Chromatography Forum

Hi All

I've been working on an anion exchange method containing Alendronate, phosphate and phosphite.

Mobile phase is 0.5mM Nitric acid
Column is the same as that used in the EP - PSDVB packing
UV Detector

The problem is my peak shape is awful; particularly for Alendronate but phosphate and phosphite are poor too. All peaks start very gradual fronting to an apex where they then return to zero very quickly. This gives me a peak width (half height) of ~4.0mins but a height of ~0.06 AU. So components detected at 100% nominal but unusable - especially when going to those lower rel sub levels.

- Conditioned >24hrs
- Conditioned while injecting my components >50 injections
- I've tried reducing injection volume and sample concentration
- Different brand/type of anion exchange columns
- A wide range of pH (also different buffer salts to control said pH)
- Increased column temp

I cannot get the components off of the column quicker to form a "sharper" peak, coincidentally none of the peaks are made "much worse" by all of the above.

I'm completely stumped any help would be greatly appreciated.

I would suggest controlling your pH. You have no buffer control. Try pH phosphate buffer (pH 3).

My initial conditions do not control pH but I have tried using a buffered solutions @ pH 3.0, 4.0 and 5.0 with no success. (I also tried one @pH 9.0 in desperation; Alendronate distorted and no phosphate or phosphite detected.)

My observations are that pH has an effect of increasing and decreasing the retention and the effect is consistent the RRt between the peaks stays the same but it has absolutely no effect on the peak shape.

Thanks for getting back to me.

Hi MFarr02,

Did you try 0.2% formic acid in water, pH 3.5 and a column temperature of 35 degrees Celsius?