Chromatography Forum

I am considering separating aminoglycoside antibiotics on alternate media to avoid the long run times and gradients common with typical C18 columns. Most literature methods employ C18 and end up with long gradients or poor peak shape or both. C18 seems to be used because it is familiar to all and can be forced to work with a gradient. It seems to me that aminoglycosides should provide shorter run times and good selectivity if we try to focus on the carbohydrate portion rather than the lipophilic portion of the molecules.

My first approach would be to try an ACN/basic buffer mobile phase on an amino column to accomodate the salt forms of the antibiotics and keep the column regenerated without going too basic.

My second approach would be to try a mixed mode ion exchange in which I try to balance the RP effects by adjusting the ionic strength and organic content to allow the ion exchange effects to be dominate.

Does anyone have any experience with either of these alternate approaches or suggestions on where to start my screening conditions?

Here are few applications for aminoglycoside antibiotics using mixed-mode SIELC columns:
http://www.sielc.com/compound_357.html (amicasin)
http://www.sielc.com/compound_142.html (gentamicin)
http://www.sielc.com/compound_003.html (proprietary amino sugar)
http://www.sielc.com/compound_313.html (tylosin)

If you have aminoglycoside with two or more amino group you can go with Obelisc R column.

Contact me if you have questions.

I would avoid NH2 phase for this application. ODS works quite
well for these types of compounds:

http://www.silvertonesciences.com/files/TI426E.pdf

The method we currently have is a 90-minute isocratic run on a C18, which is better than the original 120-minute gradient with a terrible baseline. I don't consider a 90-minute injection "working quite well" in this day and age.

I am not satisfied with traditional wisdom and letting the overnight run solve a problem that some science and thought could do better.

Amino columns have been used for saccharides for years, as have ion loaded ion exchange columns and ion exchange. The compunds are modified disaccharides, so that is a reasonable starting point. The gentamicin separation sounds like a good starting point for a mixed mode column.

If I stick with a "traditional" approach, I will not discover my "positive Black Swan" that dramatically improves the testing. If we don't try something new, we will only discover what we already know and will be running 90-minute runs for years, or 45-minute run s on Acquity systems. We can do better! We just have to learn how.

I have had good luck with SIELC columns in some other separations at a previous company and the risk is worth buying a column for this company to see if it would work.

Hi Rick -

Thanks for your comments. I think it will be difficult to achieve good
peak shape for these compounds on NH2 phase. Just my opinion,
I could be wrong.

The data I posted shows a nice separation for gentamicin
sulfate on Cadenza CW-C18 (30nm pore size).

Anyways, please keep us posted on your progress.
Welcome to the forum!

In my opinion, this sounds like an excellent application for a silica HILIC column, due to the weak cation-exchange properties of this packing.

Rick,

make sure that you will get a right SIELC column. For aminosugars with three and more amino groups you can use Obelisc R column. Our other columns which you probably tried (Primesep 100, 200 or C) will give too long of retention. Here is application which shows the strength of two column for rtetention of compound with several amino groups. You can see that even 10 mm Primesep 200 column provides with good retention:
http://www.sielc.com/compound_140.html

Basically for this application you can try any C18 column, just need to play with carbon chain on ion-pairing reagent, problem is that for aminosugars with 3 or 4 amines you will have very long retention, you can try to C3-C4 IP reagent.
silica will work too, but I am afraid that you can suffer with long retention and some tailing with polycharged amino sugars.