HSS T3 column

<p>WE use ACQUTY UPLC HSS T3 column for the analysis of the drugs from urine, however we always find the rentention time of the compounds shifting from time to time and the curves were not very stable. What PH value is good for this column? (For example, using formic acid adjusts the mobile phases)</p>


  • If you take a look at:


    you will find that the HSS T3 column is allows a pH range of 2-8.


  • Yes, I know the range, but would like to know if anybody had the experience for using what exactly ph value to optimize the performance of the column. I tried the low ph value but it seems not working well for my compounds

  • We also hade problems with drifting retention times in the paston our system. Onces it turned out that the seal rings in the primary were leaking due to extensive use of the system.

    For some compounds we see also that the pH is indeed important to get a reproducable retention time (especially amphoteric compounds turned out to be sensitive to this, we are also using 0.1% FA).

  • We use this column for the analysis of vitamins with excellent results. Our mobile phase is sodium hexansulphonate 5 mM at pH 3.00 and methanol. I wonder if you have under control the column temperature, because we have noticed that temp environment changes produces inestability on the column.

  • I am using this column with 0.1% formic acid (in water and acetonitrile). Stability of retention time is excellent.

    When retention time in my system shifts, it's always a sign for problems as a leak, unstable temperature, eluent problems, exhausted pre-column, ...

  • Have you checked the Column Lot QC to see if there are changes from lot to lot for your columns?  We see our compounds occasionally broaden/elute much later on different lots of column...

  • yes, I found a little retention time change from different lot of column, especially when the PH is higher (>4). However, lower PH seems not working well for the standard curves of the compounds. So far we did not find any leaking form the system. I am guessing the RT changes may also relate to the solubility of the compounds. (We have 20 compounds analyzed together). There are a lot of things to be adjusted regarding the solubility and separation of compounds…….

  • Are they basic or acidic drugs?  What is the pKa range of your drugs of interest?

  • They are basic compounds, includiing opiates and amphatamines.........

  • I've found that the columns themselves do have somewhat shifting retention times (from lot to lot... and from run to run)... especially if your gradient does not sufficiently elute compounds from the column.  There are ways to clean your column you might want to try flushing the column with 100% water for 20 minutes at a flow rate of 0.25 mL/min, then flushing it with 100% ACN at 0.25 mL/min for 20 minutes... and then starting your normal flow again and let it equilibrate for a little bit.  The lot to lot variation there is nothing we can really do but get a different lot of column and make sure there is no dead volume with your extracolumn peek/ferrule connections...

    Other than that, I would try another pH for the buffer... but if things are sticking to your column then that could be a culprit for things not retaining consistently.  I would maybe keep the flow going at a low flow rate even when samples aren't running... proper equilibration matters.

  • and are you using a buffer solution? ammonium formate? or just adding 0.1% fa...

  • Thanks! We just use 0.1% formic acid. We tried buffer, but did not get good separation for all the compounds.  Besides the HSST3, any other column from Waters is good for the drugs analysis? ( for example opiates, amphetamines.....)

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