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UV Spectroscopy
Research Skills For Biology
Bath Spa University
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UV-Visible Spectroscopy
According to Maxwell, light is an electromagnetic field characterised by a frequency (f), velocity (v), and wavelength (gamma). Light obeys the relationship f=v/gamma
Absorbance is set to 0% or light transmitted using a solvent blank in a cuvette (distilled water usually). This compensates for absorbance by the cell container and solvent and ensures that any absorbance registered is solely due to the component under analysis. The sample to be analysed is placed in a cuvette. Qualitative analysis is achieved by determining the radiation absorbed by a sample over a range of wavelengths. The results are plotted as a graph of absorbance/transmittance against wavelength, which is called a UV/visible spectrum.
Although the light absorbed is dependent on path length through a cell, a usual standard 1cm path length is used so it can effectively be ignored. The absorption of a sample at a particular wavelength (chosen by adjusting a monochromator) is measured and compared to a calibration graph of the absorptions
of a series of standard solutions.
What can be analysed? - in its qualitative form, UV-visible spectroscopy can be used to detect coloured species in solution, eg. bromine, iodine, and organic compounds or metal ions that are coloured, or can be converted into a coloured compound.
Lambert’s Law, now commonly known as Beer’s Law gives a direct relationship between absorbance and concentration of a solution.
In the equation above, A is the absorbance, c is the molar concentration, usually in mol/cm^3, backwards 3 is the molar coefficient, 1 is the pathlength of the cuvette (cell holder), Io is the intensity of radiation before passage through the solution, and I is the intensity of radiation transmitted through the solution. The molar coefficient is constant for a particular compound in a particular solvent at a particular wavelength. In order to determine the concentration of an unknown analyte it is important to plot a calibration curve of absorbance vs concentration for standard solutions.
Chemical applications of UV-visible spectrophotometry - Quantitative analysis (identification of the concentration of an unknown substance) - Unknown concentration of nucleic acid and proteins are a good example. Nucleic acids absorb at 254nm (or 260nm) and proteins at 280nm. Nucleic acid absorption depends on the aromatic rings of purines and pyrimidines while that of proteins at 280nm depends on the number of amino acids - tyrosine and tryptophan content and a little due to phenylalanine content. - Qualitative analysis (identification of an unknown substance)
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- Enzyme assay
- Enzyme assay can be easily, quickly and conveniently calculated when the substrate or the product is coloured or absorbs light in the UV range. In these cases, the rate of appearance or disappearance of light
UV Spectroscopy
Module: Research Skills For Biology
University: Bath Spa University
Students also viewed
- Microbial Growth & Nutrition
- Food Poisoning and you
- Mass Movement Hazards
- Complex Conflicts in Hazard Management
- Exam - Information regarding the exam that took place at the end of the year, provided
- GEO5100-20 Lecture 1 - An introduction to the course, outlining the key concepts and what will be expected