peak shape optimisation for a very hydrophobic compound

[IUpuppy]

Hi,

Firstly I just want to say that our e2695-2998 is back to normal after the Waters PM. The leaking injector was fixed by replacing seal pack and the needle by the service engineer... I had done the same before but I couldn't fix it-probably because I didn't use proper seal insertion tool to install the seal...

I have been working on optimising the peak shape for a very hydrophobic compound. It is an organometallic material comprising of 6 aromatic rings, 2 long alkyl chains and a heavy metal center. It is heavy and its molecular weight is 1022.21.

I am using sunfire C8 column and the best gradient that I figured out in terms of retention, selectivity and peak shape is 65% ACN, 0.1% TFA, 35%H2O 0.1% TFA.

However, the peak shape is still a bit broad and it has got a bit peak fronting too. The compound is only fully soluble in DMF, DMAc. It can barely dissolve in ACN or MeOH. I am using DMAc as injection solvent and I believed that the sample crushed when it met the mobile phase-this could be the reason for bad peak shape.

I have tried using Xbridge phenyl column to analyse this compound but it didn't help improving the peak shape and reducing the peak fronting. I guessed that phenyl column is only selective towards aromatic compound but can't optimise the peak shape.

Since it is soluble in acetone in low concentration, I am going to try acetone/H2O as mobile phase as well as injection solvent on Monday. If it still doesn't work, will THF help?

Can anyone give me some suggestions on improving the peak shape for this material? I will provide more details if required. I only have 1 week to keep working on this apart from my busy routine analysis.

Thank you!

[M Farooq]

Fronting occurs whenever the analyte is more strongly retained than the mobile phase and its components on any give stationary phase. This is implies that ACN is a weak mobile phase and it is confirmed by the fact that your analyte is barely soluble in ACN.

Is it possible for you to do to polar mode chromatography (sort of normal phase) using hexane and ethanol/IPA on bare silica column?

[IUpuppy]

Hi Farooq,

Thanks a lot for your suggestion! I don't have a normal phase column in the lab and in fact we have never had normal phase HPLC in the past...

Will there be any other RP HPLC mobile phase that is stronger than ACN?

Best Regards,

Hyuna

[M Farooq]

Hi Farooq,

Thanks a lot for your suggestion! I don't have a normal phase column in the lab and in fact we have never had normal phase HPLC in the past...

Will there be any other RP HPLC mobile phase that is stronger than ACN?

Best Regards,

Hyuna

Any pure silica phase can be tried, if you have one for normal phase. For RPLC, you can do a quick solubility experiment in small vials with ACN, THF, Ethanol, and Isopropanol etc. See which solvent quickly or easily dissolves the metal-complex without much agitation. You can try that particular solvent as an additive with acetonitrile and see if this helps in improving the peak shape from fronting to typical Gaussian. Can you post a picture of the peak or tell us the extent of fronting? Is it severe fronting? You just need a small amount of a strongly adsorbing additive to change the peak shape from fronting to normal.
Regards.

[IUpuppy]

Hi Farooq,

Thanks so much for your prompt reply. I will post the chromatogram tomorrow night after work.

I will do a solubility test using solvents other than DMAc or DMF tomorrow. What I did one thing before is that, I used a tiny amount of DMAc just to dissolve all the complex, and then use ACN or MeOH to dilute it again, it helps reducing the fronting a lot but it doesn't eliminate the problem.

Best Regards,

Hyuna

[Gerhard Kratz]

Try to get a better solubility of your compound by using a micro wave. Just some seconds.

[IUpuppy]

Hi,

Thanks for your reply Gerhard, we don't have microwave in the lab...

I just want to show the best peak shape of the compound I got so far. The gradient is 65%ACN 0.1%TFA, 35%H2O 0.1%TFA. And the column that I used is sunfire C8.

The following link shows the original chromatogram for the complex

http://hwwebmail.mail.126.com/js6/read/ ... pen_attach

The second link shows the 'zoom in' version for the complex

http://hwwebmail.mail.126.com/js6/read/ ... pen_attach

I hope everyone can open the link without any problems... As you can see the fronting is quite severe.

I have tried the acetone/H2O gradient and the result is quite similar to what the above chromatograms show. I guess water crushes the sample when the sample is injected.

We don't have any silica phase column as I have checked today and it would be too late to order one by now. Does anyone have any ideas about NARP HPLC?

I will have to try using THF by this Wednesday if nothing is going to work...

Thanks all for the valuable suggestions!

[TwistedHelix]

THF gives excellent separation however it has a BHT stabiliser which will give you a lovely negative peak (chasm quite frankly) in the middle of your Chromatogram. I suppose if you manage to get the stabiliser out in the solvent front it could be used......