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Spectrophotometer.pptx

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Spectrophotometer.pptx

  1. 1. Spectrophotometer
  2. 2. A spectrophotometer is an instrument that measures the amount of light absorbed by a sample. Spectrophotometer techniques are used to measure the concentration of solutes in solution by measuring the amount of the light that is absorbed by the solution in a cuvette placed in the spectrophotometer .
  3. 3. Lambert’s Law • The proportion of light absorbed by a medium is independent of the intensity of incident light. • A sample which absorbs 75% (25% transmittance) of the light will always absorb 75% of the light, no matter the strength of the light source. • Lambert’s law is expressed as I/Io=T Where I = Intensity of transmitted light Io = Intensity of the incident light T = Transmittance • This allows different spectrophotometers with different light sources to produce comparable absorption readings independent of the power of the light source.
  4. 4. Beer’s Law • The absorbance of light is directly proportional to both the concentration of the absorbing medium and the thickness of the medium. • In Spectrophotometry the thickness of the medium is called the pathlength.
  5. 5. The spectrophotometer technique is to measures light intensity as a function of wavelength. It does this by: 1. diffracting the light beam into a spectrum of wavelengths 2. direct it to an object 3. receiving the light reflected or returned from the object 4. detecting the intensities with a charge-coupled device 5. displaying the results as a graph on the detector and then the display device .
  6. 6. 1)Measure the concentration of the solution A spectrophotometer optically determines the absorbance or transmission of characteristic wavelengths of radiant energy (light) by a chemical species in solution. Each molecule absorbs light at certain wavelengths in a unique spectral pattern because of the number and arrangement of its characteristic functional groups, such as double bonds between carbon atoms. According to the Beer-Lambert law, the amount of light absorbed at these wavelengths is directly proportional to the concentration of the chemical • species.
  7. 7. 2) Identify organic compounds by determining the absorption maximum. Spectrophotometers are used to identify organic compounds by determining the absorption maxima (which for most compounds and groups of compounds have very distinct fingerprints (that's what the absorption curves and peaks are called). 3) Used for color determination within the spectral range If one is working in the range of 380 to 700 nm, the spectrophotometers can also be used for color determination within this spectral range
  8. 8. Observed Color of Compound Color of Light Absorbed Approximate Wavelength of Light Absorbed Green Red 700 nm Blue-green Orange-red 600 nm Violet Yellow 550 nm Red-violet Yellow-green 530 nm Red Green 500 nm Orange Blue 450 nm Yellow Violet 400 nm
  9. 9. 1)Light source The function of the light source is to provide a sufficient of light which is suitable for marking a measurement. The light source typically yields a high output of polychromatic light over a wide range of the spectrum.
  10. 10. Tungsten Lamp • the most common light source used in spectrophotometer. • This lamp consists of a tungsten filament enclosed in a glass envelope, • Wavelength range of about 330 to 900 nm • used for the visible region. • It has long life about 1200h.
  11. 11. II) Hydrogen / Deuterium Lamps • For the ultraviolet region • hydrogen or deuterium lamps are frequently used. • range is 200 to 450 nm. • Deuterium lamps are generally more stable and has long life about 500h. • This lamp generates continuous or discontinuous spectral.
  12. 12. III) Xenon flash lamps 1)Their range between ( 190nm - 1000 nm) 2) Emit both UV and visible wavelengths 3) Long life 4) Do not heat up the instrument 5) Reduce warm up time
  13. 13. 2) Dispersion devices a)Monochromator Accepts polychromatic input light from a lamp and outputs monochromatic light. Monochromator consists of three parts: I) Entrance slit II) Exit slit III) Dispersion device (includes prism, filter, diffraction grating )
  14. 14. • 3)Focusing devices Combinations of lenses, slits, and mirrors. Variable slits also permit adjustments in the total radiant energy reaching the detector.
  15. 15. • Optical Materials • 1)Mirrors Type of rays Mirror material • X-rays – Ultraviolet(UV) Aluminum • Visible - Aluminum • Near infrared - Gold • Infrared (IR) - Copper or Gold
  16. 16. • 2)Lenses • Rays -Material • X-rays Ultraviolet -Fused silica , Sapphire • Visible - Glass • Infrared - Glass
  17. 17. • 4)Absorption cells(Cuvettes) A cuvette is a kind of cell (usually a small square tube) sealed at one end, made of Plastic, glass or optical grade quartz and designed to hold samples for spectroscopic experiments. For measurements in the visible region, cuvettes of optical glass are sufficient; however, optical glass absorbs light below 350 nm , and more expensive quartz or fused silica must be used for these wavelengths
  18. 18. 5)Detectors Any photosensitive device can be used as a detector of radiant energy .The photocell and phototube are the simplest photodetectors, producing current proportional to the intensity of the light striking them .
  19. 19. 6)Display devices The data from a detector are displayed by a readout device, such as an analog meter, a light beam reflected on a scale, or a digital display , Or liquid crystal display(LCD) .The output can also be transmitted to a computer or printer.
  20. 20. Types There are two classes of spectrophotometers: 1)Single beam The single beam spectrophotometer was the first invented, and all the light passes through the sample. In this case, to measure the intensity of the incident light, the sample must be removed so all the light can pass through. This type is cheaper because there are less parts and the system is less complicated.
  21. 21. The advantages of the single beam design are low cost, high throughput, and hence high Sensitivity , because the optical system is simple. The disadvantage is that an appreciable amount of Time elapses between taking the reference (I) and Making the sample measurement (Io) so that there can be problems with drift. This was certainly true of Early designs but modern instruments have better electronics and more stable lamps, so stability with single beam instruments is now more than adequate for the vast majority of application.
  22. 22. 2)Double beam * The double beam instrument design aims to eliminate drift by measuring blank and sample virtually simultaneously. A "chopper" alternately transmits and reflects the light beam so that it travels down the blank and the sample optical paths to a single detector.. * To measure a spectrum with a double beam instrument the two cuvettes, both containing solvent are place in the sample and reference positions and a "balance” measurement is made. This is the difference between the two optical paths and is subtracted from all subsequent measurement. The sample is then placed in the sample cuvette and the spectrum is measured. I and Io are measured virtually simultaneously as described above.
  23. 23. The advantage of the double beam design is high stability because reference and sample are measured virtually at the same moment in time. The disadvantages are higher cost, lower sensitivity because throughput of light is poorer because of the more complex optics and lower reliability because of the greater complexity.
  24. 24. Applications • Nucleic acid estimation:  spectrophotometry can be used to estimate DNA or RNA concentration and to analyze the purity of the preparation. Typical wavelengths for measurement are 260 nm and 280 nm.  Purines and pyrimidines in nucleic acids naturally absorb light at 260 nm. For pure samples it is well documented that for a pathlength of 10 mm, an absorption of 1A unit is equal to a concentration of 50 µg/ml DNA and 40 µg/ml for RNA. For oligonucleotides the concentration is around 33 µg/ml but this may vary with length and base sequence.  So for DNA: Concentration (µg/ml) = Abs 260 × 50.
  25. 25. Direct UV measurement: A260/A280 Ratio The most common purity check for DNA and RNA is the A260/A280 ratio Any protein contamination will have maximum absorption at 280 nm For DNA the result of dividing the 260 nm absorption by the 280 nm needs to be greater or equal to 1.8 to indicate a good level of purity in the sample. For RNA samples this reading should be 2.0 or above. Results lower than this are indicative of impurities in the sample
  26. 26. A260/A230 Ratio • An increase in absorbance at 230 nm can also indicate contamination, which may in turn affect the 260 nm reading for DNA and RNA. A number of substances absorb at 230 nm, as this is the region of absorbance of peptide bonds and aromatic side chains • Phenol contamination also increases a sample’s absorption at 280 nm and therefore can be identified through a lower A260/A280 ratio. An A260/A230 ratio of 2 or above is indicative of a pure sample.

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