Chromatography Forum

I was given a method. It called for a Hypersill Phenyl. The Mobile phase is 5ml TriEthylAmine into 1000ml of DI water. pH with phosphoric acid to 3.0. That's it.

My problems have been instantaneous, but have persisted for 3 days. I cannot achive a stable baseline for more than one shot.

The baseline likes to climb for an hour flatten for 20 min then drop. I guess you would call it a sinwave sort of baseline but it's so big that I've never actually seen it. Obviously i could extend the run time to 300 min and see it but that's alot of time.

What I know is that this mobile phase is not the greatest for the stationary phase in the method.

Am I just Sisyphus at this point??? Any ideas, I'd be glad to hear them.

Thanks

Chuck

I do not see a fundamental problem.

I would start with washing the column with 100% methanol until the baseline is flat and then reequilibrate it with the intended mobile phase. This would wash off everything that may have hydrolyzed during column storage.

Uwe,
So do you believe that this kind base line drifting is due to an unclean column under isocratic run if there is no bubble, or column bleeding? Thanks

Some brands of Phenyl columns (and I am not sure if this one falls in this catagory) do nothing but continue to bleed (i.e. the bonded phase is not stable). However, as was pointed out by a previous poster, you may be able to get it to a point that the baseline drift is not interfering with your assay. Just remember though, when you buy a new column of the same brand and try and duplicate your results, the new column (having more bonded phase on the particles), it will give you different retention charateristics thatn the old one that you pulled out of the drawer and used after several years of storage. I know that when budgets are tight, the expense of new columns can be a burden, but starting off with a new column (this mostly applies to the less stable phases) often is well worth the money when you factor in your development time and then redevelopment time, not to mention the validation and method trasfer headaches that come along for the ride.

If future users of your method (within your company, or those who might try you method if it reaches the scientific journals, I assume people still read these, but I'm not so sure) can not duplicate it, it is worthless.

Just something to think about.

AA

There were/are several different Hypersil phenyls, one with end-capping ( phenyl-2 ), one without ( phenyl ), one on base-deactivated silica with endcapping etc. - could be even more, I haven't checked.

My understanding is that some brands of phenyl column use a short C3 ( propyl ) linker that may reduce phase stability in presence of acid or base, whereas some others use longer carbon linkers and very dense bonding to reduce hydrolysis, such as Phenomenex's Luna phenyl-hexyl, which is the only phenyl phase I've regularly used recently. If a brand works for me, I use it to benchmark other columns that might be specified by clients.

I would want to see more details of the method, especially the mobile phase absorption at the chosen detector wavelength, and the quality of the TEA and acids used to prepare the mobile phase, as well as the quantities used. I'd like to know how the samples are prepared, and their solvent, and what happens if you just inject 10x volume of the sample solvent.

Bruce Hamilton

Chuck and YM3142:
As Bruce has pointed out, there are many different phenyl phases around. We have in our product lines several ones as well. For example, the XBridge Phenyl is a hexylphenyl packing, and the XTerra packing is a short-chain packing. However, I believe that the difficulties that you have seen are associated with the shipping solvent of your column. My assumption was that the column was shipped in the column test solvent mix.

I am just peeking in from vacation on this, so I can not follow up on it. But Charles´statements confused me. He can not get a stable baseline for more than one shot, and his baseline instability is an extremely broad peak. Sounds like he is injecting persistent dirt? Or air? I saw another chain here, today, where air could have been the culprit. One shouldn´t neglect such things.

Charles,
Have you used triethylamine before with another phase?
I tried it once and got all sorts of baseline wobbles and chunks.
I ended up sourcing a different mobile phase configuration.
WK