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.
  • Spectrophotometry

    1. 1. Presented by Clarinda Clare Linus Diploma in Medical Sciences January intake 2011
    2. 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. 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. 4.  Spectrophotometer Distilled water Colored solution (Red, Blue, Green & Yellow) Cuvettes
    5. 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.  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. 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. 8. 42 distilled water red0 green distilled water 425 450 yellow 475 500 525 550 575-2 600 625 650 675 blue-4
    9. 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. 10.  Highest absorbance : Green Lowest absorbance : Yellow
    11. 11.  According to the result green has the higest absorbance. This is due to the combination of color which is blue and yellow.
    12. 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. 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. 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. 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. 16.  Absorbance is measured to see the relationship between concentration of a compound and its absorbance.

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