1. An introduction to Ultraviolet/Visible Absorption Spectroscopy
Lectures 21 and 22

2. In this chapter:
Absorption by molecules, rather than atoms, is considered.
Absorption in the UV and Vis regions:
occurs due to electronic transitions from the ground state to excited state.
Broad band spectra are obtained:
Because molecules have vibrational and rotational energy levels associated with electronic energy levels.
The signal is either absorbance or percent transmittance of the analyte solution where:

3. Where T = P/Po always a fraction)
Absorption measurements based upon ultraviolet and visible radiation:
Find widespread application for the quantitative determination of a large variety species.
Beer’s Law:
A = -logT = logP0/P = bc
Where T = P/Po always a fraction)
A = absorbance
 = molar absorptivity [M-1 cm-1]
c = concentration [M]
P0 = incident power
P = transmitted power (after passing through sample)
b= path length in cm

6. UV-Vis Absorption Spectroscopy

7. Some losses of radiation power

8. Measurement of Transmittance and Absorbance:
The power of the beam transmitted by the analyte solution is usually compared with the power of the beam transmitted by an identical cell containing only solvent.
An experimental transmittance and absorbance are then obtained with the equations.
P0 and P: refers to the power of radiation after it has passed through the solvent and the analyte.
Because T is a fraction so generally multiplied by 100 to give %T

9. Beer’s law and mixtures
Each analyte present in the solution absorbs light!
The magnitude of the absorption depends on its e
A total = A1+A2+…+An
A total = e1bc1+e2bc2+…+enbcn
If e1 = e2 = en then simultaneous determination is impossible (difficult to separate con. of substances. seam to be same substance).
Need to measure A at nl’s (get n2 e’s) to solve for the concentration of species in the mixture
For 3 substance we need to measure at 9 e
Where  is max for each substance

10. Large slope = large e and good sensitivity
b=1cm
Large slope = large e and good sensitivity

11. Limitations to Beer’s Law
Deviation from linearity of the A and C relationship
(remember no interpolation and extrapolation of the calibration:
It is regions of deviation from linearity)
Types of deviations:
Real limitations
Chemical deviations
Instrumental deviations

12. 1.      Real Limitations
 a. Beer’s law is good for dilute analyte solutions only.
High concentrations (>0.01M) will cause a negative error since as the distance between molecules become smaller the charge distribution will be affected which alter the molecules ability to absorb a specific wavelength.
The same phenomenon is also observed for solutions with high electrolyte concentration, even at low analyte concentration.
The molar absorptivity is altered due to electrostatic interactions.

13. b. In the derivation of Beer’s law we have introduced a constant (e) which depends on refractive index.
The refractive index is a function of concentration.
Therefore, e will be concentration dependent.
However, the refractive index changes very slightly for dilute solutions and thus we can practically assume that e is constant.
c. In rare cases, the molar absorptivity changes widely with concentration, even at dilute solutions.
Therefore, Beer’s law is never a linear relation for such compounds, like methylene blue.

14. 2.  Chemical Deviations:
 This factor is an important one which largely affects linearity in Beer’s law.
It originates when an analyte dissociates, associates, or reacts in the solvent, or one of matrix constituents.
For example:
An acid base indicator when dissolved in water will partially dissociate according to its acid dissociation constant:

15. Hin (color 1)= H+ + In- (color 2)
It can be easily appreciated that the amount of HIn present in solution is less than that originally dissolved where:
CHIn = [HIn] + [In-]
Assume an analytical concentration of 2x10-5 M indicator (ka = 1.42x10-5) was used, we may write:

16. 1.42x10-5 = x2/(2x10-5 – x)
Solving the quadratic equation gives:
X = 1.12x10-5 M which means:
[In-] = 1.12x10-5 M
[HIn] = 2x10-5 – 1.12x10-5 = 0.88x10-5 M
Therefore, the absorbance measured will be the sum of that for HIn and In-.
If a 1.00 cm cell was used and the e for both HIn and In- were 7.12x103 and 9.61x102 Lmol-1cm-1 at nm, respectively, the absorbance of the solution can be calculated:

17. A = AHIn + AIn
A = 7.12x103 * 1.00* 0.88x x102 * *1.12x10-5 = 0.073
However, if no dissociation takes place we may have:
A = AHIn
A = 7.12x103 * 1.00 * 2x10-5 = 0.142
If the two results are compared we can calculate the % decrease in anticipated signal as:
% decrease in signal = {(0.142 – 0.073)/0.142}x100% = 49%
As a result of dissociation

18. However, at 430 nm, the molar absorptivities of HIn and In- are 6. 30
However, at 430 nm, the molar absorptivities of HIn and In- are 6.30*102 and 2.06*104, respectively.
A = AHIn + AIn
A = 6.30*102 * 1.00* 0.88x *104 * 1.00 *1.12x10-5 =
Again, if no dissociation takes place we may have:
A = AHIn
A = 6.30*102 * 1.00 * 2x10-5 = 0.013
If the two results are compared we can calculate the % increase in anticipated signal as:
% increase in signal = {(0.236 – 0.013)/0.013}x100% = V. large
Error as a result of dissociation

19. Comparison between results obtained at 570 nm and 430 nm show large dependence on the values of the molar absorptivities of HIn and In- at these wavelength.
At 570 nm:
A = AHIn + AIn
A = 7.12x103 * 1.00* 0.88x x102 * *1.12x10-5 = 0.073
And at 430 nm:
A = 6.30*102 * 1.00* 0.88x *104 * *1.12x10-5 = 0.236

20. Chemical deviations from Beer’s law for unbuffered solutions of the indicator Hln.
Note that there are:
positive deviations at 430 nm
negative deviations at 570 nm.
At 430 nm:
The absorbance is primarily due to the ionized In- form of the indicator and is proportional to the fraction ionized, which varies nonlinearly with the total indicator concentration.
At 570 nm:
The absorbance is due principally to the undissociated acid Hln, which increases nonlinearly with the total concentration.

21. +ve error
-ve error

22. Calculated Absorbance Data for Various Indicator Concentrations

23. A = eCrO4 *b*CCrO4 + eCr2O7 *b*CCr2O7 If the two e are equals no error
An example of association equilibria:
Association of chromate in acidic solution to form the dichromate according to the equation below:
2 CrO H+ D Cr2O72- + H2O
The absorbance of the chromate ions will change according to the mentioned equilibrium and will thus be nonlinearly related to concentration.
A = eCrO4 *b*CCrO4 + eCr2O7 *b*CCr2O7
If the two e are equals no error
+ve error

24. 3.      Instrumental Deviations
a.  Beer’s law is good for monochromatic light only since e is wavelength dependent.
It is enough to assume a dichromatic beam passing through a sample to appreciate the need for a monochromatic light.
Assume that the radiant power of incident radiation is Po and Po’ while transmitted power is P and P’.
The absorbance ( at two ) of solution can be written as:

25. A = log (Po + Po’)/(P + P’) at the two 
P = Po10-ebc, (from the log Eq.) substituting in the above equation:
A = log (Po + Po’)/(Po10-ebc Po’10-e’bc) for diff. e
Assume e = e’ = e
A = log (Po + Po’)/(Po + Po’) 10-ebc
A = ebc
However, since e’ # e, since e is wavelength dependent, then A # ebc
Result:
Beers law is good for monochromatic radiation. But for different lamps or polych. there is a deviation

27. The effect of polychromatic radiation on Beer’s law.
In the spectrum at the top, the molar absorptivity of the analyte is nearly constant over band A.
Note that in Beer’s law plot at the bottom, using band A gives a linear relationship.
In the spectrum, band B corresponds to a region where the absorptivity shows substantial changes.
In the lower plot, note the dramatic deviation from Beer’s law that results.

28. Sign. change in e
No sign. change in e

29. Therefore, the linearity between absorbance and concentration breaks down if incident radiation was polychromatic.
In most cases with UV-Vis spectroscopy, the effect is small especially at the wavelength maximum.
The small changes in signal is insignificant since e differs only slightly.

30. UV-Vis Absorption Spectroscopy
Lecture 23

31. Error due to Ps: Ps is added not multiplied
b.      Stray Radiation أشعة ضالة
Stray radiation resulting from scattering or various reflections in the instrument will reach the detector without passing through the sample.
The problem can be severe in cases of:
1- High absorbance.
2- When the wavelengths of stray radiation is in such a range where the detector is highly sensitive as well as at wavelengths extremes of an instrument.
The absorbance recorded can be represented by the relation:
A = log (Po + Ps)/(P + Ps)
Solvent sample
Error due to Ps: Ps is added not multiplied
Where; Ps is the radiant power of stray radiation.

33. Instrumental Noise as a Function in Transmittance
The uncertainty in concentration as a function of the uncertainty in transmittance can be statistically represented as:
sc2 = (dc/dT)2 sT2
 sT is uncertainty of transmitance
A = -log T = ebc = ln T
c = -(1/eb)*0.434 ln T (1)
dc/dT = /ebT
sc2 = (-0.434/ebT)2 sT2 (2)

34. Dividing equation 2 by the square of equation 1
(sc /c)2 = (-0.434/ebT)2 sT2/{( ln T)2/(eb)2}
sc/c = (sT / T ln T)
 Therefore, it is clear that the uncertainty in concentration of a sample is nonlinearly related to the magnitude of the transmittance.
Substitution for different values of transmittance and assuming sT is constant, we get:

36. Very lower unc. in con
at : Abs:

37. Therefore, an absorbance between 0. 2-0
Therefore, an absorbance between may be advantageous in terms of a lower uncertainty in concentration measurements.
At higher or lower absorbances, an increase in uncertainty is encountered.
It is therefore advised that the test solution be in the concentration range which gives an absorbance value in the range from for best precision.
However, it should also be remembered that we ended up with this conclusion provided that sT is constant.
Unfortunately, sT is not always constant which complicates the conclusions above.

38. Effect of bandwidth on spectral detail for a sample of benzene vapor.
EFFECT OF bandwidth which is related to slit width
Effect of bandwidth on spectral detail for a sample of benzene vapor.
Note that as the spectral bandwidth increases, the fine structure in the spectrum is lost.
At a bandwidth of 10 nm, only a broad absorption band is observed.

39. Effect of slit width (spectral bandwidth) on peak heights.
Here, the sample was a solution of praseodymium chloride.
Note that as the spectral bandwidth decreases by decreasing the slit width from 1.0 mm to 0.1 mm, the peak heights increase.
لتحسين الاشارة يتم التضييق تدريجيا للحصول على أحسن امتصاص اي ثبات للاشارة قبل النزول

40. Effect of Scattered Radiation at Wavelength Extremes of an Instrument
Wavelength extremes of an instrument are dependent on:
Type of source
Type of detector
Type of optical components used in the manufacture of the instrument.
Outside the working range of the instrument:
It is not possible to use it for accurate determinations (at extremes unc. increases).
However, the extremes of the instrument are very close to the region of invalid instrumental performance and would thus be not very accurate.

41. An example:
A visible photometer which, in principle, can be used in the range from nm.
It may be obvious that glass windows, cells and prism will start to absorb significantly below 380 nm and thus a decrease in the incident radiant power is significant.

42. What defines the instrumental wavelength extremes?
Three main Factors:
Source
Detector
Optical components (lenses, windows, etc)
Measurements at wavelength extremes should be avoided since errors are very possible due to:
Source limitations
Detector limitations
Sample cell limitations
Scattered radiation

43. B: UV-VIS spectrophotometer
A: VIS spectrophotometer
EFFECT OF SCATTERED
RADIATION
Spectrum of cerium (IV) obtained with a spectrophotometer having glass optics (A) and quartz optics (B).
The false peak in A arises from transmission of stray radiation of longer wavelengths.

44. The output from the source at the low wavelength range is minimal.
Also, the detector has best sensitivities around 550 nm which means that away up and down this value, the sensitivity significantly decrease.
However, scattered radiation, and stray radiation in general, will reach the detector without passing through these surfaces as well as these radiation are constituted from wavelengths for which the detector is highly sensitive.
In some cases, stray and scattered radiation reaching the detector can be far more intense than the monochromatic beam from the source.
False peaks may appear in such cases and one should be aware of this cause of such peaks.

45. (Abs. Inst.) Instrumentation
Light source
 - selector
Sample container
Detector
Signal processing
Light Sources (commercial instruments)
D2 lamp (UV: 160 – 375 nm)
W lamp (vis: 350 – 2500 nm)

46. Sources (UV) 1) Deuterium and hydrogen lamps (160 – 375 nm)
D2 + Ee → D2* → D’ + D’’ + h

47. (a) A deuterium lamp of the type used in spectrophotometers and (b)
UV region
(a) A deuterium lamp of the type used in spectrophotometers and (b)
its spectrum.
The plot is of irradiance Eλ (proportional to radiant power) versus
wavelength.
Note that the maximum intensity occurs at ~225 m.
Typically: Instruments switch from deuterium to tungsten at ~350 nm.

48. 2) Tungsten lamp: Visible and near-IR region
A tungsten lamp of the type used in spectroscopy and its spectrum.
(b) Intensity of the tungsten source is usually quite low at wavelengths shorter than about 350 nm.
Note that the intensity reaches a maximum in the near-IR region of the spectrum.

49. The tungsten lamp is by far the most common source in the visible and near IR region with a continuum output wavelength in the range from nm.
The lamp is formed from a tungsten filament heated to about 3000 oC housed in a glass envelope.
The output of the lamp approaches a black body radiation where it is observed that the energy of a tungsten lamp varies as the fourth power of the operating voltage.

50. Tungsten halogen lamps are currently more popular than just tungsten lamps since they have longer lifetime.
Tungsten halogen lamps contain small quantities of iodine in a quartz envelope.
The quartz envelope is necessary due to the higher temperature of the tungsten halogen lamps (3500 oC).
The longer lifetime of tungsten halogen lamps stems from the fact that sublimed tungsten forms volatile WI2 which redeposits on the filament thus increasing its lifetime.
The output of tungsten halogen lamps are more efficient and extend well into the UV.

51. Tungsten lamps (350-2500 nm) Why add I2 in the lamps? W + I2 → WI2
Low limit: 330 nm
Low intensity
Glass or quartz envelope

52. 3. Xenon Arc Lamps:
 Passage of current through an atmosphere of high pressured xenon excites xenon and produces a continuum in the range from nm with maximum output at about 500 nm.
Although the output of the xenon arc lamp covers the whole UV and visible regions, it is seldom used as a conventional source in the UV-Vis.
The radiant power of the lamp is very high as to preclude the use of the lamp in UV-Vis instruments (very low amount absorbed not detected).
However, an important application of this source will be discussed in luminescence spectroscopy which will be discussed later.

54. UV-Vis Absorption Spectroscopy
Lecture 24

55. Instrumental Components
Source
 - selector (monochromators)
Sample holders
Cuvettes (b = 1 cm, typically)
Glass (Vis)
Fused silica (UV+Vis)
Detectors
Photodiodes
PMTs

56. Sample Containers
Sample containers are called cells or cuvettes and are made of either glass or quartz depending on the region of the electromagnetic spectrum.
The path length of the cell varies between 0.1 and 10 cm but the most common path length is 1.0 cm.
Rectangular cells or cylindrical cells are routinely used.
In addition, disposable polypropylene cells are used in the visible region.
The quality of the absorbance signal is dependent on the quality of the cells used in terms of matching, cleaning as well as freedom from scratches.

57. Instrumental designs for UV-visible photometers or spectrophotometers.
Types of Instruments
Instrumental designs for UV-visible photometers or spectrophotometers.
In (a), a single-beam instrument is shown.
Radiation from the filter or monochromator passes through either the reference cell or the sample cell before striking the photodetector.

58. 1. Single beam
Place cuvette with blank in place in the instrument and take a reading  100% T
Replace blank with sample and take reading  % T for analyte (from which absorbance is calc’d)

59. Most common spectrophotometer Spectronic 20
On/Off switch and zero transmission adjustment knob
Wavelength selector/Readout
Sample chamber
Blank adjustment knob
Absorbance/Transmittance scale

61. End view of the exit slit of the Spectronic 20
spectrophotometer pictured earlier

62. Single-Beam Instruments for the Ultraviolet/Visible Region

63. Single-Beam Computerized Spectrophotometers
Inside of a single-beam spectrophotometer connected to a computer.

64. 2. Double beam (most commercial instruments)
Light is split and directed towards both reference cell (blank) and sample cell
One or two detectors; electronics measure ratio (i.e., measure/calculate absorbance)
Advantages:
Compensates for fluctuations in source intensity and drift in detector
Better design for continuous recording of spectra
Faster

65. General Instrument Designs Double Beam: In – Space (not populated)
Needs two detectors 65

66. General Instrument Designs
Double Beam: In - Time
To give zero difference between the two Ref cells
Half the radiation pass

67. We can choose any of the two sources
Self collimation

69. Merits of Double Beam Instruments
Compensate for all but the most short term fluctuation in radiant output of the source.
Compensate drift in transducer and amplifier.
Compensate for wide variations in source intensity with wavelength.
Much faster than single beam.

70. 3. Dual Beam Instruments (single beam)
Measure the ratio of the two beams at the two detectors

71. 4. Multichannel Instruments
Photodiode array detectors used (multichannel detector, can measure all wavelengths dispersed by grating simultaneously).
Advantage:
scan spectrum very quickly “snapshot” < 1 sec.
Powerful tool for studies of transient intermediates in moderately fast reactions.
Useful for kinetic studies.
Useful for qualitative and quantitative determination of the components exiting from a liquid chromatographic column.

72. A multichannel diode-array spectrophotometer
Sample located before monochromator as the sample exposed to radiation at very short time
A multichannel diode-array spectrophotometer

74. Location of Sample cell
In all photometers and scanning spectrophotpmeters described above, the cell has been positioned after the monochromators.
This is important to decrease the possibility of sample photodecomposition due to prolonged exposure to all frequencies coming from the source.
However, the sample is positioned before the monochromator in multichannel instruments like a photodiode array spectrophotometer.
This can be done without fear of photodecomposition since the sample exposure time is usually less than 1 s.
Therefore, it is now clear that in UV-Vis:
Where photodecomposition of samples can take place, the sample is placed after the monochromators in scanning instruments.
While positioning of the sample before the monochromators is advised in multichannel instruments.

75. 5. Probe Type Instruments
These are the same as conventional single beam instruments but the beam from the monochromators is guided through a bifurcated optical fiber to the sample container where absorption takes place.
The attenuation in reflected beam at the specified wavelength is thus measured and related to concentration of analyte in the sample.

76. A fiber optic cable:
Can be referred to as a light pipe where light can be transmitted by the fiber without loss in intensity (when light hits the internal surface of the fiber at an angle larger than a critical angle).
Therefore, fiber optics can be used to transmit light for very long distances without losses.
A group of fibers can be combined together to form a fiber optic cable or bundle.
A bifurcated fiber optic cable: has three terminals where fibers from two separate cables are combined at one end to form the new configuration.

80. Fiber optic probe instrument

82. 6. Double Dispersing Instruments
The instrument in this case has two gratings where the light beam leaving the first monochromators at a specified wavelength is directed to the second grating.
This procedure results in better spectral resolution as well as decreased scattered radiation.
However, double dispersing instruments are expensive and seem to offer limited advantages as compared to cost; especially in the UV-Vis region where exact wavelength may not be crucial.

84. Optical diagram of the Varian Cary 300 double-dispersing spectrophotometer.
A second monochromator is added immediately after the source.
Used for Qualitative analysis.
Note: not needed for quant. Analysis; as we measure at max