Ultraviolet Spectrophotometry To Characterise Vitamin B12 And Lysozyme Biology Essay

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Spectroscopy originated in 1801, from the work of William Wollaston, a British scientist who discovered dark lines in the solar spectrum. Wollastons work was repeated in 1814 by Jospeh Von Fraunhofer, whom speculated these dark lines were caused by the absence of certain wavelengths of light [19].

In 1859, the German physicist Gustav Kirchoff made a great breakthrough when he successfully purified substances and proved that each pure substance produced a distinctive light spectrum. This discovery led to the birth of analytical spectroscopy, which Kirchoff later developed into a tool to determine the chemical composition of matter. In fact, shortly after, Kirchoff liaised with Robert Bunsen to uncover the chemical constitution of the sun [13; 9].

At the start of the 20th century, developments in spectroscopy were accelerated, when Johann Balmer and Johannes Rydberg developed equations to explain the frequency spectrum of hydrogen. Working with this recent discovery, in 1913 Niel Bohr worked out that the energy levels of hydrogen could be calculated, but his famous model fell through when he learned that it could not be applied to elements containing more than one electron. However, when Erwin Schrödinger and Werner Heisenberg developed Quantum mechanics in 1925, the spectra of most elements were explained, and this has paved the way to modern spectroscopy[16].

The three main variations of current spectrophotometry include absorption, emission, and scattering spectroscopy. Ultraviolet- visible (UV-visible) spectroscopy is a form of absorption spectroscopy, and works by measuring wavelengths of light that a substance absorbs, to give information about the structure [15]. It is a very simple, reliable and reproducible technique used to identify, characterise and quantify many biological molecules. REFERENCE!

What is Spectroscopy?

UV/visible spectroscopy is associated with the region of the electromagnetic spectrum that emits or absorbs ultraviolet and visible waves. The wavelength of this region is typically between 200-800nm [3]. However, deep- and near- UV (DUV and NUV) spectrometry is concerned with a spectral region of 250-400nm [2]. In addition, recent advances have demonstrated single 8.7 femtosecond DUV pulses with a refined spectral range of 255-290nm [12].

UV-vis spectroscopy pertains to the excitation of molecular species, where there is absorption of visible and/or ultraviolet radiation. This excitation instigates rovibrational states in ground and excited electronic states. The type of excitation can determine whether or not charge is transported, evoking the excitation of delocalized electrons, and a potential ionizing process [1]. The Royal Society of Chemistry describes the excitation of electrons as the electronic translocation from lower to higher energy levels, within both atoms and molecules [3]. The complication in molecules is owing to the fact that the relative ease of electronic excitation in various bonding situations is reflected in the frequency of the radiation absorbed. To exemplify, typically the excitation of π-bonded electrons occur at a lower frequency (higher wavelength) than σ-bonded electrons [21]. Energy levels associated with electron orbitals are precisely determined by quantum mechanics and solutions follow the laws of the Shrödinger equation, with limited transitions. Thus, taking into account the fact that energy levels of matter are quantized, the only light absorbed is that with a significant and very unique energy to cause transition from one level to another [3]. Figure 1 shows the possible transition of electrons caused by light.

Figure 1. Light provoked electronic transitions

Each case shows an electron excited from a full orbital from ground state or a low energy level, into an empty anti-bonding (lumo) orbital of higher energy (i.e. excited state). The corresponding wavelength of the particular light energy that is sufficient to cause one of these transitions, is absorbed. Larger gaps between energy levels equate to a greater required energy to promote electronic excitation to a higher energy level, affecting light of a higher frequency, and thus absorption of a shorter wavelength [3].

Electronic excitation occurs in all molecules upon absorption of light, but frequently very high energy known as vacuum ultraviolet is required (<200nm). Hence light absorption in the UV-visible region will only result in the transition shown in figure 2 [3].

Figure 2. Absorption of light in molecules

Only these transitions occur in molecules. For light to be absorbed within the range of UV-visible light i.e. 200-800nm, the molecule must contain either ς bonds or atoms with non-bonding orbitals. Non-bonding orbitals consist of one or more lone pairs of electrons[3].

Table 1 discusses the variation of molecules yielding different electronic excitation.

Table 1. Molecules of different excitatory states and colours


Extent of Excitation and colour



ChemSpider 2D Image | ethene | C2H4

Both ethene and buta-1,3-diene absorb light in the UV region and are therefore both colourless.

Π bonds are formed by an overlap of half-filled p orbitals on the 2 carbon atoms of the double bond. Both electrons in the electron cloud are found in the resulting π bonding orbital in the ground state[3].


ChemSpider 2D Image | butadiene | C4H6

Conjugated system :. Single and double bonds alternate, causing delocalization of electrons where p-orbitals overlap in double bonds. This delocalization increases the energy gap between π orbitals, subsequently causing π* to decrease in size, favouring the absorption of light with lower energy and longer wavelengths [3].

Octahedral copper complex [Cu(H2O)6]2+


Highly colourful

Transition metal complexes - d orbitals split when ligands approach and bond to the central metal ion. Extent of splitting depends on central metal ion and ligands. D-orbitals can gain or lose energy. Energy difference between new energy levels determines the amount of energy that will be absorbed when an electron is raised to a higher energy level. This energy will decide the colour of light to be absorbed. E.g. [Cu(H2O)6]2+ absorbs yellow light [3].

Adapted from Spectroscopy in a Suitcase, The Royal Society of Medicine [3]

Applications of UV-visible spectroscopy

The fundamental core for any biologist, is the ability to understand biochemical techniques in the analysis of biological extracts. Basic biochemical knowledge is essential for researchers studying many different aspects of biology, including biochemical pathways involved in the synthesis of cell structures and metabolites; molecular basis of gene expression and regulation [18; 17; 7]; secretion of pathogenic molecules by bacteria and fungi [20]; diagnosis of disease [8; 11; 10; 14]; and the metabolism of drugs within the human body [6; 4; 22; 5]. Even more interesting, this technique can be used by a Zoologist in a quest to determine the pregnancy status of females in packs of wolves, by collecting droppings and analysing the hormonal content in the laboratory.


This article aims to identify and quantify vitamin B12 and lysozyme by using UV-visible spectroscopy. The technique will enable absorption spectra to be produced, and following the Beer-lambert law will enable allow the concentrations to be derived from the colorimetric data.

Vitamin B12, otherwise known as cyanocobalamin,