INSTRUMENTAL ANALYSIS CHEM 4811 CHAPTER 5 DR. AUGUSTINE OFORI AGYEMAN Assistant professor of chemistry Department 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 λ max Color Observed Green-yellow Yellow Orange Red Purple-red Violet Violet-blue Blue Blue-green Green Color Absorbed Violet Violet-blue Blue Blue-green Green Yellow-green Yellow Orange Red UV-VIS SPECTROSCOPY

Complementary Colors UV-VIS SPECTROSCOPY

Complementary Colors Ru(bpy) 3 2+ λ max = 450 nm Color observed with the eye: orange Color absorbed: blue Cr 3+ -EDTA complex λ max = 540 nm Color observed with the eye: violet Color 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 = hν ΔE = E 1 – E o E 1 = excited state energy E o = 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

Energy 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* σ ΔEΔE s orbital 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*σ* π*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 λ max 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 λ max 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 -1 cm -1 or Lmol -1 cm -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 - ε max implies ε at λ max (see table 5.4 for some ε max values) - ε is 10 4 – 10 5 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 (D 2 ) Arc Lamp - Made of deuterium gas (D 2 ) in a quartz bulb - D 2 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 electrons shifts the λ max 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 ε max ) Hypochromism - A decrease in intensity of an absorption band (decrease in ε max ) 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 A T = ε 1 b 1 c 1 + ε 2 b 2 c 2 + ε 3 b 3 c 3 + …. For the same sample cell b 1 = b 2 = b 3 = b A T = b(ε 1 c 1 + ε 2 c 2 + ε 3 c 3 + ….)

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 = kP o c k is a proportionality constant P o 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

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, then an 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 (S o ) - 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 transition Absorption > Fluorescence > Phosphorescence

MOLECULAR EMISSION SPECTROSCOPY Excited Singlet State (S 1 ) Ground Singlet State (S o ) Excited Triplet State (T 1 ) Relative Energy Absorption Fluorescence Phosphorescence ISC Radiationless transition to the lowest vibrational level in the excited state Intersystem crossing (radiationless)

Source λ selector sample monochromator (λ selector) readout detector Instrumentation Components of the Fluorometer 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