Spectroscopic Techniques Used On Uv And Fluorescence Spectroscopy Biology Essay


Spectroscopic techniques investigate the relations of electromagnetic radiation with substance. Since light is considered as a wave with electric and magnetic fields, which are mutually perpendicular and radiate out from a source in all directions, it is therefore a form of electromagnetic radiation. This description of wave was an experimental result of Maxwell et.al in the nineteenth century. They established a relationship between a wavelength ÊŽ (i.e. the distance between one parts of the wave to the corresponding position on the next wave), and frequency, v (the number of times a wave passes through a fixed point in space per second). Wavelength and frequency are directly related to the energy, E, of the wave.

E= hc/ÊŽ= hv: where h= plank's constant, c= the speed of light. High energy radiation has a short wavelength and high frequency, and low energy radiation in the reversed form. There are several different techniques of spectroscopy, such as UV-light spectroscopy, Fluorescence spectroscopy, Electron Spin Resonance (ESR) spectroscopy, Nuclear Magnetic Resonance (NMR) spectroscopy, Infrared spectroscopy, etc. Each technique measures different type of interactions using precise wavelength of light. This provided different information on the biological molecule being investigated. Spectroscopic techniques are very important in pharmaceuticals industry and in particularly biochemical research areas. This essay will focus on UV-visible absorbance and Fluorescence spectroscopy.

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The UV-visible spectroscopy

It is observed that light from the sun appears white, but when impacted on a colour object, some of it is absorbed and it is the reflected or non-absorbed light that gives out the colour awareness. When light in ultraviolet/visible region of passes through a sample solution, some light energy may be absorbed. Fig1 below shows absorption spectrum of Cyclotene* 4024-40 Resin.

UV/Visible Spectrum of CYCLOTENE* 4024-40 Resin


A spectroscopy experiment of particular frequencies at which light is absorbed is affected by both the structure and the environment of the chromophore (molecules or parts of molecules capable of absorbing light). To promote electron from the ground state to different excited states as shown in the diagram below, light energy is required.

Fig 2


Transition is the change between energy levels which represent the energy needed to move one electron from one orbital to another.

Each chemical structure's absorption differs in frequency of light absorbed due to their characteristic electronic structure. The ground and excited electronic energy levels of each contains numerous vibration energy levels differing from each other by smaller energy increments than these between the electronic energy levels (ΔE). Excited electrons can return to the ground state via vibration transitions, by means of excess energy lost in collision with solvent molecules. A light energy absorbed in solution therefore appears to heat (kinetic energy).

The absorption phenomenon is quantified by the Beer-Lambert law: log (I°/I) = εcl

Where I° is the intensity of incident light, I is the intensity of transmitted light, c is the molar concentration and l is the length of the path (usually 1cm). Ƹ is the molar extinction coefficient. The absorbance (Aλ) at particular wavelength/frequency is termed (log10 (I0/I). An absorption spectrum is the plot of probability of photon absorption against wavelength. Refer back to fig 1.

The amplitude is the maximum value the electric or magnetic vector can have. As shown on fig 3 below.


The wavelength corresponding to the maximum absorption is called ÊŽmax. As each electronic energy level consists of many vibrational energy levels, a scope of wavelength is absorbed rather than on fixed wavelength.

Under standard conditions, this spectrum is a fixed property of a pure chromophore and may therefore, as a reference point of concentration provided that a standard curve for that chromophore is also available. Absorbance is a suitable method of measuring concentration of a solution. There is a linear relationship that can reach a maximum of infinity. Absorbance are best accurately measured between 1 and 3, above 3 is not usually accurate. Knowing that amino acids have a strong absorbance around 210 nm, this is frequently used to detect peptides. Aromatic amino acids, like tyrosine and tryptophan have relatively strong absorbance at 280 nm while nucleic acids absorb strongly at 260 nm. These wavelengths are subsequently widely used in studies of proteins and nucleic acids respectively. The reduction of NAD to NADH causes major increases in absorbance at 340 nm which is taken advantage of in assay of oxidareductase enzymes.

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Since the absorbance spectrum is only partly determined by it chemical structure under standard conditions, the environment of the chromophore affecting its precise spectrum should be considered. The most important factors affecting the absorption spectra are: pH, polarity of the solvent or its neighbouring molecules, and relative orientation of nearby chromophores. These factors are particularly important in studies of biopolymers like proteins and nucleic acids in determining their environments where chromophore acts like receptor molecules.

The protonation/deprotonation effects from pH changes or oxidation/reduction effects on chromophore those have a spectacular differences between the absorption spectra on the chromophore.

Evaluation of spectra for protonated and deprotonated tyrosine reveal isosbestic points where absorbance of both forms of the chromophore are identical. It is frequently possible to work out a wavelength where only one from of the chromophore has a strong absorbance and the other does not. Measurements of absorbance over the varieties of pH range may be used to calculate the pKa of the relevant group.

Solvent polarity also affects the absorption spectrum determined for chromophore.

Alternative solvent to water in aqueous solution give a slightly different spectrum known as perturbation.

Orientation effects: hyperchroism of nucleic acids. Absorbance of nucleic acids decreased when the nucleotide is in a single stranded polynucleotide and decreases further in a double stranded polynucleotide. In most cases changes in spectra as a result of environmental influence are obvious in experiment.

Equipments used in absorption spectroscopy.

Absorption spectrum is measured using a spectrophotometer, a light source provided by an electromagnetic radiation, a monochrometer or grating, a cuvette (sample holder), a detector and a recorder. There are varieties of spectrophotometers available including single beam, split (double) beam and dual beam instruments.

Fluorescence Spectroscopy

Some molecules emit a new lower energy called fluorescence after an absorption of a photon, and the molecule is called a fluorophore.

Due to the rigidity and inflexibility of some molecules, their vibrational energy level range may be limited. In such molecules returning to the ground state means undergoing a series of radiative transitions. The excited molecules return to ground state by dissipation of the absorbed energy as heat (kinetic energy) by emitting light in non-radiative means. This characteristic fluorescence or emission spectrum is evident in the absorbance spectrum.

Figure 4 shows excitation of three different fluorophore at different wavelengths (EX 1, EX 2, EX 3). Though, this does not have any impacted on the emission shape but does impacted variations in fluorescence emission intensity (EM 1, EM 2, EM 3) that match to the amplitude of the excitation band".

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If the excited state and the ground state vibrational level overlaps, energy can be transfer from higher energy level to lower energy level by non-radiative transfer. Non-radiative transfer to neighbouring molecules competes with fluorescence (quenching).

Fluorescence measurements are performed in a specrofluoriment. The structural rearrangement (internal quenching) and the interaction of excited molecule with another molecule in a sample (External quenching), affect the amount of light energy emitted. All forms of quenching result in non-radiative loss of energy. Fluorescence is emitted in all directions and commonly measured at 90o or as backscatter. Biochemistry usually quantified fluorescence by Quantum yield, Q.

Q= number of photon emitted/number of photons absorbed.

An intrinsic fluorophore is one contained within the macromolecule, e.g. Tyrosine, Tryptophan and Phenylalanine residues. Extrinsic fluorophore is one added to the macromolecule, ideally should bind to a single site in the macromolecule. Fluorescence spectra are mostly influenced by exposure to solvent and or quenchers present in the solvent. This fact underlies much of the practical usefulness of fluorescence spectroscopy in biochemistry. Ligand binding often caused quenches to the three-dimensional structures of protein. Such changes of fluorescence can be determined at a particular wavelength provided the fluor has a unique location. Overlaps of absorption peaks of fluorophore can transfer energies by resonance energy transfer. Resonance Energy Transfer (RET) is proportional to distance (R) between the fluors: E= KT/KT + Kf + K1;

Where; KT, Kf, and K1 are respectively the rates of transfer of excitation energy, and the sum of other deexcitation energies. E can be determined from fluorescence intensity (f) or the excited state lifetime (T) of the donor determines in the presence of (da) and absence of (d) of the acceptor as follows:

E= 1-(Fda /Fa)

E= 1- (Tda/ Td)


Both UV-visible and Fluorescence are affected by the follow factors: pH of the solvent, orientation effects, temperature of their environment and polarity. Fluorescence is affected further by ligand binding and quenching. Both methods used Beer-Lambert law, though there may positive or negative deviations in some cases which violate the Beer-Lambert law. UV-visible determines the concentration of the biomolecule, assay on chemical and enzymatic reactions, identified unknown substances, determine pka from the pH titration curve, determined binding site of protein and hypochromicity of nucleic acids.

Fluorescence determined the three-dimensional structure of protein. It is very sensitive, and used to determine emitted light from firefly luciferase and peroxidises enzymes respectively.


Spectroscopic techniques are of paramount important in both biochemistry research and in the pharmaceutical industry. Identifying both the chemical and three-dimension studies of protein helps in developing new drugs and modifying protein structures for gene manipulation. Beer-Lambert law is used in all techniques.