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Spectrophotometry

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  • Plug in and power on the spectrophotometer. Run the machine for five up to 10 minutes to allow it to warm up.Prepare colored solution (red, blue, green & yellow.Fill cuvette with colored solution.Place the cuvette in the spectrophotometer.Adjust the wavelength from 425nm. Repeat steps by using the other coloured solution.
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    • 1. Presented by Clarinda Clare Linus Diploma in Medical Sciences January intake 2011
    • 2.  Measure the wavelengths of visible light that various colores dolutions absorb. Understand reason/s as to why each solution appears as a particular color after gathering results. Learn and appreciate the concepts of spectrophotometry.
    • 3.  A spectrometer (spectrophotometer, spectrograph or spectroscope) is an instrument used to measure properties of light over a specific portion of theelectromagnetic spectrum, typically used in spectroscopic analysis to identify materials.
    • 4.  Spectrophotometer Distilled water Colored solution (Red, Blue, Green & Yellow) Cuvettes
    • 5.  1. Turn on the power switch, set the desired wavelength and allow to warm up at least 15 minutes. 2. Set the transmittance to zero. 3. Place the blank into the sample compartment with the vertical line on the cuvette aligned with the mark on the front edge of the sample compartment, and close the lid. 4. Remove the blank from the sample compartment. Change to "Absorbance" using the "mode" button.
    • 6.  6. Place a cuvette containing color sample into the sample compartment, close the lid, and read the absorbance from the meter. 7. Record readings and remove sample. 8. Repeat steps 1 – 7.
    • 7.  Special light filters may be required on some spectrophotometers if working at certain wavelengths. The machine must be zeroed out after each sample or if changing the wavelength. Failure to allow the spectrophotometer enough time to warm can result in erroneous results. Make sure cuvettes are free of any particles, smudges or fingerprints, as these can throw the machines calculations off.
    • 8. 42 distilled water red0 green distilled water 425 450 yellow 475 500 525 550 575-2 600 625 650 675 blue-4
    • 9.  Distilled water – The highest spectrum is at 425nm Red – The highest spectrum is at 525nm Green – the highest spectrum is at 625 nm Yellow – the highest spectrum is at 425nm Blue – The highest spectrum is at 425nm
    • 10.  Highest absorbance : Green Lowest absorbance : Yellow
    • 11.  According to the result green has the higest absorbance. This is due to the combination of color which is blue and yellow.
    • 12.  The concentration of colored solute in a solution is directly proportional to the intensity of its color, which in turn is proportional to the amount of absorbance of light at the wavelength that the color absorbs. The color, or absorbance, of a solution is also proportional to the path length that the light passes through. This is often expressed as the Beer-Lambert Law, or Beer’s Law:
    • 13.  A=εCl Where A is absorbance at a given wavelength of light, ε is the extinction coefficient (amount of color of the solute per mole) C is the concentration of solute in the solution (doubling the concentration doubles the amount of light absorbed) and l is the pathlength (if you double the length of the path of light, you double the opportunities for it to be absorbed)
    • 14.  The sequence of events in a modern spectrophotometer is as follows: The light source is imaged upon the sample A fraction of the light is transmitted or reflected from the sample The light from the sample is imaged upon the entrance slit of the monochromator The monochromator separates the wavelengths of light and focuses each of them onto the photodetector sequentially
    • 15.  Spectrophotometric techniques are used to measure the concentration of solutes in solution by measuring the amount of light that is absorbed by the solution in a cuvette placed in the spectrophotometer. Spectrophotometry takes advantage of the dual nature of light. Namely, light has: 1. a particle nature which gives rise to the photoelectric effect 2. a wave nature which gives rise to the visible spectrum of light
    • 16.  Absorbance is measured to see the relationship between concentration of a compound and its absorbance.