ULTRAVIOLET AND VISIBLE SPECTROSCOPY Chap 6&7-T1 Chap 1-T2 Chap 13&14-R1

when a molecule is subjected to UV (or Vis) radiation transitions of valence electrons occurs in the molecule. i.e excitation of an electron from a filled molecular orbital to the next higher energy orbital. since UV energy is quantized, the absorption spectrum arising from a single electronic transition should consist of single discrete line. but due to superimposition of rotational and vibrational sublevel a discrete line never obtains

Lambert-Beer law: For monochromatic radiation absorbance is directly proportional to the pathlength and the concentration of the absorbing species. Absorbance A of a sample is defined as: A = -log10 T = log P0/P = ε c l where, Po = intensity of incident radiation energy P = intensity of transmitted radiation energy c = molar concentration of solute l = internal length of the cell ε= molar absorptivity

Molar absorptivity/Molar extinction coefficient: if the concentration of species is expressed in molar concentration and pathlength is in centimeter, the constant is c/a molar extinction coefficient Its unit = liter mole-1cm-1 and it is constant for a given absorbing molecule For Bovine Serum Albumin(BSA) ε2801% =6.67

Deviation from BL law: fall into 3 categories: Real deviation:At high concentration, - the average distance between the species molecules decreases affecting the charge distribution of neighboring molecule - the refractive index of solution (>10-3M) varies considerably. 2. Instrumental deviation: arise from finite bandpass of filters or monochromators. Means truly monochromatic radiation( single wavelength) is seldom practical

3. Chemical deviation: arises when a species dissociates,associates or react with a solvent to form a product having different absorption. ex. dichromate ion absorbs in the visible region at 450 nm. 2CrO42- + 2H+  2HCrO4-  Cr2O72- + H2O Upon diluting the equilibrium shifts to the left.The equilibrium can be controlled by converting all the chromium to CrO42- by making the solution of 0.05M in KOH

Instrumentation Radiation sources: Hydrogen or Deuterium Lamps for UV regions, Tungsten Filament lamps for vis and near-IR Wavelength selector:filters and monochromators Cells: for UV quartz, for visible plastic etc Photodetector: photomultiplier tubes Readout device

Type of Instrument Single beam instrument: simplest and least expensive one consists of a battery-operated tungsten bulb as the source, a set of glass filters for wavelength selection, test tubes for sample holders, a photovoltic cell as the detector and a small microammeter a the readout device

b) double beam instrument: In double-beam-in-space instrument two beams are formed in space by a V-shaped mirror c/a a beam splitter one beam passes through the reference cell and other through the sample cell, simultaneously, to respective detector

b) double beam instrument: In double-beam-in-time instrument two beams are separated in time by a rotating sector mirror that direct entire beam first through the reference cell and then sample cell

Application: 1. Qualitative analysis have some what limited application for qualitative analysis because the number of absorption maxima and minima are relatively few nevertheless it is useful for detecting the presence of certain functional groups that act as chromophore a weak absorption band in the region of 280 to 290nm, which is shifts towards shorter wavelengths with increased solvent polarity, strongly indicates the presence of the carbonyl group.

Application: 2. Quantitative analysis one of the most useful and widely used tools available for quantitative analysis. (95% in the health field) any organic compound containing one or more chromophore can easily be quantitated a number of inorganic species also absorb and can be directly determined nonabsorbing species can also be quantitated by reacting selectively with certain reagents

Procedural Details:involve establishment of working conditions and the preparation of a calibration curve 1. Selection of wavelength: λmax from spectrum 2. Variables that influences absorbance: solvent, pH temperature, interfering substances. 3. Cell handling: lens paper soaked in spectrograde methanol,careful reposition a cuvette 4. Preparation of calibration curve: from a series of standard solutions that bracket the conc. range expected for the unknown sample

= εMcMl + εNcNl … A2 = ε2M c2M l + ε2N c2N l (at λ2) Analysis of Mixtures of absorbing substances: total absorbance of a solution is equal to the sum of absorbances of the individual component present Atotal = AM + AN… = εMcMl + εNcNl … By measuring the absorbance at 2 wavelength (λ1 and λ2) A1 = ε1M c1M l + ε1N c1N l (at λ1) A2 = ε2M c2M l + ε2N c2N l (at λ2)

4 molar absorptivities ε1M ε2M ε1N ε2N can be obtained from individual standard solutions of M and N l is the cell thickness, constant Absorbance of mixture at 2 wavelengths (λ1 and λ2) are experimentally determined Thus from the above two equations conc. of individual species, cM and cN, can be calculated

Photometric Titration: photometric titration curve is a plot of absorbance vs. volume of titrant and can be employed to measure the equivalence point of a titration change in absorbance of a solution is used to follow the change in concentration of a absorbing species during a titration absorbance is directly proportional to the conc. of the absorbing species in ideal condition the plot consist of two straight lines that intersect at the end point Some of the typical titration curves are:

a)Where titrant alone absorbs ex: titration of arsenic(III) with bromate-bromide, where absorbance is measured at the wavelength where the bromine absorbs b)Where the product of the reaction absorbs ex: titration of Copper(II) with EDTA c) Where species is converted into non-absorbing product ex: titration of p-toluidine in butanol with perchloric acid d) Where a colored species is converted to a colorless product by a colored titrant ex: bromination of a red dyestuff a b c d

Advantages over direct photometric determination: presence of other absorbing impurities at the analytical wavelength does not cause interference accuracy is more because data from several measurements are used to calculate the end point relatively dilute solution can be used nonabsorbing species can also be quantitated

Application: Determination of the dissociation constants of weak acids or bases Determination of the composition of complex ions Determination of chlorine in water Determination of the total, calcium and magnesium hardness of water samples by photometric titration Determination of silicon, calcium, magnesium, iron and aluminum in cements by photometric titration of the solubilized product Analysis of jams, fruit and vegetable juices and their concentrates