Chromatography Forum

Hi,
I am working on a project to purify an organic ionic dye. We try to achieve gram scale PrepLC. The compound was successfully separated with 25% acetonitrile. The problem is that the column 30x150 mm Waters T3 column does not have enough capacity to make large quantity. Each injection can only do a few mg. At flow rate about 100 ml/min, it will generate lots of waste solvent. This difficulty in sample size and waste generation limits the development.

I have tried activated carbon solvent recycling, the impurity will not stay on the activated carbon.

I would like to seek some advices from the professionals in the community.

The impurities may be non ionic, I tried anionic column, did not achieve separation. I imagine I can load column in aqueous solution, then use gradient to rinse the impurities out. The compound loaded on to the column alright, but there is no separation.

anyone can make some suggestions?

Thanks for your help.

What is your sample dissolved in?

Have you considered using column chromatography or FLASH chromatography?

I helped purify dyes in this manner decades ago to assist in chosing synthesis parameters. LC columns became fouled with impurities and was too expensive a solution. We could watch the separations using column chromatography and it was easy to modify the mobile phase to achieve the quickest and most efficient separation.

There are inexpensive columns which can process gram to scores of gram samples using the new FLASH hardware. There are also a wide variety of supports available.

There are many vendors of such systems, including of course, my employer, Supelco.

Perhaps a call will enable you to find a similar application already developed.

techservice@sial.com
1-800-359-3041

best wishes,

Rodney George
Supelco

Is it possible to convert your dye into a non-ionic form at high pH?

What are you using to control the pH? Maybe a change there would improve the lodability.

What are you observing when you inject a larger amount?

What state is the sample in (solvent, pH)?

You can achieve 10 times higher capacity with mixed-mode approach. Here is comparison of Atlantis T3, ZORBAX and Obelisc mixed-mode:
http://www.sielc.com/Products_Obelisc.html


It is very good approach for prep separation: you are not limited by solubility (like in HILIC) or overloading (RP)
http://www.sielc.com/Technology_Prepara ... raphy.html

Your compound will retain based on reverse phase and ion-exchange mechanisms. You don't need to go to high pH and can use TFA, formic acid or ammonium formate in the mobile phase. Also if you compound is quaternary amine there is no way to have it in non-ionized form.

Also here is an article for loading capacities of SIELC columns compare to RP:
"A study of retention and overloading of basic compounds with mixed-mode reversed-phase/cation-exchange columns in high performance liquid chromatography" by Nicola H. Davies, Melvin R. Euerby and David V. McCalley (Journal of Chromatography A, Volume 1138, Issues 1-2, 5 January 2007, Pages 65-72).

Contact me if you have questions. If you can share your structures I will advise you on conditions and column

Others have addressed the issue of other columns and conditions. I'd like to suggest that you could reduce solvent usage by using recycle, and perhaps even improve separation ( and thus amount loaded ) with that technique. You could also look to reducing flowrate, increasing column temperature, and changing solvent polarity to reduce solvent volumes.

It does seem overkill to use a Prep HPLC to separate compounds that sound so dissimilar, and I'd suggest trying some of the suggestions, eg pH, flash, mixed mode ( I've no experience of that technique, but it seems like it might be very helpful to you ).

Please keep having fun,

Bruce Hamilton

Hi,
Thank everyone's suggestions. I will definitely look into the new terms I am not so familiar. I wish I had consulted with expert here before I purchased the PrepLC system for this project.

The compound is meta cresol purple, a pH indicator. At pH above 6, it exists in HI- and I2- form. The H2I form shows up at low pH (pH 2) and will precipitate.

I do not have access to Mass spec to resolve the impurities, just a rough guess from the reactants to make the compound.

My mobile phase is 10 mM NH4CH2COOH.

_________________________________
My stock solution is 8 mM, dissolved in milliq water, the injection for analytical column is only 20 ul. The PrepLC injection is 400 ul. Only mg of mcp is injected. far from the Prep scale I am dreaming about. The separation is ok, but the pure, main component peak broadens and tails. The reagent consumption is a killer. The reagent recycling scheme does not seems to work. I read a design using activated carbon. at 25% level of Acetonitrile, the color could not be retained on the column.

Thanks again for everyone's help.

The 10 mM additive to the mobile phase is not very useful, whether it is glycine or ammonium acetate. Use 10 mM acetic acid, and redevelop the separation, if needed.

There is not a good way to make the analyte non-ionic, which would improve the loadability. However, it might be worth your while to explore higher concentrations of acetic acid (at least 100 mM or even 1 M) to see if this improves the loadability. My bet is that you will get better loadability at 10 mM acetic acid, but I could be wrong.

Since it precpitates at pH 2 it might be possible to crystallize it out with high purity, the mother liquor could then be chromatographed if there is enough left.

It was my initial thought. However due to the coprecipitation, there is no separation at all, no increase of purity.

Dyes are not very pure in most cases (at least the one from Aldrich), but they serve the purpose even with 80% purity (just used few dyes for summer camp shirts for my kid). HPLC will show bunch of peaks and it might be hard to isolate them.

zhensr, maybe you were simply too heavyhanded with your precipitation. One could think of a recrystalization as something different than a precipitation. I have encountered few substances which show repeatable co-crystallization.