Aqueous Normal Phase with mobile phase 100% buffer

[AMH]

Hello,

I would like your help to find a washing method to the HPLC column I'm using.

My compound is polar, highly water soluble and my column is a Zorbax NH2 with a mobile phase of 100% 0.08M KH2PO4 (isocratic).

The column is new, but the compound is being eluted faster after each run, having moved to half of the retention time after one week.

I tried to wash it with 100% water, but it didn't solve the problem.

Many thanks,

AMH

[carls]

100% aqueous mobile phase with an amino column is anion exhange, not HILIC (or ANP). Does your compound have an acidic functionality/group? What is the pH of the buffer?

[Andy Alpert]

The fact that retention is decreasing steadily implies that this compound is in fact acidic. The decrease probably reflects the poor stability of many silica-based amino phases; the amine group creates a high pH in its microenvironment that results in the hydrolysis of the Si-O-Si link that anchors the silane ligand. Try a Shiseido CapcellPak NH2 column; they seem to be among the more stable such materials and also afford good chromatography.

[AMH]

The compound is acidic and has one group NH2 and one group P=O.

The pH of the buffer is 4.5.

[ym3142]

I am sorry, like a thousand other readers, I am jumping in:

KHPO4 has pKa 2.2, 7.2 and 12.3. so your pH4.5 buffer is not a buffer.

[carls]

Since your compound is acidic this separation is based on ion exchange. Is the NH2 group next to the P=O?

As Andy stated, a slow loss of retention with an amino column in anion exchange mode likely indicates loss of bonded phase.

However, as was also mentioned by ym3142 the pH you are using does not provide enough buffer capacity to ensure a stable pH. It's possible the pH of your mobile phase is decreasing over time (adsorption of atmospheric CO2) and the ionization of your acid is being reduced. This would also show up as a slow decline in retention.

Try 50mM pH 6.5 HPO4 with 20% ACN and let us know what happens.