• Measures the light that passes through a liquid sample
• Spectrophotometer gives readings in Percent Transmittance (%T) and in Absorbance (A)
The Electromagnetic Spectrum = c / E = h
Absorbance and Complementary Colors
I o I Cell with Pathlength, b, containing solution light source detector blank where I o = I concentration 2 concentration 1 b with sample I < I o The process of light being absorbed by a solution As concentration increased, less light was transmitted (more light absorbed).
The law states that the amount of light absorbed by a solution (colored) is proportional to the concentration of the absorbing substance and to the thickness of the absorbing material (path length). Absorbance is also called optical density
A = abc
where a – molar absorptivity, b – pathlength, and c – molar concentration
BEER - LAMBERT'S LAW
I – intensity where I o is initial intensity
T – transmission or %T = 100 x T
(absorption: Abs = 1 – T or %Abs = 100 - %T)
T = I/ I o
A – absorbance
A = - log T = -log I/ I o
The blank contains all substances expect the analyte.
Is used to set the absorbance to zero:
A blank = 0
This removes any absorption of light due to these substances and the cell.
All measured absorbance is due to analyte.
Conventional Spectrophotometer 1. A stable and cheap energy source. 2. A monochromator to break the polychromatic radiation into component and wavelength/bands of wave length. 3. Transparent vessels (cuvettes) to hold the sample. 4. A photo sensitive detector and associated amplifier and recorder
Conventional Spectrophotometer Optical system of a split-beam spectrophotometer
prisms and diffraction gratings. Simple glass prisms are used for visible range. For UV region silica, fused silica or quartz prism is used. Fluorite is used in vaccum UV range.
Gratings are often used in the monochromators of spectrophotometers operating in UV, visible and infra red regions. Their resolving power is far superior to that of prisms & they yield a linear resolution of spectrum.