1. INSTRUMENTATION An Insight Into UV – Visible Spectroscopy
N V SARMA RETIRED CHIEF CHEMIST CAL NAGPUR

2. UV & VIS Spectrophotometers - in use since last 40 years
> Imp. analytical tool because of
Simplicity
Versatility
Speed
Accuracy
Cost effectiveness

3. ELECTRO MAGNETIC RADIATION
Light as a wave form
Waves
Wave = a way of shifting energy from one place to another - whether waves on the sea or waves in light.
In water, the energy is transferred by the movement of water molecules in a roughly circular path. The wave builds to a crest; and a trough as they move down again.
The energy in light travels because of local fluctuating changes in electrical and magnetic fields - hence "electromagnetic" radiation.

4. WAVELENGTH, FREQUENCY AND THE SPEED OF LIGHT
Wavelength - the distance between two crests or two troughs.
Frequency = the number of crests passing through a particular point per second measured as "cycles per second", now called Hertz, Hz. (Cycles per second and Hertz are same).
Example - Orange light has a frequency of about 5 x 1014 Hz (often quoted as 5 x 108 MHz - megahertz). That means that 5 x 1014 wave peaks pass a given point every second.
This is actually the speed of electromagnetic radiation

5. Relationship between the Wavelength ,Frequency and the Speed of light:
V = c 

6. These relationships mean that if you increase the frequency, you must decrease the wavelength.
If the wavelength is longer, the frequency is lower and if wavelength is shorter, the frequency is higher.
For example, red light has a wavelength of 650 nm, and green light has a wavelength of 540 nm,
It means the green light with a shorter wavelength has a higher frequency. (Note:  nm = nanometer = 10-9 meter.)

7. THE FREQUENCY OF LIGHT AND ITS ENERGY
Each particular frequency of light has a particular energy associated with it,
given by another simple equation:
NOTE Higher the frequency, higher the energy of the light.
Violet colour has a wavelength around 380 – 435nm.
Red colour has a wavelength around nm.
The light with the highest energy will have the highest frequency Highest frequency means the one with the smallest wavelength.
In other words, violet light at the 380 nm.

8. THE ELECTROMAGNETIC SPECTRUM
Visible light
The diagram shows an approximation to the spectrum of visible light.
The main colour regions of the visible spectrum are approximately as given below:
Colour region
Wavelength (nm)
VIOLET
380 – 435
INDIGO
435 – 500
BLUE
500 – 520
GREEN
520 – 565
YELLOW
565 – 590
ORANGE
590 – 625
RED
625 – 740

9. Placing the visible spectrum in the whole electromagnetic spectrum
The electromagnetic spectrum doesn't stop with the colours seen by the human eye. It is perfectly possible to have wavelengths shorter than violet light or longer than red light and this can be extended even further into -rays and radio waves.
The diagram shows the approximate positions of some of these on the
spectrum.
NOTE - The energy associated with the various kinds of radiation increases as the frequency increases.

10. Light spectrum varies from 10 nm (wavelength)
to nm (wavelength) expressed in nms.
But for Spectroscopy - only UV & visible regions are
considered.
Other units not in use now but used earlier were -
Angstrom (A) & milimicron (mm)
1 nm = 1 mm = 10A = 10.9 meters.

12. Spectral Regions (as set by Jt
Spectral Regions (as set by Jt. Committee on Nomenclature in Applied Spectroscopy
(Also called electromagnetic spectrum/band)
Region Wave length (nm)
1. Far ultraviolet
2. Near ultraviolet
3) Visible
4) Near infrared – 3,000
5) Middle infrared , ,000
6) Far infrared ,000-3,00,000
7) Microwave ,00,000-1,000,000,000

13. Human eye can visualize only between 380-780 nm (i.e. visible range)
All colours (VIBGYOR) fall within this range.
Wavelength band ranges from Gamma waves to Microwaves.
On a linear scale if range (from Gamma to microwave) is 25 kms visible
range is only 1 cm.

14. COLOUR CODE IN ABSORPTION SPECTROPHOTOMETER
If you pass white light through a coloured substance, some of the light gets
absorbed.
Ex A solution looks blue because the solution absorbs yellow colour.
This is because blue and yellow are complementary colors.

15. Some colourless substances also absorb light - but in the ultra-violet region. Since we can't see UV light, we don't notice this absorption.
Different substances like metal ions or particular functional groups absorb different wavelengths of light. This helps to identify the substance.
The absorption depends on the concentration and the thickness of the substance in the solution.
Measurement of the amount of absorption helps to find concentrations of even dilute solutions.

16. Quantum Theory
Consider radiation as a stream of particles (photons) (i.e. light energy) instead of waves considered earlier.
Atoms/molecules have a defined energy state/level.
Change of state/level of energy requires absorption or emission of a certain quantity of energy called quantum (i.e. here) a certain quantity of photons.
Energy of a photon absorbed/ emitted by atom/molecule during transition from one energy level (ground) to another energy level (excited) is given by
E = h u .
h = Planck’s constant,
u = frequency of photon

17. What happens when an EMR hits a molecule in a solution ?
1.The molecule as a whole may move this is called transition and the energy associated with this movement is called transinational energy. (Etrans)
2.The part of the molecules, that is atom or groups of atoms, may move with respect to each other. This motion is called vibration and the associated energy is called vibrational energy. (Evib)
3.The molecule may rotate about an axis. And such rotation is characterized by the rotational energy. (Erot)
4.Besides these modes of movements, the molecule possesses an electronic configuration and the energy associated with this configuration is called electronic energy. (Eele)
E total = E Trans + E Vib + E Rot + E Ele

18. Shorter wavelength = Greater the energy of the photon.
Longer wavelength = Lesser the energy of the photon.
Any Molecule has its own internal energy because of –
Energy of its electrons
Energy of the vibration between its constituent atoms
Energy because of rotation of the molecule.

19. Energy levels of electrons are widely separated.
In case of simple molecules –
Energy of electrons
Energy levels of electrons are widely separated.
Hence they require absorption of high energy (i.e. very short wavelength) to excite it from ground level to higher (excited) level.

20. In case of complex molecules Energy levels of electrons
Are more closely spaced and requires photons of UV & visible
(i.e.) low energy (i.e. longer wavelength) to excite it from ground
level to higher (excited) level.
Hence these molecules absorb light energy (photon) from near
UV & visible range.

21. Energy of vibration between its constituents atoms
Atoms of molecule are more closer than the electronic energy
levels as stated in (a)
Photons of lower energy (longer wavelength) are sufficient to
bring about vibrational changes.
Light energy (photon) absorption due to vibrational changes
occur in the IR region.

22. Energy due to rotation of molecule
Rotational energy state/level of molecules are closely packed.
Therefore photons of far IR and microwave region of
electromagnetic spectrum is sufficient to cause changes from
ground level to excited level.

24. 4] It can be transmitted
5] It can excite florescence
Scattering - Radiation is first absorbed and almost instantly
completely scattered uniformly in all directions but uncharged in energy.
Fluorescence - Photon is first absorbed .Takes the molecule to a higher
Energy state. But drops back to an intermediate energy level or
almost ground state by re-emitting the photon.
Since some energy of the incident photon is retained by the
molecule, the emitted photon has less energy (longer
wavelength) then the absorbed photon. Like Scatter,
Fluorescent radiation is also emitted uniformly in all Directions.
In case of absorption Spectrophotometers –
Focus is only on absorption & transmission. Therefore Reflection, Scatter & fluorescence are kept to the minimum negligible by choosing suitable chemicals/Solvents/Wavelengths etc.
UV/ Vis Spectrometers are used for quantitative Analysis. (i.e.) estimation of the amount of a compound in the sample.

33. Important Terms to Remember –
1. Chromophore : - It is a group of molecules, which is responsible for the absorption of light by molecules. It is the minimum structural requirements for the absorption of radiation in UV range.
2. Auxochrome : It is a saturated group containing unshared
Electrons which when attached to a Chromophore changes both intensity as well as the wavelength of the absorption maxima. E.g. OH, NH2, Cl etc.
3. -max : It is wavelength at which there is a maximum absorption intensity. It is a physical constant and characteristic of structure and so useful for identification of compounds. It is independent of concentration.

34. Bathochromic shift : The shifting of absorption to a longer wavelength (right hand side) due to substitution or solvent is called as bathochromic shift. It is also called as Red shift ex.  max of Ascorbic acid = 243 nm.
 max of Ascorbic acid in alkali medium = 299 nm.
Hypsochromic shift (Blue shift) : Shifting of  max to
lower value (left hand side) due to substitution, solvent,
pH etc is called as Hypsochromic shift.
ex  max of Phenol in basic media = 297 nm,
 max of Phenol in acidic media = 277 nm.
Hyperchromism : Increase in absorption intensity (e) due
to solvent, pH or some other factors is called hyperchromic
effect.
7. Hypochromism : Decrease in absorption intensity due to
substituent, solvent, pH etc. is called hypochromic effect.

35. A1% 1 cm (A one percent one centimeter): Is the absorbance of the solution having concentration 1 gm per
100 ml of the solution.
9. Molar absorptivity :(Є) : Is the absorbance of the
solution having concentration gm.mol. weight/1000 ml of
the solution [Є = (A 1% 1 cm X Mol. Wt.)/ 10]
10. Transmittance (T): is the ratio of IT/10 and %
Transmittance(%T) is given by %T = 100 IT/10
11. Absorbance (A): Is the degree of absorption of light by a
medium through which the energy passes. It is expressed as the logarithm of the ratio of light transmitted through a
pure solvent to the intensity of light transmitted through the medium.
A= logI0 / IT
A= logI0 – logIT
A= 2- log % T

39. Lambert (Bouger’s) Law
1] Each layer of equal thickness of any absorbing medium absorbs an
equal fraction of the radiant energy passing through it.
2] The radiant energy transmitted is independent of the incident
radiation.
3] If the incident radiation is Io and the transmitted radiation is I then
the fraction transmitted.
= T= I/IO
The % T = I/Io x 100
Assume a series of glass cuvettes of equal thickness placed
in parallel. Let each cuvette absorb ¼ of incident light. Then Amount of transmitted radiation will be 75%
i.e. ( 1- ¼ )/ 1x100 = 75 % Ex /100x100
This also means that if a cuvette (10mm ) is filled with an absorbing Solution and 75% of the light incident on it is transmitted on to another mm cuvette placed next to it, the transmitted light will be reduced to 56.25%
If the contents of the two containers are evaporated to half their volume (i.e) doubling its concentration and then measured in a single container it will be found that transmitted light will again be reduced to 56.25/.

40. ] Ref. To the example
100% - ¼ = 75% - ¼ = 56.25% - ¼ = %
Transmittance of sample fell from 75% to 56.25% when conc. is doubled.
What happens to absorbance under the same circumstances?
A = log (100/T) = log 100 – log T = 2-log T
When T = 75%  A = 2 – = 0.125
When T = 56.25%  A = 2 – = 0.250
23] This proves that absorbance doubles with the conc. Therefore it
is far more convenient to the work in Absorbance (also called as
‘extinction’ or optical Density (OD.)) rather than Transmittance for
Quantitative analysis.
Remember - 0% T = Infinite A
0.1 % = 3.0 A
1.0 = 2.0 A
10% = 1.0 A
100% = 0.0 A.

41. ¼ ¼ ¼ Io I I I 100% 75% 56.25% 42.19% ( 1- ¼) (75 - ¼) (56.25% - ¼)
( ¼)
(56.25% - ¼)
(10mm )
(10mm )
(10mm )

43. The Beer-Lambert Law
Different symbols are given for some of the terms in the equation - the concentration of the solution is "c" and the length is "l".

44. The expression on the left of this equation is what we have just defined as the absorbance, A. and is written as
It can also be expressed as
The Greek letter epsilon in these equations is called the molar absorptivity - or sometimes the molar absorption coefficient.

45. Molar absorptivity
If you rearrange the simplest of the equations above to give an expression for epsilon (the molar absorptivity), you get:

46. A DOUBLE BEAM SPECTROPHOTOMETER
(1) The Light Source
a combination of two light sources – a deuterium lamp for the UV part of the spectrum, and a tungsten / halogen lamp for the visible part to get a wavelength between 200 to 800 nms. The combined output of these two bulbs is focused on to a diffraction grating.

47. (2) Grating:
Most modern UV spectrophotometer uses diffraction grating as a Monochromator.
It consists of a very large number of equispaced lines ( per mm) ruled on a glass plate.
They can be used either as transmission grating or when aluminized, as
reflection grating
(3) Slit: (Radiation intensity controlling device):
Enough light must pass through the sample to elicit a measurable response from the detector.
(4)Monochromator: (wavelength selecting device).
It converts polychromatic light in monochromatic light (light having one wavelength).
a) Filters:
Glass filters are pieces of colored glass, which transmit limited wavelength ranges of the spectrum. The color is produced by incorporating oxides of such metals
b) Prisms:
When a beam of light passes through a prism, it is bent or refracted. The amount of deviation is dependent on the wavelength.
The prism is made up of quartz for use in the UV light, since glass absorbs
wavelengths shorter than about 330 nm.
Glass prism is preferable for the visible region of the spectrum, as the dispersion is much greater than that obtained with quartz.

48. (5) The rotating discs
The disc is made up of three different sections –
The light coming from the diffraction grating and slit hits the rotating disc and one of three things can happen.
If it hits the transparent section, it goes straight through the cell containing the sample. It is then bounced by a mirror onto a second rotating disc.

49. This second disc is rotating such that when the light arrives from the first disc, it meets the mirrored section of the second disc. That
bounces it onto the detector.
(See the red path in the diagram.)

50. If the original beam of light from the slit hits the mirrored section of the first rotating disc, it is bounced down along the green path. After the mirror, it passes through a reference cell and gets to the second disc which is rotating in such a way that it meets
the transparent section. It goes straight through to the detector.
(See the green path in the diagram.)

51. Rotating Disc
If the light meets the first disc at the black section, it is blocked - and for a very short while no light passes through the spectrometer. This just allows the computer to make allowance for any current generated by the detector in the absence of any light.

52. The sample and reference cells
Are small rectangular glass or quartz containers of 1 cm. thickness.
The sample cell contains a solution of the test substance.
The solvent chosen shouldn’t absorb any significant amount of light in the wavelength range we are interested in ( nm).
The reference cell just contains the pure solvent.

53. The detector and computer
The detector converts the incoming light into a current. Greater
the intensity of the light higher the current.
The light passing through the reference cell is measured and is referred
to as Io (I =Intensity.)
Light passing through the sample cell is measured and is referred to
as I.
If I is less than Io, then obviously the sample has absorbed
some of the light.
Absorbance of the sample is represented by the symbol, A.
An absorbance of 0 at some wavelength means that no light of
that particular wavelength has been absorbed. The intensities
of the sample and reference beam are both the same, so the
ratio Io/I is 1. Log10 of 1 is zero.
An absorbance of 1 happens when 90% of the light at that wavelength has
been absorbed - which means that the intensity is 10% of what it would
otherwise be.
In that case, Io/I is 100/I0 (=10) and log10 of 10 is 1.

55. Absorbance isn't very good for making comparisons
The importance of concentration
The proportion of the light absorbed will depend on how many molecules it interacts with. Suppose you have got a strongly coloured organic dye. If it is in a reasonably concentrated solution, it will have a very high absorbance because there are lots of molecules to interact with the light.
However, in an incredibly dilute solution, it may be very difficult to see that it is coloured at all. The absorbance is going to be very low.
Suppose then that you wanted to compare this dye with a different compound. Unless you took care to make allowance for the concentration, you couldn't make any sensible comparisons about which one absorbed the most light.

56. The importance of the container shape
Imagine this time that you had a very dilute solution of the dye in a cuvette so that the light travelled 1 cm through it. The absorbance isn't likely to be very high. On the other hand, suppose you passed the light through a cuvette 100 cm long containing the same solution. More light would be absorbed because it interacts with more molecules.
Again, if you want to draw sensible comparisons between solutions, you have to allow for the length of the solution the light is passing through.
Both concentration and solution length are allowed for in the Beer-Lambert Law.

57. 2] Qualitative Analysis - What analyte is in the sample
Applications of UV – visible Spectrometry.
1] Quantitative Analysis -How much analyte is in the sample.
2] Qualitative Analysis - What analyte is in the sample
3] Single Component Analysis –
a] Direct Analysis –
To measure compds having conjugated double bonds or aromatic rings as well as
many inorganic species because these absorb light in the UV-visible regions.
The substance is dissolved in a suitable solvent, diluted to the required Conc. &
absorbance is measured.
b] Indirect Analysis
(i.e. Analysis after addition of some reagent )
The analyte is converted by a chemical agent because the analyte otherwise
absorbs weakly in the UV region or the interference of irrelevant compds can be
avoided by converting the analyte to some derivative which absorbs in the visible
region where absorption of irrelevant compounds is negligible.
4] Cost effective Method.