I am using C18 HPLC columns to analyze nucleotides but have the problem that their lifetime is so short.
I have tried a lot of variatns of ACE columns (C18, C18aq, C18, hexyl-phenyl etc) from Advanced Chromatography Technology. The mobile phase consists of 7% methanol, 50 mM potassium dihydrogen phosphate (Scharlau >99.5%) and 2.8 mM tetrabutylammonium hydrogensulfate from Sigma (>99%) adjusted to pH 5.6 with KOH. I can run many samples continuously without losing performance, but as soon as I change conditions or I try to store the column, it deteriorates. I can only store it in 7% methanol. If I increase the methanol content, the column gets much worse. I don´t think it is the samples that destroy the column since the change is during storage and I don´t think it is the tatrabutylammonium ions that destroy the column since it is only the top of the column that is destroyed. If I turn the column around I get good performance again. Guard columns have also extremely short lifetimes. I have been thinking about if it perhaps can be impurities in the tetrabutylammonium hydrogen sulfate or in the potassium phosphate. Both of them have high purities but are not HPLC grade.
The short answer is that tetrabutylammonium hydrogen sulfate (TBAHS ) is an ion-pairing agent. While it is useful because it forms a complex with the analyte, the downside is that it permanently changes the surface chemistry of the column after use. This makes the column unusable for any other type of work (always label and dedicate them to a specific application). This is a very common effect seen in their use and one of the reasons we never use them unless we "have too" to get the job done (Nucleotide analysis is one of those times).
You might be able to find another mode of chromatography which yields good retention or perhaps find another chemical additive which is not so aggressive. Also, you can try a different brand of column (don't stick with the same supplier, there are hundreds of others). However, if you are using good column and system wash techniques after each run, not overloading the column, then you may find that you might just have to live with the short lifetimes.
- Remember that COLUMNS are consumable items (not capital items). They have a finite lifetime and sometimes it is a short one.
Thanks for the feedback. I am analyzing nucleotides and the main reason that I am using the procedure is that I get much higher resolution than on ion exchange columns. However, the column lifetime is extremely short. If I have not used the column for a week, it has often lost its performance. My feeling though is that it is not the tetrabutylammonium ions themselves that destroy the column because then the whole column should be destroyed, not only the top. That is the reason why I have started to think about that if it could be impurities in the TBAHS that cause the problem rather than the TBAHS itself. I have contacted Sigma about if there is any difference between HPLC-grade >99% TBAHS and the >99% TBAHS that is not HPLC grade but they could only answer that the HPLC grade has gone through more tests.
I am about to try a column from another brand next week (HALO C18).
I would definitely recommend other brands of columns than what you are using. There are nearly one thousand to chose from and some have much better stability than others.
Hello Andreas Hofer,
in the past we had problems with precipitations of TBAHS after some time. The TBAHS we prepared were (about 1/3) under solubilty in water. The organic modifier changes the solubility dramatically. And after some time we saw precipitation in the eluent. Perhaps the cristallisation is slow. This also happend on the column an closed it. The way it woks was reduction of TBAHS concentration to 1/10 of solubility.
This was a really informative answer. I am not using a high concentration (2.8 mM) but it could anyway be like you suggest since I assume that the concentration may be much higher on top of the column since it binds to it. I will now try to use two guard columns instead of one, one before the injector to catch most of the precipitate and one on top of the column.main In-line filters do not help, that I have tried already.
Anders: You should not have any issues with precipitate formation. The ultra low concentration that you are using (2.8 mM) plus the fact that your mobile phase has only a tiny amount of organic component (7% MeOH) should eliminate that outcome. TBAHS has excellent solubilty in aqueous systems (at very high concentrations). Also, I do not recommend guard columns in general (yes, there are of course exceptions), and esp 2 guard columns sounds like a really bad idea! Complicating things rarely helps. (1) Keep it simple. (2) Don't invest time in fixing things that are not broken. (3) Find the cause first, then implement changes/solutions as needed.
One type of column that did wonders for me is XSELECT C18 CSH 3.5 micron column from Waters. It is a "hybrid" silica-polymer packing, has excellent stability.
A question I have is about your guard columns... do they actually contain any stationary phase or are they a filter type?
Good to know for the future if the column I have ordered now does not work. The guard columns I am using are real guard columns, filters do not solve the problem. I still think that the idea about precipitation makes sense assuming that the concentration of tetrabutylammonium ions is much higher on the column than in solution. That could explain why the problem gets so much worse when I try to wash the column with organic solutions.
Like Bill, I do not like complicated systems. Can you use a different column and perhaps avoid the tetrabutylammonium hydrogen sulfate altogether?
I think I can never get that separation I have now with a new setup and the results with the first precolumn was so promising so it is worth trying. Without precolumn, the column gets destroyed even after a slight increase in organic content but with a precolumn I can change to 100% acetonitrile and only destroy the precolumn. If I then have a pre-precolumn, I might be able to protect the precolumn as well and I am free to store my column in solutions recommended by the manufacturer. If the pre-precolumn is destroyed it does not matter since the pressure not is affected, only the resolution.
In that case, it would be nice to understand how the stationary phase is "destroyed"... I would do research on this topic.
Surprisingly, I have not found anything written about it but if someone knows I would be interested to hear more.
Anders wrote: " That could explain why the problem gets so much worse when I try to wash the column with organic solutions." - NO, that is because the salt is not soluble in the highly concentrated organic wash solution you are using. Washing must be done using solutions that everything is soluble in (and stays soluble in). *Perhaps your column wash and/or sample prep steps are causing the precipitation and problems?
I am never going directly to highly concentrated organic solutions, actually I lost some of the performance already when going from 7% methanol to 10%.
Hello Anders Hofer,
another possible parameter is your buffer-concentration. You have about 7 g/L KH2PO4 in your solution. The anion of the phosphate also makes an ion-pair with TBA an reduces the stability of your target ion-pair. (The stability of an ion-pair is an balance of concentations.) So reduce your phosphate concentration at least to 2 mM, lower than your TBAHS concentration.
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