large peak of glutamate

[andrewn]

I have two Alliance 2695 separation modules attached to separate Waters 474 fluorescence detectors. when I inject glutamate stds onto them they give similar sized peaks. When I inject biological samples onto them system A gives a peak 6 times larger than system B. If I cross over the biological samples system A still gives a peak 6 times larger. So it is not the sample variation. Also, as the standards are the same it cannot be the detector.

Could it be that system A is not producing the correct gradient and so a couple of peaks are merging? (the peak shape is good though, suggesting this is not the case). How could I check this?

I cannot apply logic to this problem - can anyone help?

thanks
nick

[Chris Pohl]

Nick,

Even though your standards are getting similar responses on the two systems, it's still possible that the variation is detector related. Your extraneous peak might be on a sharply rising/falling edge for your excitation wavelength (for example) which would make it quite sensitive to calibration accuracy. Your standards might not be as sensitive to calibration error if the excitation and emission wavelengths were properly chosen. I would suggest swapping detectors and seeing if the discrepancy follows the detectors.