Spectrophotometry basics and principle.pptx

Spectrophotometry is a method to measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through sample solution. The basic principle is that each compound absorbs or transmits light over a certain range of wavelength. This measurement can also be used to measure the amount of a known chemical substance. Spectrophotometry is one of the most useful methods of quantitative analysis in various fields such as chemistry, physics, biochemistry, material and chemical engineering and clinical applications.

1. Spectrophotometry

2. Light Electromagnetic radiation is described as the photons of energy traveling in waves. The Electromagnetic waves spectrum is a wide variety of radiation with different wavelength.

3. Planck’s formula Relation between energy, frequency and wavelength is described by Planck’s formula “E = hv” E - energy h - Planck's constant v - Frequency. Whereas, V = 1/ λ λ – is wavelength of the radiation.

4. V=1/ λ Thus it states that frequency is inversely proportional to wavelength. And so the Energy is also inversely proportional to wavelength.

5. Interaction of light with matter. As the radiant energy with particular frequency is provided to an atom, ion, molecule with a right vibration/frequency, the energy is absorbed which bumps the electron to higher energy valence orbit, Excited electron to higher orbit is unstable, it returns to ground/stable state by emitting energy.( which can be light, vibrational, kinetic energy) – this principle is used in flame photometry or fluorometry.

6. Colours and complementary colours of visible spectrum

7. Beer-Lambert law Absorbance of radiant energy is directly proportional to the concentration of the solute/substance. % transmittance of radiant energy is inversely proportional to the concentration of solute/ substance . Absorbance of radiant energy is directly proportional to the concentration of the solute/substance. % transmittance of radiant energy is inversely proportional to the concentration of solute/ substance

8. Beers law deviation/ limitations Things that can cause deviation from Beer’s law – 1). Incident radiation being non monochromatic. 2). Sides of the cuvette. 3). Stray radiation (diffracted part of radiation, which are not in wavelengths set my the monochromator). In solution that that has a mix of two different solution which have different absorptivity of the wavelength of light impeded, will not follow the Beer’s law. If absorbance of a fluorescent solution is measured, Beer’s law will not be followed.

9. Instrumentation Single beam spectrophotometer Major components Stable radiant energy source Entrance slit Wavelength selector Exit slit Cuvette holder Radiant energy detector Electric signal reader

10. Radiant energy source. Tungsten filament lamp- ( 360-950nm ). Tungsten iodide used as a visible and near uv radiant energy source. (tungsten halide filament are longer lasting). Deuterium discharge lamp ( 220-360nm ). Mercury lamp (line spectrum - 313, 365, 405, 436, 546nm ). Selection of lamp based on the requirement for specific analysis is of utmost importance.

11. Wavelength selector. Filter – simple device where it has a material that transmit specific wavelength and absorb rest. 1)glass and Wratten filter, 2) Interference filter. Monochromator: 1) Prism, 2) Grating. Glass filter – contains two or more coloured glass. Wratten filter – contains coloured gelatin between two glass plates. Interference filter -->

12. Monochromator – a device used to isolate a particular wavelength . 1) Prism- has more optical efficiency- which is because light comes out as single order of emerging spectrum. 2) Grating – has number of parallel lines equally spaced on a surface.

13. Entrance and exit slits, Cuvettes Importance of entrance slit is to focus the source radiation on the dispersion device (filter, prism etc) While exit slit is important in determining the bandwidth of emerging light from dispersed spectrum onto the cuvette (sample tube). Cuvettes – receptacle of sample for analysis, also known as cells, Glass cuvettes for wavelength 320-950nm. Quartz cuvettes for wavelength below 320nm. .Square cuvettes give more accuracy than normal test tube cuvettes.

14. Detectors Barrier layer cell or photocell – composed of light sensitive (copper oxide or selenium) over iron plate and over it is a thin transparent layer of silver. When radiation hits the light sensitive material it generates electron movement which is transmitted to less sensitive layer and just producing current by electron movement. This current is recorded by ammeter .

15. Photodiode – a semiconductor which give a change in voltage when struck by light. Photomultiplier tube- detects and amplifies the radiant energy. . As the light hits a cathode electron is emitted, and it bumps off on subsequent cathodes with charge one greater than other. And in end giving an amplified levels which is read by ammeter.

16. Quality Control Check of Spectrophotometers Wavelength accuracy – wavelength indicated on the control dial is the actual wavelength of the light passed by monochromator. Checked using standard solution or filters. Mercury vapor lamp and deuterium lamp, Didymium or holmium oxide (241, 279, 287, 333, 361, 418, 453, 536, 636nm ), in glass is frequently used filters. As the filters can deteriorate over time it must also be checked at particular time interval to know its accuracy.

17. Linearity of detector response : A good working machine Will show the absorbance vs concentration graph when plotted, Appears as a straight line, as discussed under Beer’s law. Some solutions which follow Beer’s Law, with increasing concentration is used to check this response ( eg : nitrophenol at 405nm, Oxyhemoglobin at 415nm, etc).

18. Stray radiation: Radiation whose wavelength is outside the splitting wavelengths of monochromator. Can be caused due to damages to the optical surfaces or dust. If any stray light radiation Is present certain filters can be used to assess it. ‘cut off filters’ can be used where the radiation beyond its limit is cut off from passing through. Examples: Acetone( below 320nm), Lithium carbonate(below 250nm). The transmittance is shown using these filter and the percentage is calculated, if it is beyond 1% machine must be calibrated or cleaned based on the issue.

19. Photometric accuracy- To check the absorbance photometric accuracy a standard that has absorbance for particular wavelength which doesn’t get affected by spectral bandwidth. Examples for standard– SBM 930 filter, which has a absorbance to 4 wavelengths, suggested by (NIST)national institute of standards and technology. Materials used frequently are – potassium dichromate solution, cobalt ammonium sulfate solution, potassium nitrate solution.

20. Thank You.