Chromatography Forum

Dear All,

Has anyone done any analysis using Xterra (MS and non-MS) C18 column with mobile phase set at pH~9? I am using this column under gradient condition but with flow rate and pH remaining constant all throughout the run. Only buffer/water and organic modifier proportion changes with time. My early analytical runs produced very nice symmetrical peaks but after 2 weeks or so, the peaks suddenly deteriorated forming very ugly, wide and unsymmetrical peaks.

Thanks and hope you can help me on this problem.

Hello Jasmine,
We've used XTerra RP18 with phosphate buffer at pH 7.1 and 35°C (LC-UV buffer - acetonitril gradient)
Nice peaks for about 100 injections, than peaks start splitting.
It's always nice to see claims for high pH stability, but be sure to stay away from phosphate buffer.
What kind of buffer are you using?
Jan

Hi Jasmine,

I first tried running high pH with Xterra as soon as the chemistry was launched. Even after a lengthy dialogue with Waters I could not get the column longevity that Waters claim - even though I was told that no other users had experienced this etc etc...

The long and the short of this is that I now use an Agilent Zorbax Extend C18 instead and this seems to work better than the Xterra both in terms of efficiency and longevity.

I think that the longevity issue may be application specific (?) as I have been assured (by Waters) that there plenty of users who routinely run 1000s of injections through Xterra columns at pH10 (10 mM ammonium bicarbonate, with ammonium hydoxide added to pH 10) without the longevity issue that I experienced (at around 1000 injections the chromatography became unaceptable and the column was not recoverable by backflushing or any other means).

I would be very interested to hear from anyone who routinely runs large numbers of samples at high pH on Xterra who finds that it really does work - more than 2000 injections without column failure? according to Waters there should be lots of you out there!

How stable the column is, depends a lot on the details of the operating conditions. I have run myself the XTerra column at pH 11.5 at 30 degrees C for 50 days for about 3200 injections. We have also been very comfortable with ammonium bicarbonate buffers in the range of pH 9 to 10. This buffer has been used very routinely in our labs.

Column lifetime depends on a lot of things. What are you injecting? What is the temperature? What is the buffer that you are using?

I am from Waters, and I have worked with XTerra for many years now. We did not claim good lifetime at high pH without data to support this. We also have comparisons to other surface chemistries available, which I however will not post here.

We had a good experience with the XTerra when working with alkylamines as mobile phase additives (pentyl to octylamine, pH 9-10.5).

I do not know if it makes any sense announcing number of injections, I think that thousants of "void volumes" would make more sense.

Also, from an emperical point of view, I always thought that organic acids and organic bases are more "gentle" -provided higher column lifespans- when operating in extreme pH than inorganic alternatives. I remember having seen a relevant article in Journal of Chromatography where the authors proved in a way the above statement (or at least their data implied the above statement). I'll try to dig it out later tonight...

Dear Jan, Richard, Uwe and Kostas,

Thank you for your replies. I am running highy polar compounds which have pka's around 9. I injected mixed stds (as is, unextracted) and extracts for about two weeks and got good results (peak shape, linearity). I used a gradient condition which starts from 100%water (containing 10% of 0.005M Ammonium Bicarbonate and changed to 40%water (containing 10% of 0.005M Ammo. Bicarbonate)/60% Acetonitrile later, all at a constant flow rate. All runs were done under room temperature. I was only able to get good results for about two weeks and then the peaks just deteriorated. I believe I was only able to make less then 500 injections.

Please advice.

Hi Jasmine,

If I understand well, you just use 0.2-0.5 mM of ammonium bicarbonate in your mobile phase. This is quite small concentration with a very small buffering capacity. May I ask if you have measured your mobile phase pH?

Also, what is your matrix (I do not quite understand as you talk about extracted and unextracted compounds). Maybe the problem is not in the mobile phase pH after all...

Hi Kostas,

My mobils phase comprise of three different solutions:(A) Water (B) 90% ACN in water (C) 0.005M Ammo. Bicarbonate in water (pH 9.5). I ran the gradient starting with 90A/10C. Then, changed to 30A/60B/10C, all at constant flow rate, switiching to initial conditions later. No, I did not check my final pH after the A/B/C mixing step. My matrix is plasma but all underwent SPE clean up prior to injection. The unextracted solutions are just mixtures of stds in dilute aqeous solution and did not undergo extraction.

I hope these info can help. Thanks.

Well - there is nothing wrong with getting about 500 injections out of a column with an extracted plasma sample. That is about what we are getting as well - independent of the pH of the mobile phase. If one is lucky, one can get 1000 injections from such a matrix, but I would not panick about 500 injections at all.

Considering that all your other conditions are rather mild, my opinion is that this has nothing to do with the stability of the column, but is just a question of contamination from the matrix.

If you want to get more out of your column, you can use a guard column that contains the same material. They are quite effective, if you replace them early enough. Based on what you are describing, I would replace the guard column after about 250 to 300 injections.

I personally am a great fan of guard columns, based on our experience in the lab, but this is entirely up to you.

Jasmine, I suspect that you have nicely behaving plasma (healthy or nearly healthy creatures) if you get something like 500 injections with samples only cleaned by SPE. This was mentioned before: With samples from highly catabolic patients we have had disfunctioning columns after one injection (pretreatment: at least a 4.6x250mm restrictive access column). (Partially described in J Chromatogr B, 678, 137 (1996). Right now I don´t remember whether it is already mentioned there that "fatal" incidences of this type became very seldom if some proteins were precipitated with Na2SO4 as first step, prior to restrictive access.... or an ultrafiltration).
In very obstinate cases the columns could almost be restored by treatment with a solution of thiothreitol and Li-dodecylsulfate.

Dear Uwe and HW,

Out of all the injections that was made into this recent Xterra column, about three quarters of them extracts from SPE treated plasma, and the rest are "clean" mixtures of std in aqeous matrix (unextracted). I thought SPE treated samples are supposed to be cleaner then extract from liq-liq extraction. In our past experience with other routine assays, with sample extracts from liq-liq extraction (plasma and whole blood matrices), we were able to get >>>>1000 injections in our other columns.

We have also previously used Xterra MS columns for large batches of samples, using simple li-liq extraction for sample treatment of plasma or whole blood and we were able to use it for long period of time. This is the first time that I have observed a column degrading very fast. That was why I was wondering if the column was indeed stable at high pH.

Please advice. Thanks!

Hi Jasmine,

Although I suspect that Uwe may disagree with me, I would suggest that you give your samples a go with a different column. I have tried several different sample preps to resolve the Xterra issue that I experienced and none of them resolved my problem, the same samples run under identical HPLC conditions did not cause column longevity or poor chromatography issues. I run with the Extend column because after running samples in parallel (with Xterra and Extend) this column worked best for my application.

There are other columns out there that you may wish to consider too, I have not tried the new Gemini from Phenomenex but that is supposed to be the best thing since sliced bread (yet again!)...

Jasmine,
did you run the columns after solvent extraction (as mentioned before, this is not really a liqu/liqu extraction) with the same mobile phase (pH...) as after the SPE? If yes that rules out a pH problem.
On SPE: Proteins are individuals, though generally SPE might be a bit better, one can imagine that you may have samples were a problematic protein has only been removed by solvent extraction. Also, I can imagine that one can botch a SPE more easily than solvent extraction.

Richard,
did you really handle all the columns which you mentioned ~ identically? Because if you did, it would be a highly interesting result. It would mean that some manufacturers can´t even do a relatively simple pH stability test.

Dear HW,

In the first test I ran standards and the second test I ran "real" samples on the same system with the same mobile phase, gradient and injection volume for both Xterra and Extend columns - sample decomposition was ruled out by separate analysis. The results were conclusive and have been presented to both Agilent and Waters. The longevity test was run after it became apparent that column lifetime was an issue - we needed to use pH as a selective tool combined with good column lifetime.

I think that the real point here is that if you talk to the major column manufactures they all tell you that their columns are the best and they will also tell you why their competitors columns don't work. Each manufacturers stability test will show their column to out perform their rivals column. In order to find out what really is the best you have to try it out for yourself, in your lab and for your particular application and always take manufacturers claims with a pinch of salt.

Jasmine,

Unfortunately, your perception that SPE-treated samples are cleaner than liquid-liquid extraction samples is incorrect (if indeed you mean liquid-liquid extraction and not protein precipitation).

In addition, the cleanliness of SPE samples depends on the methodology used for the clean-up. I can send you a thorough article on how to optimize sample clean-up from plasma samples, and what the different options are that are available to you.

Of course, there are a few other columns out there that claim to have good stability at high pH. My point was and is that the conditions that you are running were not what I consider to be a high pH for XTerra. Due to that and together with my own experience, I do not believe that the problem that you have is with your column. It is also of course your decision, how you would like to proceed...