Chromatography Forum

Hi there
i'm developing a new method for a pharmaceutical product which has 4 impurities to be separated. The problem is that one these impurities is a base and at the pH i run the analysis, the compound is in it's ionic form. This makes it elute near the dead volume even if the mobile phase is 95/5 (v/v) (KH2PO4 10mM/ACN). I can't use MeOH instead, because the analysis runs at 205nm and it absorbs. I use a CN-column 15cmx 0,46cm 5μm. Is it possible to use a mobile phase of 100% buffer? I have read that for the most columns it is wise to use at least 5% of organic solvent to extent the lifetime of the column. Is this constraint exist in this situation too? Could a hypercarb column be more effective in retaining more time the compound in the column so it wouldn't elute at the dead volume?
The basic problem is that i have also non polar compounds, and polar compounds both bases and acids.
Thanks for any suggestion.

Try SIELC mixed-mode columns. They are designed to retain ionized and neutral compounds. Mechanisms of retention: reverse phase, cation and anion exchange:

http://www.sielc.com/pdf/SIELC_June_2004.pdf

http://www.sielc.com/pdf/SIELC_May_2007.pdf


Please contact me if you need help with column and mobile phase selection.

Regards,

Vlad

Thanks for the suggestion. I'll check it out.
But is it possible anyway of using 100% buffers as mobile phases? Is is acceptable?
Thanks

It is possible to run a CN column in 100% water/buffer. No problem.

For hydrophilic basic compounds, it is often quite diffcult to achieve adequate retention on a RP column alone. Therefore, RP column with an anionic ion-pairing agent can be considered.
The alternative is to use a RP/cation-exchange mixed-mode column. One of the benefits of such column is flexible selectivity adjustable by mobile phase ionic strength, pH, and/or organic solvent content. Attached link leads you to the datasheet of Acclaim Mixed-Mode WCX-1 column which you might find helpful in your case.
http://www1.dionex.com/en-us/webdocs/65 ... PN2024.pdf

Very often, mixed-mode columns can solve the problems that a RP column can't solve and are suited as valuable method development tool complementary to RP columns.

If you are looking for other suggestions, here is another option to try.

Get the Discovery HS F5 column from Supelco (this is a fluorophenyl phase). I suggest 4.0 X 50 mm, 3 um. Use a mobile phase of 0.01% phosphoric acid and acetonitrile (try 70/30 first) at a flow of 1.25 mL/min and injection volume of 5 uL.

Under these conditions, this phase will retain cations. You can then adjust acn concentration (more acn increases retention), or acid concentration (less acid increases retention). This works very well for some bases, but I have seen some tailing for others. I am not sure why it works the way it does, but it does work.

If you are able to try this, let me know how it works.

Since everybody is giving suggestions on other columns to use, let me suggest my options:

1. Use Atlantis T3, a fully endcapped C18 column designed for use in 100% water without phase collapse.
2. Alternatively, use a packing with an embedded polar group (which also can be used in 100% water): SymmetryShield RP18 or RP8, Xterra RP18, XBridge RP18.

Thank you very much for the suggestions.

Ultimately i used the CN-column i mentioned before (150mmx4,6mm 5μm) and a mobile phase of 100% water/buffer KH2PO4 25mM.

The working solutions of the substances were prepared with methanol.
It was very crucial to prepare the final solution with as little MeOH as possible. As a result, the retention time of the first compound was shifted enough from the dead time and i managed to have a clear peak.

Thanks again

Hi Project 44,

Given the latest information, it is obvious now that your problem has been caused by the sample solvent – all along.
I’m almost certain that you can achieve even better separation if you change to a C8 or C18 column and keep the rest as it is now. You might even get to start the separation with 5 – 10 % solvent B.

Best Regards

Hi Danko,
well yes i can say that the final dilution was one of the most critical problems for the achievement of a good separation.
However, i had already used a C8 column (but not at 100% water/buffer mobile phase) and the result didn't differ too much from the C18 column. The reason i couldn't use a 100% water/buffer mobile phase was the fact that i had to separate both polar (acids and bases) and non-polar compounds, so i wouldn't be able to elute the later compounds (only if i used a gradient mode).
I tried this, and i managed to separate all these compounds (5 at all) with gradient in a C18 column 250mm x 4,6mm 5μm, starting from 5% ACN to 80% ACN. The last compound was eluted at 13 min. I finally chose to use the method using the CN column i mentioned in the previous message, but it was a good experience just to try other columns and test their capabilities and limits!
Best Regards