3. Spectroscopy Fundamentals

Spectroscopy

Analytical methods based on interaction between electromagnetic radiation and matter.

Electromagnetic relation

c = νλ; c = speed of light, ν = frequency, λ = wavelength.

Photon energy

E = hν; h = Planck constant.

Energy-wavelength relation

Higher frequency means higher energy and shorter wavelength.

UV/VIS transition type

Mostly electronic transitions.

IR transition type

Mostly molecular vibrations and rotations.

Microwave transition type

Molecular rotations.

Absorption

Matter absorbs radiation; measured as decrease in detected light relative to incoming light.

Absorption process

M + hν → M*.

Emission

Excited species emit radiation when returning to lower energy; excitation often by heat/flame/plasma, no optical source required.

Emission process

M* → M + hν.

Luminescence

Light emission from an excited state after excitation by radiation or reaction, not thermal emission.

Fluorescence

Prompt luminescence: molecule absorbs photon then emits longer-wavelength light.

Phosphorescence

Luminescence involving spin change; emission lasts longer than fluorescence.

Why emitted fluorescence has longer wavelength

Some energy is lost before emission, so emitted photon has lower energy and longer wavelength.

Transmittance formula

T = P/P0; P0 = incident radiant power, P = transmitted radiant power.

Absorbance formula

A = log(P0/P) = -log T.

Beer’s law molar form

A = εbc; ε = molar absorptivity, b = path length, c = molar concentration.

Beer’s law mass concentration form

A = abc; a = absorptivity when c is e.g. g/L.

Path length b

Distance light travels through sample, usually in cm.

Molar absorptivity ε

Substance- and wavelength-dependent measure of how strongly analyte absorbs.

Spectrophotometer general setup

Radiation source → sample → analyzer/monochromator → detector → readout/registration.

Absorption instrument diagram

Source and analyzer illuminate sample; detector measures transmitted light; reference/blank often used.

Fluorescence instrument diagram

Source → analyzer → sample; emitted light passes second analyzer; detector placed at angle to excitation light.

Emission instrument diagram

No lamp source; excitation by heat/flame/plasma; emitted light selected by analyzer and measured by detector.

Analyzer/monochromator function

Selects wavelength or wavelength band before detection.

Examples of analyzers

Filter, prism, grating monochromator.

Bandwidth

Range of wavelengths passed by analyzer.

Narrow bandwidth consequence

Better spectral resolution/selectivity but lower light intensity.

Wide bandwidth consequence

Higher light intensity but more spectral overlap and poorer selectivity.

Stray light

Unwanted light reaching detector; causes deviation from Beer’s law.

Beer’s law chemical deviations

High concentration, analyte interactions, association/dissociation or reactions.

Beer’s law instrumental deviations

Polychromatic light, stray light, detector/instrument limitations.

Other absorbance losses

Reflection and scattering can reduce transmitted light and distort absorbance.

Reason analyte gives no spectrophotometric signal: no chromophore

Fix by derivatization or forming colored/absorbing complex.

Reason analyte gives no spectrophotometric signal: wrong wavelength

Fix by choosing λmax or suitable wavelength.

Reason analyte gives no spectrophotometric signal: concentration too low

Fix by preconcentration, more sensitive method, longer path length or fluorescence.

Reason analyte gives no spectrophotometric signal: matrix/background

Fix with blank, separation, different wavelength or method.

Chromophore

Part of molecule that absorbs UV/VIS radiation.

Auxochrome

Group that modifies/intensifies chromophore absorption.

VIS cuvette material

Glass or plastic can work if transparent and inert at selected visible wavelength.

UV cuvette material

Quartz is needed because ordinary glass/plastic can absorb UV.

IR sample cell material

IR-transparent salts/windows such as KBr/NaCl; ordinary glass is unsuitable.

Diode array detector

Disperses light onto many detector elements, allowing many wavelengths/spectrum to be measured quickly.

Main advantage of diode array detector

Fast simultaneous wavelength measurement and spectrum collection.

Atomic absorption spectroscopy AAS

Atoms absorb element-specific radiation from lamp; requires atomization.

Atomic emission spectroscopy AES

Atoms are excited thermally/electrically and emit element-specific radiation.

X-ray spectroscopy phenomenon most often used

Luminescence/fluorescence: X-ray fluorescence after inner-shell excitation.

How to obtain X-rays

Accelerate electrons into a metal target or use X-ray tube; inner-shell processes produce X-rays.

Chemical interference in atomic spectroscopy

Matrix changes atomization or forms stable compounds, reducing/freeing analyte atoms differently.

Reduce chemical interference

Use releasing/protective agents, hotter flame/plasma, matrix matching, standard addition, or chemical separation.

Spectral interference

Overlapping emission/absorption lines or background at analytical wavelength.

Ionization interference

Analyte atoms ionize, reducing neutral atoms for AAS/emission; controlled with ionization suppressor.

Matrix interference

Matrix changes signal through viscosity, transport, atomization, scattering, background or chemical effects.