Equilibration/resolution problems with new Acquity columns

[HW Mueller]

If I thought a peptide would be a good test for the state of a column I wouldn´t have suggested another testing standard.
Similarily, if I knew that you have this problem only after sample injection and whether you have an equilibrium problem or not , I would not have asked what a ACN/H2O wash does prior to injecting sample.

[Uwe Neue]

Hi Ronald,

Not sure if the Snyder paper truthfully applies. We have run peptides many, many times in many different departments, without encountering the problem that you described. Nevertheless, if you follow my rules on how to keep the column equilibrated, your problem will go away.

I am working in the R&D group in the US, Feel free to contact me at the email adress associated with my posts.

Uwe

[rhaefe]

For my knowledge, the BEH C18 column and the BEH PST 130 C18 are the same stationary phase. Waters declared that they produce a BEH 130 C18 material. Then they perform a test for the requirement for PST (Peptide Separation Technology). If the test is passed, the column becomes a BEH 130 PST C18 column. If the test fails, the column becomes a "normal" BEH C18 column.

Can anyone confirm/refute this statement? Or maybe the better question would be: Has anyone the authority to confirm/refute this statement?

Slow column equilibration seems to be the most likely cause for the phenomenon. And the columns obviously can handle this forced euilibration step. At least when it comes to the peptides at hand. Would the columns pass the initial QC tests developed by Waters?

[Uwe Neue]

I have not verified it myself, but the procedure to run a second test specific to peptides is a logical way to qualify a packing material for peptide separations. So what is the mystery?

Slow column equilibration is not the only issue here. The columns with the RP packing are not stable under the extremely harsh washing conditions, and even for the Phenyl packing, I have expressed my doubts. I do not see how one could come to a different conclusion!!!

[rhaefe]

No mystery. I guess the wording of the original explanation sounded a bid curious to me. Probably that's just me being paranoid.

My point was if the columns are being damaged beyond repair by Nicki-Nitro's procedure it should be very easy to show that.

[HW Mueller]

If I understand this correctly then the cooked/damaged columns work again with peptide. If this is correct I can only understand this if he has a "all or nothing" situation and that some disequilibrium and or deposit cause the deteioration by drastically changing adsorption behavior, more so than the damage of the column.
That is a bit mysterious to me, though, and smacks of a observation/communication problem to boot.

[Peter Skelton]

Just a small question for Nicki-Nitro

You say you are having trouble with new columns, have you by any chance done method development using Aquity columns with the black RFID tags which you are now trying to make work on columns with the yellow RFID tags?

If so, send the columns back to whoever you bought them from and get them to send you some from a newer batch. Waters changed the packing process between the black tag columns and the yellow tag columns, and the first few batches of yellow tag columns didn't work very well. However the issue seems to be ironed out now.

[Nicki-Nitro]

Hello everybody,

I was terribly busy during the last couple of weeks. Therefore I could not make any further experiments. But now I have time today and received the required test columns from Waters (Acquity PST 130 BEH C18; 150x2.1mm; 1.7µm; batch 193). I set up a design of experiments, that I discussed with Uwe (Neue) and I want to start today:

At first I want to analyzed my special peptide method, that is sensitive for this equilibration problem. This method will be run with all columns to obtain chromatograms for T0 (not well equilibrated columns).

Method parameters: 0.02% TFA in water/acetonitrile mixture; temp: 60°C, flow rate 0.25 mL/min

Starting position:
30min equilibration at starting conditions
22min gradient run without injection (inject immediate samples)
22min blank run
22min sample run

This will be performed for 3 new columns (0 injections) and one well equilibrated column of the same batch (145 injections).

After that, the 3 new columns will be stored different:
1: flush column with water/acetonitrile 3/7 + 0.02% TFA for 30min, stop flow, storage for 1 week at 25°C
2: flush column with water/acetonitrile 3/7 + 0.05% TFA for 30min, stop flow, storage for 1 week at 25°C
3: flush column with water/acetonitrile 3/7 + 0.05% TFA for 30min, stop flow, storage for 1 week at 40°C (stability chamber)

After the first week the sequence for the starting position will be repeated again to see, which of the different slow equilibration procedures was successful.

I keep you updated.

[Nicki-Nitro]

Hello everybody,

It was a while ago since I've started the experiments but I was busy and I was not able to take care for this issue. The outcome of my experiments were:

One week equilibration with water/acetonitrile 3/7 + 0.02% TFA at room temperature was not enough to reach the required resolution of the column.
One week equilibration with water/acetonitrile 3/7 + 0.05% TFA at room temperature was sufficient to reach the required resolution of the column.
One week equilibration with water/acetonitrile 3/7 + 0.05% TFA at 40 °C in a stability chamber was as good as equilibration with water/acetonitrile 3/7 + 0.05% TFA at room temperature to reach the required resolution of the column.

My conclusions are:
Temperature is not necessary for the equilibration of the columns. 0.05% TFA is better than 0.02% TFA for the equilibration of UPLC columns to use them for ion-paaring chromatography with TFA. To retain the resolution with an well equilibrated column, we never flush the column without TFA. We do also store it in water/acetonitrile 3/7 + 0.05% TFA. Otherwise, we have to start again the long equilibration procedure.