Calculating Absorbance - Waters ACQUITY UPLC Operator's, Overview And Maintenance Manual

Optics assembly components (Continued)
Component
Photodiode array

Calculating absorbance

The detector computes absorbance by subtracting the dark current (see
current" on page
Absorbance is based on the principles of Beer's law.
Beer's law
The relationship between the quantity of light of a particular wavelength
arriving at the photodiode and the concentration of the sample passing
through the flow cell is described by the Beer-Lambert law (commonly called
Beer's law). Beer's law is expressed as A = εlc where
A = dimensionless quantity measured in absorbance units
ε = constant of proportionality, known as the molar extinction coefficient
l = path length, in centimeters (1.0 cm in the detector's normal flow cell)
c = concentration, in moles per liter
Beer's law applies only to well-equilibrated dilute solutions. It assumes that
the refractive index of the sample remains constant, that the light is
monochromatic, and that no stray light reaches the detector element. As
concentration increases, the chemical and instrumental requirements of
Beer's law can be violated, resulting in a deviation from (absorbance versus
concentration) linearity. The absorbance of mobile phase can reduce the linear
range by the amounts shown in
4
Function
An array of 512-pixel photodiodes arranged linearly.
The diode width (50-µm), together with a 100-µm slit
(for the PDA) or 50-µm slit (for the eλPDA), yield single
wavelength resolution of 1.2 nm.
12) and reference spectrum from the acquired spectrum.
"Mobile phase absorbance" on page
"Dark
105.