Chromatography Forum

I want to separate basic salt API (A) with acidic API (B) by using C-18 column, API (A) is unstable at pH <3 , API (A) and API (B) has no separation at pH>3.

To separate API (A) and API (B), I chose the following HPLC conditions:
C18 column 150x 4.6 mm 5u, mobile phase of acetonitrile: water: TFA (70:30:0.05), pH = 2, gradient elution at 1ml/min. Since API (A) is not stable at pH<3, on-column degradation was observed, an extra degraded peak was observed (<0.4%). To control the on-column degradation, API (A) was dissolved in 50 mM ammonium acetate buffer (pH=6.9).

It was noticed dissolving API (A) in pH=6.9 buffer minimized the on-column degradation. But the on-column degradation was not completely eliminated by doing so. Is there any suggestion for eliminating the on-column degradation? Any suggestion is appreciated!

I don't know what all you tried in an attempt to get them separated at pH > 3, but you could go back to the higher pH and replace ACN with methanol, and see if they are separated. Or try a different stationary phase at the higher pH, such as phenyl, cyano, PFP, etc., if you have them on hand. Or a combination of changing the mobile phase and stationary phase.

Or a column with an embedded polar group...

I would try to get the maximum resolution I can get between these two compounds by alternating the chromatographic conditions (i.e. ion pairing reagents, mixed mode columns, HILIC etc) and then speed the analysis time as much as possible.

I would try to use the appropriate chromatographic conditions in order to elute first the API (A) as it is the most unstable one. That would propably minimize if not eliminate the degradation... (?).

Don't assume the world knows what API is. I presume acid and basic API is not simply something protonated or not? If so it can't be separated.

The rest of my comments might be more relevant if I knew what the chemistry was. But never-the-less, I am bothered by the statement that a degraded product peak was observed. If degredation occures during elution and not in the sample vial there should be a degraded smear, but I see no mechanism to generate a degraded peak unless the parent compound as well as product does not move at some time during the separation.

As the unstable peak moves down the column, degredation product is continually formed and it will lead or trail the original component. Since the formation of this product occurs all along the length of the column, it never elutes as a peak but rather a tail or front to the main peak that extends from the parent peak to the elution time of the degraded product peak, had it been present in the original sample. It will be impossible to estimate its concentration because the signal will appear as baseline.

degredation is a time and temperature dependent reaction. Speed up separation and lower temperature to minimize problem. I

Bill hit on the key point: Are API(A) and API(B) the same compound, except in salt and free acid forms, are are you dealing with two different compounds?

If they are simply the acid and salt form of the same compound, then they will not be separated in any aqueous system.