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SPECTROSCOPY Study of the molecule structure and dynamics through absortion emission and light dispersion

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

ABSORPTION SPECTROSCOPYThe 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 SPECTROSCOPYIt happens when the matter releases energy coming back from the excited to the basal state

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Absorption spectroscopyLight: it is considered as a wave that consists of a magnetic and an electric field (electromagnetic) which are perpendicular among then and oscillate as a sinusoid while propagate in the space. This wave is characterized by a frecuence (ν) and a wavelength (λ)

Characteristics of the electromagnetic radiation

Undulatory and corpuscular NatureCharacterized by:Energy defined by:

Frecuence (ν: s-1 units),wavelength (λ;nm units) or the wave number (ν in cm-1)

Intensity (I) the number of photons.

What are the nature of

these absorptions?

Example: * transitions responsible for ethylene UV absorption at ~170 nm calculated with semi-empirical excited-states methods :

HOMO u bonding molecular orbitalLUMO g antibonding molecular orbital

h 170nm photon

π

π

n

π

π

n

π*

π

π

n

π*π*π*π*

π*π* π*

π*

-*; max=218 =11,000

n-*; max=320 =100

The UV Absorption process• * and * transitions: high-energy, accessible in vacuum UV (max <150 nm). Not usually observed in molecular UV-Vis.

•n * and * 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 corrresponding to a transition from ground to excited state.

•Energies correspond to a 1-photon of 300 nm light are ca. 95 kcal/mol

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The quantitative picture

• Transmittance:T = P/P0

B(path through sample)

P0

(power in)P

(power out)

• Absorbance: A = -log10 T = log10 P0/P

• 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 cmc is the concentration of the compound in solution, expressed in mol L-1 (or

M, molarity)

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7Array of wavelengths to determine the wavelength of maximum absorbance

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A Chromophore isa compound that absorbs UV or VISlight:● Doubke bounds (alterned, the delocalization of electrons reduces the increment of energy between levels (resonant structures) and makes easier the jump between levels

●Aromatic and heteroaromatic●

●Peptidic bounds●

●Carbonyl bounds

Factor that affect to the absorbance properties of a chromophor

PH effect polaridad of the solvent Orientation effect

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Benzenoid aromatics

From Crewes, Rodriguez, Jaspars, Organic Structure Analysis

UV of Benzene in

heptane

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APLICATIONS OF SPECTROPHOTOMETRY IN BIOLOGY

Protein spectra with Prostetic groups

Hemo-proteins

D-amino-acid transferase. Prostetic group

B6 vitamin

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PROTEIN STUDY

SIDE CHAIN FREE AATrp long max: 280nm 219nm coef max 5600Tyr long max 274nm 193, 222nm coef max 1400Phe long max 257nm 188, 206nm coef max 200

●CONCENTRATION MEASUREMENT●SPETRA OF PERTURBATION BY THE SOLVENT●PROTEIN DENATURATION●SPECTROPHOTOMETRIC TITTER USING THE pH OF A PROTEIN

Cromophors

Peptidic groups (-200 nm)Aromatic amino acids (250-300 n)Prostetic groups: hemo, metallic centers, (absorbing in VIS):

NAD+ / NADH (absorb in the UV region)

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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 coloured compound.

Oxidized guayachol extintion molar coeficient at 470 nm is

2.66 · 104 M-1 cm-1.

V máx

[E ]Tot

Pte = K = nº recambio2

0,5 1 1,5 2,52 3 T (min)

A (ua)470

1ml

2 ml

4 ml

3 ml

V máx

E (ml)Tot1 2 3 4

Pte 1

Pte 2

Pte 3

Pte 4

(Ua/min)

A595

[BSA]Real

Speed r

eact

ion

Vmáx

[S]

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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-BCAThe most used are Lowry and Bradford

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Bradford's method; consists of a change of colour in the reagent Coomassie brillant blue G-250 in response to different con contrartions of proteins. This compound interacts with basic amino acids and aromatics thus the instensity of absortion depends on their proportion. Tere 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

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.

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DETERMINATION OF NUCLEIC ACIDS

DETERMINATIO OF BACTERIAL COLONIES

MONITORING OF CHEMICAL REACTIONS

TRANSSITION FROM HELIX INTO UNFOLDED STATE

STUDIES OF PROTEIN STRUCTURES BY SOLVENT PERTURBATION