chem604 (10)

Applications of Spectroscopy & Luminescence – Comprehensive Notes

Spectroscopy has many important applications in:

• Chemical analysis

• Environmental monitoring

• Medical diagnostics

• Biosensors

• Food analysis

• Molecular biology

One major application involves:

• Luminescence spectroscopy

Luminescence includes:

• Fluorescence

• Phosphorescence

These techniques are extremely sensitive and can detect:

• Very low concentrations

• Single molecules in some cases

Sources:

Luminescence

Definition

Luminescence is:

• Emission of light from a substance after energy absorption

Unlike incandescence:

• Luminescence does not require high temperatures

Types of Luminescence

Two important forms are:

1. Fluorescence

2. Phosphorescence

Fluorescence

Definition

Fluorescence occurs when:

• A molecule absorbs energy

• Electrons are excited to a higher energy state

• A photon is emitted during return to the ground state

Transition:

$<br>S_1 \rightarrow S_0<br>$

Characteristics of Fluorescence

Fluorescence:

• Occurs rapidly

• Has short lifetime

• Usually stops immediately when excitation source is removed

Typical lifetime:

$<br>10^{-9} - 10^{-5}\ s<br>$

(nanoseconds to tens of microseconds)

Phosphorescence

Definition

Phosphorescence occurs when:

• Excited molecules undergo intersystem crossing

• Molecules enter a triplet state

• Photon emission occurs during return to ground state

Transition:

$<br>T_1 \rightarrow S_0<br>$

Characteristics of Phosphorescence

Phosphorescence:

• Occurs more slowly

• Continues after excitation source is removed

• Has longer lifetime

Typical lifetime:

$<br>10^{-5} - 10^2\ s<br>$

(tens of microseconds to hundreds of seconds)

Why Phosphorescence Is Slower

Phosphorescence involves:

• Change in spin quantum numbers

This transition is:

• Spin-forbidden

• Less probable

Therefore:

• Emission occurs slowly

Relative Intensity of Fluorescence and Phosphorescence

For the same molecule:

• Phosphorescence occurs at lower energy

• Phosphorescence is weaker than fluorescence

Often:

• About 10 times weaker

Phosphorescence is commonly observed only when:

• Samples are cooled

Persistent Phosphorescence

Some materials phosphoresce for:

• Hours after light exposure

Example material:

$<br>SrAl_2O_4:Eu:Dy<br>$

(strontium aluminate doped with europium and dysprosium)

Application of Persistent Phosphorescence

Used in:

• Emergency exit signs

• Glow-in-the-dark materials

These continue glowing:

• After power loss

Nonradiative Processes

Internal Conversion

Definition

Internal conversion is:

• Nonradiative transition between states with same spin multiplicity

Example:

$<br>S_1 \rightarrow S_0<br>$

Energy is released as:

• Heat

Intersystem Crossing

Definition

Intersystem crossing is:

• Nonradiative transition between states with different spin multiplicities

Example:

$<br>S_1 \rightarrow T_1<br>$

Why Luminescence Is Highly Sensitive

Luminescence is generally:

• More sensitive than absorption spectroscopy

Stadium Analogy

Absorption Analogy

Imagine:

• Stadium lights on

• 50,000 people holding candles

• 500 extinguish candles

Change is:

• Difficult to detect

This is similar to:

• Measuring tiny absorbance changes

Fluorescence Analogy

Now imagine:

• Completely dark stadium

• 500 candles suddenly lit

Change becomes:

• Very obvious

This resembles:

• Detecting fluorescence against dark background

Absorbance Example

Changing transmittance from:

$<br>100$

corresponds to absorbance:

$<br>A = -\log(0.99)<br>$

$<br>= 0.0044<br>$

This small signal is difficult to measure.

Sensitivity of Luminescence

Luminescence is sensitive enough to:

• Detect single molecules

Rhodamine 6G Experiment

Highly fluorescent dye:

• Rhodamine 6G

was used to observe:

• Random motion of individual molecules

These experiments confirmed:

• Einstein’s random walk theory

proposed in:

$<br>1905<br>$

Relation Between Absorption and Emission Spectra

Key Principle

Fluorescence and phosphorescence occur at:

• Lower energy

• Longer wavelength

than absorption.

Emission at Longer Wavelength

Molecules emit radiation at:

• Longer wavelengths

than the radiation they absorb.

This means:

$<br>\lambda_{emission} > \lambda_{absorption}<br>$

Mirror Image Relationship

Absorption and emission spectra often show:

• Approximate mirror-image relationship

This occurs because:

• Vibrational energy levels are similarly spaced

Absorption Process

During absorption:

• Molecules start in ground vibrational level of:

$<br>S_0<br>$

Absorption promotes molecules to:

• Vibrational levels within:

$<br>S_1<br>$

Vibrational Relaxation

After excitation:

• Molecules rapidly relax to lowest vibrational level of:

$<br>S_1<br>$

before emitting radiation.

Emission Process

Emission then occurs from:

• Lowest vibrational level of:

$<br>S_1<br>$

to:

• Various vibrational levels of:

$<br>S_0<br>$

Stokes Shift

Definition

The energy difference between:

• Absorption

• Emission

is called:

• Stokes shift

Cause of Stokes Shift

Immediately after absorption:

• Molecule still has ground-state geometry

Shortly afterwards:

• Geometry and solvent arrangement reorganize

• Excited state becomes more stable

This lowers excited-state energy.

Therefore:

• Emission occurs at lower energy

Solvent Effects

Polar Solvents

In polar solvents:

• Vibrational structure becomes broadened

Only broad absorption bands are observed.

Nonpolar Solvents

In nonpolar solvents:

• Vibrational fine structure is easier to observe

Solution vs Gas-Phase Spectra

Solution Spectra

Solution spectra are broadened because:

• Molecules interact with solvent molecules

• Many slightly different environments exist

Gas-Phase Spectra

Gas-phase molecules:

• Have fewer interactions

• Produce extremely sharp absorption lines

Example:

• Different water isotopes can be distinguished by very small wavelength differences.

Excitation and Emission Spectroscopy

Emission Spectroscopy

Principle

A fixed excitation wavelength is selected.

Emission intensity is measured over:

• Range of emission wavelengths

Emission Spectrum

An emission spectrum is:

• Graph of emission intensity vs emission wavelength

Instrument Setup

Usually:

• Detector positioned at:

$<br>90^\circ<br>$

to incident light

This reduces:

• Scattered light

Excitation Spectroscopy

Principle

Excitation wavelength is varied while:

• Monitoring emission at one fixed wavelength

Excitation Spectrum

An excitation spectrum is:

• Graph of emission intensity vs excitation wavelength

Relationship to Absorption

Excitation spectra resemble:

• Absorption spectra

because:

• Greater absorption produces greater emission

Fluorimetric Assay of Selenium in Brazil Nuts

Importance of Selenium

Selenium is:

• Essential trace element

Biological Role

Selenium-containing enzyme:

• Glutathione peroxidase

destroys harmful:

• Peroxides

Toxicity

Although essential:

• Excess selenium is toxic

Sample Preparation

Brazil nuts are digested using:

$<br>70\ wt$

in:

• Teflon bomb

• Microwave oven

Chemical Reactions

Hydrogen selenate:

$<br>H_2SeO_4<br>$

is reduced to hydrogen selenite:

$<br>H_2SeO_3<br>$

using:

• Hydroxylamine

Fluorescent Product

Selenite is derivatized to form:

• Fluorescent compound

which is extracted into:

• Cyclohexane

Measurement Conditions

Maximum fluorescence occurs at:

Excitation wavelength

$<br>378\ nm<br>$

Emission wavelength

$<br>518\ nm<br>$

Concentration Effects

Fluorescence is proportional to concentration only up to approximately:

$<br>0.1\ mg\ Se/mL<br>$

Self-Absorption

At high concentration:

• Fluorescence decreases

because:

• Emitted light is reabsorbed

This is called:

• Self-absorption

Luminescence in Analytical Chemistry

Naturally Fluorescent Compounds

Some compounds are naturally fluorescent, including:

• Riboflavin (vitamin B2)

• Polycyclic aromatic hydrocarbons

Fluorescent Labelling

Most compounds are not naturally luminescent.

Sensitivity can be improved by:

• Attaching fluorescent groups

Example:

• Fluorescein

Calcium Analysis

Calcium ions:

$<br>Ca^{2+}<br>$

can be measured using:

• Fluorescent complex with calcein

Chemiluminescence

Definition

Chemiluminescence is:

• Light emission from a chemical reaction

No external light source is required.

Examples of Chemiluminescence

Examples include:

• Fireflies

• Glow sticks

Chemiluminescence in Analysis

Chemiluminescence detectors are used for:

• Sulfur compounds

• Nitrogen compounds

in:

• Gas chromatography

Luminol Reactions

Luminol reactions can detect:

• Nitric oxide

• Quorum sensing molecules

at:

• Parts-per-billion levels

Organosulfur Compounds

Organosulfur compounds in water can be measured at:

• Parts-per-trillion levels

using chemiluminescence.

Biosensors

Definition

A biosensor is a device that combines:

• Biological recognition element
  with

• Signal detection system

to detect specific analytes.

Biological Recognition Elements

May include:

• Enzymes

• Antibodies

• DNA

• RNA

• Carbohydrates

FRET Biosensors

Fluorescence Resonance Energy Transfer (FRET)

FRET occurs when:

• Energy transfers between nearby chromophores

without photon emission.

FRET Dependence on Distance

FRET efficiency decreases with:

• Sixth power of distance

Relationship:

$<br>FRET \propto \frac{1}{r^6}<br>$

Where:

• $r$ = distance between donor and acceptor

FRET Biosensor Mechanism

No Analyte Present

• Tethered analogue binds recognition element

• Donor and acceptor chromophores remain close

• Efficient energy transfer occurs

• Strong fluorescence observed

Analyte Present

• Analyte displaces tethered analogue

• Chromophores separate

• Energy transfer decreases

• Fluorescence decreases

TNT Detection Example

FRET biosensor can detect:

• TNT (trinitrotoluene)

Detection limit:

$<br>0.1\ mg/L<br>$

or

$<br>0.1\ ppm<br>$

Immunoassays

Definition

Immunoassays use:

• Antibodies

to detect:

• Specific analytes (antigens)

Antibodies

Antibodies are:

• Proteins produced by immune system

They bind specifically to:

• Antigens

ELISA

Full Name

ELISA stands for:

• Enzyme-Linked Immunosorbent Assay

ELISA Principle

Step 1

Antibody 1 attached to solid support binds:

• Target analyte

Step 2

Surface is washed to remove:

• Unbound material

Step 3

Antibody 2 containing enzyme label binds:

• Another region of analyte

Step 4

Substrate added.

Enzyme converts:

• Colourless reactant
  into

• Coloured or fluorescent product

Signal Amplification

One enzyme molecule catalyses:

• Many reactions

Therefore:

• Signal becomes amplified

Sensitivity

ELISA methods can detect:

• Less than nanogram quantities

Pregnancy Tests

Home pregnancy tests are:

• Immunoassays

that detect:

• Placental proteins in urine

Environmental Immunoassays

Commercial kits detect:

• Pesticides

• Explosives

• Industrial chemicals

• Microbial toxins

Advantages of Field Immunoassays

Compared with chromatography:

• Faster

• Cheaper

• Portable

Comparison with Chromatography

Immunoassays:

• Require small samples

• Can be completed in:

$0.3 - 3\ h$

Chromatography:

• Usually performed in laboratory

• Often requires days

because:

• Samples need extraction/concentration

Summary of Key Concepts

Sources: