Chromatography Forum

Fellow board users,

I'm looking for some assistance and guidance in the analysis of a quaternary ammonium bromide. Some details which describe how this compound has been tripping me up follow.

I'm not certain of the analytes' inherrent hydrophobicity as although retention appears adequate on a 'regular' C18 it shows a large degree of tailing on well endcapped columns such as Luna C18(2) with H2O/MeCN/0.1% TFA. As we had a new column technology in house, we decided to give it a go on a Primesep 100 but have been troubled by an incomplete understanding of retention control on this phase. The first opinion I am looking for from fellow users is can I be certain I am visualising the correct analyte peak? The compound is an aromatic ammonium bromide salt; when I extract the UV spectrum of the (one) peak I have so far been observing it displays a broad maxima around 210-215 nm and has reduced to a negligible value by 254 nm. Does bromide ion have a strong chromophore?

Is Primesep 100 too strong of a cation exchanger for this application? The peak we have been observing has not been sensitive to the amount of TFA in the mobile phase (0.1 - 0.25% v/v), is retained at very high MeCN (is HILIC complicating matters?) and fronts at above ambient temperatures. Should I continue to pursue my method development on this column, or switch to an alternative; perhaps a simple polar embedded alkyl phase.

Regards,
JA

Your UV data sound about right for a benzyl group. Yes bromide absorbs UV, and it elutes ahead of the void on a cation exchange column. Using both hydrophobicity and ion exchange to retain a quaternary surfactant is excessive, and will give you the kinds of troubles you are seeing.

May I recommend an Acclaim Surfactant column? It produces reasonable retention times with moderate concentrations of acetonitrile, and the peaks are well shaped for quats (and most other surfactants too). The user's manual also explains how to control retention and selectivity for different situations.
http://www1.dionex.com/en-us/columns_ac ... 25924.html

Dear JA,

I will take the liberty of clarifying some issues on Primesep 100 use. First of all, bromide ion is absorbing in UV and you will see the bromide peak at 210 nm. On Primesep 100 column bromide will not retain and you will observe it close to the void. Your quaternary amine will retain based on hydrophobic and ion-exchange properties. Because of positive charge of quaternary amine you will have strong cation-exchange interaction on Primesep 100 column. Reverse phase interaction depends on hydrophobicity of your compound. In mixed mode chromatography you have two interactions at the same time.

If both interactions contribute to retention you need strong mobile phase which will reduce retention by RP mechanism (high organic concentration) and IE mechanism (high buffer concentration).

You pointed out that your compound is retained well on RP column, meaning that you have enough hydrophobicity in your analyte. The only problem is peak shape, which can be fixed with another mixed mode column such as Primesep B or Primesep B2. These are reverse phase and anion-exchange columns at the same time. The column surface has a positive charge which improves peak shape for positively charged analytes due to ion-exclusion phenomena.

In both cases you can control elution by playing with buffer concentration. Here are few applications which show excellent peak shape for basic analytes (amines and quaternary amines)) retained/separated based on reverse phase and anion-exclusion mechanisms:

http://www.sielc.com/compound_113.html

http://www.sielc.com/application_065.html

Bromide ion will retain well on these columns. The example below shows an application with both bromide and basic compounds retained at two different compositions of the mobile phase.

http://www.sielc.com/application_066.html

You explore benefits of Primesep mixed mode cation-exchange columns to the full when you analyze hydrophilic quaternary amines with insufficient retention on RP column even with no organic in the MP. See example below:

http://www.sielc.com/application_007.html

For quaternary amines, a weaker ion-exchange column such as Primesep C would be more appropriate. Most likely, you are not eluting your quaternary amine from Primesep 100 column. As a first step, I would inject your sample without column and measure peak area (UV 210-215). After that, you need to inject you compound with Primesep 100 column as see if you are observing two peaks –one for bromide ion and another one for quaternary amine. Sum of the peak areas for bromide ion and second peak must match you peak area without column (plus minus 5%). If with the column installed you have smaller peak area (50%???) then you are not eluting your quaternary amine. You need to either increase your buffer concentration or use a weaker Primesep column

If you can provide me with your email I will send you a few other applications and can help you with optimization of your separation

Kind regards,
Vlad

Ja,

I have separated different kinds of weak and strong bases with Primesep.Without knowing your compound logP and pka-values I will
assume that Primesep 100 may not be perfect for you.You could try Primesep
200 or Primesep C.With these mixed mode columns I have been succesful
with some separations but I have also realized some basic facts:
-good backgroundknowledge of you compounds chemical nature helps a lot
-choosing right buffer concentration and PH-value is essential

Have you tried both isocratic and gradient separation?Have you tried isocratic separation with keeping buffer concentration constant but changing MECN concentration.Elevated temperature will not give you
many possibilities(Max.temp +50C).

Hope this helps,
Weba

Just very roughly out of memory: A benzene ring also has characteristic absorptions near 300nm. As few other substances have lines there these should be a good marker for your compound. The only thing is: these high nm absorptions are about 100 x lower in intensity than the other ones (but even that fact can be used in identification).

If you have followed this forum closely you will know that my recommendation is to use RP if you get retention with it.

Dear all,
Thank you for the responses. On moving to the Primesep 100 column it was my hope that peak shape would be improved (as I was only considering R4N+ and SiO-). We found however that this wasn't necessarily the case for a quat and the retention by ion exchange is a bit excessive. A learning experience. We went on to confirm that the compound is certainly retainable by hydrophobicity alone, and that there were no appreciable HILIC effects under high MeCN conditions - retention went up considerably on increasing the water content to 60%.

Although I now feel that the methodology could be simplified and allieviate the peak shape problems with something like a polar embedded or endcapped phase (presumably a similar masking effect as using ion exclusion?) my colleague who is working on this project seems happy with the Primesep 100 at high organic / low temperature (instrument is an Agilent 1100). The discussion about bromide ion was an interesting side note but we don't need to retain or quantify it in this project. Nonetheless, thanks for the comments.

Regards,
JA