CH223_Ch_27_Fluorescence

Chapter 27 – Molecular Fluorescence Photoluminescence

Definition

A molecule or ion (M) is excited by absorbing a UV-visible photon (hn1) and then emits a photon (hn2) while returning to the ground state. The reaction can be summarized as:

M + hn1 → M* → M + heat + hn2

Key Concepts

  • Resonance Fluorescence: Occurs when the wavelength absorbed equals the wavelength emitted (no heat loss). For useful analytical fluorescence, the absorbed and emitted wavelengths differ.

  • Heat: Refers to infrared photons; photons mentioned in this context relate to UV-visible photons.

Pathways for Energy Release

  1. Collisional Deactivation: A non-radiative process where energy loss occurs through vibrations and collisions with other molecules. This is typically the fastest method for many species to return to lower energy states.

  2. Fluorescence: Some energy is dissipated non-radiatively, followed by the emission of a UV-visible photon. This process occurs on timescales ranging from less than 10^-5 seconds to 10^-10 seconds and is useful analytically, though not all molecules exhibit fluorescence.

  3. Phosphorescence: Involves an electron spin flip into an excited triplet state, making the return to the ground state forbidden and slow. This process can take longer than 10^-4 seconds and even hours, which limits its analytical usefulness.

Electronic States

The transitions between energy states include the ground state, fluorescence, and phosphorescence, with associated timescales of:

  • Fluorescence: 10^-5 to 10^-10 seconds

  • Phosphorescence: 10^-4 seconds to hours

Jablonski Energy Diagram

The Jablonski energy diagram visually represents transitions between energy states, highlighting processes such as absorption, non-radiative relaxation, fluorescence, and depicting vibrational relaxation and internal conversion.

Vibrational Modes in Molecules

There are various types of vibrations in molecules:

  • Stretching Vibrations: Symmetric and asymmetric stretching.

  • Bending Vibrations: This includes in-plane rocking, scissoring, and out-of-plane motions, with directionality indicating symmetrical or asymmetrical movements.

Absorption and Relaxation Processes

  • Absorption: Electron transitions to a higher electronic energy level, typically within ~10^-15 seconds.

  • Vibrational Relaxation: Transfers excess energy non-radiatively through collisions, occurring between ~10^-15 to 10^-12 seconds.

  • Internal Conversion: Non-radiative relaxation between electronic levels, taking about 10^-9 to 10^-6 seconds.

  • Fluorescence: Photon emission primarily from the v=0 level of an excited state, occurring from 10^-10 to 10^-5 seconds.

  • Intersystem Crossing: Electron spin flip from S1 to T1 leads to phosphorescence.

Additional Concepts

  • Resonance Fluorescence: Identical absorption and emission wavelengths.

  • Stokes Shift: Emission photons have lower energy than excitation photons due to vibrational relaxation. The peaks in the emission spectrum reflect vibrational energy levels.

Fluorescence Quantum Yield

  • Quantum Yield (Φ): Measures the effectiveness of fluorescence, defined as:

    Φ = (Number of emitted photons) / (Number of absorbed photons) = (Rate of Fluorescence) / [(Rate of Fluorescence) + (Rate of Radiationless Relaxation)]

Φ values range from 0 (no fluorescence) to approximately 1 (excellent fluorescer).

Factors Affecting Fluorescence

  • Conjugated Pi Bonding: Aromatic or conjugated systems exhibit better fluorescence due to favorable π → π* transitions.

  • Rigidity: Rigid structures reduce non-radiative relaxation, thus enhancing fluorescence.

  • Temperature: Lower temperatures typically enhance fluorescence by reducing molecular motion.

  • Solvent: Increasing the viscosity of solvents can also lead to improved fluorescence by minimizing molecular movement.

Relating Fluorescence to Concentration

Fluorescence intensity (F) is proportional to concentration and is given by:

F = 2.3K′P0εbc(Where P0 is the power of incoming light, ε is molar absorptivity, b is path length, and c is concentration.)

Beer’s Law Like Relationships

Methods for determining concentration include:

  • Calibration curves

  • Standard addition

  • Spectrophotometric titration

Common units used are ppm and ppb.

Self-Quenching

At high concentrations, emitted photons may be reabsorbed by other molecules, leading to a reduction in detected fluorescence (self-quenching). Halide ions are noted as common quenchers.

Fluorimeters

Fluorimeters resemble UV-visible spectrometers but are designed with angled monochromators to optimize light paths for fluorescence detection.

MicroPlate Reader Fluorometers

These devices are adaptable to UV-visible spectroscopy with slight modifications to the monochromator position, allowing for quicker analyses compared to cuvette-based methods, although there may be trade-offs in accuracy.

Key Features of Fluorescence

  • Selectivity: While fewer species fluoresce compared to UV-visible techniques, fluorescence offers higher selectivity due to the requirement for both excitation and emission wavelengths.

  • Detection Limits: Fluorescence techniques often achieve detection limits that are 10-1000 times lower than UV-visible spectroscopy, reaching down to ppb levels.

  • Analyzing Main Group Metals: Ligand complexes can facilitate fluorescence analysis of metals that do not fluoresce well on their own, e.g., the interaction of 8-hydroxyquinoline with Zn2+.

Fluorescence Probes in Biochemical Analysis

Common fluorescent tags and their respective excitation/emission characteristics include: FITC, PE, APC, Cascade Blue, Texas Red, TMR, among others.

Chemiluminescence Examples

Chemical reactions that yield electronically excited species that emit light as they return to their ground state include notable examples like glow sticks and luminol reactions, with iron in hemoglobin catalyzing these processes.