Chromatography Forum

Hello

I am looking for a suitable column for the analysis of dequalinium chloride and its impurities in a pharmaceutical preparation. The European Pharmacopoeia uses a waters symmetry. I also use this column, but the separation is not very good. Also a strong tailing occurs. Can anybody recommend me very good endcapped columns to include them into a column screening program.

Thank you very much
Florian

Nearly a year ago, Waters introduced the Sunfire column. It is a step forward over Symmetry.

While this sample is very tough, I am somewhat surprised that tyhe Symmetry column should give you a lot of tailing. Is it a rather new column, or has it seen many mobile phases? Also, what is the mobile phase used for this assay?

Hi Uwe

The mobile phase is prepared as follows:

Dissolve 2.0 g sodium heptane sulfonate in 335 mL water, mix with 665 mL methanol and adjust the pH of the mixture to 3.5 with glacial acetic acid.

Florian

For me this method seems to be not very robust regarding the pH. 3.5 is just a little bit of the buffer range of an acetate buffer. So maybe that is the reason for the poor peak shape. By the way what is the pH of the sample ?

Regarding column screening I can make the following two suggestions to have not only Waters columns in your screening.

You could try a Luna C18(2) from Phenomenex which has a very good endcapping. And something really new would be the Gemini C18 also from Phenomenex which is not based on a classical silica.

Nevertheless if you want to have this method robust, check what are the tolerances of European Pharmakopeia regarding the pH and the buffer. I believe that any of the by Uwe and me mentioned columns will be able to provide you with a good chromatography (under the appropriate conditions).

Hello,

I have forgotten one detail. How long have you equilibrated your column with the ion-pair reagent ? Maybe you should increase the equilibration time. But if this is an isocratic method and you have done several runs and nothing has changed you can forget this suggestion.

Dirk

Hi Dirk

Thank you for your answer.
Our method is isocratic and we use the column only for this analysis. The pH value of the mobile phase is the one that EP says; the sample is dissolved in mobile phase.

I also thought to take a Gemini, we managed a big problem with a Gemini column (separation of a corticosteroid from its degradants and the parbens).

So I already have three column suggestions: Gemini, Luna and Sunfire.

Any other suggestions ? Or experience with dequalinium chloride ?

Florian

Florian,

Here is another alternative. If you have enough hydrophobicity you can analyze your compound on Primesep B or Primesep B2. The links below show comparisson of peak shape for Primesep to lead brand columns (Z and W)
http://hplcmethods.com/application_065.php

Also you can check method for cetylpyridinium chloride (quarternary amine):
http://hplcmethods.com/application_047.php

Your tailing comes from silica interactions, ligand on Primesep masks the silica.

Can you provide me the source of dequalinium chloride, I will try to see if we can buy it and do a few runs for you.

regards,

Vlad

Hi Vlad

The manufacturer of Dequalinium chloride is LEBSA (www.lebsa.com)

Regards
Florian

Florian,

I found Dequalinium Cl in Aldrich and I am going to order it. What is the objective of your experiments? What is your detection technique? What mobile phase you prefer?
What else is in your sample (by-products, decomposition products, impurities, etc.) Let me know, if you provide us with this information it might be much easier for us to develop method for you. There is no obligation from you to do anything; we are doing free screening/method development for our customers. Usually (if we are not bound by secrecy agreement) we are posting our methods on our website. Check the link below, you can sort methods by name, by date, by application and by column (Primesep):

http://hplcmethods.com/Applications_By_Publish_Date.php

The substance contains usually three impurities, which are described in European pharmacopoeia. Impurity A is commercially available. I would prefer an isocratic method using a mobile phase containing acetonitrile or methanol and no THF. The omitting of ion pair reagent is desirable, but I think a method won't work without it. Detection should be possible with UV (240 nm). The product is a tablet containing mainly cellulose / lactose; another product is a cream containing mainly vaseline / paraffins /emulgator / water. Degradation products should not be present in the sample, the substance seems to be very stable.
At the moment, the main problem is the separation of dequalinium chloride from its directly followed peak (poor resolution because of the bad peak shape of the dequalinium chloride), the second disadvantage is that the first impurity peak interferes with the "first eluating peak" (retention is too less) and finally the third disadvantage is the late elution of the third impurity (run time up to 30 minutes).

Now, you are informed in detail about the problem.

Florian

I don't have access to EP, would you please send me structures (ChemDraw, jpeg, power point, etc.) by email (mail@sielc.com)

We are not going to use ion-pairing reagent, the core idea of Primesep columns is to eliminate the use of ion-pairing reagents. In general you can use three parameters to adjust elution/retention on Primesep columns-pH of the mobile phase, buffer concentration and amount of organic component. With these parameters you are controlling reverse phase, ion-exchange (ion-exclusion), polar interactions (HILIC), etc.

I will let you know on results.
If you would like we can take this conversation to direct emailing.

Regards,

Vlad

Vlad

I'm at home at the moment and have no EP access too. Monday, I will be able to provide you with the information.

Have a nice weekend
Florian

I do not see anything fundamentally wrong with the mobile phase. With ion-pair reagents, you need a long equilibration time until the column is equilibrated with the ion-pair reagent, and any time you take it out of this mobile phase and say wash it with an organic solvent for storage, this equilibration starts over again.

It could be that a thorough equilibration with the ion-pair reagents is all you need to get a good peak shape, nearly independent of the column that you are using.

Florian,

You have received several recommendations that look good. Let me also suggest that you add a buffer salt to your mobile phase. Since you seem to need a relatively high amount of Methanol (65%), you could try (NH4)H2PO4 at a 0.02M level, adjust the pH with H3PO4 and keep everything else the same. I have the feeling that this composition will give you a better peak shape. If you have time, try also decreasing the pH to 3 or 2.5, this change is also very likely to improve peak symmetry.

If you need to follow EP methods, then you are sort of tied up to the ion-pair conditions. Any change to other columns without ion-pair reagents may require extensive studies and/or method validation.

Good Luck,

Josebenjamin