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Electronic Absorption Spectroscopy

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Electronic Absorption Spectroscopy.

Exploration

Introduction.

Electron absorption spectroscopy can be useful in the detection of small molecules that are studied in chemistry and life sciences. These include macromolecules such as nucleic acids and proteins. A common everyday use of electron absorption spectroscopy in biochemistry labs is for the quantization of DNA concentration in solutions. Learning the concentrations of certain molecules is important in laboratories that require precise concentrations. Besides that, the principles of electron absorption spectroscopy can be applied to simply detect the presence of certain molecules such as carotene, which is useful in a synthesis lab if not already interesting itself.

The Beer Lambert Law.

The Beer-Lambert law applies the concept of the absorption of light in order to determine composition of chemical samples. This law can be used to determine the concentration of components in solutions by examining their relative absorption values to standardized values. This also allows one to determine certain properties of a sample through a simple spectroscopic observation.

This law relates the transmissivity (T) to the percentage of the light transmitted. The incident light I_0,is the amount of light transmitted without a sample in place, whereas (I) is the amount of light transmitted with the sample. Therefore T is the ratio of the transmitted light. This can also be written as an expression of the absorbance of the solution (α) and the path length (l) or the molar absorbity (ε), the concentration (c) and the path length (l). Using these expressions, one can effectively calculate and express the absorption. This expression is useful for determining the concentrations of unknown solutions given any amount of known values.

The law also defines the Absorbance (A) of a component as follows:

Absorbance of a solution with multiple compounds can be represented as a linear combination of the absorbances of its components.

Different Kinds of Absorption.

The nature of the excitation of the electron determines the wavelength of the light absorbed by the excitation. Electron absorption spectroscopy is only valid for the absorption of visible light, which falls in the range between 200 nm and 700 nm. Therefore, for an absorption to be useful, it is important for it to be close to that range. There are three major forms of electron excitations that are studied for spectroscopy.

The first is the excitation of a sigma-bonding orbital to a sigma-antibonding orbital. However, sigma orbital bonds are generally hard to break due to their high potential energy, this is because the bonds are in the same plane as the nuclei and are therefore held much more tightly. Thus, the energy of such excitations is very high. sigma-bonding orbital excitations generally exhibit wavelengths of absorption that are far too high and are not useful to the spectroscopy field.

Another kind of electron excitation is from nonbonding electrons to antibonding sigma bonds. This requires less energy than the transition from bonding to antibonding since nonbonding electrons are not in their lowest potential energy state by being bonded. The lower energy required to excite the electrons shows in the absorption, which generally falls between 150 and 250 nm for organic molecules.

The most applicable form of electron absorption (due to its absorption falling between 200 nm and 700 nm) is the excitation of pi bonding orbitals and nonbonding electrons to antibonding pi orbitals. The absorption of nonbonding excitations are generally low, they range from 10 to 100 1/mol cm, whereas the absorption of pi excitations are much higher and range from 1000 to 10,000 mol^-1cm. As a result, the most majorly studied form of electron absorption is the excitation of pi electrons to pi antibonding electrons.

The nature of such bonds explains the importance of conjugated systems in spectroscopy. For an excitation of pi electrons (double bonds and triple bonds) to antibonding, there must be pi electrons, which imply some form of resonance so that double bonds can change into single bonds. This transition shows why most chromophores (molecules that absorb light) have conjugated systems that allow double bonds to transition to single bonds: this represents a pi to pi antibonding transition.

Validity and Usefulness in a Non-Uniform Medium.

When photons enter an absorbing medium, they are absorbed according to the Beer-Lambert Law. The absorption coefficient is related to the complex index of refraction by the following equation:

ε = 4K/λ

Where ε is the absorption coefficient and λ is the wavelength of the light used in the experiment. The Beer-Lambert law, as described above, only holds true when the spectroscopy is conducted in a uniform medium. If the medium in which the spectroscopy is conducted is NOT uniform, then the rate at which the medium transmits power will not be constant throughout the experiment, and thus the law cannot be exactly followed.

This being said, the information obtained from the spectroscopy is still valid in many applications. The result of using information obtained from the use of a non-uniform medium (which is most often the case - completely uniform mediums are often only available in the ideal case) can be likened to the use of scrambled information. In transmission, light passes through a slab of material. There is little or no scattering (none in the ideal case; but there are always internal reflections from the surfaces of the medium).

Although the presence of a non uniform medium slightly affects the results of the equation above, the scattering of information can still be used at a slight differential from the values provided by the theoretical Beer-Lambert Law.

Importance of Electronic Absorption Spectroscopy to Chemistry and Life Sciences.

Absorption spectroscopy is important in analytical chemistry in determining the presence of, concentration of, and identity of organic molecules. Though absorption spectroscopy may occur across any portion of the electromagnetic spectrum, three particular types of spectroscopy are most commonly used: 1) ultraviolet-visible, 2) infrared, and 3) nuclear magnetic resonance spectroscopy.

1) Ultraviolet-Visible Spectroscopy

UV-Vis Spectroscopy is used in the analysis of highly conjugated compounds and induces electronic transitions in these molecules. Absorption of the electromagnetic radiation by the molecule of interest results in the excitation of electrons in pi-bonding orbitals from the ground state into an excited state (see figure below).

Absorption will only occur if the energy of the photon exactly corresponds to the energy needed for the transition. The strength of the bond is indicated by the wave number necessary for excitation. For example, higher energy, shorter wavelength light is necessary to excite σ-bonds than is necessary for the excitation of a corresponding π-bond. The portion of the molecule responsible for the absorption is referred to as the chromophore. Applying Beer-Lambert Law, UV-Vis spectroscopy can be used to determine the concentration of a particular molecule in a solution by measuring how quickly the absorbance changes with concentration (determined from a calibration curve). The predicted absorptions in UV-Vis spectroscopy can be approximated using particle in a box quantum theory (see equation below).

2) Infrared Spectroscopy

IR spectroscopy measures the vibrational frequencies of molecules for the detection of functional groups. However, this technique is unable to give specific structural details such as connectivities. Though IR measures both bending and stretching vibrations (see below), the stretching vibrations are more valuable in the detection of functional groups.

As with UV-Vis spectroscopy, IR spectroscopy is indicative of the strength of the bonds. Stretching vibrations are well modeled by Hooke’s law (see equation below). The stronger a bond, the greater its ‘force constant’ will be.

Here, μis the reduced mass (the effective inertial mass) of the molecule. By using the harmonic oscillator as a model for the potential energy of the system, Schrödinger’s equation may be solved to,

This in turn can then be used to create selection rules for energy absorption to predict the behavior of these molecules until a certain point (at which the molecules exhibit anharmonicity, aka non-harmonic behavior).

3) Nuclear Magnetic Resonance Spectroscopy

NMR is a powerful spectrographic technique, based on the electronic spins of the nuclei of carbon and hydrogen atoms, which maps out the connectivity of a particular molecule. This type of spectroscopy has an advantage over UV-Vis and IR spectroscopy because it correlates individual atoms within the molecule rather than groups of atoms. Recently, protein NMR has been developed, with the goal of achieving similar resolution as that of x-ray crystallography. Unfortunately, protein NMR is usually limited to smaller proteins, as the spectra soon becomes too crowded with overlapping signals for any useful information to be directly elucidated.

References.

http ://www 2.chemistry .msu .edu /faculty /reusch /VirtTxtJml /Spectrpy /nmr /nmr 1.htm