Chromatography Forum

Hi all,

We are running a transfer of a validated method that involves quantifying the following solvents: methanol, ethanol, acetone, dichloromethane, ethyl acetate and cyclohexane.

A standard is prepared by dissolving the required amounts in Dimethylformamide.

The analysis is performed by GCHS using a Perkin Elmer Clarus 500 with the following column ZB-624, 30m x 0.32 mm x 1.8 um.

The first 5 solvents all have the expected retention time & response as in the validation of the method; however cyclohexane has an extremely low response when compared and also its retention time has shifted to after the DMF peak whereas in the validation report the cyclohexane elutes before DMF.

The method was transferred to an alternate lab with cyclohexane response & retention time as in the validation report.

Can anyone help us in this issue.

Jane

There is something very wrong here (but you knew that, right? )

It is quite unlikely that cyclohexane elutes after DMF. Really, it is.

It is possible you have lost a peak due to co-elution (not lost but confused and hidden) or from a sealing problem with your vials. This you may have misidentified your peaks.

Run solvent solutions without your matrix. Run cyclohexane alone without the other solvents.

Without knowing your matrix and its chemistry we are shooting in the dark. Of course, the parameters of your HS analysis and injection onto your column might be slightly helpful in diagosing your difficulty.

But if you do the investigation you should be able to solve the puzzle.

Good luck, Jane.

Rod

Thanks for your quick reply! and yes I had realised that something was wrong

We have tried investigating this problem, mainly by injecting spiked vials with cyclohexane - one time with DMF & another time in water but the retention times were the same.

I had run another method with a similar column that involved similar solvents (IPA & Cyclohexane dissolved in DMF) and cyclohexane eluted after DMf in this case too.

If there was a sealing problem with the vials then surely there would be some indication of this in the responses of all the peaks - the RSD for peak area was more than fine for all the solvents.

The method is to analyse for residual solvents in a particular pharmaceutical compound; the headspace instrument parameters are as follows:

Headspace parameters:
Thermostatting: Temp 130C; time 60min
Needle Temp: 110C
Transfer line temp: 130C
Pressurize time: 3.5min
Withdraw time: 0.5min
Injection time: 0.05min

Thanks a lot!

Jane

Hi all....the problem has been solved.

The issue is due to our supplier...turns out that what we believed to be Cyclohexane is not.

For obvious reasons this supplier has now been black-listed!

Thanks anyway for anyone who tried to think up a reason!

Jane