Chromatography Forum

Hello all -

Trying to develop a DPPC Content/Assay method using an ELSD on an Acquity H-Class and we have been having issues with repeatability/precision. Basically, recovery of Check Standards and/or Accuracy solutions is very poor, often off by up to 10%.

We are calibrating using a five point log-log linear standard curve and have obtained an R^2 value > 0.995 pretty consistently. Diluent is 75:25:1 MeOH:Chloroform:H20, MP A is 1% Formic in water, MP B is 1% Formic in MeOH. Gradient starts at 10:90, transitions to 2:98, then back to 10:90. We are currently using an Acquity BEH C18 1.7 µm column (We previously tried an Xterra RP8 column with a slightly modified gradient but observed bad peak splitting and decided we were better off with the Acquity). For the ELSD, gas pressure is at 45 psi, nebulizer is at 80% (48C I think), and drift tube is at 40C.

I suspect there is some sort of inherent flaw with the method as we currently have it set up. Maintenance/Calibration was just performed on the detector a few weeks ago. Tailing and plate count look fine, but the appearance of the peak itself is as bad as I've ever seen. I guess I would call it "lumpy". We are using smoothing in our processing method to make it a bit more palatable.

Any suggestions? Is it just something simple that I am completely overlooking?

Thanks.

First of all, did you check solvent miscibility? If I had to shot I would say that solvent from sample wont mix with you mobile phase.

Futhermore I started to work with phosholipids this year and I would consider solvent changing. Dont really remeber but I think that phosphocholine doesnt dissolved in MeOH:CHCl3 2:1 very good. I would go with MeOH:CHCl3 1:2 or n-Hex:IPA 8:2.

You must remember that choline always have a positive charge so interaction with silanols in C18 column will be substiential. I would recomend going to normal phase chromatography - if You want I can give you a nice normal pahse method on Diol column or Silica.