Spectrophotometry

Spectrophotometry

• 1.
  Presented by ClarindaClare 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.
• 5.
   Spectrophotometer  Distilled water  Colored solution (Red, Blue, Green & Yellow)  Cuvettes
• 6.
   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.
• 7.
   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.
• 8.
   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 machine's calculations off.
• 9.
  4 2 distilled water red 0 green distilled water 425 450 yellow 475 500 525 550 575 -2 600 625 650 675 blue -4
• 10.
   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
• 11.
   Highest absorbance : Green  Lowest absorbance : Yellow
• 12.
   According to the result green has the higest absorbance.  This is due to the combination of color which is blue and yellow.
• 13.
   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:
• 14.
   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)
• 15.
   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
• 16.
   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
• 17.
   Absorbance is measured to see the relationship between concentration of a compound and its absorbance.