedure could give a cleaner chromatogram.

We are using precipitation with 30% perchloric acid - I have tried some other protein precipitation methods but that one is resulting in the cleanest chromatograms so far. Any other ideas - preferably not involving SPE?

Merrin, try 1 M ammonium sulphate at appox. neutral pH. It should precipitate the proteins in your sample.

Best Regards

Which other protein precipitation methods have you tried?

Why not SPE? There are well designed, proven and well established procedures SPE around. SPE gives you always a cleaner extract, which results in longer column life etc...

It's only no SPE because my boss has told me no SPE - otherwise I'd be happy to give it a go.

As for precipitation methods, I have tried ACN, MeOH, acidified MeOH, ethanol and trichloroacetic acid. I am now giving the 1M ammonium sulphate a go.

Incidentally changing the flow rate as suggested is looking promising!