Chromatography Forum

Just wanted to start a topic for peoples experiences with reverse phase columns at high pH

I work with small molecules that contain a basic nitrogen, and are best analysed at high pH for both selectivity and sensitivity. To date I have developed a number of methods for these molecules that operate around pH 10, which in comparison to acidic or neutral methods are far superior.

The problem is column lifetime. The first high pH method I developed used the Gemini column …. not good, peaks which had a resolution of 4 initially dropped to below 2 in under 100 injections (35 minute run time). In an attempt to rectify the problem I substituted a XTerra into the method, this doubled column life (200 injections). Please note that the columns are not failing catastrophically in this time, just losing resolution between critical pairs.

As far as I can tell the difference between the two columns above is the Gemini uses a ‘protective’ polymeric layer to protect the silica, while the Xterra is a crosslinked hybrid particle. I presume that once the protective layer on the Gemini particle is breached, the column dies quite quickly, whereas the hybrid particle is more stable.

I have now tried XBridge columns with the hybrid BEH particle, and have increased column life to 300 injections. This was also helped by a reduction in column temperature (now 30 deg C).

Can anyone share their experiences under similar conditions?, has anyone tried the new Gemini column?, or can recommend a good high pH column. I also have not tried the Agilent Extend columns, although am sceptical about how base resistant these would be.

Perhaps a zirconia or polymeric column would be the way to improve column life, I am curious about the possible loss in efficiency in comparison to silica based columns, and am very interested to hear what peoples experiences with zirconia has been.

In anticipation to requests for more information…. I have tried vendor recommended regeneration procedures, and no, I do not use a guard column. I am fortunate enough to be analysing nice clean pharmaceutical actives. The mobile phase is ammonium formate for one method and triethylamine bicarbonate for another, with no difference in column life between the two.

Thanks

You might want to read these previous articles:

http://www.sepsci.com/chromforum/viewtopic.php?t=5005

http://www.sepsci.com/chromforum/viewtopic.php?t=4081

http://www.sepsci.com/chromforum/viewtopic.php?t=2950

http://www.sepsci.com/chromforum/viewtopic.php?t=899

Also you might want to consider porous graphitic carbon columns as an alternative for high pH applications (stable at pH 1-14)...

Thanks Kostas,

What are your experiences with the porous graphitic carbon columns you are recommending?

My experience is good, method development is not as straight forward as silica based columns, sometimes it is hit or miss (in terms of peak shapes) in high pH for no good reason. The column exhibits some anion exchange properties as well...

How do you know the deterioration is due to the high pH?

Hi Lusby (welcome to the board)

Quick question - are your methods isocratic or gradient? I've often found that although high pH methods do give me the retention, selectivity and peak shape I'm after, baselines are too poor for effective gradient low level impurity analyses (especially at the 0.05% level where baseline rise swamps the detection of such low level impurities).

HW: Fair question…. I don’t have direct evidence that it is high pH other than observations such as:

>Increasing the pH further reduces column life.

>Substituting more ‘pH resistant’ columns into the method results in increased column life.

>The columns seem to last much longer under neutral or slightly acidic conditions.

>Column regeneration makes absolutely no difference, and there is no significant rise in backpressure. So I’m thinking it isn’t contamination.

I should also point out that I am not particularly disappointed with 300 injections (~200 hours) before changing column, if I was using a method in the traditional silica pH range the selectivity would require 2 methods to achieve what can be done with one at high pH.

My post was to see if my experiences with the latest hybrid particle columns are typical, and just in case someone would read my post and say “I did this….., and my column life doubled!â€

I've been using the new Gemini-NX intermittently since April, usually around pH=10 to pH=11 with ammonia-based buffers.

I haven't seen any obvious performance degradation, and I currently don't use Waters columns in similar apps - so I can't offer any relative experiences.

I'd hope the more expensive Waters column would be more durable, but two Gemini-NX at less than the price of one Waters column locally was my imperative for purchasing the -NX and, so far, the column serves my purposes well.

According to the Phenomenex Literature, the -NX is supposed to last 4 - 5 times longer at pH=10.5 than the earlier Gemini, which I never used.

Bruce Hamilton

Lusby43, ok your evidence seems good, but in view of what Bruce said, as one example, I still wonder whether something other than pH is behind this (or did someone say this already here?: the pH is different from what you think it is?).

We typically get much higher column lifetimes from at least the XBridge column, which is the most durable of all these column types. Most of our experience is in continuous use of the same column.

When storing the column, the high-pH buffer should be removed.

Silica dissolves at higher pH, so even if it is protected, the tendency is always there. If it is simple pH, then look at non-silica phases.

Someone else suggested carbon. Polymeric options are also available, although performance is not as good. Zirconia is another option. Zirchrom has published a number of high pH applications (www.zirchrom.com); this stuff just doesn't dissolve in basic solution.

Merlin,

XBridge is not silica. It is hybrid material thoughout the entire particle. The Phenomenex particles have a silica core, and your assessment applies to such particles indeed.

Uwe:
Yes, I am aware of the BEH technology, and it is certainly designed to be more resistant to high pH attack. But in looking at the structure, I still see some Si-O bonds, which, if in contact with a strong base, could possibly hydrolyze. Clearly the particles do not do this under normal circumstances.

Bruce, how much work have your Gemini NX columns done?. I was ‘burnt’ by the first generation of this column, and it seems the new one still uses a protective layer rather than a complete hybrid particle like the XBridge (thanks for confirming this Uwe). At the time initial efficiency was much better for the Gemini than the XTerra (pre XBridge release), but performance of the Gemini degraded rapidly.

Looking at the Waters chromatograms for XBridge high pH stress testing, it actually looks like a similar thing happened in their test as I am observing. The vendor used a peak eluting in the middle of the chromatogram to assess performance, the elution of this peak was quite stable. However there is a late eluting peak in the examples for which the retention time changes quite a bit, this is what I am observing…. as the column ages one of the late eluting peaks changes retention time and co-elutes with another impurity.

My theory on this is that the compound that changes RT can interact with unbonded particle surface that gets exposed as the column degrades / ages (flame away!). I can change the RT for this compound with TEA in the MP, furthering my suspicion that it can interact with degraded stationary phase (the other compounds are not affected by TEA).

Still keen to hear experiences on substituting zirconia into traditional and hybrid silica C18 methods, if indeed it is possible….

Interesting observations. You may just have a system that is a little too much for most phases.

Zirconia is stable under these conditions. ( I am told that you can clean the columns with 1 M base!) The zirconia is the actual substrate; it is not a hybrid. The phases are attached or coated to the surface. But the selectivity is much different than silica, and it is sometimes hard for us silica-based chromatographers to make progress. But it is worth considering if the other things aren't working.

Applications are available at: http://www.zirchrom.com/PDFNotes.asp

Note: While I know many of the Zirchrom staff, I do not have any contractual or financial arrangements with them.