Chromatography Forum

I am fairly new to liquid chromatography and have been trying to set up the Agilent 1260 for a new method. Prior to setting up the Poroshell 120 EC-C18 column for sodium benzoate analysis, the column was flushed in its appropriate solvent for long term storage. The old mobile phase bottles were switched out for the new bottles of buffer (7.8 mM ammonium acetate buffer in acetonitrile pH 4.2 (72:28) and (15:85), degassed) for the new method. The system was flushed overnight to remove any salt buildup from the previous method with 10% isopropanol (column compartment heated to 60C), then rinsed with water. With the Poroshell column placed on the HPLC and the column temp off, the system was purged with the new mobile phase for at least an hour, then the purge valve was closed. The column was equilibrated with the new buffer, but the DAD baseline is still noisy (~5 mAU difference). Can anyone suggest what else I can do to reduce the baseline? I can provide more details if needed.

Is this random noise or do you see a regular pattern? Could be the pump then...

My response is not directly related to the detector, but that seems like a complicated mobile phase for detection of sodium benzoate (as benzoic acid). I think if I was doing such assay, I'd simply use 0.5% phosphoric acid/water and ACN (or methanol).

What wavelength are you using?

Is this random noise or do you see a regular pattern? Could be the pump then...

It appears to be random noise. The scientist who worked on this had a clear baseline in their analysis. If it is a pump issue, how would I be able to tell?

My response is not directly related to the detector, but that seems like a complicated mobile phase for detection of sodium benzoate (as benzoic acid). I think if I was doing such assay, I'd simply use 0.5% phosphoric acid/water and ACN (or methanol).

What wavelength are you using?

This method was worked on by a different scientist before they left, and they had very clean baseline and clear separation in their analysis. The wavelength is set at 225 +/- 4nm, reference 360 +/- 20nm.

ammonium acetate buffer... wavelength is set at 225 +/- 4nm, reference 360 +/- 20nm

Wrong combination. Acetate absorbs well at 225 nm; hence, the signal is noisy.

This method was worked on by a different scientist before they left, and they had very clean baseline

Doubtfully.