Chromatography Forum

I'm trying to work out a good RP hplc separation method for two related compounds, they are differ by the halogens attached to the aromatic ring, the molecules themselves also have positive charges, an analytical C18 column can resolve them as two peaks side by side, when acetonitrile, water, & TFA were used as solvent, both isocratic & gradient runs. Has anyone had any experience in method development of similar compounds? What columns & conditions did you use to get an adequate good separation, I mean possibly 1 to 2 minutes apart between the two peaks, possibly more.

Thanks for sharing.

You can always try Phe-Hex column or PFP column. With different halogens attached to aromatic ring there will be different electron cloud, different pi-pi interaction so there is a chance to saperate those compounds.

I would try a HILIC mode column

I'm a simple person, so if me, I'd start simple. I'd use my standard mixture of the two, and same mobile phase conditions. So I'd run like you have been, then I'd try 15C cooler column temperature, and 15C higher column temperature. Look at the chromatograms, and see for each whether the separation is better, worse, or the same. This could help point you to the solution, and is easy to try.

We have several assays where resolution was improved by just using temperature modification.

an analytical C18 column can resolve them ........ I mean possibly 1 to 2 minutes apart between the two peaks, possibly more.

Thanks for sharing.

Why do you want the extra separation ? - if they are resolved they are resolved, nothing will improve if you move them further apart.

Peter

Hi everyone, thanks for your suggestions, the reason that I need to separate the compounds further is beacause I need to purify the compound, one of the compounds is the precursor of the other, upon making it, we need to separate the precursor from the product. and on a prep scale the peaks were too close to be separated completely and cleanly, so we need an adequate separation better be about 2 mins apart. so the precursor and product does not merge into each other.
yes I have tried PFP column, the peaks were broad.

thanks for your insights

How about methanol instead of acetonitrile in the mobile phase ?

The discussion might go quicker if you can tell us what you have tried that did not work.

Peter

The compounds I am trying to separate have got aromatic rings with Br (precursor compund) and I (product) attached to it and also a positively charged N atom on the ring. I have attempted to separate them by using analytical size coreshell PFP column with Acetonitrile/water/TFA, peaks were broad and tailing. also tried C18 charged surface hybrid column, peak shape was symmetrical bell shape but broad, and not enough separation, peaks were resolved side by side, but not enough separation in my case.

thanks

The compounds I am trying to separate have got aromatic rings with Br (precursor compund) and I (product) attached to it and also a positively charged N atom on the ring. I have attempted to separate them by using analytical size coreshell PFP column with Acetonitrile/water/TFA, peaks were broad and tailing. also tried C18 charged surface hybrid column, peak shape was symmetrical bell shape but broad, and not enough separation, peaks were resolved side by side, but not enough separation in my case.

So did you even try column temperature variations, or just assume that such investigation would not help?

The compounds I am trying to separate have got aromatic rings with Br (precursor compund) and I (product) attached to it and also a positively charged N atom on the ring. I have attempted to separate them by using analytical size coreshell PFP column with Acetonitrile/water/TFA, peaks were broad and tailing. also tried C18 charged surface hybrid column, peak shape was symmetrical bell shape but broad, and not enough separation, peaks were resolved side by side, but not enough separation in my case.

thanks

If you want to go to prep scale you absolutely must confine yourself to phases that are available in prep scale columns (which for all I know these might be).

Peter

Probably, the reason you have broad peaks is the nitrogen atom in aromatic ring. It interacts with silalons gruops on silica surface. Maybe you can try to use TEA or some short chain aliphatic sulfonic acid like 1-butanesulfonic acid, or try changing pH value to more alkaline one.

Your answer is mixed-mode chromatography if your compounds are ionic and hydrophobic. It offers alternative selectivity and allows to use minimal difference in ionic and hydrophobic properties to separate compounds:
http://www.sielc.com/Technology_2D_Properties.html

here are few examples of separation of closely related compounds:
http://www.sielc.com/Compound-Toluidines.html
http://www.sielc.com/Compound-2-Aminobenzoic-Acid.html

If you want to isolate compounds by prep chromatography it will give you 10-50 times capacity of regular RP column:
http://www.sielc.com/Technology_Prepara ... raphy.html
http://www.sielc.com/Products_Obelisc.html

Contact me if you have questions.

Hi Vlad

Are these phases available at prep scale ?

Peter

mix mode separation sounds interesting, and its definitely new to me, will look into it. As about variation of temperature, we don't have a column heater for the system, but will keep that in mind. so is it true ion pairing chromatography ( such as adding TEA) is not suitable for prep scale?

thanks for all input

Peter,

All columns are available in prep size (10, 21.2 and 30 mm in ID), but since capacity is much bigger than regular column, you might need a smaller ID column. We have people who did prep scale isolation on analytical column.

Cathj,
We offer free method development for our customers. If you send me few mg of each compound we can do screening for you. There is no obligation to buy a column. Turn around time is usually less than a day. I have about 100 NDA with different companies.

Column temperature will have a minimum effect of selectivity. You peak might be a little bit sharper, but don't expect drastic improvement.