Chromatography Forum

Currently I am trying to measure Neomycin Sulfate and it's related compound according to Ph.Eur.
My problem with the Ph.Eur is the mobile phase in combination with the used column.

Mobile phase: 20 ml TFA, 6 ml NaOH (50%) ad. to 1000.0 ml with water (pH = 1.1)
The mobile phase is brought to a higher pH value by post column addition of a 0.5 M NaOH solution, since oxidation (in the ECD cell) does not take place at such low pH values.

My guess is that the low pH value is chosen to prevent auto-oxidation of the Neomycin before the post column.

On the "knowledge base" on the EDQM site I found the column used for the monograph is a Hypersil BDS 250 x 4.6 mm 5 um
According to column vendors, this column has a pH range of 2 - 7. According to EDQM, this monograph is being reviewed because this method is quite aggresive for both HPLC system and column.

I tried separation with a GraceSmart C18 but destroyed one column using pH 1 mobile phase.
Now I brought the pH of the mobile phase to 3.0, separation of two neomycine peaks (B and C) is good (resolution 2.3) but the reproducability is quite bad (RSD% = 6 (n = 6)). I think this poor reproducability is caused by the slightly higher pH value of the mobile phase.

Maybe someone on this forum is more familiar with ECD detection and/or Neomycin sulfate acc. Ph. Eur?
And is willing to share some thoughts on this matter.

I found a lot of info here:
http://www.myantec.com/download.php?fil ... RydWcucGRm

Dear Artsjeroen

We did the analysis using a carbohydrate column and pulsed amperometric detection. Please read our Application Note.

Hi,

I also do some neomycin analysis with the Eur. Ph. method, and we also raise the pH to keep our column working, but also to maintain our resolution.

I think your high %RSD has nothing to do with the pH raise, but more with the nature of ECD.
Keep your system working for a few days, and try injecting routinely.
This way you can see if your solution is not stable, or your system is getting stable.

Good luck

Ace

Thanks for the usefull replies!
I definitly will try the Metrohm Application. But one more question about that.

The Application Note states that the mobile phase is 10 mM NaOH, doesn't the high pH value of this mobile phase promote the oxidation (too much)? I thought the post column addition of NaOH-solution was to obtain a neutral environment, which is favourable for the oxidation in the ECD-cell.

Jeroen

The post-column addition of NaOH mainly eases the amperometri detection of the compound. The gold electrode works best in the alkaline pH. To get enough sensitivity the eluent is set to a high pH prior to analysis. The pH of 10 mmol/L NaOH would be too less alkaline.
On-column oxidation is no problem in this application.