INSTRUMENTAL ANALYSIS CHEM 4811

CHAPTER 5

DR. AUGUSTINE OFORI AGYEMANAssistant professor of chemistryDepartment of natural sciences

Clayton state university

CHAPTER 4

ULTRAVIOLET AND VISIBLE

MOLECULAR SPECTROSCOPY

(UV-VIS)

UV-VIS SPECTROSCOPY

- Solutions allow a component of white light to pass through and absorb the complementary color of the component

- The component that passes through appears to the eye as the color of the solution

- This chapter deals with molecular spectroscopy (absorption or emission of UV-VIS radiation by molecules

or polyatomic ions)

- The spectrum is absorbance or transmittance or molar absorptivity versus wavelength

Complementary Colors

λmax

380-420420-440440-470470-500500-520520-550550-580580-620620-680680-780

Color Observed

Green-yellow YellowOrange

RedPurple-red

VioletViolet-blue

BlueBlue-green

Green

Color Absorbed

VioletViolet-blue

BlueBlue-green

GreenYellow-green

YellowOrange

RedRed

UV-VIS SPECTROSCOPY

Complementary Colors

UV-VIS SPECTROSCOPY

Complementary Colors

Ru(bpy)32+

λmax = 450 nmColor observed with the eye: orange

Color absorbed: blue

Cr3+-EDTA complexλmax = 540 nm

Color observed with the eye: violetColor absorbed: yellow-green

UV-VIS SPECTROSCOPY

UV RADIATION

- Wavelength range is 190 nm – 400 nm

- Involved with electronic excitations

- Radiation has sufficient energy to excite valence electrons in atoms and molecules

- Vacuum UV spectrometers are available that uses radiation between 100 Å – 200 nm (also electronic excitations)

VISIBLE RADIATION

- Wavelength range is 400 nm – 800 nm

- Involved with electronic excitations

- Similar to UV

- Spectrometers therefore operate between 190 nm and 800 nm and are called UV-VIS spectrometers

- Can be used for qualitative identification of molecules

- Useful tool for quantitative determination

ELECTRONIC EXCITATION

- Electrons in molecules move in molecular orbitals at discrete energy levels

- Energy levels are quantized

- Molecules are in the ground state when energy of electrons is at a minimum

- The molecules can absorb radiation and move to a higher energy state (excited state)

- An outer shell (valence) electron moves to a higher energy orbital

ELECTRONIC EXCITATION

- Is the process of moving electrons to higher energy states

- Radiation must be within the visible or UV region in order to cause electronic excitation

- The frequency absorbed or emitted by a molecule is given as

ΔE = hνΔE = E1 – Eo

E1 = excited state energyEo = ground state energy

ELECTRONIC EXCITATION

Three Distinct Types of Electrons Involved in Transition

Electrons in a Single Bond (Alkanes)- Single bonds are called sigma (σ) bonds

- Amount of energy required to excite electrons in δ bonds are higher than photons with wavelength greater than 200 nm

- Implies alkanes and compounds with only single bonds do not absorb UV radiation

- Used as transparent solvents for analytes

ELECTRONIC EXCITATION

Three Distinct Types of Electrons Involved in Transition

Electrons in Double or Triple Bonds (Unsaturated)- Alkenes, alkynes, aromatic compounds

- These bonds are called pi (π) bonds

- π bond electrons are excited relatively easily

- These compounds absorb in the UV-VIS region

ELECTRONIC EXCITATION

Three Distinct Types of Electrons Involved in Transition

Electrons Not Involved in Bonding Between Atoms- Called the n electrons (n = nonbonding)

- Organic compounds containing N, O, S, X usually contain nonbonding electrons

- n electrons are usually excited by UV-VIS radiation

- Such compounds absorb UV-VIS radiation

En

ergy

s orbital

- Two s orbitals on adjacent atoms overlap to form a σ bond- Two molecular orbitals is the result

- Sigma bonding orbital (σ) is of lower energy than the atomic orbitals (filled with the two 1s electrons)

- Sigma antibonding orbital (σ*) is of higher energy than the atomic orbitals (empty)

ΔE = energy difference between σ and σ*

σ*

σ

ΔE

s orbital

ELECTRONIC EXCITATION

ELECTRONIC EXCITATION

- p orbitals of atoms can also overlap along axis to form sigma bonds

- There are three p orbitals in a given subshell

- One of these p orbitals from adjacent atoms form sigma orbitals

- The other two p orbitals can overlap sideways to form π orbitals

- The result is pi bonding (π) and pi antibonding (π*) orbitals

- p orbital filled with 2 electrons has no tendency of forming bonds

ELECTRONIC EXCITATION

σ

π

n

σ*

π*

Relative Energy Diagram of σ,π, and n electrons

ELECTRONIC EXCITATION

- Energy required to excite electrons from σ to σ* is very high(higher than those available in the UV region)

- UV radiation is however sufficient to excite electrons in π to π* and n to π* or σ* antibonding

- Molecular groups that absorb UV or VIS light are called chromophores

ABSORPTION BY MOLECULES

- Review quantum mechanics (beyond the scope of this text)

- Quantum mechanical selection rules indicate that some transitions are allowed and some are forbidden

- Electrons move from highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO)

during excitation

- LUMO is usually an antibonding orbital

- π electrons are excited to antibonding π* orbitals

ABSORPTION BY MOLECULES

- n electrons are excited to either σ* or π* orbitals

π → π* Transition- A molecule must possess a chromophore with an unsaturated bond

(C=O, C=C, C=N, etc)

n → π * or n → σ* Transition- A molecule must contain atoms with nonbonding electrons

(N, O, S, X)

- Lists or organic compounds and their λmax are available (table 5.3)

TRANSITION METAL COMPOUNDS

- Solutions are colored

- Absorb light in the visible portion of the spectrum

- Absorption is due to the presence of unfilled d orbitals

λmax is due to - The number of d electrons

- Geometry of compound- Atoms coordinated to the transition metal

ABSORPTIVITY (a)

- Defines how much radiation will be absorbed by a molecule at a given concentration and wavelength

- Is termed molar absorptivity (ε) if concentration is expressed in molarity (M, mol/L)

- Can be calculated using Beer’s Law (A = abc = εbc)

- If units of b is cm and c is M then ε is M-1cm-1 or Lmol-1cm-1

- Magnitude of ε is an indication of the probability of the electronic transition

ABSORPTIVITY (a)

- High ε results in strong absorption of light

- Low ε results in weak absorption of light

- ε is constant for a given wavelength but different at different wavelengths

- εmax implies ε at λmax (see table 5.4 for some εmax values)

- ε is 104 – 105 for allowed transitions and 10 – 100 for forbidden transitions

UV ABSORPTION CURVES

- Broad absorption band is seen over a wide range of wavelengths

- Broad because each electronic energy level has multiple vibrational and rotational energy levels associated with it

- Each separate transition is quantized

- Vibrational energy levels are very close in energy

- Rotational energy levels are even closer

- These cause electronic transitions to appear as a broad band

SOLVENTS

- Many absorbing molecules are usually dissolved in a solvent

- Solvent must be transparent over the wavelength range of interest

- Solute must completely dissolve in solvent

- Undissolved particles may scatter light which will affect quantitative analysis

- Solvent must be colorless

- Examples: acetone, water, toluene, hexane, chloroform

INSTRUMENTATION

- Radiation source

- Monochromator

- Sample holder

- Detector

- Computer

- Constant intensity over all wavelengths

- Produce light over a continuum of wavelengths

- Tungsten lamp and deuterium discharge lamp are the most common

RADIATION SOURCE

Tungsten Filament Lamp

- Just like an ordinary electric light bulb- Contains tungsten filament that is heated electrically

- Glows at a temperature near 3000 K- Produces radiation at wavelengths from 320 to 2500 nm

- Stable, robust, and easy to use

- Modern lamps are tungsten-halogen lamps (has quartz bulb)

Disadvantage- Low radiation intensity at shorter wavelengths (< 350 nm)

RADIATION SOURCE

Dueterium (D2) Arc Lamp

- Made of deuterium gas (D2) in a quartz bulb

- D2 molecules are electrically excited and dissociated

- Produces continuum radiation at λs from 160 to 400 nm

- Stable, robust, and widely used

- Emission intensity is 3x that of hydrogen at short λs

RADIATION SOURCE

Xenon Arc Lamps

- Electric discharge lamps

- Xenon gas produces intense radiation over 200 – 1000 nm upon passage of current

- Produce very high radiation intensity

- Widely used in visible region and long λ end of UV

RADIATION SOURCE

- Disperse radiation according to selected wavelengths

- Allow selected wavelengths to interact with the sample

- Diffraction gratings are used to disperse light in modern instruments

Refer chapter 2

MONOCHROMATORS

- Earlier detectors were human eye observation of color and intensity

- Modern instruments make use of photoelectric transducers (detection devices that convert photons into electric signal)

Examples- Barrier layer cells

- Photomultiplier tubes- Semiconductor detectors

DETECTOR

Barrier Layer Cells

- Also called photovoltaic cells

- A semiconductor (selenium) is joined to a strong metal base (iron)

- Silver is coated on the semiconductor

- Current is generated at the metal-semiconductor interface(requires no external electrical power)

- Response range is 350 nm – 750 nm

DETECTOR

Photomultiplier Tubes

- The most common

- Photoemissive cathode is sealed in an evacuated transparent envelope

- Also contains anode and other electrodes called dynodes

- Electrons from cathode hit dynodes which causes more electrons to be emitted

- Process repeats until electrons fall on anode (collector)

DETECTOR

Semiconductor Detectors

- Silicon and germanium are the most widely used elements

- Others include InP, GaAs, CdTe

- Covalently bonded solids with λ range ~ 190 nm – 1100 nm

Photodiode Array- Consists of a number of semiconductors embedded in a single

crystal in a linear array

- Used as detector for HPLC and CE

DETECTOR

- Called sample cells or cuvettes or cuvets

- Different types of sample holders are designed for solids, liquids, and gases

- Cells must be transparent to UV radiation

- Quartz and fused silica are commonly used as materials

- Glass or plastic cells can be used for only VIS region

- Material must be chemically inert to solvents

SAMPLE HOLDER

- HF and very strong bases should not be put in cells

- Standard cell is the 1 cm pathlength rectangular cell

- Holds about 3.5 mL sample

- Flow through cells are available (for chromatographic systems)

- Larger pathlength or volume cells are used for gases

- Thin solid films can be analyzed using a sliding film holder

SAMPLE HOLDER

Fiber Optic Probes

- Enables spectrometer to be brought to sample for analysis

- Enables collection of spectrum from microliter samples

- Can collect spectrum from inside almost every container

- Useful for hazardous samples

SAMPLE HOLDER

Chromophore- A group of atoms that gives rise to electronic absorption

Auxochrome- A substituent that contains unshared electron pairs (OH, NH, X)

- An auxochrome attached to a chromophore with π electronsshifts the λmax to longer wavelengths

ABSORPTION DEFINITIONS

Bathochromic- A shift to longer wavelengths or red shift

Hypsochromic - A shift to shorter wavelengths or blue shift

Hyperchromism- An increase in intensity of an absorption band (increase in εmax)

Hypochromism- A decrease in intensity of an absorption band (decrease in εmax)

ABSORPTION DEFINITIONS

- Molecules with absorption due to π → π* transition exhibit red shift when dissolved in polar solvents as compared to

nonpolar solvents

- Used to confirm the presence of π → π* transitions in molecules

- Molecules with absorption due to n → π* transition exhibit blue shift when dissolved in solvents that are able to form

hydrogen bonds (same with n → σ* transition)

- Used to confirm the presence of n electrons in a molecule

- Blue shift of n → σ* puts molecules into the vacuum UV region

SOLVENT EFFECTS

- A compound that contains both π and n electrons may exhibit two absorption maxima with change in solvent polarity

- π → π* transitions absorb ~ 10x more strongly than n → π* transition

- n → π* transition occur at longer wavelengths than π → π*

- Such a compound will exhibit two characteristic peaks in a nonpolar solvent such as hexane

- The two peaks will be shifted closer to each other in a polar and hydrogen bonding solvent such as ethanol

SOLVENT EFFECTS

ANALYSIS OF A MIXTURE

- Occurs when there is more than one absorbing species

- All absorbing species will contribute to absorbance at most λs

- Absorbance at a given λ = sum of absorbances from all species

AT = ε1b1c1 + ε2b2c2 + ε3b3c3 + ….

For the same sample cellb1 = b2 = b3 = b

AT = b(ε1c1 + ε2c2 + ε3c3 + ….)

APPLICATIONS

- Environmental monitoring

- Industrial quality control or process control

- Pharmaceutical quality control

- For measuring kinetics of a chemical reaction

- For measuring the endpoint of spectrophotometric titrations

- For spectroelectrochemistry in which redox reactions are studied by measuring the electrochemistry and spectroscopy simultaneously

OTHER TECHNIQUES

- Methods for nontransparent particles suspended in a liquid(colloidal suspensions, precipitates)

- Used for analyzing the clarity of drinking water, liquid medications, beverages

Nephelometry- Measures the amount of radiation scattered by the particles

Turbidimetry- Measures the amount of radiation not scattered by the particles

LUMINESCENCE

- Molecular emission

- Includes any emission of radiation

Emission Intensity (I)

I = kPoc

k is a proportionality constantPo is the incident radiant power

c is the concentration of emitting species

- Only holds for low concentrations

Photoluminescence (PL)- Excitation by absorption and re-radiation (very short lifetime)

- Examples are fluorescence and phosphorescence

Chemiluminescence (CL)- Excitation and emission of light as a result of a chemical reaction

Electrochemiluminescence (ECL)- Emission as a result of electrochemically generated species

Bioluminescence- Production and emission of light by a living organism

LUMINESCENCE

LUMINESCENCE

Fluorescence

- Instantaneous emission of light following excitation

- Excitation by photon absorption to a vibrationally excited singlet state followed by relaxation resulting in emission of a photon

- Emitted photon has lower energy (longer λ) than absorbed energy(due to the radiationless loss)

- Called the stokes fluorescence (excited state lifetime ~ 1-20 ns)

- A molecule that exhibits fluorescence is called fluorophore

LUMINESCENCE

Phosphorescence

- Similar to fluorescence

- Excited state lifetime is up to 10 s

- Excitation by absorption of light to an excited singlet state, thenan intersystem crossing (ISC) to the triplet state, followed

by emission of a photon

- Photon associated with phosphorescence has lower energy than fluorescence

MOLECULAR EMISSION SPECTROSCOPY

- Two electrons occupying a given orbital have opposite spins

- There are two possible electronic transitions

- The excited state is known as a singlet state if one of the electrons goes to the excited state without changing its spin

- The excited state is known as a triplet state if one of the electrons goes to the excited state and changes its spin for both

to have same spin

MOLECULAR EMISSION SPECTROSCOPY

- Singlet state energy levels (S) are higher than triplet state energy levels (T)

- Ground state is a singlet state (So)

- Excited state singlet can undergo radiationless transition to excited state triplet (ISC)

Transition from ground state singlet to excited state triplet is forbidden

Relative energy of transitionAbsorption > Fluorescence > Phosphorescence

MOLECULAR EMISSION SPECTROSCOPY

Excited Singlet State (S1)

Ground Singlet State (So)

Excited Triplet State (T1)

Rel

ativ

e E

nerg

y

Ab

sorp

tion

Flu

ores

cen

ce

Phosphore

scence

ISC

Radiationless transition to the lowest vibrational level in the excited state

Intersystem crossing (radiationless)

Sourceλ selector

sample

monochromator

(λ selector)readout detector

InstrumentationComponents of the Fluorometer

MOLECULAR EMISSION SPECTROSCOPY

MOLECULAR EMISSION SPECTROSCOPY

Applications- Analysis of clinical samples, pharmaceuticals,

environmental samples, steroids

Advantages- High sensitivity and specificity

- Large linear range

Disadvantage- Quenching by impurities and solvent

- Temperature, viscosity and pH must be controlled to minimize quenching