Chromatography Forum

Hello,
I want to no whether it is really possible to retain thiourea in Reverse phase HPLC analysis (it elutes very early). I want to check its content in drug substance. I do not have a HILIC column to try and therefore tried with the Diol. I tried DIOL 250x4.0mm,5µ column with ACN: Formic acid buffer in the ratio 90:10 isocratic . The peak elutes @ RT around 4.0mins and the drug substance also elutes closely and even on changing the composition there is no separation and peak shape is not good.Please let me know whether it is possible to retain thiourea in modes other than HILIC.
Thank You,
Santosh

Thiourea is retained (k' ~ 0.5) on Atlantis dC18 in 10 mM ammoniumformat buffer pH 3 (the latter is probably not needed. Please see the Atlantis column brochure at the Waters website. Atlantis columns have been designed to maximize retention in fully aqueous mobile phases. If you need more retention, this can be accomplished as well.

Hello Mr.Uwe Neue,
Can I use a polar embedded C18 Column . I have Inertsil EP (Embedded polar) column whether it is comparable to Atlantis C18. I want a better retention for thiourea because I want to quantify the content of thiourea,a in drug substance.
thank you,
Santosh Gandhi

In our studies of retention of various phases, the Atlantis phases gave typically a 2- to 3-fold higher retention than embedded polar phases.

You mention that changes to the composition of the mobile phase have been evaluated on your HILIC column. By this, I wonder if you mean that the ratio of buffer-acetonitrile have been adjusted, or whether you have considered other development strategies such as,

- Alternate organic solvents, e.g. THF, ethyl acetate, acetone, IPA, EtOH, MeOH.
- Use of salt buffer in place of acid modifier. Examples are ammonium formate, acetate and phosphate.
- Variations of pH in the aqueous portion. The above anions would be useful over most of the range from approximately pH 3 to 8, which likely sits well with the column's capability.

I'm not advocating that HILIC is the better technique as I have no idea as to the identity of your drug molecule and the other requirements of the separation.

Santosh,

try any polar embedded column with 100% water. I don't expect too much difference in retention of thiourea in any reverse phase column (including Atlantis). You will get K' less than 0.5 in reverse phase approach. You need to go to HILIC to be better retention. Try any silica column with 95% ACN, if retention is too long reduce ACN to 90% or 85%.

Well here is one example (two comparisons, same conditions, same analytes):
http://tinypic.com/view.php?pic=fky5va&s=5
http://tinypic.com/view.php?pic=xgmr7k&s=5

Forgot to mention that peak#1 is thiourea...

When you tried with the Diol column, did you use 90% ACN or 90$ water, as from your email, it looks like you used 90% ACN - which means any hydrophobic compound can elute fast.

Please clarify.

I tried Diol with 90% ACN and 10% buffer. The retention of thiourea increases comparably to other trials that I tried so far but the drug substance elutes very near to it. Same result is obtained on using Silica columns and using 90% ACN.

Vlad, is this "You will get K' less than 0.5 . . . ." also an "expectation" or is it knowledge?

Hans,

This is based on runs on several polar embedded columns including Atlantis T3 for this particular compound (thiourea). Thiourea is very polar non-ionizable and there is no additional mechanisms to retain it on reverse phase column (or even reverse phase mixed-mode column).

Santosh,

What is nature of your drug (basic, acidic, zwitter-ionic or neutral). You can probably try to retain urea by HILIC mode and your drug by ion-exchange on HILIC mixed-mode column (Obelisc)

Uwe,

We did some runs on Atlantis T3 column with no organic/or low organic. What is interesting is that compounds which do not have pi-pi properties retain much less than the one with some pi-pi. I have explanation but may be you can elaborate:
http://www.sielc.com/pdf/SIELC_August_2008.pdf

I would think that pi-pi interactions on a C18 are a rather weird idea. Lack of steric hindrance and related things due to the low ligand density, yes, but pi-pi????

Uwe,

In no way I am claiming that you can get pi-pi on C18 column, but I believe that Atlantis T3 has something which provides pi-pi. We compared several polar embedded column and that what stood out for Atlantis T3. We did a lot of compounds on Atlantis and what we noticed is if you don't have aromatic properties you don't get retention. If you look at our comparison data you can see what I am talking about You know that when column manufacturers describe their stationary phases they leave some details....understandably.

As I said, there is no pi in this pie. If there are any effects as you describe, they are structural, such as access to the bonded phase, which is different for the Atlantis packings then for a standard C18 (which is why it works...).

But, anyway, it would be easy to find the pi in the pie, if you think there is one. Go ahead and try...

[img]I%20tried%20Diol%20with%2090%%20ACN%20and%2010%%20buffer.%20The%20retention%20of%20thiourea%20increases%20comparably%20to%20other%20trials%20that%20I%20tried%20so%20far%20but%20the%20drug%20substance%20elutes%20very%20near%20to%20it.%20Same%20result%20is%20obtained%20on%20using%20Silica%20columns%20and%20using%2090%%20ACN.[/img]

Hi guys,

I don't get it. Santosh used 90% ACN (strong organic modifier) that should literally elute most hydrophobics well into the void.

Diol is still a reversed phase column and according to RP methodology, ACN is strong and it should elute the compounds pretty fast, that they do not have good retention and start to co-elute.

Silica is a different story as ACN would be weak on this phase owing to a HILIC mechanism.

Now Vlad's claim about a pi-pi interaction may be true on a Atlantis T3 column. Although it may not be true aromatic pi-pi interactions, it could have been induced by the nature of the bonding and/or endcapping on the Atlantis phase.