Fluorescence Spectroscopy Dr AKM Shafiqul Islam School of Bioprocess Engineering

Spectroscopy The basis for the microscopic description of spectroscopy is a system composed of two energy levels. In any spectroscopy, it is necessary to induce a transition between two energy levels to detect the absorption/emission of electromagnetic energy. Light is emitted or absorbed if this system is exposed to a frequency:

When we have a population of atoms or molecules (as we always do when we do macroscopic measurements), each state is populated with a certain number of electrons n1 and n2 according to Boltzmann’ distribution: The energy absorbed is proportional to the difference:

then essentially all the molecules will be in their ground state; the radiation field will slightly perturb this situation increasing the average number n2 and decreasing n1 by the same amount. the net absorption of energy will be very small because the rate of upward transitions is equal to the rate of downward transitions.

How Does Matter Absorb Radiation We see objects as colored because the transmit or reflect only a portion of light in this region. When polychrometic (white light), which contains the whole specrum of light, we see objects as colored because certain wavelengths are absorbed while others are reflected. The reflected colors impinge on our eyes, and we perceive these colors.

Fluorescence Thus far we have only considered absorption processes. If resonant absorption occurs, a system will eventually return to the ground state. There are a number of pathways that can be followed to return to the ground state. Important mechanisms are radiationless transfer (molecular collisions), fluorescent emission, and phosphorescence.

Fluorescence is probably the most important form of spectroscopy in biology, think about GFP and all sort of microscopies that are done now using fluorescence as a probe of biological structure or localization. The reason it is so widely used is that it is a very sensitive technique (even single molecules can be detected by fluorescence).

What is Atomic Fluorescence? Atomic fluorescence spectroscopy (AFS) is the optical emission from gas-phase atoms that have been excited to higher energy levels by absorption of radiation. AFS is useful to study the electronic structure of atoms and to make quantitative measurements of sample concentrations.

Fluorescence Spectroscopy In fluorescence spectroscopy, the signal being measured is the electromagnetic radiation that is emitted from the analyte as it relaxes from an excited electronic energy level to its corresponding ground state. The analyte is originally activated to the higher energy level by the absorption of radiation in the UV or VIS range. Generally one to three orders of magnitude more sensitive than corresponding absorption spectroscopy.

The Electromagnetic wave Electromagnetic radiation can be considered a form of radiant that is propagated as a transverse wave. It vibrates perpendicular to the direction of propagation, and this imparts a wave motion to the radiation. The wave is described either in terms of its wavelength, the distance of one complete cycle, or in terms of the frequency, passing a fixed point per unit time. The reciprocal of the wavelength is called wavenumber and is the number of waves in a unit length or distance per cycle.

Wave propagation >780 nm 380 nm – 780 nm <380 nm Near IR: 800 nm-2,500 nm Mid-IR: 2,500-15,000 nm Far IR: 15,000-100,000 nm

Electromagnetic Wave The relationship between the wavelength and frequency is Where l is the wavelength in centimeters (cm), n is the frequency in reciprocal seconds (s-1), or hertz (Hz), and c is the frequency of light (3 x 1010 cm/s). The wavenumber is represented by n, in cm-1:

Electromagnetic Radiation Electromagnetic radiation possesses a certain amount of energy. The energy of unit of radiation, called the photon, is related frequency or wavelength by Where E is the energy of the photon in ergs and h is Plank’s constant, 6.62 x 10-34 joule-second (J-s).

PHOTOLUMINESCENCE Fluorescence : Does not involve change in electron spin; short lived (less than microsecond). Can be observed at room temperature in solution. 2. Phosphorescence: Involves change in electron spin. Long lived (seconds). Can be observed at low temperature in frozen or solid matrices. 3. Chemiluminescence: Light emission due to a chemical reaction.

SINGLET AND TRIPLET STATES Electronic transitions responsible for absorption spectra will give rise to singlet and triplet states and hence to fluorescence and phosphorescence. Singlet State: Electron spins in the ground and excited electronic states are paired. Net spin S is zero. Spin multiplicity 2S + 1 = 1. Triplet State: Electron Spins in the ground and excited electronic states are not paired. Net spin S = 1. spin multiplicity 2S + 1 = 3.

Excited states Producing Fluorescence and Phosphorescence Ground Singlet State A molecule at room temperature normally resides at in the ground state. The ground state is usually singlet state (So), with all electrons paired. Excited Singlet State Excited Triplet State

Jablonski Diagram

Atomic Fluorescence Spectroscopy optical emission from gas-phase atoms that have been excited to higher energy levels Enhancement of sensitivity over AA Examine electronic structure of atoms Light source Hollow Cathode Lamp Laser Detection Similar to AA

Hallow cathode Tube

Fluorescence Spectroscopy Excitation beam Emission beam

Fluorescence Spectroscopy

Fluorescence Spectroscopy PF =  (P0-P) (1) Where PF= radiant power of beam emitted from fluorescent cell  = constant of proportionality (or quantum efficiency) Beer’s law: P=P010-bc (2) PF=  P0 (1-10-bc) (3) PF=kP0c (4) Where k=constant of proportionality Linear concentration range Factors: T, solvent, pH, impurity

Example: Pyrene K. Kalyanasundaram and J. K. Thomas, J. Am. Chem. Soc., 1977, 99, 2039

Example: Dansyl