Lecture 5 - Spectroscopy

Spectroscopy

Study of the production, measurement and interpretation of spectra arising from the interaction of electromagnetic radiation [EMR] with matter

• Types of spectroscopy

  • Ultraviolet (UV)

  • Visible (Vis)

  • Infrared (IR)

  • Atomic Absorption

  • Atomic Emission

  • Ramen

Electromagnetic Radiation (EMR)

• Particles of energy that move through space with wave-like properties

• Travels through space (vacuum) at the speed of light

• Described by:

  • 𝝀 = wavelength (nm)

  • 𝝂 = frequency (1/time)

Wavelength (𝝀)

Distance between 2 consecutive crests (peaks) on any given wave

𝝀*𝝂 = c (c= 2.99792 × 10⁸ m/s)

Frequency (𝝂)

Number of oscillations per second (Hz = 1/s)

• Determined by the source of radiation (light) and it remains constant as EMR travels through space and different media

𝝀*𝝂 = c (c= 2.99792 × 10⁸ m/s)

Amplitude (A)

Height of the wave

Particle theory of light

• Light can also be described as particles of energy that move through space with wavelike properties

  • Photons

• Energy of a photon is quantized; it exists in discrete steps

• Energy of a photon (E) is equal to the frequency of the wave (𝝂) x Planck’s constant (h = 6.6262 × 10^-34 J/sec)

• E = h𝝂

• Photons with a higher frequency will have more energy

Quantized energy states of atoms and molecules

• Energy of atoms and molecules is not continuous but exists in discrete steps (quanta)

• Under normal conditions atoms & molecules exist in the lowest energy state (ground state)

• Energy can be absorbed by atoms and molecules and they can be elevated to higher energy levels

• The potential energy of an atom and molecule will correspond to the energy differences between excited and the ground state

Electronic energy states of an atom

When the electron relaxes to ground state, light is emitted as a photon with a wavelength that is associated with that specific energy transition

UV-Vis Spectroscopy

Involves EMR between ~100-780 nm

• UV from 100-400 nm (colorless)

• Visible range from 400-780 nm

• Types:

  • Absorption spectroscopy

  • Emission spectroscopy

  • Fluorescence spectroscopy

Types of UV-Vis Measurements

• Qualitative - molecular identification based upon the whole absorbance spectrum (fingerprint)

  • Identification is done by comparing the absorption spectrum with the spectra of a known standard compound

• Quantitative (measuring) - determining the exact concentration of an analyte

  • Requires a standard (Beer’s law)

  • measurement of the amount of light absorbed as it passes through a sample solution

Principles of UV-Vis Spectroscopy

• Absorption of EMR by a molecule causes electrons to move from ground to excited states

  • Absorption is restricted to certain functional groups: CHROMOPHORES - contain valence electrons of low excitation energy that can absorb energy in the UV-Vis range

  • SHORTER 𝝀 = more energy

Molar Absorptivity (ε)

Measurement of how strongly chromophores absorb light at a given wavelength

• Think double bonds

Spectrophotometer Components

• Light source

  • Emits a continuous band of EMR encompassing a wide range of ERM

• Monochromator (monochromatic light)

  • Isolates the specific 𝝀_max to be measured

• Sample cell/reference cell

• Detector

  • Converts the light transmitted through the sample cell (photons) into an electric signal

• Computer

Fluorescence Spectroscopy

• 3 step process that describes the absorption and emission of ERM by certain highly conjugated molecules

• Fluorophore: a molecule that upon absorption of EMR will emit a photon of a lower energy (longer 𝝀)

• HIGHLY SENSITIVE technique

  • More sensitive than UV

• Very selective technique because there 2 𝝀 of light involved:

  • Excitation wavelength

  • Emission wavelength

Excitation

A photon of energy is supplied by an external source (lamp) and is absorbed by the fluorophore, creating an excited electronic state

Excited state

• Exists for a finite time (quick)

• Fluorophore undergoes conformational changes and part of its energy is dissipated

Emission

Photon of energy is emitted, returning the fluorophore to its ground state

• Due to energy dissipation during the excited-state lifetime, the energy of this photon is lower and the 𝝀 light emitted is longer