By ela on Friday, February 23, 2001 - 08:08 am:
I'm working in a lab specialized mainly on plants extracts. After two months of using a column C18 and another C18e on a system ( gradient Water: Acetonitrile both with 0.1% orto-fosforic acid, from 10:90 to 0:100), everything it started to be crazy. Our column is not reponding anymore like at the beginning, not even close. We tried to wash the column for many days methanol, cloroform, methanol, acetonitrile; we also tried injections with dimethylsulfoxide. Nothing works! Does anyone have an idea about how can we get our column back. We are in a lab from Eastern Europe and we can not afford another column very soon.
By Anonymous on Friday, February 23, 2001 - 05:20 pm:
What do you mean by "the column is not responding any more like at the beginning"?
(PS: my columns never talk to me...) :)
By H W Mueller on Sunday, February 25, 2001 - 11:52 pm:
Could it be that the acid ruined your column? If so, with the next column try a higher pH, maybe you can then also get away from such a high organic %? If you really need to buffer your mobile phase it would be advantageous to increase the buffering capacity (maybe 10x your concentration). Also if there is a precipitation problem. there are other buffers.
By Anonymous on Monday, February 26, 2001 - 08:15 am:
FYI: at my lab, one of our HPLC analyses uses 8% H3PO4/H2O and acetontitrile issues on an old-style regular C18 column, lots higher in phosphoric acid than the 0.1% described above, and we don't have any . We then use the same column for all our normal day-to-day assays.
By tom jupille on Wednesday, February 28, 2001 - 04:30 pm:
My experience has been that it's hard to generalize about column lifetime issues, because it depends on how "non-robust" your separation is with respect to column changes.
My first suggestion would be to try to find the original manufacturer's test chromatogram and re-run that. Those tests usually use things like toluene or benzophenone to check for a properly paked column; as a result, they are relatively insensitive to column variability (or degradation). If your re-run comes close to the initial results, then that says that the column is not too badly damaged, which implies that your assay may simply be non-robust.
If the re-run of the manufacturer's test looks very different from the initial results, then the column is, in fact badly damaged and will have to be replaced. There is a tremendous variability in column lifetimes at low pH ("end-capped" columns, for example, change quickly at first and then settle down once all the end-capping has been removed).
2 months is not an unusually short lifetime for a heavily used column.
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