Analytical methods Prepared By Dr. Biswajit Saha

Spectroscopy Spectroscopy is the branch of science which deals with the determination of the structure of a compound through a the interactions of various types of electromagnetic radiation with the matter. What is electromagnetic radiation ? Example: Light Light interacting with matter Exactly how the radiation interacts with matter is directly dependent on the energy of the radiation. Energy , E= hv Wave length, frequency etc.

Electromagnetic Spectrum Electromagnetic Waves

Visible region Violet: 400 - 420 nm Indigo: 420 - 440 nm Blue:   440 - 490 nm  Green:   490 - 570 nm  Yellow:   570 - 585 nm  Orange:   585 - 620 nm  Red:   620 - 780 nm

Energy level diagram Different colors observed when the white light was dispersed through the prism Energy level diagram E2 vibrational levels v2 v1 rotational levels r2 v0 r1 E1 Pure electronic transition Eelec >> Evib >> Erot v2 DE = hn v1 r2 v0 E0 r1 electronic levels 5

Absorption & Emission Electron exist in a certain number of possible states corresponding to definite amounts of energy. Electron can absorb energy and change to a higher energy level called the excited state. The amount of energy absorbed in this transition is exactly equal to the energy difference between the states.

E2 E1 E0 ENERGY Energy Level Diagram for an Atom of Sodium 590 nm 330 nm E0 Energy Level Diagram for an Atom of Sodium Ground State

Atomic energy level

Different Spectroscopies UV-vis – electronic states of valence e/d-orbital transitions for solvated transition metals Fluorescence – emission of UV/vis by certain molecules IR – Vibrational transitions of molecules X-Ray Spectroscopy – Electronic transitions of core electrons NMR – Nuclear spin transitions

UV-Visible Spectroscopy Ultraviolet-visible spectroscopy involves the absorption of ultraviolet/visible light by a molecule causing the promotion of an electron from a ground electronic state to an excited electronic state. Ultraviolet/Visible light: wavelengths (l) between 190 and 800 nm UV-visible spectrum The two main properties of an absorbance peak are: Absorption wavelength lmax Absorption intensity Amax

UV-Visible Spectroscopy When a beam of monochromatic radiation passes through a homogeneous medium, A part of the incident light is reflected, a part of it is absorbed within the medium and the remainder is transmitted. Let the intensity of Incident light (I0), absorbed light (Ia ), transmitted light (It) and Reflected light (Ir). Then I0=Ia+It+Ir 106 photons 0.7x106 photons  500 nm

Beer-Lambert Law and UV-Visible Spectroscopy Lambert Law: the rate of the intensity of light decrease with thickness of the medium is proportional to the Intensity of the incident light. -dI/dx α I Beer-Lambert Law: the rate of the intensity of light decrease with thickness of the medium is proportional to the Intensity of the incident light as well as concentration of the solution. -dI/dx α cI Mathematical derivation x

Beer-Lambert Law log(I0/I) = exc A = exc I A = log = e xc = - log T I I0 = Intensity of incident light I = intensity of transmitted light = molar absoptivity coefficient in cm2 mol-1 c = concentration in mol L-1 x = pathlength of absorbing solution in cm-1 A = absorbance = log(Io/I) T=transmittance (I/I0) log(I0/I) = exc A = exc I A = log = e xc = - log T I

Transmittance, Absorbance, and Concentration

Numerical problem The percentage transmittance of an aqueous solution of unknown compounds is 20 % at 25 oC and 300 nm for 4x10-5 M solution in a 2 cm cell. The absorbance (A) The molar extinction or absorption coefficient Ans. 0.699, 8.7x103 M-1 cm-1 For a solution of camphor in hexane in a 10 cm cell, the absorbance (A) was found to be 2.52 at 295 nm with molar absorptivity (ε) 14. What is the concentration of the camphor?

Limitation of Beer-Lambert Law

Five types of transitions are possible: *, *, n *,  * n * Molecular energy level Five types of transitions are possible: *, *, n *,  * n *

Effect of conjugation on UV spectra The addition of conjugation in naphthalene, anthracene and tetracene causes bathochromic shifts of these absorption bands,

Lycopene from Tomatoes Visible region

UV-visible spectroscopy definitions Chromophore: Any group of atoms that absorbs light whether or not a color is thereby produced. Auxochrome : A group which extends the conjugation of a chromophore by sharing of nonbonding electrons. Bathochromic shift: The shift of absorption to a longer wavelength. Because the red color in the visible spectrum has a longer wavelength than most other colors, this effect is also commonly called a red shift. Hypsochromic shift : The shift of absorption to a shorter wavelength. (blue shift)