spectrophotometry in UV-VIS
Biophysics Dept. UMF C. Davila 2015
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Biophysics Dept. UMF C. Davila 2015
c
E = hν = h
Biophysics Dept. UMF C. Davila 2015
λ
h = 6.62 · 10-34 J·s (Planck constant)
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Spectrometry/Spectroscopy
UV-VIS molecular absorption spectrum
SPECTROMETRY –
determining quantities/amounts
(quantitative analysis)
SPECTROSCOPY –
determining the nature of a
Biophysics Dept. UMF C. Davila 2015
substance (qualitative
analysis)
Classification of spectroscopic/spectrometric techniques
M + hν → M**
o The molecule will not stay in the excited state M*, it will return to
its fundamental state M either by non-radiative processes, or by
radiative (emission). In the latter case, ν’ ≤ ν
EM domain Transitions
Microwave Rotational
Infrared Vibrational +
Rotational
range.
Shows the relation between the intensity of the absorbed light and the
concentration of a solution.
− εcl
I = I o10
I0 – intensity of the incident light
I – intensity of the transmitted light
ε – molar absorption coefficient
(characteristic for a substance)
c – concentration of the substance in
the cuvette
l – length of the cuvette
Biophysics Dept. UMF C. Davila 2015
ε = f (λ)
Quantities used in spectrometry
Transmittance
I I
T = = 10 −εcl T % = ⋅100 = 10 −εcl ⋅100
I0 I0
Absorbance
I0
A = − log(T ) = log( ) = εcl
I
A = ε λ cl
Biophysics Dept. UMF C. Davila 2015
1. RADIATION SOURCE
2. MONOCHROMATOR – chooses one wavelength of radiation
3. SAMPLE COMPARTMENT
4. DETECTOR
5. COMPUTER
o One has to measure the intensity of the light that passes through a
solution as close as possible to that which we analyze, but which does
not contain the substance of interest – blank (solution).
Single/double beam spectrometer
Single beam
Double beam
Biophysics Dept. UMF C. Davila 2015
c (μM) A (… nm)
3
5
Ax
…
Aλmax
X
N xy − x y c (μM)
b=
N x − ( x )
2 2
X=?
y − b x
Biophysics Dept. UMF C. Davila 2015
a=
N
Observations