Aminoglycoside preparative HPLC

[Hassel Hoff]

I am trying to do some purification runs for an aminoglycoside. I'll most likely be using a 50 x 250 prep column. Most analytical methods out there use HFBA on a C18. HFBA makes it an undesirable method for prep. Any suggestions? I would like to do this at high pH using volitile modifiers. I want to avoid any post prep run clean up.

[Uwe Neue]

I would use a silica HILIC column with 70% acetonitrile / 30% water, and maybe a dash of formic acid. If the retention is too short, add more acetonitrile.

[Hassel Hoff]

Thanks for the suggestion. We've persued HILIC. It works great for trace analysis but not for prep. The aminoglycoside is not very soluble in high organic. Loading is an issue since we are trying to purify in as little runs as possible.

I'm getting a bit desperate, there is not much out there on this!

[Patrik Appelblad]

I would also vote for HILIC. (Perhaps not to surprising :-)..
However, as you eventually intend to set up your application for semi-prep/prep e, I'd be a bit thoughtful in choosing experimental conditions.
Your analytes are easily retained, hence I'd try to set it up using either methanol or any cheaper solvent compared to acetonitrile.
Secondly, as always I would look for the trueness in scalability and this might be different from vendor to vendor...
I've done my share of upscaling on our ZIC-HILIC phases and I can guarantee trouble-free upscaling.

If you need more info about HILIC, we can send you our HILIC guide, where these issues and other important issues are dealt with.

/Patrik

[SIELC_Tech]

You can use our mixed mode column for retention/separation of aminoglycosides. Here is method for gentamicin (one of aminoglycosides). Mobile phase is prep compatible: ACN-water-TFA. You have retention of 15 minutes on 50 mm column.
Method is on Primesep 200 column and you will end up with TFA salt of your compound. You can try a weaker column (Primesep C or Primesep 500) and instead of TFA use formic or even acetic acid. As you can see the column separates even isomers of gentamicin and bunch of small impurities

http://www.sielc.com/compound_142.html
http://www.sielc.com/compound_003.html (prep separation of amino sugar was performed on this one)

Your interaction comes from amine in aminoglycoside and carboxylic acid on the surface of Primesep column. You can also use the following application as guidance.

http://www.sielc.com/compound_140.html

Primesep C has carboxylic acid with pKa of 3 and you can suppress this by running gradient of pH from 4 to 3 (or less). With this approach you can use the prep column in SPE mode. Run on one mobile phase with pH-4 and another one with pH-3. The more amino group you have, the harder it is to remove it from the column. By using right column, buffer and buffer pH you will separate aminoglycosides on a shorter column (50 or 100 mm)

We have few other applications for aminoglycoside but they are covered by secrecy agreement.

Contact us if you have questions or would like to try analytical column for your separation.

Regards,

Vlad

[Uwe Neue]

A fair possibility might be to use the silica as an ion-exchanger. Give it a shot in maybe 50/50 MeCN/Water around pH 7. If you have a Spherisorb silica column, it would be a good column to try.