SPECTROSCOPY

Study of the molecule structure and dynamics through absorption

emission and light dispersion

The interaction with radiation affects the energy levels of the matter.

ABSORPTION SPECTROSCOPY
The transition from a basal to the excited state takes place when the energy of the
radiation has the corresponding value to the difference between both states. It
happens when the matter absorbs the energy to pass from the basal to the excited
state.

EMISSION SPECTROSCOPY
It happens when the matter releases energy coming back from the excited to the
basal state 1
Absorption spectroscopy
Light: it is considered as a wave that consists of a magnetic and an electric field
(electromagnetic) which are perpendicular among them and oscillate as a sinusoid while
propagate in the space. This wave is characterized by a frequency (ν) and a wavelength
(λ)
Characteristics of the electromagnetic
radiation

Ondulatory and corpuscular Nature

Characterized by:
Energy defined by: Frequence (ν:
s-1 units),wavelength (λ;nm units)
or the wave number (ν in cm-1)

E=h.ν

Intensity (I) the number of

photons.

2
 What are the π*
π*
π*
π*
π*
π*

nature of π*
n
π*
n
π*
n

these π
π
π
π
π
π

absorptions? -*;  max=218 n-*;  max=320

=100
=11,000
 molecular orbitals are formed when atomic perpendicular ‘p’ orbitals are combined
n molecular orbitals are formed between heteroatoms like N or O, the electrons occupy atomic orbitals
instead.
s molecular orbitals are formed when atomic frontal ‘p’ orbitals are combined

Example:  → * transitions responsible for ethylene UV absorption at

~170 nm calculated with semi-empirical excited-states methods :

h 170nm photon

HOMO u bonding molecular orbital LUMO g antibonding molecular orbital

The UV Absorption process
•s → s* and s → * transitions: high-energy, accessible in
vacuum UV (max <150 nm). Not usually observed in molecular
UV-Vis.
•n → s* and  → s* transitions: non-bonding electrons (lone
pairs), wavelength (max) in the 150-250 nm region.
•n → * and  → * transitions: most common transitions
observed in organic molecular UV-Vis, observed in
compounds with lone pairs and multiple bonds with  max =
200-600 nm.
•Any of these require that incoming photons match in
energy the gap corresponding to a transition from ground to
excited state.
•Energies correspond to a 1-photon of 300 nm light are ca. 95
kcal/mol
 The quantitative picture

• Transmittance:
P 0 P
T = P/P0 (power in) (power out)
• Absorbance:
A = -log10 T = log10 P0/P B(path through sample)

• The Beer-Lambert Law (Beer’s Law):

A = bc
Where the absorbance A has no units, since A = log10 P0 / P
 is the molar absorbtivity with units of L mol -1 cm-1
b is the path length of the sample in cm
c is the concentration of the compound in solution, expressed in mol L-1 (or
M, molarity)

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6
 Array of wavelengths to determine the wavelength of maximum absorbance
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http://www.jove.com/science-education/5038/introduction-to-the-
spectrophotometer
 A Chromophore is a compound that absorbs UV or VISlight:
⚫ Double bonds (alternate, the de-localization of electrons reduces the increment
of energy between levels (resonant structures) and makes easier the jump between levels
⚫Aromatic and heteroaromatic

⚫Peptidic bonds

⚫Carbonyl bonds

Factor that affect to the absorbance properties of a chromophore

PH effect
polarity of the solvent
Orientation effect

8
 Benzenoid
aromatics
UV of
Benzene in
heptane

From Crewes, Rodriguez, Jaspars, Organic

Structure Analysis

9
APLICATIONS OF SPECTROPHOTOMETRY IN BIOLOGY

Protein spectra with Prostetic

groups

Hemo-proteins

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 PROTEIN STUDY
Cromophors ⚫CONCENTRATION MEASUREMENT
⚫SPECTRA OF PERTURBATION BY THE SOLVENT

⚫PROTEIN DENATURATION
Peptidic groups (-200 nm)
⚫SPECTROPHOTOMETRIC TITTER USING THE pH
Aromatic amino acids (250-300 n)
Prostetic groups: hemo, metallic centers, (absorbing in VIS): OF A PROTEIN
NAD+ / NADH (absorb in the UV region)

SIDE CHAIN FREE AA

Trp long max: 280nm 219nm coef max
5600
Tyr long max 274nm 193, 222nm coef max
1400
Phe long max 257nm 188, 206nm coef max
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200
 Kinetic studies of enzymatic acitivity and quantification of the protein amounts

The speed reaction can be measured since it is

proportional to the appearing of colored compound.
Oxidized guayacol extinction molar coefficient at 470
nm is 2.66 · 104 M -1 cm-1.

V máx

Vmáx
Speed reaction

Pte = K 2 = nº recambio

[S]
[ETot ]
595
A
A470(ua)
4 ml

3 ml

2 ml

1ml

0,5 1 1,5 2 2,5 3 T (min) [BSA]Real

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Methods for protein quantification

Absorption methods:
-They can be used directly in samples without the application of
any additional reagent.
-They are fast and do not need incubation.
-The ration between protein concentration and absorbance is
lineal .

Colorimetric methods
We can distinguish 4 different methods :
-Biuret
-Lowry
-Bradford
-BCA
The most used are Lowry and Bradford

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Lowry's method, the protein is determined by measuring the amount of
total proteic N taking into account that this element represents aprox the
16% of the protein weight.

Bradford's method; consists of a change of colour in the reagent

Coomassie brillant blue G-250 in response to different concentrations
of proteins.

This compound interacts with basic amino acids and aromatics thus the
intensity of absorption depends on their proportion. There are two
different forms of colour in the reagent, blue and red. The red form turns
into blue after binding the protein.
The complex reagent-protein has a high extintion coeficient therefore it
has a high sensitivity in the measurement of the protein.
The binding reaction is very fast (it just needs 2 min incubation). The
complex is stable for a long period (about 1h) this makes the process
fast and accurate

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DETERMINATION OF NUCLEIC ACIDS

DETERMINATION OF BACTERIAL COLONIES

MONITORING OF CHEMICAL REACTIONS

TRANSSITION FROM HELIX INTO UNFOLDED STATE

STUDIES OF PROTEIN STRUCTURES BY SOLVENT PERTURBATION

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 The images used are either owned
or belong to another source and
represents less than 10% of the
work. Otherwise url are listed at
the end of the chapter.

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404986/

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