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Spectrophotometer Introduction Spectrophotometry is a technique used to determine the amount of protein or nucleic acid present in a given solution; this can be achieved by determining amount of light (electromagnetic radiation) that is absorbed by the solution. In addition, the solution used in this experiment was cytochrome-C. The amount of light that passes through the solution unabsorbed is measured by the photocells on the other side of the cuvette. Spectrophotometer is a useful technique in measuring the concentration of an unknown analyse, this is done by measuring the amount of light absorbed by the sample. Absorption of light by a sample Photons are absorbed as light passes through a solution that contains light absorbing species; as a result, the radiant power of the monochromatic beam decreases. The radiant power of a beam of light is the number of photons per second at right angles to the beam. Diagram showing the pathway of the beam of light through a sample. The radiant power of a beam of monochromatic light can be referred to as 'Io' (original intensity of light sample), and the radiant power of light that exists in the solution can be reoffered to as 'I' (intensity of light after passing through the sample). The Io measurement is obtained by firstly placing a cuvette containing a blank solution into the spectrophotometer. ...read more.


The general trend within the results displayed was that the absorbance increased with a higher wavelength up to a certain wavelength and then the absorbance started to fall (values were decreasing). This trend was plotted in a graph; refer to the absorbance spectra graph . Table 2: Shows how the serial dilutions of the stock 0.1mg/ml were made using a 5ml pipette. Cell Number Stock Vol. of cyt.c (V1) (mg/ml) Diluent's Vol. of de-ionised water (ml) Working vol. (V2) (mg/ml) Dilution Factor 1 4.0 1.0 5.0 4/5 = 0.8 2 3.0 2.0 5.0 3/5 = 0.6 3 2.0 3.0 5.0 2/5 = 0.4 4 1.0 4.0 5.0 1/5 = 0.2 The stock solution is the concentration of the solution being diluted. The working solution is the diluted solution, which is ready for use. The dilution factor is a number that describes the strength of the dilution. It is equal to the volume of stock solution used (V1), divided by the total working volume (V2). So (V2) = (V1) + volume of dilutent used. The equation for the dilution factor can be expressed as V1 = V2. Table 3 The value 0.003 was determined by filling the cuvette with water in order to determine the absorbance. This was carried out to see if the two cuvettes differed appreciatebly in amount of light transmitted. ...read more.


* Other causes may have been due to carrying out of incorrect procedures, such as some students may have pipetted directly from the stock solution *students may have washed out the pipette with distilled water instead of using the stock solution. *failure to wipe the surface of the cuvette clean of fingerprints before making an absorbance measurement *not ensuring that all solutions measured were free of bubbles. *Not making sure that the cuvette was dry before usage and holding the cuvette from the sides instead of holding only the top edge of the ribbed sides. All of the factors outlined above may have been a cause of the irregularity of the calibration curve. An absorbance spectrum is the relative absorbance value of the solution (cyt.C) at different wavelengths within the visible range. The absorbance spectrum plotted correlating to the results obtained is shown in graph 1. The reason why the absorbance was measured at a wavelength that corresponded to maximum absorbance reading was because the greatest change in the concentration of the absorbing species is seen at the maximum wavelength and therefore, the capability of the spectrophotometer to detect small changes in the concentration of the absorbing species maximises. Not only this but as explained above, after the peak absorption had been reached at the maximum wavelength possible, the changes in wavelength after these values are very small. Beer's law will no longer be followed. ...read more.

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