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Molecular absorbtion

spectrophotometry in UV-VIS
Biophysics Dept. UMF C. Davila 2015

Dr. Octavian Călinescu

Electromagnetic radiation (light)
Biophysics Dept. UMF C. Davila 2015

Where: λ = wavelength, ν = frequency, T = period


Spectrum – all values that a physical quantity can take

Continuous – can take any value

Discrete – can have

only certain values
Biophysics Dept. UMF C. Davila 2015
Electromagnetic (EM) spectrum

E = hν = h
Biophysics Dept. UMF C. Davila 2015

h = 6.62 · 10-34 J·s (Planck constant)
UV-VIS molecular absorption spectrum

determining quantities/amounts
(quantitative analysis)

IR molecular absorption spectrum

determining the nature of a
Biophysics Dept. UMF C. Davila 2015

substance (qualitative
Classification of spectroscopic/spectrometric techniques

Atomic spectroscopy Molecular spectroscopy

 Atomic absorption spectrometry  IR spectrometry
 UV-VIS spectrometry
 Atomic emission spectrometry
 Molecular fluorescence spectrometry
 Atomic fluorescence spectrometry  X ray fluorescence spectrometry
 Mass spectrometry
 Raman spectrometry
 NMR spectroscopy
 Electronic paramagnetic resonance (EPR)
Biophysics Dept. UMF C. Davila 2015

Needs atomization of the sample In general spectra contain bands

Line (discrete) spectra
Interaction of a molecule with EM energy –

o A molecule will absorb electromagnetic radiation, going to an

excited state:

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

M** → M* (without light emission)

M* → + hν’
Biophysics Dept. UMF C. Davila 2015
Energy levels of a molecule

Total energy of a molecule – the sum of energies: electronic, vibrational, rotational

ΔE = ΔEel + ΔEvib + ΔEr
ΔEel > ΔEvib >ΔEr

EM domain Transitions
Microwave Rotational
Infrared Vibrational +

Ultraviolet and Electronic +

visible Vibrational +
Biophysics Dept. UMF C. Davila 2015
Absorption of energy by molecules

Various functional groups (chromophores) in Chromophore Absorption peak

molecules absorb light at different wavelengths.

When the molecule contains a system of
conjugated double bonds – absorption at lower
energies (higher wavelengths).

Depending on the wavelength at which a

substance absorbs, it will appear as coloured in
its complementary colour (non-absorbed
radiation is reflected).

The substance will be coloured in the opposite

colour on the wheel from the absorbed colour.
White substance – insignificant VIS absorption;
black substance – strong absorption in the VIS
Biophysics Dept. UMF C. Davila 2015


Eg : absorption at 420 nm (in blue) → yellow

Beer-Lambert law

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

A substance absorbs differently at different wavelengths:

ε = f (λ)
Quantities used in spectrometry


T = = 10 −εcl T % = ⋅100 = 10 −εcl ⋅100
I0 I0

Nonlinear dependence on concentration – more difficult to work with. A

linearly dependent quantity is preffered.

A = − log(T ) = log( ) = εcl
A = ε λ cl
Biophysics Dept. UMF C. Davila 2015

Alternate form of the

Beer-Lambert law concentration concentration
Application – analysis of biological fluids

• Biological fluids contain a large number of dissolved substances

• Spectrophotometry cannot provide qualitative or quantitative
information about the dissolved substances unless they are first
separated from each other
• Separation – obtained using chromatography (HPLC/GC)$file/hplc.gif Călinescu et al., J. Chrom. Sci 2012:50

• Detection is ensured via spectrophotometry (usually at a single

Biophysics Dept. UMF C. Davila 2015

wavelength) – a chromatogram results (absorbance as a function

of the time that the substance spends in the chromatographic
Absorption spectrophotometer

2. MONOCHROMATOR – chooses one wavelength of radiation

o The detector measures the intensity of the transmitted radiation. To

measure the absorbance one needs also the intensity of the incident
Biophysics Dept. UMF C. Davila 2015

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

The double beam construction allows a much faster analysis, without

needing to exchange the sample for a blank each time we change the
wavelength. Disadvantage – higher cost.
Experimental part

1. Drawing the absorption spectrum for a solution of food dye Sunset

Yellow (E110) in the range 400 – 700 nm by reading the
absorbance every 20 nm
2. Choosing the wavelength where absorbance is maximum (λmax)
3. Measuring the absorbance at this wavelength for solutions of
Sunset Yellow of known concentration (3, 5, 10, 20, 30, 40 μM)
4. Drawing the calibration curve A = f (c) and finding its parameters
5. Measuring the absorbance of a solution of Sunset Yellow of
unknown concentration at λmax
6. Finding the unknown concentration by interpolation on the
calibration curve
Biophysics Dept. UMF C. Davila 2015
Experimental procedure

1. The spectrometer is turned on and one has to wait until the

screen shows the absorbance.

2. The wavelength is selected.

3. As our spectrometer uses a single beam, it is necessary to bring

the absorbance to 0 every time the wavelength is changed. To
do this a cuvette filled with the sample solvent (water) is inserted
and the ZERO button is pressed.

4. The cuvette with the solution of interest is inserted in the

spectrometer and the absorbance is read.
Biophysics Dept. UMF C. Davila 2015
The absorption spectrum Sunset Yellow (E110)

The absorption spectrum of Sunset Yellow is obtained by representing A = f (λ)

Finding the absorption maximum

in the selected interval
Biophysics Dept. UMF C. Davila 2015
Drawing the calibration curve

c (μM) A (… nm)


The parameters of the line y = a + bx

are obtained using Excel/Origin or the
equations :

N  xy −  x  y c (μM)
N  x − ( x )
2 2
 y − b x
Biophysics Dept. UMF C. Davila 2015


1. In quantitative determinations we only use the absorbance

measured at a wavelength where there is a maximum in

2. The Beer-Lambert law is valid when molar or normal

concentrations are used.

3. Absorbance is additive. If the solution would contain another

species that would absorb at the same wavelength as our dye, the
absorbance would be the sum of the individual absorbances of
both components.

4. The spectrophotometrical method is sensitive down to ~ 1 ppm (1

Biophysics Dept. UMF C. Davila 2015