REVIEW OF RELATED LITERATURE Spectrophotometer Ultraviolet and visible spectrometers have been in general use and have become

the most important analytical instrument in the modern day laboratory. In many applications other techniques could be employed but none rival UV-Visible spectrometry for its simplicity, versatility, speed, accuracy and cost-effectiveness. A spectrophotometer is an instrument for measuring the transmittance or absorbance of a sample as a function of the wavelength of electromagnetic radiation (ThermoSpectronic, n.d). When a beam of radiation strikes any object it can be absorbed, transmitted, scattered, reflected or it can excite fluorescence. The processes concerned in spectrometry are absorption and transmission. Usually the conditions under which the sample is examined are chosen to keep reflection, scatter and fluorescence to a minimum. Ultraviolet and visible spectrometry is almost entirely used for quantitative analysis; that is, the estimation of the amount of a compound known to be present in the sample (ThermoSpectronic, n.d). The key components of a spectrophotometer are: (1) a source that generates a broad band of electromagnetic radiation; (2) a dispersion device that selects from the broadband radiation of the source a particular wavelength (or more likely, a waveband); (3) a sample area; (4) one or more detectors to measure the intensity of radiation; and (5) other optical components, such as lenses or mirrors which relay light through the instrument (Owen, 2000). The ideal light source would yield a constant intensity over all wavelengths with low noise and long-term stability. Unfortunately, however, such a source does not exist. Two sources are commonly used in UV-visible spectrophotometers. The first source, the deuterium arc lamp,

yields a good intensity continuum in the UV region and provides useful intensity in the visible region. The second source, the tungsten-halogen lamp, yields good intensity over part of the UV spectrum and over the entire visible range. Most spectrophotometers used to measure the UVvisible range contain both types of lamps. In such instruments, either a source selector is used to switch between the lamps as appropriate, or the light from the two sources is mixed to yield a single broadband source (Owen, 2000). Dispersion devices cause different wavelengths of light to be dispersed at different angles. When combined with an appropriate exit slit, these devices can be used to select a particular wavelength (or, more precisely, a narrow waveband) of light from a continuous source. Two types of dispersion devices, prisms and holographic gratings, are commonly used in UV-visible spectrophotometers. Samples may be contained in a vessel made of transparent material such as silica, glass or plastic, known as a cell or cuvette. The faces of these cells through which the radiation passes are highly polished to keep reflection and scatter losses to a minimum (ThermoSpectronic, n.d). Detector converts a light signal into an electrical signal. Ideally, it should give a linear response over a wide range with low noise and high sensitivity. Spectrophotometers normally contain either a photomultiplier tube detector or a photodiode detector. Either lenses or concave mirrors are used to relay and focus light through the instrument (Owen, 2000).

Figure 1| Schematic diagram of a conventional single-beam spectrophotometer.

Energy Transitions
Chemical samples or compounds involves various orbitals (refer to figure 1) referring the relative energies of the electrons present in the valence shell of the atoms considered in the molecule.

Figure 2. Orbitals present valence electrons of atoms and molecules. EM has wave property thus wavelength can be measured. Given its specific wavelength, wherein frequency can also be calculated, and the Plancks constant, energy can be obtained.

(Equation 1)

(Equation 2)

When EM wave passes through the compound, energy from the EM wave is used to promote an electron from a bonding or non-bonding orbital into one of the empty anti-bonding orbitals.

Figure 3. Possible energy transitions upon electron excitation. Each wavelength of light has a particular energy associated with it. If that particular amount of energy is sufficient to cause one of the above mentioned energy transitions, then that wavelength will be absorbed its energy will have been used in promoting an electron into a higher energy (Clark, 2007). Typical samples used in UV-Vis spectrophotometric analyses, compounds that are able to have energy transitions within the UV-Visible region of the electromagnetic spectrum, are those that have double bonds or lone pair of electron thus allowing and n transitions to higher energy states.

Beer-Lambert Law Spectrophotometry is primarily based on the principle of Beer-Lambert Law. The law states that the concentration of a substance in solution is directly proportional to the 'absorbance ', A, of the solution.

The law is only true for monochromatic light that is, a light of a single wavelength or narrow band of wavelengths, and provided that the physical or chemical state of the substance does not change with concentration. Hence, when monochromatic radiation passes through a homogeneous solution in a cell, the intensity of the emitted radiation depends upon the thickness (l) and the concentration (C) of the solution.

Figure 2| Diagram of absorption of light by sample. Io is the intensity of the incident radiation and I is the intensity of the transmitted radiation. The ratio I/Io is called transmittance. This is sometimes expressed as a percentage and referred to as %transmittance. Mathematically, absorbance is related to percentage transmittance T by the expression: ( ) ( )

where b is the length of the radiation path through the sample, c is the concentration of absorbing molecules in that path, and a is the absorptivity ( as molar absorptivity)- a constant dependent only on the nature of the molecule and the wavelength of the radiation. For quantitative analysis, it is normally chosen to use radiation of a wavelength at which a, the absorptivity, is a maximum, i.e. at the peak of the absorption band, for the following reasons: (1) the change in absorbance for a given concentration change is greater, leading to greater sensitivity and accuracy in measurement; (2) The relative effect of other substances or impurities is smaller; and (3) the rate

of change of absorbance with wavelength is smaller, and the measurement is not so severely affected by small errors in the wavelength setting (ThermoSpectronic, n.d). Other limitations of the Beers law include that the concentrations of the samples must be low. The analyte solution must be neither fluorescent nor heterogeneous and the solute must not undergo photochemical transformations. Lastly, the solute must not undertake variable associations and reactions with the solvent (Skoog et. al., 2007) Having looked, albeit very briefly, at the theory and origins of UV-Visible spectra the technique has great deal of application to chemical analyses starting with consideration of sample state. With solid sample it is usually found that the material is in a condition unsuitable for direct spectrometry. Unless the sample can be easily made as a homogenous polished block or film, it is usual to eliminate these interfaces by dissolving the sample in a transparent solvent. Liquids may be contained in a vessel made of transparent material such as silica, glass or plastic, known as a cell or cuvette. The faces of these cells through which the radiation passes are highly polished to keep reflection and scatter losses to a minimum. Gases may be contained in similar cells which are sealed or stoppered to make them gas-tight (ThermoSpectronic, n.d). It is relatively rare to find a practical problem in which one has a mixture to be analyzed with only one component which absorbs radiation. When there are several such components which absorb at the same wavelength their absorbances add together, and that the absorbance of the sample is proportional to the concentration of one component is no longer true. In these cases, several approaches can be adopted with the most important being chemical reaction and multi-wavelength measurements. This specific study would emphasize solely on the multiwavelength measurement or method.

In a mixture of components, the observed absorption at any wavelength is the sum of the individual absorption spectra of the components thus: Measure absorbance at first wavelength (1)

Similarly at a second wavelength (2)

where

and

are the molar absorptivities of component x at wavelength 1 (1) and

wavelength 2 (2) and c is its concentration. The cell path length b is generally constant and therefore cancels. If absorbance measurements are taken at a number of different wavelengths equal to the number of components, and if the value of the absorptivities are known by measurements of the pure components at each of the wavelengths concerned, then the required or the corresponding concentrations of each components can be solved equations (ThermoSpectronic, n.d). using simultaneous

Ultraviolet Radiation UV radiation penetrates the ozone layer over two wavelength regimes, UVB (290320 nm) and UVA (320400 nm). UVB acts directly on biological molecules, causing skin cancer, skin aging, and the familiar delayed sunburn that arises 1224 hours after exposure. In contrast, UVA acts indirectly through reactive oxygen species, causing immediate sunburn that diminishes within 2 hours after exposure, and potentially plays a role in skin cancer and delayed sunburn (Hiller, 1995).

Sunscreen or Sunblock Limiting exposure and covering the skin protects the body from sunburn. Modern chemists have developed two basic types of sun protection formula, sunscreen and sunblock (Kunin 2005). Sunscreen is a chemical solution, classified by the Food and Drug Administration as a drug, which absorbs ultraviolet radiation before it can harm the skin.
Table 1. Active ingredients of Suncscreens by SPF rating and Brand

Sunblock, however, protects the skin by reflecting and scattering the sun's rays. The bestknown example of a sunblock is zinc oxide, the thick white substance that lifeguards apply to their noses. Sunblock are highly effective, but are typically sticky creams that are impractical for full body use.

Sun Protection Factor (SPF)

A sun protection factor (SPF) for rating the UVB (i.e.,delayed sunburn) protection provided by sunscreens was developed in the 1970s by Plough. The SPF is defined by the ratio

MED is the smallest dose (in J/m2) of UV radiation that produces delayed sunburn on skin. In the equation, protected skin means skin covered with 2 mg/cm2 of sunscreen, and unprotected skin means uncovered skin. The SPF also can be estimated from absorption measurements as the reciprocal of the effective transmission of UV radiation through the sunscreen. Because UVA is not a primary cause of delayed sunburn, UVA protection is not well represented by the SPF. Industry attempts to develop simple, absorption-based methods to quantify UVA protection, such as directly measuring the fraction of blocked UVA rays, have met with resistance (FDA, 1993).

Skin Diseases In recent years, exposure to ultraviolet (UV) radiation has been linked to a number of human health problems, including sunburn, skin cancer, premature aging of the skin. At the same time, evidence of aerosol-induced damage to the earths primary UV filter, the ozone layer, has increased and skin cancer rates have risen to more than 900,000 cases per year further intensifying concern over exposure to UV radiation (Runger, 1995).

Literature Cited

Kunin, A., M.D. 2005. The DERMAdoctor Skinstruction Manual. New York, NY: Simon & Schuster. Hiller, M. R. 1995. Sunscreen Education Key for Kids. Orange County Register. Runger, T. M.; Epe, B.; Moller, K. 1995. Recent Results Cancer Res. FDA. Fed. Regist. 1993, 58. Owen, T (2000). Fundamentals of Modern UV-Visible Spectroscopy Primer. Germany: Agilent Technologies. Skoog, D. A., D. M. West, F. J. Holler, and S. R. Crouch. 2004. Fundamentals in Analytical Chemistry. 8e. New York: Brooks/Cole Publishing. Skoog, D. A., F. James Holler, and S. R. Crouch. 2007. Principles of Instrumental Analysis. 6e. New York: Brooks/Cole Publishing. ThermoSpectronic. N.d. Basic UV-Vis Theory, Concepts and Applications.