Column temp and sample temp

[michaelcarolus]

Hi everyone

I have an HPLC method that has a column temp. of 40 Deg C. This column temp was chosen to allow the actives to elute faster. When I injected my samples I discovered that my samples were only stable for ± 5hrs. After about 5 hrs the sample has degraded by about 2%. I then decided to set the sample temp at about 5 deg C. The samples at 5 deg C were stable for more than 30 hrs. Do you think I should set the sample temp at 5 deg C and the column temp at 40 deg C. The alternative is to state in the method of analysis: "inject samples immediately." - this will allow for minimal degradation.

Any comments/suggestions will be appreciated.

Many thanks
Mike

[Uwe Neue]

If you do not observe any new problems such as retention time shifts or on-column degradation, there is nothing wrong with keeping the sample at 5 deg C and the column at 40 deg C.

[JA]

Uwe,
I struggled to come up with a reason of my own for why changes in retention might occur from a "cold" sample. Can you think of any cases, or give me an idea? If there were epi-/isomerisation which didn't reach equilibrium in the "ambient" MP before hitting the column for example?

Michael,
to add to Uwe's suggestions it might be worth confirming that the cold sample recovery is ok just to check its not [slowly] dropping out of solution.

[Uwe Neue]

JA, I would need to know the type of problems that you have encountered in order to troubleshoot it. If you see a retention time shift between a cooled injector and an injector at room temperature, and if the retention is higher with the cooled injector, one would conclude that the post-injector temperature equilibration was not good enough. In such a case, the peak may get broader as well. If you can't discuss it in public, you can contact me privately.

[michaelcarolus]

Thanks Uwe and JA

The method I'm using is from the USP with a slight modification (i.e column temp of 40 deg c as apposed to 23 in the USP). The USP states thats the sample must be prepared immediately prior to use and remain refrigerated until use. So, I guess it will be better to set my sample temp to 5 deg C. In this way I'll be able to analyse many more samples.

Thanks for the help.

Mike

[Uwe Neue]

If there is no problem, there is nor problem...

[JA]

Uwe,
I don't have a problem per se, I was just curious to hear of any intreguing examples where injecting a sample from a refridgerated sample compartment had caused a retention time shift. The more I think about it, the less of a retention time problem I expect.. Either a cold sample plug will equlibrate with the MP in the precolumn volume or, given that the analyte is retained, the cold solution passes on and dissipates down the column while the analyte partitions from the head of the column into some "ambient" MP.

I wonder if the assumption that samples at ~5C, injected in tens of microlitres, will equilibrate with an "ambient" MP before reaching the column on a typical analytical scale system is correct..
I wonder if low-volume optimised systems display a more frequent problem..
I wonder about the Acquity system..?