Chromatography Forum

I've been using XBridge columns for a particular long gradient assay

100%A-35%A over 40' where A=15% ACN/0.1% TFA, B=50% ACN in 0.1% TFA.

The first of 3 columns ordered failed a tailing factor spec. for the main peak right out of the box but gave otherwise good, if uniformly earlier (than a previous lot used in method development) peaks. The second column was better with respect to peak shape and was a better match to the first lot with respect to retention times but it seems to have died after having run just 3 sample sets. It was stored in ~75% ACN/water for a few weeks between its last sample set and its recently failed attempt to run more. Another XBridge that was abused in storage (was stored in ~50%organic/acetate buffer for 3-4 months) was utterly useless for this particular assay.

Waters states a preference for ACN as the storage solvent for this column but they are not adamant about it.

My questions:
Have others had similar experiences?

Is this particular phase *that* sensitive (damaged by long term storage in anything but straight ACN)?

Is Waters addressing lot to lot variation on this packing?

The batches have rather tight specifications, as you can see from the certificate of analysis. The certificate of analysis will also tell you, if you are looking at different batches, or if the differences are exclusively due to storage problems. If the columns are made from the same batch, the differences are due to use or storage.

I personally would not panick about retention differences between columns that have been used and a brandnew column. 3-4 month storage under aqueous conditions definitely changes the surface of a packing. How much? Don't know, depends on your analytes and your assay...

You can contact me at the email below and we can troubleshoot the problem together. It is always better to see some chromatograms to understand what is going on.

We had exactly the same problem with xBridge - reproducibility, chemical stability and tailing on basic analytes (hydrophobic quaternary amines). Our lab had 5 columns (for around 6 month) from three different lots. We have tried to play with pH to achieve a better peak shape but the best we could do was a tailing factor of 2. The was a problem with storage conditions in ACN/water and I don’t understand why (pH-7 with ACN/water??) Waters claims stability range which is much wider than traditional RP columns.
After struggling with these problems for few weeks we ended up with Zorbax Eclipse Plus with a good peak shape (tailing factor <1.5) and good reproducibility (RSD below 1%).

You can contact me at the email below and we can troubleshoot the problem together. It is always better to see some chromatograms to understand what is going on.

Thanks, you have mail (please do not post the pic. here, I have a safer version that I may put up later). I do plan on reinjecting some of my problem samples on another system to rule out a hardware problem.

Hopefully the rest of us will get something out of this?

I want to confirm that there is not something stupid (hardware issue) at the root of this before I go much further, I'm sure Uwe wants to have a look at the chromatograms before anyone gets into any serious muck-raking.


Patience please.

Hey DR,

An alternative column to try for this particular application is
our Cadenza CD-C18 column. It's polyfunctional ligand bonding and
novel polymeric endcapping allow for long column lifetime under these harsh acidic conditions. In addition, it gives great peak shape for
basic compounds:

http://www.imtakt.com/TecInfo/TI062E.pdf

Thank you.

I ran a marker solution on another system and still have a weird 1st peak shape...