Chromatography Forum

Okay, here's a rather simplistic question, so please bear with me. If I inject the same volume of a solution on two different instruments, should I expect the mAU response to be identical? They are both Agilent HPLCs, one is an 1100 and the other a 1200. I am using the same method, the same type and brand of c18 column; gradients are a little different. I would think the mAU response should be the same, or at least pretty darn close, but this is not the case. Also, if I inject half the volume of the solution, shouldn't the response be about half of the full volume response? I am not seeing this with these instruments. Comments/thoughts?

Thanks.

When you're talking about "mAU response", are you referring to peak height or peak area?

Sorry....yeah I guess that little detail would help. Peak height.

So, two separate issues: 1. difference between two instruments and 2. response linearity with volume.

1. The absorbance maximum will depend on the flow cell path length (may not be the same between your 1100 and your 1200 -- for that matter, there are different models in the 1200 series), on the width of the peak (which can vary depending on the column used, on the gradient conditions and flow rate, and to some extent on the system plumbing), on the detector wavelength and bandpass (especially if you are working on a sloped part of the absorbance spectrum), to some extent on the data system settings (especially things like time constant, sampling rate, or bunching which can "smear" peaks somewhat), on whether the absorbance your seeing is still within the linear range of the detector, and on the injection volume accuracy. Bottom line is that I would not expect it to be the same on *any* two instruments; that's why a calibration curve is only valid for that instrument on that day.

2. The peak *area* should be directly proportional to the volume injected. If it's not, then there is a problem somewhere: most likely either with the autosampler or a gross overload. If the area proportionality is OK but the peak height is not, that suggests overload (the increased volume is making the peak wider instead of taller); you should be able to see the effect on peak width and/or shape.