Chromatography Forum

Hi, I have a problem with my Atlantis T3. I have used Atlantis T3 3um 2.1x100mm with UPLC system coupled with mass spectrometer in order to identify some compounds. After that analysis was done using HPLC system. In order to maintain similar gradient elution conditions I bought Atlantis T3 3um 4.6x150 mm column (bought longer column to achieve better separation in comparison with 4.6x100mm). First chromatogram was very similar to UPLC, peaks were not identical but separation was very good. But after 30-40 injections the efficiency was very low and peaks were very wide - efficiency become terrible, but retention times were the same.

I used two solvents: A: 10mM TEAA pH7 and B: ACN. Gradient conditions: -10min 95 A and 5 B, 0min 95 A and 5 B, 11min 0 A and 100 B, 16min 0 A and 100 B (linear gradient).
After some injections I washed column 95 H2O and 5 ACN for 15 min (in order to eliminate TEAA from column) and then 100 ACN for about 15 min. After washing loss of efficiency was observed. But efficiency declined from inject to inject, too.

I guess the problem could be too low ACN conc. in washing eluent or the part of gradient where 100 ACN is used for 5 min (11-16) caused efficiency loss (maybe triethylammonium acetate precipitates). System backpressure is stable and analytes in the mixture is stable too.

I would like to read other opinions and suggestions. Maybe someone used Atlantis columns and could help me with proper washing and equilibrating conditions of this column.
Sorry for my English, it is not my mother tongue.

Hi Andrius,

Your English is fine to me. I've used the Atlantis dC18 and T3 for many years...my feeling is that, while these are excellent phases for polar compound resolution (no, I Don't Work for Waters, but Atlantis phases have treated me well over the years), your eluent pH may be incompatible with them, perhaps too basic. Something you could try would be to place a "sacrificial" column in front of the Atlantis so that the eluent can "chew" on that instead of your Atlantis column's stationary phase, and swap the sacrificial columns out fairly frequently.

Remember, the strength of Atlantis is limited C18 ligand density...once these ligands are cleaved from the head of the column and the silica starts to disappear a bit...there will go your peak shape at first...and then resolution and everything else. Remember too, mixing ACN (or MeOH) with an eluent...the pH will become a bit more alkaline, your pH in practice is likely closer to 7.3-7.4 in the mixed eluent with ACN...and now, while more acidic than 8 (the danger value for silica dissolution), it's pretty close.

Anyhow, that's my opinion, and I'm sticking too it. Best wishes, see what you think.

Hi Andrius,

Generally I agree with Matt. What Matt means is to use a saturator column, packed with a "cheap" C18 packing material, what ever you can get in bulk will work, but remember to install the saturator column BEFORE the injector.

If their is a too fast strip off of the C18 Alkylchaines first thing you will see is an increase of retention time. Many years ago I worked for Waters....... due to an increase in active silanol groups on the surface.
Did you messure the pH of your mobile phase A?
Please check pH first. Do you work under constant temperature conditions? Remember, pH is depending on the temperature!
Good luck
Gerhard