Chromatography Forum

Hello

I am new to HPLC.
I would like to know if the mAU measure is in absolute numbers?

thank you

I'm not quite sure what you mean by "absolute numbers" in this context, but the answer I would give is "probably not". What the A/D converter in your detector or data system is measuring is the output voltage from the detector, so the units of the actual meassurement would be volts (V). The data system translates that into absorbance units (A or AU) with an appropriate scaling factor, most commonly 1 V = 1 AU although sometimes that is adjustable.

Wait, it gets even more interesting!

UV/Vis absorbance detectors don't actually measure absorbance, they measure transmittance (T), the fraction of the incident light that gets through the flow cell. The absorbance is calculated as the negative log of the transmittance.

So, the detector measures the transmittance, calculates the absorbance, outputs a voltage that is proportional to the absorbance, which the data system then measures and converts back to absorbance.

Thanks Tom, I think that answers it quite well

If you're using a typical modern system where the whole system was made by one manufacturer who also supplied the software, and the software says the absorbance is 1AU, then it really ought to be 1AU (i.e. 90% of incident light being absorbed). Anything otherwise would be a lie. If it says 1000mV, that's another issue altogether (but even then, if, somewhere, it's allowed you to specify that 1AU = 1V, then the result should still be, in absolute terms, correct).
What this means is another matter altogether: you'd need to know the flow-cell's path-length if you want to make a comparison with a proper spectrophotometer. Also you need to consider the absorbance of solvent alone (which may be continuously changing in a gradient method), and unlike a spectrophotometer, you have no guarantee that the material in the flow-cell is homogeneous (if you think about a light-pipe cell with appreciable length, at the start of a peak, there is more absorbing material in the start of the pipe than the end...), so the whole concept of path-length is potentially a bit untidy (only a bit, because a decent system will have been designed with a flow cell volume very small compared to the peak volume).
How absorbance relates to quantity of material will also depend on the dwell-time of solvent in the flow cell, as the peak area (which is what you'll normally be looking at) has a time-axis as well as an absorbance axis.
All of which adds up to mean this: an average chromatographer with average brain power, faced with a molar extinction coefficient, prefers to make up standards and run a calibration curve.