Chromatography Forum

Hi All,

Hope to get an advice on how to clean up a sample or reviwe a HILIC column(not sure what is causing a problem).

I am developing a method for Imidurea assay in Shampoo. The method 90%ACN: 10% Water isocratic works excellent on standard, however, I am having troubles with my sample. It contains surfactants (one of them is triethanolamine lauryl sulfate) which were not interfering with imidurea in my early experiments.

Right now TLS coelutes with imidurea in a very random way - it may overlap with first peaks or last peaks leaving only one out of 6 not overlapped.

I have tried column wash 90% ACN 1 hour, then 14 hours 90% water, then 90% ACN 2 hours. Doesn't help.
I have played with %organic (going down to 70 and up to 98), tried different filters, SPE cartridges, increasing and decreasing pH of Mobile Phase, prolonging a run time (I use 15 min isocratic 90:10 ACN water, then switch to 90:10 water: ACN for 20 min, and then 20 min to equilibrate to initial conditions). No luck as well.
I am concerned about two things:
First, I had a nice separation of imidurea from sample excipients on different days on different samples. Then, all of a sudden it started getting worse and worse and is not consistent. In the meantime, chromatograms of standards are unaffected.
Second, is there any way to remove surfactant from sample or mask it if it's so similar to imidurea in polarity?

Any suggestions are wellcome.

Regards,
Julia

What column are you using and what is your sample dissolved in when you inject it? Also, if you're using an automatic sample injector, then what solvent are you using for the needle wash?

Sample solvent - MeOH 100%, no needle wash, column Cosmosil HILIC 250x4.6, silica (bonded triazole).

you can do some sample prep with anion-exchange SPE or use mixed-mode RP anion-exchange guard to trap your surfactants (or significantly increase retention for it). You should no see effect of these manipulation on your target.

Imidurea itself is neutral, I believe. Its degradation products are charged. You're running these samples on a column with a (+) charge in the triazole ring. Whether or not the degradation products experience electrostatic attraction or repulsion by the column - a force that's superimposed on the hydrophilic interaction - depends on whether or not you have enough electrolyte present to titrate the ionizable groups in the coating. I suspect that's why you're getting irreproducible results when ionized surfactants are present. Try including 20 mM salt overall in the mobile phase and in the sample solvent; 20 mM suffices to form an electrical double layer of counterions on the stationary phase. That means you can't use 100% MeOH as the sample solvent; at the least you'll have to dilute it with 1 or 2 volumes of the starting mobile phase.

Vlad: Would an anion-exchange SPE cartridge trap a neutral analyte like imidurea, or would the objective be to trap some component of the contaminants?

What detector are you using? Imidurea should be detectable by UV and TLS has very weak chromophore.
What is the retention of Imidurea in your current method? It looks that a raw silica HILIC column or other HILIC column with some cation-exchange property may work better.
Andy was right that you need to add electrolyte (buffer) to keep the charged analytes behave themselves. It is challenging to obtain reproducible result using HILIC for real-life samples even if you do everything right on your part, mainly becuase in many cases the sample matrix are aqueous based. As the result, the retention time may depend on injection volume, different samples, column conditions, etc. For this reason, a cation-exchange column may be a viable choice.
Can you tell me what brand of Shampoo you are interested so that I can give it a try in my lab using the RP/WCX mixed-mdoe column we developed at Thermo (formerly Dionex)? I will share my findings.