UV-Visible spectroscopy provides qualitative access to information on the nature of the bonds present in the sample but also to quantitatively determine the concentration of absorbing species in this spectral range. Non-destructive and fast, this spectroscopy is widely used in practical chemistry as well as in chemical or biochemical analysis.
For many centuries, the nature of the atom has been the subject of many attempts at modeling. From the XX th century, the quest for the infinitely small was marked by the frantic race between theorists teams, seeking to unify the various fundamental interactions, predict the existence of many elementary particles, and those experimenters who build complex devices to prove their presence. The last elementary particle, the Higgs boson, has been observed very recently by CERN, putting an end to months of suspense and validating the unified theory of fundamental interactions.
The electromagnetic interaction is one of the interactions concerned by this unified model. It reports on the interaction between an electromagnetic wave and a charged particle. The interaction between matter and radiation is a perfect illustration. At the atomic scale, the material is not continuous but constituted of an assembly of elementary particles, the energy is not it either and can take only discrete values. The total energy of an atomic building can be in the form of the following sum:
E = E el + E via + E rot + E trans
Eel represents the electron energy, E vib vibrational energy, E rot the rotational energy E and trans energy translation system.
The first three are quantum in nature and therefore quantified, while the trans- E term corresponds to a macroscopic movement of the center of gravity of the building. The latter is consequently not quantified and can take its values in a continuum of energy.
The electromagnetic interaction characterizes the ability of an atomic building to see its energy modified by the action of electromagnetic radiation. Let an atomic system be characterized by two quantified energy levels E 1 and E 2 (with arbitrarily E 1 <E 2 ). If the electromagnetic radiation makes it possible to pass from the level E 1 to the level E 2, the system must acquire energy. This is called absorption. On the contrary, the transition from level E 2 to level E 1leads to a release of energy, it is about emission. The absorption or emission of energy is then in the form of an electromagnetic wave, the energy of which depends strongly on the order of magnitude of the difference in energy between the two states, denoted by ΔE, and therefore intrinsically the nature of the levels concerned.
In the table below are collected different orders of magnitude concerning energy transitions and the field of electromagnetic radiation concerned.
|Electronic transition||Vibrational transition|
|Order of magnitude ΔE (in eV)||1 – 10||0.1 – 1|
|Order of magnitude ΔE (in kJ.mol -1 )||100 – 1000||10 – 100|
|The wavelength of emitted or absorbed radiation||300 – 800 nm||One μm|
|Spectral domain||UV – Visible||Infrared|
The orders of magnitude of the energy transitions presented in the preceding table illustrate the fact that the rotational levels are sub-structures of the vibrational levels, themselves sub-divisions of the electronic levels. This complex structuring of the energy levels thus allows a very large number of transitions under the effect of electromagnetic radiation.
The frequency ν of the emitted or absorbed radiation and the Planck-Einstein relation: Δ relates the energy difference ΔE between the initial and final levels? = ℎν ?, with h the Planck constant (h = 6.63.10 -34 Js).
Now, in a vacuum, frequency ν and wavelength λ are linked by the celerity of light c:? Ν = c? / Λ. We then deduce the relation between ΔE and λ: Δ? E = ℎc? / Λ. We will focus here only on energy transitions absorbing or emitting in the UV – Visible, that is to say involving transitions between electronic levels (but modifying the vibrational and rotational substructures). These electronic levels are corresponding to different electronic configurations; the absorption mechanism will thus be due to the excitation of valence electrons and emission to their de-excitation.
From an experimental point of view, the wavelength (or frequency) of absorbed electromagnetic radiation is therefore characteristic of the energy difference between two electronic levels. Absorption spectroscopy, experimentally leading to the determination of absorbed wavelengths, thus makes it possible to obtain the ΔE differences between electronic levels and consequently information on the electronic structure of the building. We will only be interested in absorption spectroscopy.
Apparatus and Operation
The use of a spectrophotometer determines the wavelengths of absorbed electromagnetic radiation. The most used device in high school is the single-beam spectrophotometer, whose schematic diagram is presented below:
A polychromatic source (emitting in the UV or visible) is placed in front of a prism. This dispersive system will decompose the polychromatic radiation emitted by the source. By correctly orienting the diaphragm-sample-photodetector system, the solution contained in the tank will be irradiated with almost monochromatic radiation. The diaphragm, a simple thin slot, makes it possible to illuminate the sample with a beam of small width, and therefore of good monochromatic quality, the photodetector measuring the intensity of the radiation transmitted after passing through the sample solution, denoted I t, λ.
From a practical point of view, the sample consists of the building studied, dissolved in a solvent and contained in a tank. It is, therefore, necessary that solvent and tank do not interfere in the measured data. So we will choose transparent in the chosen field. In the trade, there are different tanks adapted to the different spectral domains encountered (plastic for the visible, quartz of more or less good quality for the UV). As for the solvent, its influence is neutralized by producing a blank, that is to say by measuring the intensity of the radiation transmitted after passing through the tank containing the only solvent. The samples must be transparent to avoid any diffusion phenomenon: only clean solutions can be analyzed in clean tanks.
Experimentally, the apparatus extracts as raw data the intensity I t, λ, obtained after passing through the solution. Since this is dependent on the source, it is preferred to calculate two derived quantities: the absorbance A and the transmittance T.
T defines the transmittance T = I t, λ / I 0, λ. It is expressed as a percentage.
The absorbance A is calculated by: A = log (I 0, λ / I t, λ ) = – log. It is a positive magnitude.
A spectrophotometric study in UV-Visible
In a UV-Visible spectrophotometric study, it is customary to plot the graph of the absorbance A as a function of the wavelength λ.
For example, the spectrum of (1E, 4E) -1,5-di-2-thienylpenta-1,4-diene-3-one (D2TDO) is shown below (25 ° C, acetonitrile):
The spectrum is made up of wide bands, not peaks. Many energetically close transitions are thus realized. However, if the electronic transitions are indeed responsible for these absorptions, the vibrational and rotational substructures, within the same electronic level, can lead to transitions energetically of the same order of magnitude, thus resulting in the same electronic levels but putting at play different vibrational and rotational levels. Different electromagnetic radiations of slightly different wavelengths then lead to different energetically very close transitions and thus to absorption bands.
The analysis of such a spectrum leads to the determination of the wavelength of the maximum absorption λ max. In the previous example, this is 360 nm. However, the data of such a wavelength does not give information on the intensity of the absorbance. Intensive and quantitative data is needed. The Beer-Lambert law provides this: for a solution containing a single absorbent solution, A = ε.lc, with 1 the width of the vessel containing the sample (hence the length of the optical path), c molar concentration of the sample and ε the molar extinction coefficient (usually expressed in mol -1 .L.cm -1if l is expressed in cm). This law is valid for transparent solutions, little concentrated and in these conditions, it is also addictive. Thus, the linearity relation is valid as long as the absorbance keeps low values (typically A less than 1.5-2).
The Beer-Lambert relationship thus gives access to the molar extinction coefficient ε which characterizes the absorption of the building under the conditions of the experiment. Thus, it depends on the temperature, the building and the solvent in which the spectrum is recorded. At the wavelength of the absorption maximum, the coefficients ε max can be calculated. The data of these two magnitudes (λ max , ε max ) is characteristic of the absorption of a building under given experimental conditions, but does not depend on the apparatus used.
The absorption comes from an energy transition between two electronic levels whose nature plays strongly on the two magnitudes λ max and ε max . In the case of organic molecules the electronic levels concerned by transitions in the UV-Visible roughly correspond to the valence orbitals of the building and their energy is dependent on their nature (σ, π) and their character (binding, anti-boring , non-binding). Schematically, the relative order of the electronic levels is as follows:
Many transitions are possible but only those with lower energies lead to absorption in UV-Visible. The nature σ or π of the levels involved reflect the nature of the functional group present in the building. Some organic functions will, therefore, cause absorption, they are chromophores. The recording of a UV-Visible spectrum can, therefore, like infrared spectroscopy, lead to the identification of the functions present in an organic molecule. The orders of magnitude of many characteristic chromophores are tabulated:
|chromophore||Transition||λ max||log (ε max )|
|Alcohol||N- σ *||180||2.5|
|Aldehyde – Ketone||π-π *
|Carboxylic acid||n-π *||205||1.5|
The presence of multiple bonds and nonbinding doublets generally allows good absorption in the UV-Visible. Moreover, the conjugation of the π system leads to a tightening of the levels π and π * and consequently an increase of λ max. This is the bathochromic effect. If the alkenes absorb typically in the UV, the polyenes see their λ max increase with the number of conjugated π bonds to eventually reach the visible range for the large conjugated molecules. Thus, β-carotene, containing 11 C = C conjugated bonds, has its maximum absorption around 450 nm.
A solution of β-carotene is absorbing in blue; it allows only poorly absorbed radiation to pass and then appears in its complementary color, orange. The color wheel makes it easy to find.
In the case of transition metal complexes, the electron transitions are carried out between orbitals d, whose degeneration has been lifted by the ligands. It is then dd transitions, generally not very intense and which often lead to absorptions in the visible. This is the case, for example, of hexa-aqua complexes of metals: [Cu (H 2 O) 6 ] 2+ (blue), [Ni (H 2 O) 6 ] 2+ (green) …
UV-Vis spectroscopy thus provides qualitative access to information on the nature of the bonds present within the sample (via the order of magnitude of λ max and ε max ), but also to quantitatively determine the concentration of absorbing species in this spectral domain (via the Beer-Lambert law). Non-destructive and rapid, this spectroscopy is widely used in practical chemistry (after the construction of a calibration line and transfer of an experimental measurement) as well as in chemical or biochemical analysis. Examples include the determination of nitrate ions in pool water (after adding a colored complex additive) or the determination of the purity of DNA and certain proteins after their extraction.