INSTRUMENTATION An Insight Into UV Visible Spectroscopy N

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

UV & VIS Spectrophotometers - in use since last 40 years > Imp. analytical tool because of ØSimplicity ØVersatility ØSpeed ØAccuracy ØCost effectiveness 2

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. 3

• 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 4

• Relationship between the Wavelength , Frequency and the Speed of light: 1) SPEED 2) WAVELENGTH =c v 3) FREQUENCY V = c 5

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. ) 6

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 – 435 nm. Red colour has a wavelength around 625 - 740 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. 7

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 8

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. 9

Light spectrum varies from 10 nm (wavelength) to 100000 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 = 10 A = 10. 9 meters. 10

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Spectral Regions (as set by Jt. Committee on Nomenclature in Applied Spectroscopy (Also called electromagnetic spectrum/band) Region Wave length (nm) 1. Far ultraviolet 10 -200 2. Near ultraviolet 200 -380 3) Visible 380 -780 4) Near infrared 780 – 3, 000 5) Middle infrared 3, 000 - 30, 000 6) Far infrared 30, 000 -3, 000 7) Microwave 3, 000 -1, 000, 000 12

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. 13

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. 14

• 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. 15

• • 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=hu. h = Planck’s constant, u = frequency of photon 16

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 17

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 – Ø Energy of its electrons Ø Energy of the vibration between its constituent atoms Ø Energy because of rotation of the molecule. 18

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. 19

In case of complex molecules a) 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. 20

b) 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. 21

c) 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. 22

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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. 24

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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, NH 2, 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. 33

4. 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. 5. Hypsochromic shift (Blue shift) : Shifting of max to lower value (left hand side) due to substitution, solvent, p. H etc is called as Hypsochromic shift. ex max of Phenol in basic media = 297 nm, max of Phenol in acidic media = 277 nm. 6. Hyperchromism : Increase in absorption intensity (e) due to solvent, p. H or some other factors is called hyperchromic effect. 7. Hypochromism : Decrease in absorption intensity due to substituent, solvent, p. H etc. is called hypochromic effect. 34

8. A 1% 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= log. I 0 / IT • A= log. I 0 – log. IT • A= 2 - log % T 35

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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 )/ 1 x 100 = 75 % Ex 100 -25/100 x 100 Ø This also means that if a cuvette (10 mm ) is filled with an absorbing Solution and 75% of the light incident on it is transmitted on to another 10 mm cuvette placed next to it, the transmitted light will be reduced to 56. 25% Ø 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/. 39

] Ref. To the example 100% - ¼ = 75% - ¼ = 56. 25% - ¼ = 42. 19% 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 = • When T = 56. 25% A = 2 – 1. 875 2 – 1. 750 = = 0. 125 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 0. 1 % 1. 0 10% 100% = = = Infinite A 3. 0 A 2. 0 A 1. 0 A 0. 0 A. 40

Io 100% ¼ (10 mm ) I 75% ( 1 - ¼) ¼ (10 mm ) I ¼ 56. 25% (75 - ¼) I 42. 19% (56. 25% - ¼) (10 mm ) 41

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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". 43

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. 44

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

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. 46

(2) Grating: Most modern UV spectrophotometer uses diffraction grating as a Monochromator. It consists of a very large number of equispaced lines (200 - 2000 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. 47

(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. 48

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. ) 49

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. ) 50

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. 51

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 (200 - 800 nm). The reference cell just contains the pure solvent. 52

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. Log 10 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/I 0 (=10) and log 10 of 10 is 1. 53

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• 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. 55

• 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. 56

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. 57

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