Chromatography Forum

Hi there. Currently I am assaying Benzalkonium chloride using a radial compression Nova-pak CN type cartridge column, and would like to change over to using a stainless steel Waters CN column. Cetylpyridinium chloride is used as an internal standard. I tried to use the current mobile phase on the stainless steel column, which is 1000mL MeOH, 20 mL glacial acetic acid, and 1.0g hexanesulfonic acid sodium salt. It gave me horrible peak shape, using 10uL injection volume, and 1mL/min flow rate. In an attempt to rectify this, I added 200mL water to the phase. This significantly improved the peak shape, however it also caused the peaks to elute too close together. Any advice to help me get better resolution? Thanks, James

CN columns are not reproducible. They should be a last resort.
Benzalkonium chloride has been retained on Unison UK-C8:

http://www.silvertonesciences.com/files/TI197E.pdf

Correction - a more appropriate description would be that CN columns
are "unstable" as opposed to "not reproducible".

JFrancis,

I don't remember all the details of the benzalkonium chloride analysis method that I used a while back at a previous company, but I will give what details I can remember.

I think that we used a Zorbax SB-CN column. The mobile phase may have been a simple methanol/water mixture. I can't recall if we used a buffer or an ion pair reagent, but I don't think that we did.

We did not use an internal standard. We did get the reference standard from the USP.

You may already know, but the benzalkonium chloride does not come as a pure material. The material contains a mixture of compounds with different alkyl chain lengths from C12 to C20. If I remember correctly, the USP reference standard contained compounds with the C12, C14, C16 and C18 alkyl chains (maybe C20 also) while our material had only the C14, C16 and C18. You need to quantitate using peak area grouping for all of the benzalkonium peaks, in the standard and sample.

So your chromatogram should give you 3 or 4 peaks for the benzalkonium chloride. Perhaps, you had bad peak shape due to incomplete resolution.

Sorry, I can't be more complete on the method details. It has been a while and I can't just go look it up. As the USP has a reference standard, there may be a monograph method that you can look up. I don't have a USP to search.

Regards,
Dan

The Acclaim Surfactant column gives excellent peak shapes and resolution for quaternary-amine surfactants, including benzalkonium salts. No ion-pairing reagents needed. Look at page 5 of this data sheet: http://www1.dionex.com/en-us/webdocs/25 ... et_V24.pdf

Thanks a lot for your help guys. The existing method with the radial compression cartridge only separates one peak for all of the homologs of Benzalkonium chloride, and I would like to keep this as being the case, if at all possible, rather than split them all up to get resolution for all of the homolog peaks.

There could be an equilibration issue. The Radial-Pak Cartridge is dry, when it arrives in your hands. The steel column is shipped wet, if I recall correctly, it is shipped with a mixture of acetonitrile and a salt solution used to test the column. Before you will be able to use the column in 100% organic, you need to remove the salt. Most likely, you have done this now by flushing the column with an eluent containing water. I would go back to the original mobile phase and see if the separation is now what you had anticipated.

The CN columns hydrolyze in general, so I am not a fan of them, but they should give you reasonable results in the non-aqueous medium that you are using.