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Huge tailing during SEC HPLC on 1260/1290 but not 1100 machines?

jli

I'm running SEC HPLC and getting these very long broad tails consistently - the peak doesn't broaden, but it also does not return to baseline except as a very gradual slope. The issue seems to get worse the longer the machine has been running (see below), and switching from steel to PEEK tubing and swapping out different flow cells has not helped. What's most unusual is that the problem only happens on our 1260/1290 series hplcs - the exact same sample, method, buffer, and column on a much older 1100 series does not give any issues.

 

Blue line is the first run, teal line is the exact same sample after 2 more repeat injections.

 

Does anyone have any idea what's causing this? And why the older series instrument doesn't have the same issue? We are using an advancebio SEC.

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  • +1

    I have similar problem but with reversed-phase HPLC-UV. The newer instrument 1260 II has tailing in early peaks. Difference in delay volume between the old and new instrument is only +26 µL. As suggests, these observations do point to extra-column effects.I am also considering to change capillaries from autosampler to heat exchanger from 0.17 mm to 0.12 mm. 

      Did changing to thinner capillaries solve your problem? 

  • 0 in reply to mst87nk

    For clarification, is the tailing observed only on the 1260 and not on an older instrument (e.g. 1100)? Is the analyte a protein or other large molecule? With RPLC, especially at the intact level, commonly issues are more chromatographic (i.e. secondary interactions). If the issue is indeed instrument- thus likely detector specific- a similar phenomenon might be observed here, as with this SEC example.

    With SEC and other high salt separation modalities, secondary interactions with flow cell (teflon) leads to many tailing issues. Effectively, you have a huge increase in baseline that makes integration of peak difficult, as well as discrepancies in peak asymmetry measurements.

    Cleaning the flow cell (IPA, passivation) seems to work well, though in reality simply swapping out for a titanium flow cell will be the most impactful.

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  • 0 in reply to mst87nk

    For clarification, is the tailing observed only on the 1260 and not on an older instrument (e.g. 1100)? Is the analyte a protein or other large molecule? With RPLC, especially at the intact level, commonly issues are more chromatographic (i.e. secondary interactions). If the issue is indeed instrument- thus likely detector specific- a similar phenomenon might be observed here, as with this SEC example.

    With SEC and other high salt separation modalities, secondary interactions with flow cell (teflon) leads to many tailing issues. Effectively, you have a huge increase in baseline that makes integration of peak difficult, as well as discrepancies in peak asymmetry measurements.

    Cleaning the flow cell (IPA, passivation) seems to work well, though in reality simply swapping out for a titanium flow cell will be the most impactful.

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