Spectrophotometry Spectroscopy is the study of interaction of spectrum of light with a substance to be analysed, for its identification (i.e qualitative analysis) as well as determination of its amount (i.e quantitative analysis). It is one of the different forms of radiant energy, such as, sunlight, radio waves and X-rays, which have similar properties and are called electromagnetic radiations (EMR) due presence of both electric and magnetic components. Light

Dual nature of light Light exhibits wave property during its propagation and energy particle during its interaction with matter. The double nature of light (Waves and particles) is known as dualism. EMR display the property of continuous waves and can be described by the characteristics of wave motion. Such wave motion in conveniently classified according to the wavelength. Wavelength ( ) 1. Wave property It is the linear distance measured along the line of propagation between crest of one wave to the next. Wavelength (l)

Unit of Length Micron (  ) = 1 x10 -6 m. = 1 x cm = 1x mm. Millimicron (m  ) = or nanometer (nm) = 1 x m. = 1 x cm = 1 x mm. Angstrom (A o ) = 1 x m = 1 x cm = 1 x mm.  = 1000 m . Frequency[][] is the number of waves/ second or number of cycles occuring/ second (CPS) or Hertz (Hz) or waves vibration/ second. Relation between &  a) C=.  = C / where c is the velocity of light cm/s. C= 3.0 x cm/s

Wave number (  ): which is the number waves/ cm 2-Particle property (light as energy) Light consist of energy packets, known as photons. The energy (E) of photons is proportional to the frequency i.e related to c and. It can be expressed by max plank relation: E = h  h = max plank constant = 3.63 x erg., sec.) i.e E   or E   (  )  = C /  = 1/ 1/ Therefore energy of a beam of EMR increase as wave length decreases.

Interaction of a substance with EMR When a molecule of certain substance interact with radiant energy (EMR), a molecule may absorb energy in three ways: 1-When molecule absorb visible and U.V region, Raising electrons to a higher energy level (transitional energy 3-When the molecule absorb in F.I.R region. Raising the Vibration of the constituent nuclei (Vibrational nergy) 2-When the molecule absorb I.R region. Increasing rotation of the molecule around the axis (rotational energy)

When a molecule interact with radiant energy in the visible and U.V region, leads to displacement of an outer electron (valence electron) and the molecule is said to be excited because electron undergo transition from original energy level (ground state = Eg) to an excited state (Es). Excited state E s Ground state E g The energy of transition is given by the following equation.  E = Es - Eg = h 

For analytical purposes we use the region of I.R, visible and U.V radiations Visible radiation part (day light) consist of some colored radiations, which are, red, orange, yellow, green, blue, indigo and violet (ROYGBIV).

Types of electronic transition The outer electrons in an organic molecule may occupy one of three different energy levels, as follows. 1-Sigma () electrons: they are bonding electrons posses the lowest energy level ( the most stable). 2-Pi () electrons :the bonding electrons constituting the pi bonds (double bonds) 3-Non-bonding (n) : electrons: don’t participate in bonding, they usually occupy the highest level of ground state.

In excited state  electrons occupy an ant bonding energy level denoted as * and the transition is termed -* transition.  -electrons occupy the ant bonding * level, while n electrons occupy either * or *.

Cut off  max of solvent Far UV near UV - Absorption: Compounds containing only -electrons are the saturated hydrocarbons which absorbs <170nm in the far UV. They are transparent in the near UV ( nm) making them ideal solvents for other compounds to be studied in this region. n-electrons absorption in saturated compounds: containing heteroatoms ( or halogens), the majority of these compounds show no absorption in the near UV, Methanol at 177nm, triethylamine at 199nm, chloroform at 173nm. Alcohols and ethers absorb at wavelength shorter than 185nm and so they are useful as common solvents at > 200nm. However, their intense absorption usually extends to the edge of the near UV producing end absorption (Cut off wavelength) in the nm region.

Absorption spectrum: according to the electronic transition that occur in each organic molecule, absorption spectrum is obtained by plotting Absorbance (A) as a function of wavelength (). It has characteristic shape which show the of maximum absorbance (max). It is characteristic for each molecule according to its structure (i.e Number and arrangement of electrons) and consequently type of transitions Therefore it is used for identification of a chemical substance (qualitative analysis). Also max is used for quantitative measurement, in order to increase sensitivity and to minimize error of the analytical method.

Shifting of max -Bathochromic shift (or red shift) It is the shift of max to a longer wavelength due to substitution with certain functional groups (e.g. –OH and –NH2), conjugation and effect of the medium (solvent). Bathochromic shift is also produced when two or more chromophores are present in conjugation. - Hypsochromic shift (or blue shift) It is the shift of max to a shorter wavelength due to removal of conjugation by changing polarity of the solvent. - Hyperchromic effect (or shift) an increase in the intensity of absorption. It is usually brought about by introduction of an auxochrome - Hypochromic effect (or shift) It involves a decrease in the intensity of absorption

Factors affecting absorption spectrum 1-Effect of pH The spectra of compounds containing acidic (phenolic-OH) or basic (-NH2) groups are dependent on the pH of the medium. 2-Effect of redox reaction An increase in conjugation due to oxidation may increases the absorbance spectrum of the compound appears coloured and exhibits hyperchromic effect.

PhenolAniline The U.V spectrum of phenol in acid medium benzenoid form while in alkaline medium is the corresponding phenate anion. The spectrum in alkaline medium exhibits bathochromic shift (red shift), increasing the delocalization of the -electrons, leading to the formation of conjugated system; electrons become more energetic and need less energy to be excited, therefore absorb longer and hyperchromic effect its spectrum exhibits bathochromic shift and hyperchromic effect in alkaline medium due to its conversion to the quinonoid species, while in acid medium its spectrum exhibit hypsochromic shift and hypochromic effect due to its conversion to the benzenoid species.

Increase in conjugation, increases absorbance of light by a compound which appears coloured and exhibits hyperchromic effect. redox reaction

Isosbestic point At different pH, the spectrum will be shifted to different max but all spectra intersect at certain which is known as isosbestic point At isosbestic point, the same absorbance is given for the same concentration at different pH,i.e. absorbance is not pH dependent but concentration dependent Thus solutions must be buffered at specific pH or measurements are carried out at the isosbestic point.

Theory of light absorption : (Beer-Lambert Law) When a monochromatic light having intensity (Io) is allowed to pass through absorbing medium, some is absorbed (Ia), reflected (Ir), transmitted (It), refracted (If) and scattered (Is). Io = Ia + It + Ir + If + Is Is = zero for clear solution, while If and Ir may be canceled by means of control cuvette containing the solvent in which the substance to be anaylsed is dissolved Therefore, under experimental conditions. Io = Ia + It or Ia = Io - It

The diagram below shows a beam of monochromatic radiation of radiant power P 0, directed at a sample solution. Absorption takes place and the beam of radiation leaving the sample has radiant power P. Absorbance, A = log 10 P 0 / P A = log 10 1 / T A = log / %T A = 2 - log10 %T The amount of radiation absorbed may be measured in a number of ways: Transmittance, T = P / P0 % Transmittance, %T = 100 T

Beer-lambert’s law: lamberts’ law: log Io/It  b (thickness ), Beers’ law : log Io/It  C (Concentration), i.e log Io/It  bclog Io/It = a b c log Io / It = K b log Io / It = K C Absorbance (A ) = log Io/It A = a b c a : is a constant, known as absorptivity which is the bsorbance, when thickness of solution is unity (i.e 1cm) and concentration is unity. Molar absorptivity or epsilon () If the unit of concentration is 1M, (a) is known as molar absortivity or epsilon () (Unit of  is L mol-1­­ cm-1) A (1% - 1cm): If unity of concentration is 1%, (a) is known as A (1%, 1cm). - Both  and A (1%, 1cm) are characteristic for each substance - and are used for qualitative purpose.

Colorimetry Requirements for substances to be measured colorimetricaly: 1.Substance must be coloured e.g CuSO 4, organic dyes,…etc. 2-If the substance to be analysed is colourless, it must react with certain reagent (known as chromogen) to produce equivalent coloured product. 1.orthophenanthrolene which reacts with ferrous (Fe2+)in buffered medium (acidic pH) to produce intense red colour. 2+ 3

3-If the substance to be analysed is colourless and there is no suitable chromogen, it must be converted to a certain derivative which has a suitable chromogen.e.g esters, which is first converted to hydroxamic acid derivative through the reaction with hydroxylamine. Hydroxamic acid derivative gives purple colour on addition of ferric (Fe3+) due to the formation of iron chelate. OO R –C– OEt + H 2 N – OH R –C– NH – OH+ EtOH Hydroxamic acid derivative

Requirements for ideal chromogen 1-Should be colourless or easily separated 2-Should be selective. 3-Its reaction to produce coloured product, should be of known mechanism and proceed stoichiometrically. 4-The full development of colour must be rapid. 5-Produce only one colour of specified max.