jenway 7315, Operating Manual

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16
SECTION 3 – Theory and Practice of Spectroscopy Measurements
3.1 THEORY OF SPECTROSCOPY MEASUREMENT
UV-visible spectroscopy is the measurement of the absorbance of light at a specific wavelength in a
sample. This is used to identify the presence and concentration of molecular entities within the sample.
The Beer-Lambert law is used to relate the absorption of light to the properties of the sample through
which the light is travelling through. The Beer-Lambert law states that:
A is the absorbance
is the molar absorption coefficient (l mol
-1
cm
-1
)
c is the concentration (mol l -1 )
l is the path length (cm)
This law shows that absorbance is linear to concentration but this is only true for low concentrations. For
absorbance levels above 3 the concentration starts to move away from the linear relationship.
Transmittance is the proportion of the light which passes through the sample:
Therefore: T = I
t
I
o
Absorbance is inversely related to transmittance:
A = log 1
T
3.2 SPECTROSCOPY MEASUREMENT
There are four main components of a spectrophotometer. These are a light source to emit a high and
constant amount of energy over the full wavelength range; a method for separating the light into
discreet wavelengths; a sample holder and a light detector.
Where:
L
o
is the incident light
l
t
is the transmitted light
l is the path length
l
I
o
I
t
l