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Spectrophotometry

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Introduction

STUDY AND LABORATORY SKILLS - SPECTROPHOTOMETRY Introduction Much of the background to spectrophotometry and ERE (electromagnetic radiant energy) phenomena has been discussed in the schedule. The schedule states that the energy associated with ERE is calculated by the formula E=hv This is infact the incorrect terming for 'Planck's Equation' and the more widely accepted version of: E=hf Where E is the energy in joules, h is Planck's constant (6.626x10-34 Js) and f is the frequency in hertz. The reagent used is cytochrome C, which is essentially a biological extract and not a synthetic product. Aims Again the aims are already stated in the schedule. However it is necessary jus to summarise them: Determine the peak wavelength of absorption of cyt c solution Produce a calibration curve for cyt c solution (using a range of different concentrations of cyt c) ...read more.

Middle

Results The results below are the data obtained from the spectrophotometer readout. The stock solution of 0.1 mg/ml cyt c had its absorption at different wavelengths measured (from 380nm to 420nm in increments of 5nm). Table 1 The absorbance values of cyt c for a range the range of wavelengths of 380nm to 420nm Wavelength (nm) Absorbance 380 0.263 385 0.313 390 0.380 395 0.483 400 0.625 405 0.741 410 0.685 415 0.570 420 0.406 A graph of wavelength against absorbance based on table was plotted (Graph 1). Comments Graph 1 shows that the peak wavelength that cyt c absorbs at is 405nm. The relevance of identifying the peak wavelength is that it is the wavelength at which maximum absorption occurs. ...read more.

Conclusion

The peak wavelength is the wavelength of light that cause the biggest excitation of electrons in the cyt c molecules, from their ground state to the highest energy level. 2. Calibration graph of unknown Method The method is detailed as in the schedule. However some details were omitted from the data tables in the schedule. The following table shows the concentrations of the range of cyt c solutions that were used (all were dilutions of the original 0.1 mg/ml cyt c solution). Cell number 1 2 3 4 0.1 mg/ml cyt c solution (ml) 4 3 2 1 Deionised water (ml) 1 2 3 4 Final conc.of cyt c (mg/ml) 0.08 0.06 0.04 0.02 The final concentrations of the diluted cyt c were calculated by: Final concentration = (volume of deionised water/ volume of 0.1 mg/ml cyt c)/ Total volume As mentioned in the schedule, each of the different diluted cyt c solutions were tested in the absorbance ...read more.

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