Spectroscopy: Principles and EMR

• Spectroscopy is the study of interaction of matter with light or electromagnetic radiation.

Electromagnetic Radiation (EMR)

• EMR consists of discrete energy packages called photons.
• A photon is made up of oscillating electric and magnetic fields perpendicular to each other.
• Frequency (ν) is the number of oscillations per second and is measured in Hertz (Hz), where 1 Hz = 1 cycle per second.
• Wavelength (λ) is the distance between two nearest parts of a wave in the same phase, like the distance between two crests or troughs.
• The relationship between wavelength and frequency: c = νλ.
• The energy of a photon: E = hν = hc/λ.

Principles of Spectroscopy

• Spectroscopy measures the spectrum of a sample containing atoms or molecules.
• A spectrum is a graph of intensity of absorbed or emitted radiation versus frequency (ν) or wavelength (λ).
• A spectrometer measures the spectrum of a compound.

Absorption Spectroscopy

• Absorption spectroscopy measures the absorption of electromagnetic radiation.
• Examples include UV (185-400 nm) / Visible (400-800 nm) Spectroscopy and IR Spectroscopy (0.76-15 µm).

Emission Spectroscopy

• Emission spectroscopy is an analytical technique where emission is dispersed based on its properties, and the dispersion amount is measured.
• An example is Mass Spectroscopy.

Interaction of EMR with Matter

• Molecules at room temperature are typically in the lowest energy levels (So).
• When molecules absorb UV-visible light from EMR, an outermost bond/lone pair electron is promoted to a higher energy state (S1, S2, ...Sn), which is called electronic transition.
• The difference in energy is expressed as ΔE = hν = Sn - So, where n = 1, 2, 3, etc.
• Vibrational energy levels are less energetic than electronic energy levels.
• When IR radiation is absorbed, molecules are excited from one vibrational level to another or vibrate with higher amplitude.
• Rotational energy levels are quantized and discrete.
• Spacing between energy levels are smaller than vibrational energy levels: ΔErotational < ΔEvibrational < ΔEelectronic.

Lambert's Law

• Absorbance (A) or optical density is given by: A = log (Io / I).
• The typical absorbance range is 0 to 2.
• Transmittance (T) is the ratio of transmitted light to incident light: T = I / Io.
• The equation A = log (1/T) is also expressed as A = 2 - log T%.

Beer's Law

• Beer's Law states: A = ε.C.l, where:
• A is absorbance
• ε is the molar extinction coefficient
• C is the concentration
• l is the path length

Principles of UV-Visible Spectroscopy

• UV radiation extends from 10 nm to 400 nm, and visible radiation extends from 400 nm to 800 nm.
• The Near UV region spans 200 nm to 400 nm, while the Far UV region is below 200 nm.
• Far UV spectroscopy is performed under vacuum conditions.
• Common solvents for sample preparation include ethyl alcohol or hexane.

Electronic Transitions

• Possible electronic transitions include σ → σ*, π → π*, n → σ*, n → π*, σ → π*, and π → σ*.

Electronic Transition Details

• σ → σ* transition: σ electrons are excited to corresponding anti-bonding orbital σ*, requiring high energy; methane (CH4) can undergo this transition and shows absorbance maxima at 125 nm.
• π → π* transition: π electrons in bonding orbitals are excited to corresponding anti-bonding orbital π*; alkenes, alkynes, carbonyl, etc.,undergo this transition, with alkenes generally absorbing in the 170-205 nm range.
• n → σ* transition: Saturated compounds with lone pair electrons (O, N, S, halogens) can undergo this transition, requiring less energy than σ → σ* transitions; organic functional groups with peaks in UV region are small (150-250 nm).
• n → π* transition: Electrons from non-bonding orbitals are promoted to anti-bonding π* orbitals; compounds with double bonds involving heteroatoms (C=O, C=N, N=O) undergo such transitions, requiring minimum energy and absorbing at longer wavelengths around 300 nm.
• σ → π* & π → σ* transitions: Considered forbidden and only theoretically possible, these transitions show absorption in the region above 200 nm and accessible to UV-visible spectrophotometers, resulting in a few broad absorption bands in the UV spectrum.

Chromophore

• A chromophore is a molecule part which imparts color and is a covalently unsaturated group.
• It is a functional group with multiple bonds capable of absorbing radiation above 200 nm due to n → π* & π → π* transitions such as NO2, N=O, C=O, C=N, C≡N, C=C, C=S, etc.
• Non-conjugated alkenes show intense absorption below 200 nm and are inaccessible to UV spectrophotometers.
• Non-conjugated carbonyl group compounds give a weak absorption band in the 200-300 nm region.
• Acetone has λmax = 279 nm whereas cyclohexane has λmax = 291 nm.
• When double bonds are conjugated in a compound, λmax is shifted to a longer wavelength; 1,5-hexadiene has λmax = 178 nm, and 2,4-hexadiene has λmax = 227 nm.
• Conjugation of C=C and carbonyl groups shifts the λmax of both groups to a longer wavelength.
• For example, Ethylene has λmax = 171 nm, Acetone has λmax = 279 nm, and Crotonaldehyde has λmax = 290 nm.

Auxochrome

• An auxochrome is a functional group attached to a chromophore that modifies the chromophore's ability to absorb light, altering the wavelength or intensity of absorption.
• It includes a functional group with non-bonding electrons that does not absorb radiation in the near UV region but alters the wavelength & intensity of absorption when attached to a chromophore.
• For example, Benzene λmax = 255 nm, Phenol λmax = 270 nm, and Aniline λmax = 280 nm.

Absorption & Intensity Shifts

• Bathochromic Shift (Red Shift): The shift of absorption maxima (λmax) of a compound to a longer wavelength, caused by the presence of an auxochrome or a change of solvent; e.g., an auxochrome group (-OH, -OCH3).
• Hypsochromic Shift (Blue Shift) occurs when the absorption maxima (λmax) of a compound shifts to a shorter wavelength; this is caused by the removal of conjugation or by the change of solvent.
• Hyperchromic Effect increases the absorption intensity (ε) of a compound, often by introducing an auxochrome.
• Hypochromic Effect decreases the absorption intensity (ε) of a compound.

Applications of UV/Visible Spectroscopy

• Qualitative & Quantitative Analysis: Used to characterize aromatic compounds and conjugated olefins and to find out molar concentration of solutes.
• Detection of impurities: Detect impurities in organic solvents.
• Detection of isomers.
• Determination of molecular weight using Beer's law.