1. visible spectophotometry
&
Spectrophotometry

2. INTRODUCTION
Absorption Spectrophotometry in the ultraviolet and visible region is one of the most oldest methods used for quantitative analysis and structural elucidation.
The ultraviolet and visible Spectrophotometry is mainly used for quantitative analysis and used as a auxiliary tool for structural elucidation.

3. Useful Terminology
Photometer is an instrument which measuring the ratio, or some function of the two of radiant power of two electromagnetic beams.
Colorimetry is the use of the human eye
to determine the concentration of colored species.
Spectrophotometry is the use of instruments to make the same measurements. It extends the range of possible measurements beyond those that can be determined by the eye alone. it use more sensitive detectors.

4. Colorimetry
Visual Observations – Because colorimetry is based on
inspection of materials with the human eye, it is
necessary to review aspects of visible light.
Visible light is the narrow range of electromagnetic
waves with the wavelength of nm.
                                                   
ROY G. BIV
= the mnemonic used to remember the colors of the visible spectrum.

5. Visible light is only a very small portion
of the electromagnetic spectrum.
Note: Frequency (υ) and Energy (E) are directly proportional
whereas Frequency (υ) and Wavelength (λ) are inversely proportional.

6. Electromagnetic Spectrum
Type of Radiation
Frequency Range (Hz)
Wavelength Range
Type of Transition
gamma-rays
<1 pm
nuclear
X-rays
1 nm-1 pm
inner electron
ultraviolet
400 nm-1 nm
outer electron
visible
4-7.5x1014
750 nm-400 nm
near-infrared
1x1014-4x1014
2.5 µm-750 nm
outer electron molecular vibrations
infrared
25 µm-2.5 µm
molecular vibrations
microwaves 3x
1 mm-25 µm
molecular rotations,
electron spin flips*
radio waves
<3x1011
>1 mm
nuclear spin flips*

7. Where h = Planck’s constant & c = speed of light in a vacuum.
Electromagnetic radiation is characterized by its
wavelength, , Frequency,  and energy, E:
E = h= hc /  c =  
Where h = Planck’s constant & c = speed of light in a vacuum.
longer wavelength, lower energy;
shorter wavelength, higher energy.

8. Theory of spectrophotometry
When a beam monochromatic light passes through a transparent medium, part of the light is absorbed and the transmitted beam has a lower intensity than the intensity of the incident beam.

9. The TRANSMITTANCE, T of the solution is defined as the ratio of the intensities of the transmitted beam, P to the intensity, Po of the incident beam:
T = P/Po
The ABSORBANCE, A of a solution is defined as
Absorbance
A = log10 P0 / P A = log10 1 / T  A = log10 100 / %T A = 2 - log10 %T 
A = -log10T
Since A is a logarithmic function, it is dimensionless.

10. T= (I/Io) = 10-A %T = (I/Io) x 100 A = -logT = log(1/T)
Equation Summary
T= (I/Io) = 10-A %T = (I/Io) x A = -logT = log(1/T)
Sample Calculation
If %T = 95%, then A = log(100/95) = log(1/.95) = -log(.95)
A =
Note the scale for Absorbance: 9/10th of the scale is from 0-1 and 1/10th is from 1-2.
For this reason, the spectrometers have been calibrated in % Transmittance and
all readings will be taken in %Transmittance.

11. Lambert’s Law
“When a beam of light is allowed to pass through a transparent medium, the rate of decrease of intensity with the thickness of medium is directly proportional to the intensity of the light”
- dI / dt α I Or
- dI / dt = kI

12. Integrating on both sides putting I=I0 When t=0, we get
In I0 / It = kt
or It = I0 e-kt
Where k is the constant which depends on wavelength and absorbing medium used.
Converting to natural log
It = I kt = I0 10 -Kt
Where K=k/ which is absorption coefficient, which is defined as “the reciprocal of the thickness which is required to reduce the light to 1/10 of its intensity”
I0 / It = 0.1=10-kt or Kt =1 or K α 1 / t

13. Beer’s Law
Lambert’s law shows that there exists a logarithmic relationship between the transmittance and the length of the optical path through the sample.

14. Beers observed that a similar relationship between the transmittance and the conc of a solution. i.e “ the intensity of a beam of monochromatic light decrease exponentially with the increase in conc if the absorbing substance arithmetically”
It = I0 e-k’c
It = I k’t = I0 10 –K’t
Where k’ and K’ are constants and c is the concentration of the absorbing substance.

15. Beer’s Law It = I0 10 -act or log I0 / It = act
This is mathematical statement of Beer-Lambert law.
This is the fundamental for colorimetry and Spectrophotometry.
Where c is the conc expressed in mole dm-3
t is the path length which is expressed in cm.
a is replaced with Є and is termed as the Molar absorption coefficient or molar absorptivity (molar extinction coefficient)

16. Beer-Lambert law log I0 / It = act A = act A= Єct Є=A/ct
Where A is absorbance (no units, since A = log10 P0 / P )
Є is the molar absorbtivity with units of L mol-1 cm-1 b is the path length of the sample - that is, the path length of the cuvette in which the sample is contained. We will express this measurement in centimetres. c is the concentration of the compound in solution, expressed in mol L-1

17. Deviations from Beer’s Law
Beer’s law states that a plot of absorbance versus concentration should give a straight line passing through the origin with a slope equal to ab. However, deviations from direct proportionality between absorbance and concentration are sometimes encountered.
These deviations are a result of one or more of the following three things ; real limitations, instrumental factors or chemical factors.

18. Real Limitations
Beer’s law is successful in describing the absorption behavior of dilute solutions only ; in this sense it is a limiting law. At high concentrations ( > 0.01M ),the average distance between the species responsible for absorption is diminished to the point where each affects the charge distribution of its neighbors.
This interaction, in turn, can alter the species’ ability to absorb at a given wavelength of radiation thus leading to a deviation from Beer’s law.

19. Limitations (cont)
Deviations also arise because e is dependent upon the refractive index of the solution. Thus, if concentration changes cause significant alterations in the refractive index h of a solution, departures from Beer’s law are observed.

20. Chemical Deviations
Chemical deviations from Beer’s law are caused by shifts in the position of a chemical or physical equilibrium involving the absorbing species.
A common example of this behavior is found with acid/base indicators.
Deviations arising from chemical factors can only be observed when concentrations are changed.
eg:dichromate ions on dilution.
Cr2O72- + H2O -- 2HCrO4- - 2H+ + 2CrO4 2-
(orange color) (yellow color)

21. Instrumental Factors
Unsatisfactory performance of an instrument may be caused by fluctuations in the power-supply voltage, an unstable light source, or a non-linear response of the detector-amplifier system.

22. There are at least six conditions that need to be fulfilled in order for Beer’s law to be valid. These are:
The absorbers must act independently of each other;
The absorbing medium must be homogeneous in the interaction volume
The absorbing medium must not scatter the radiation - no turbidity;
The incident radiation must consist of parallel rays, each traversing the same length in the absorbing medium;
The incident radiation should preferably be monochromatic, or have at least a width that is narrower than that of the absorbing transition; and
The incident flux must not influence the atoms or molecules; it should only act as a non-invasive probe of the species under study. In particular, this implies that the light should not cause optical saturation or optical pumping, since such effects will deplete the lower level and possibly give rise to stimulated emission.

23. Instrumentation
Source: A continuous source of radiant energy covering the region of spectrum in which the instrument is designed to work.
Filter or Monochromator: Both filter and monochromator allow the light of the required wavelength to pass through but absorb the light of other wavelengths.
Sample cells: All instrument must contain a container for the sample.
Detector: It is used for measuring the radiant energy transmitted through the sample.

24. Radiation sources Requirements of a radiation sources
It must be stable.
It must be of sufficient intensity for the transmitted energy to be detected at the end if the optical path.
It must supply continuous radiation over the entire wavelength region in which it is used.

25. Tungsten filament lamp
It is most common source of visible radiation.
Its construction is similar to the household lamp. But the tungsten wire is heated at controlled atm.
Constant power supply is needed to achieve constant radiant energy.

26. Drawback of tungsten lamp
Major portion of its radiant energy in the near IR region of the spectrum, i.e only 15% at the operating temp of about 2725o c and only 1 % at 1725o c.
If the temp is increased to 2850o c and above will increase the total energy output and shift the wavelength of maximum intensity to shorter wavelength but the life of the lamp will be reduced.

27. Tungsten lamp is the most satisfactory for lamp filaments but the carbon arc is used when a more intense source is required.
The energy from a tungsten lamp can be used above 375 nm.

28. Filters and Monochromators
A Source is generally emits the continuous spectra.
We need a device to select the narrow bands from wavelengths of the continuous spectra.
For that selection we have
Filters and
Monochromators.

29. FILTERS
A light filter is device that allow light of the required wavelength to pass but absorbs light of other wavelengths wholly or partially.
Filter are two type
1) absorption filters and
2) interference filters

30. Absorption filter
It is a solid sheet of glass on which the color pigments are dispersed.
Absorption filters are classed as either cut off or band pass filters.

31. Color Wheel
(ROYGBIV)
Complementary colors lie across the diameter on the color wheel and combine to form “white light”, so the color of a compound seen by the eye is the complement of the
color of light absorbed by a colored compound; thus it
completes the color.

32. Observed Color of Compound Color of Light Absorbed
Observed Color of Compound
Color of Light Absorbed
Approximate Wavelength of Light Absorbed
Green
700 nm
Blue-green
600 nm
Violet
550 nm
Red-violet
530 nm
Red
500 nm
Orange
450 nm
Yellow
400 nm

33. Observed Color of Compound Color of Light Absorbed
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
500 nm
Orange
Blue
450 nm
400 nm

34. Interference filters
These filters works on the phenomena of interference.
It has a semi-transparent metal film is deposited on the glass plate.
Then the metal film is coated with thin layer of dielectric material like MgF2.
Followed by another metal film and then glass plate for support.

35. Interference filters

36. Monochromators
It is separate the narrow wavelength more specifically then filters.
Monochromators have the following parts
An Entrance slit
A dispersing element (Prism or grating) and
An Exit slit

38. Replica grating are cheaper than prisms.
Main disadvantage of the gratings is that they produce more then one order of diffraction.
For instance, the second order of 400 mμ may interfere with the first order of 800 mμ.
This can be removed my employing filters in front of the entrance slit to absorb interfering radiation.

39. Grating
A Grating consist of large number if parallel lines ruled on a highly polished surface such as alumina.( 15,000 – 30,000 lines per square inch)
Grating are very difficult to prepared. Therefore replica gratings are prepared from an original grating using an epoxy resin.

40. The main advantage of prisms is that they undergo dispersion giving wavelengths which do not overlap.
But the main disadvantage is that they give Non-linear dispersion.
On the other hand gratings give linear dispersion but they suffer from an overlap of spectral orders.
For this reason, filters are employed to reduce the radiation of different orders and stray radiation.

41. Cells

42. DETECTORS
PHOTOVOLTANIC CELL
PHOTOTUBES
PHOTOMULTIPLIER

43. PHOTOVOLTANIC CELL

44. Photocell or Phototubes

45. Photoemissive cell

46. Photomultiplier tube

47. Power supply The power supply serve a triple function
It decrease the line voltage to the instrument’s operating level with a transformer.
It convert alternating current to direct current with a rectifier if direct current is required by the instrument.
It smooths out any ripple which may occur in the line voltage in order to deliver a constant voltage to the source lamp and instrument.

48. Visual comparators
Intensity: For light shining through a colored solution, the observed intensity of the color is found to be dependent on both the thickness of the absorbing layer (pathlength) and the concentration of the colored species.
There are four techniques for color comparison in quantitative visual colorimetry.
Multiple standard method,
Duplication method,
Dilution methods, and
Balancing method-uses Klett Bio colorimeter or Dubosque colorimeter

49. Multiple standard method
The unknown solution is taken in a 50/100 ml Nessler tube and made up to the mark.
The color of the unknown solution is compared with the series of known amount of the known substance.
The conc of the unknown will be equal to the conc of known if the color matches exactly.

50. ←Side view
←Top view
(a.k.a. Bird’s eye view)
For One Color: A series of solutions of a single color demonstrates the effect of either concentration or pathlength, depending on how it is viewed.

51. Visual Colorimetry
←Ratio used
←Purple produced
For more than one color: the ratio of an unknown mixture can also be determined by matching the shade of the color to those produced from known ratios.
In this example, the ratio of a mixture of red and blue can be determined visibly by comparing the mixture to purples produced from known ratios of red and blue.

52. Duplication method
The unknown solution is taken in one Nessler tube followed by the development of color with appropriate reagent, and made up to the mark.
In another tube the solution of developing reagent is taken and made up a little lower than the mark on the Nessler tube.
From the burette the std solution is added to this tube until the color matches the unknown solution.

53. Dilution methods
Two Nessler tubes of equal diameter, height and made out same quality of glass are used.
To the first tube we add a standard solution and to the second with the unknown conc solution.
Light from the same source is allowed to pass through each cell and the emergent beams are the compared.

54. The more concentrated solution is progressively diluted until the intensity of the emerging light from the both the cells become equal.
At this point the concentrations per unit volume of solution in both the tubes (std and the unknown) should also be the same.

55. As the two beams are equal in intensity, the absorbance A is equal in each case.
A=εc1t1
A=εc2t2
from the above equations, we get
εc1t1 = εc2t2
But t1 = t2
Therefore c1 = c2

56. As the total quantity of material in the sample remains equal to the original concentration x original volume of sample.
c1 V2 = c(original) V original
but the final conc of the sample C1 is equal to the conc of the std, therefore
c(original) =c1 V2 / V1 or
Conc of std x volume of solution after dilution
Original volume of solution

57. Balancing method Dubosque colorimeter is used.
By varying the length of the light oath in two solutions the color of the unknown is matched with the std.

58. Intensity: When the product of the concentration and the pathlength of any two solutions of a colored compound are the same, the same intensity or darkness of color is observed.
Duboscq visual
colorimeter
Adjustable
Path Lengths

59. problems
40 ml of the 0.5 M Fe3+ std is diluted with 25 ml of water in order to match the intensity of the unknown. Calculate the Fe3+ conc of the unknown solution.
50 ml of the unknown sample is diluted to a total volume of 60 ml in order to match the intensity of the original std. the final conc of the unknown is 0.04 M. find out the original conc of the unknown.

60. Single beam instrument

61. Double beam instrument

62. Advantage of double beam
It is not necessary to continually replace the blank with the sample or to zero adjust at each wavelength as in the single beam units.
The ration of the power of the sample and reference beams is constantly obtained and used.
B’cos if the previous two factor the double beam system leads itself to rapid scanning over a wide wavelength region.