Can a new column change retention times?

I put on a new column on my UPLC binary machine last week, its a 100 x 2.1, 1.7micron C18 column and I equilibrated it by running 100% ACN through it for 10 mins followed by 50:50 ACN:Water for 10 mins. Not long I know but I had to get samples on it but when I put on a few test injections of a standard, the active peak is coming off 8 minutes later than usual (30 min run, peak usually comes off at 5 mins, now coming off at 13mins). The peak area is typical and peak shape but I cant really accept that can I?
The mixer is fine, the right pump is pumping the right mobile phase lines, the only thing I can think of is I changed the connection from B1 to mixer recently but there was no leaks coming out when I put flow through it so that leaves the column (the temperature is normal). Is this something that might settle out after a few injections or is better to put on a new column and re-equilibrate?

Best Answer

    Accepted Answer
    In my experience, your issue may be a bit excessive, but not shocking.  It is all dependent upon the method (column, analytes, mobile phase, etc.).  I've got a few methods where you could make injections instantly after getting up to temp and not have any inconsistencies over hundreds of subsequent injections and other methods where I have to run the mobile phase for about 4 to 6 hours (between low flow rate and running 2-3 condition column steps plus 2 diluent injections) to fully stabilize so I don't get any peak movement over the course of a 15-injection analysis (both in new and old column situations).  I've had situations where you had to baby a column with super low flow while warming up and then another 30 minutes or you'd destroy the column almost instantly.  Another issue I've seen is where we'd get a "contaminant" eluting off new columns and we'd have to make 100+ mobile phase injections on a column to clear it before we could use the column for real samples or else this blob would inevitably elute with each injection, slowly moving earlier in the chromatogram with each injection.


  • Hi Empower 2018 - did you try again after letting it equilibrate for a little longer?
  • Hi, yes the retention times went back to normal after I added 8 condition columns before the run, which surpised me as my test injection showed the retention times way off.Do you think a long equilibration time is needed for new columns? Its still no 100% as the baseline is a bit jagged for some injections but at least the Retention times are typical. 
  • In my experience with other columns, I've needed long equilibration times for new columns.  I'll give some time for other experts to chime in.  Then I'll ask some users within Waters to see what standard practice is here.
  • Thanks for the replies. Yes I have also worked with columns that were pretty much connect and inject away with no issues but this current one uses a gradient, a low pH and some concentrated samples so a lot of condition columns and a few trial injections of standards were required to stabilize. 
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