UV-Vis Spectroscopy Chapter 2 PDF

Analytical AnalyticalChemistry Chemistry 22 –– Introduction Introduction to to Spectroscopy Spectroscopy and and UV-vis UV-vis spectrophotometry Spectroscopy Outline 2.1. Introduction to spectroscopy 2.2. E...

Analytical AnalyticalChemistry Chemistry 22 –– Introduction Introduction to to Spectroscopy Spectroscopy and and UV-vis UV-vis spectrophotometry Spectroscopy Outline 2.1. Introduction to spectroscopy 2.2. Electromagnetic radiation (EMR) and the spectrum 2.3. Wave characteristics 2.4. The particle nature of light 2.5. Interaction of radiation and matter – Absorption and Emission of EMR 2.6. Atomic spectroscopy vs molecular spectroscopy 2.7. Energy level diagram for molecules 2.8. The Beer-Lambert law and its limitations 2.9. Quantitative analysis 2.10. UV-vis spectroscopy 2.11. Advantages of UV-vis spectroscopy 2.12. Interferences and solutions in UV-vis spectroscopy Further Reading: Analytical Chemistry: An 9/19/2024 2 Prepared By: Analytical Chemistry Team 2022 Introduction, Fifth Edition (Skoog, et. al.)  2.1. Introduction to spectroscopy Spectroscopy ▪ Spectroscopy is a technique that uses the interaction of electromagnetic radiation Light_dispersion_conceptual with matter (atoms, ions and molecules) in quantitative and qualitative analysis. ▪ The use of absorption, emission, or scattering of electromagnetic radiation by atoms, molecules or ions to study the quantity, quality or physical processes of them. 9/19/2024 Prepared By: Analytical Chemistry Team 2022 3  2.1. Introduction to spectroscopy Spectroscopy ▪ Interaction with radiation causes redirection of radiation or transitions between energy levels of atoms or molecules. ▪ It gives information about structure and properties of the molecules 9/19/2024 Prepared By: Dr. Allan 4  2.2. Electromagnetic radiation (EMR) and the spectrum Electromagnetic Spectrum A continuum of all electromagnetic waves arranged according to frequency and wavelength. pages 654-655 9/19/2024 5 Prepared By: Analytical Chemistry Team 2022  2.2. Electromagnetic radiation (EMR) and the spectrum Radiation Frequency (Hz) Wavelength (nm) Type of transition Gamma ray 1020-1024 < 1 pm Nuclear X-Ray 1017-1020 1 nm -1 pm Inner electron Ultraviolet, UV 1015-1017 400 nm-1 nm Outer electron Visible 4-7.5 x 1014 750 nm – 400 nm Outer electron Outer electron Near-infrared 1x1014-4x1014 2.5 m – 750 nm molecular vibrations Infrared, IR 1013-1014 25 µm – 2.5 µm Molecular vibrations Molecular rotations, Microwaves 3x1011-1013 1 mm-25 µm electron spin flips Radio waves < 3x1011 > 1 mm Nuclear spin flips 9/19/2024 6 Prepared By: Analytical Chemistry Team 2022  2.2. Electromagnetic radiation (EMR) and the spectrum 9/19/2024 7 Prepared By: Analytical Chemistry Team 2022  2.2. Electromagnetic radiation (EMR) and the spectrum Electromagnetic Radiation ▪ The emission and transmission of energy in the form of electromagnetic waves. ▪ Maxwell (1873), proposed that visible light consists of electromagnetic waves (Electric and Magnetic field). Light is electromagnetic radiation (EMR) Electric field Perpendicular Magnetic field page 651 9/19/2024 Prepared By: Analytical Chemistry Team 2022 8  2.2. Electromagnetic radiation (EMR) and the spectrum EMR Wave Particle Theory Theory Undergoes Sinusoidal Made up of packets of energy oscillation called photons or quanta. Travel through vacuum Photons depends upon the Has a wavelength frequency of radiations. page 651 9/19/2024 Prepared By: Analytical Chemistry Team 2022 9  2.3. Wave characteristics Wave Characteristics ▪ Wavelength (λ) - is the distance between identical points on successive waves. ▪ Amplitude - is the vertical distance from the midline of a wave to the peak or trough. ▪ Frequency (ν) - is the number of waves that pass through a particular point in 1 second (Hz = 1 cycle/s). The speed (c) of the wave = λ x ν page 652 9/19/2024 Prepared By: Dr. Allan 10 c = speed of light = 2.998 x 108 m s-1 = 3 x 108 m s-1  2.3. Wave characteristics Example A photon has a frequency of 6.0 x 104 Hz. Convert this frequency into wavelength (nm). Does this frequency fall in the visible region? λ c=λxν ν λ = c /ν λ = 3.00 x 108 m/s / 6.0 x 104 Hz λ = 5.0 x 103 m λ = 5.0 x 1012 nm Radio wave page 652 9/19/2024 11 Prepared By: Analytical Chemistry Team 2022  2.4. The particle nature of light The Particle Nature of Light ▪ Energy (light) is emitted or absorbed in discrete units (quantum). E = h x ν = h c/ λ Planck’s constant (h) h = 6.63 x 10-34 J s c = speed of light = 3 x 10 8 m s-1 Exercise What is energy of photon with , i) A wavelength of 827 nm? What type of radiation is it? ii) A wavelength of 1 nm ? What type of radiation is it? page 653 9/19/2024 12 Prepared By: Analytical Chemistry Team 2022  2.4. The particle nature of light Example When copper is bombarded with high-energy electrons, X-rays are emitted. Calculate the energy (in joules) associated with the photons if the wavelength of the X-rays is 0.154 nm. E=hxν E=hxc/λ E = 6.63 x 10-34 (J s) x 3.00 x 10 8 (m/s) / 0.154 x 10-9 (m) E = 1.29 x 10 -15 J SF???? Extra problems and information available in the book please check the references in last slide page 653 9/19/2024 13 Prepared By: Analytical Chemistry Team 2022  2.5. Interaction of radiation and matter – Absorption and Emission of EMR Interaction of Radiation and Matter Interaction with light Redirection Scattering Transitions Transitions From lower to higher level From higher to lower level Absorption Emission pages 656-655 9/19/2024 14 Prepared By: Analytical Chemistry Team 2022  2.5. Interaction of radiation and matter Absorption and Emission of EMR – Absorption and Emission of EMR ▪ The structure of the atoms and molecules cause some wavelength to be absorbed and some to be reflected. ▪ Atoms at room temperature are usually in their lowest electronic energy level known as the ground state ▪ After absorption of EMR by the atom electron goes to higher energy level known as the excited state Atom – only electronic transition Molecules – along with electronic, rotational and vibrational transition pages 655; 675 9/19/2024 15 Prepared By: Analytical Chemistry Team 2022  2.5. Interaction of radiation and matter – Absorption and Emission of EMR Atomic and Molecular Spectroscopy Atomic Measurement of spectroscopy atoms in the UV- visible region Measurement of Molecular spectroscopy molecules in the UV- visible region pages 664-668 9/19/2024 16 Prepared By: Analytical Chemistry Team 2022  2.5. Interaction of radiation and matter – Absorption and Emission of EMR Atomic spectroscopy Electrons / atoms can move from one energy level to a higher one if: 1. They absorb the right amount of energy 2. There are vacant higher energy levels C B A pages 655; 675 9/19/2024 17 Prepared By: Analytical Chemistry Team 2022  2.5. Interaction of radiation and matter – Absorption and Emission of EMR Molecular spectroscopy Energy Level Diagram for Molecules pages 655; 675 9/19/2024 18 Prepared By: Analytical Chemistry Team 2022  2.5. Interaction of radiation and matter – Absorption and Emission of EMR Absorption ▪ When light absorbed, the electrons of a molecule or atom is excited to a higher energy level. Type of excitation Electrons Vibrations Rotations promotions UV-Vis IR MW page 655 9/19/2024 19 Prepared By: Analytical Chemistry Team 2022  2.5. Interaction of radiation and matter – Absorption and Emission of EMR Excited state atoms obtained by Bombardment Absorption of E ↑ voltage Heat by flame with particles pages 655; 675 9/19/2024 20 Prepared By: Analytical Chemistry Team 2022  2.5. Interaction of radiation and matter Absorption – Absorption and Emission of EMR Absorption spectrum Transmission spectrum A T or %T Click to view enlarged λ λ A plot of the amount of radiation A continuous spectrum except that absorbed by a sample versus the it is upside down relative to the wavelength of the radiation absorption spectrum. pages 664-668 9/19/2024 21 Prepared By: Analytical Chemistry Team 2022  2.5. Interaction of radiation and matter – Absorption and Emission of EMR Emission 07_09 ▪ Excited molecule or atom decay to a lower energy level by emitting radiation. Type of emissions Atomic/optical Atomic Molecular emission fluorescence fluorescence Atoms excited Atoms excited Molecules excited by ↑T by light by light Atomic emission Atomic fluorescence Molecular fluorescence spectroscopy spectroscopy Spectroscopy pages 675-677 9/19/2024 22 Prepared By: Analytical Chemistry Team 2022  2.5. Interaction of radiation and matter – Absorption Emissions of Radiation and Emission of EMR Molecular emissions Molecular Molecular fluorescence phosphorescence Transition of Transition of Same spin different spin 220px-Phosphorescent_pigments_1_min pages 678- Fluorescence emits radiation faster while phosphorescence emits radiation slower. 679 9/19/2024 https://youtu.be/CcN8NnGGPhs Prepared By: Analytical Chemistry Team 2022 23  2.5. Interaction of radiation and matter Emission – Absorption and Emission of EMR A* 250px-AtomicLineSpEm Absorption Emission A Excited atoms generated by the use of thermal or electrical energy return to the ground state because the ground state is the lowest energy state. pages 675-677 9/19/2024 24 Prepared By: Analytical Chemistry Team 2022  2.6. Atomic spectroscopy vs molecular spectroscopy Atomic spectroscopy Molecular spectroscopy Atoms must be in free gas phase in Samples are usually in solution 1 the ground state. Absorption spectrum formed is a Absorption spectrum formed is a 2 line spectrum.. continuous spectrum. Sources of radiation are spectral Sources of radiation emit all the 3 line sources. wavelengths in the given region Highly limited, only elements Large numbers of molecules and 4 (metals) can be analysed. complex ions can be analyzed. Wavelengths of measurements are Need to sometimes scan the absorption 5 known and can be simply looked spectral in order to obtain wavelength up in books. for maximum absorption. 9/19/2024 25 Prepared By: Analytical Chemistry Team 2022  2.6. Atomic spectroscopy vs molecular spectroscopy pages 664-668 9/19/2024 26 Prepared By: Analytical Chemistry Team 2022  2.6. Atomic spectroscopy vs molecular spectroscopy pages 664-668 9/19/2024 27 Prepared By: Analytical Chemistry Team 2022 Energy Transition A + hv → A* Only the absorption of radiation in the visible and ultra-violet region of the electromagnetic spectrum can cause Eelectronic. A* Unstable Relaxes within 10-6-10-9 s Eelectronic A*→ A + hv (H or/and EMR) E0=Eelectronic + Erotational + Evibrational pages 655; 675 9/19/2024 Prepared By: Analytical Chemistry Team 2022 Evibrational 28  2.7. Energy diagram for molecules and types of orbitals Electronic Spectra and Molecular Structure ▪ When continuous wave radiation is passed through a prism a diffraction pattern is produced (a spectrum) ▪ A spectrum is made up of all the wavelengths associated with the incident radiation. ▪ When continuous wave radiation passes through a transparent material (solid or liquid) some of the radiation might be absorbed by that material. pages 710-713 9/19/2024 29 Prepared By: Analytical Chemistry Team 2022  2.7. Energy diagram for molecules and types of Electronic Spectra and Molecular Structure orbitals Radiation source Diffraction prism Spectrum Spectrum with ‘gaps’ in it Transparent material that absorbs some radiation pages 710-713 The gaps in the light spectrum caused by the absorption of radiation by the transparent 9/19/2024 material through WAR which 2011 is passed. 30  2.7. Energy diagram for molecules and types of orbitals Electronic Spectra and Molecular Structure ▪ The absorption of UV radiation of energy causes transition of bonding electrons from a low energy orbital to a higher energy orbital. The energy difference between missing’ parts of the = the orbitals involved in the transition. spectrum pages 710-713 9/19/2024 31 Prepared By: Analytical Chemistry Team 2022  2.7. Energy diagram for molecules and types of orbitals Electronic Spectra and Molecular Structure 9/19/2024 32 Prepared By: Analytical Chemistry Team 2022  2.7. Energy diagram for molecules and types of orbitals * Unoccupied Increasing energy * Energy Levels n Occupied  Energy Levels  UV Antibonding orbitals are unoccupied in the ground state and can pages 710-713 only be occupied by an electron in an excited state 1- * orbital 2- * orbital 9/19/2024 33 Prepared By: Analytical Chemistry Team 2022  2.7. Energy diagram for molecules and types of orbitals Types of orbitals that might be occupied in the ground state -bonding orbitals Non-bonding -bonding all functional orbitals orbitals groups that contain atoms that have alkanes are low lone pair(s) of double and triple energy bonds electrons C-C C=O, C=C O, N, S, Halogens A transition of an electron from occupied to an unoccupied energy level can be caused by UV radiation. to * implies that UV is useful with compounds containing double bonds. pages 710-713 Prepared By: Analytical Chemistry Team 2022 34  2.7. Energy diagram for molecules and types of orbitals Electronic Spectra and Molecular Structure H  → * N ·· C C H3 n → * H ·O· O ··  → *  → * n → * n → * Some possible Range Transition n → * 150 – 250 nm uv  → * 200 – 700 nm uv + visible n → * pages 710-713  → * less than 200 nm uv 9/19/2024 35 Prepared By: Analytical Chemistry Team 2022 Absorption of ultraviolet (UV) or visible light? ▪ When light is interacted with matter, then it can be absorbed, scattered or transmitted. T = I / I0 %T = T x 100 A = - log T = - log I / I0 I0 - is the incident radiant power or intensity I - is the radient intensity that remains b - is the path length Extra problems and information available in the book please check the references in last slide pages 658-668  2.7. The Beer-Lambert law and its limitations THE BEER-LAMBERT LAW A =   cb A = − log 10 T ▪ The negative logarithm of T is called the absorbance (A) and this is directly proportional to sample depth (called pathlength, b) and sample concentration (c). ▪ The equation is called the Beer-Lambert law. ▪ The Beer-Lambert law is more useful because the relationship pages 664-668 between absorbance and concentration is linear. 9/19/2024 37 Prepared By: Analytical Chemistry Team 2022  2.7. The Beer-Lambert law and its limitations THE BEER-LAMBERT LAW ▪ A plot of A versus c is called a Beer-Lambert plot. A = cb ▪  is called the molar absorption coefficient and has units of dm3 mol-1 cm-1 ▪ b is the path length of the cuvette in which the sample is contained (in cm). ▪ In the UV-VIS region, the maximum value of  observed is ~105 dm3 mol-1 cm-1, which is found in many natural pigments (e. g. chlorophyll) and in sunscreens. 0.5 max~45,000 O SO3H HO3S 0.4 O Absorbance UVA UVA 0.3 0.2 HO3S O O SO3H 0.1 pages 664-668 9/19/2024 Mexoryl® SX 0 38 2002022 Prepared By: Analytical Chemistry Team 250 300 350 400  2.7. The Beer-Lambert law and its limitations Limitations of Beer-Lamberts Law 1 – Real Deviations ▪ Due to increase in concentration, the linear relationship between A and c does not hold good. ▪ Absorptivity depends on the refractive index of the medium which is a function of concentration. ▪ if there is scattering of light due to particulates in the sample pages 669-673 9/19/2024 39 Prepared By: Analytical Chemistry Team 2022  2.7. The Beer-Lambert law and its limitations Concentration Effects ▪ High concentration results in non-linearity because: ▪ Strong electrostatic interactions between molecules ▪ Might cause changes in refractive index ▪ In a system in chemical equilibrium, equilibrium may shift at high concentrations pages 669-673 9/19/2024 40 Prepared By: Analytical Chemistry Team 2022  2.7. The Beer-Lambert law and its limitations CLASS PROBLEMS In a UV-VIS study of commercial alcoholic drinks, it was found that an alcopop sample had an absorbance of 0.21 at its max (630 nm) in a 1 cm cuvette. Given that the molar absorption coefficient of the dye is 50,000 dm3 mol-1 cm-1, what is the dye concentration and what colour is the dye likely to be? pages 664-668 9/19/2024 41 Prepared By: Analytical Chemistry Team 2022  2.7. The Beer-Lambert law and its limitations A =   cb A = 0.21 b = 1 cm  = 50,000 dm3 mol-1 cm-1 A c= eb 0.21 c= 50,000 dm3 mol-1 cm-1 x 1 cm c = 4.2 x 10-6 mol dm-3 max (630 nm) is Red pages 664-668 9/19/2024 42 Prepared By: Analytical Chemistry Team 2022  2.7. The Beer-Lambert law and its limitations Exercise If the solution has molar absorptivity of 0.347 L.mole-1. cm-1, and the absorbance was measured in 1 cm cell, what is the concentration if: i) the absorbance is 0.362 ? C = 1.043 M ii) the % transmittance is 63.2 ? C = 0.574 M %T = T x 100 A = - log T Extra problems and available provided in the book please check the references in last slide pages 664-668 9/19/2024 43 Prepared By: Analytical Chemistry Team 2022  2.8. Quantitative Analysis Quantitative Analysis A 1: Dilute the sample Use a narrower cuvette (cuvettes are usually 1 mm, 1 cm or 10 cm) Plot the data (A v C) to produce a calibration ‘curve’ Obtain equation of straight line (y=mx) from line of ‘best fit’ Use equation to calculate the concentration of the unknown(s) 9/19/2024 44 Prepared By: Analytical Chemistry Team 2022  2.8. Quantitative Analysis Quantitative Analysis Calibration curve showing absorbance as a function of metal concentration Absorbance ( no units) 1.2 1 y = 0.9982x 0.8 R2 = 0.9996 Correlation 0.6 coefficient 0.4 0.2 0 0 0.2 0.4 0.6 0.8 1 1.2 Concentration (mg L-1) 9/19/2024 45 Prepared By: Analytical Chemistry Team 2022  Ultraviolet (UV) – visible (vis) Spectroscopy 9/19/2024 46 Prepared By: Analytical Chemistry Team 2022  2.9. UV-vis spectroscopy Radiation range Visible range UV range UV – VIS range UV UV-VIS Instrument Colorimeter spectrophotometer spectrophotometer pages 710-713 9/19/2024 47 Prepared By: Analytical Chemistry Team 2022  2.9. UV-vis spectroscopy Instrumentation Optical Filters Photo tubes Monochromators PMT’s Continuous or Interferometers Silicon photodiodes Discontinuous Sample holder Computer display cuvette Digital or analog readout 9/19/2024 Prepared By: Analytical Chemistry Team 2022 or recorder 48  2.9. UV-vis spectroscopy UV-VIS sources Tungsten filament Deuterium lamp Xenon Arc lamp Visible & near IR UV UV-Vis 185 nm – 370 nm pages 710-713 9/19/2024 49 Prepared By: Analytical Chemistry Team 2022  2.9. UV-vis spectroscopy Source Continuous sources Discontinuous (discrete) sources Produce spectra Produce only specific over broad band wavelengths Deuterium Arc Lamp A Hollow cathode lamp pages 710-713 9/19/2024 Tungsten Lamp 50 50 Prepared By: Analytical Chemistry Team 2022  2.9. UV-vis spectroscopy Sample Holder Receptacle for Sample Material Quartz, Fused Silica (Using over 190nm) Glass (Using Over Visible Region) pages 710-713 9/19/2024 51 Prepared By: Analytical Chemistry Team 2022  2.9. UV-vis spectroscopy Instrumentation Optical path 1- Single beam 2- Double beam Light passes through Light passes through sample Sample Blank Sample beam Reference pages 710-713 9/19/2024 52 Prepared By: Analytical Chemistry Team 2022  2.9. UV-vis spectroscopy Single beam Instrument Light from monochromator passes only through sample solution before reaching the detector. pages 710-713 9/19/2024 53 Prepared By: Analytical Chemistry Team 2022  2.9. UV-vis spectroscopy Single beam Advantages Disadvantages a. Cheap a. Identification of λmax is tedious b. Useful when λ is known b. Time lag between blank & sample reading pages 710-713 9/19/2024 54 Prepared By: Analytical Chemistry Team 2022  2.9. UV-vis spectroscopy Double beam Light from monochromator is split into two parallel beams: sample beam passes through the sample reference beam pages 710-713 9/19/2024 passes through WAR 2011 the blank 55 Prepared By: Analytical Chemistry Team 2022  2.9. UV-vis spectroscopy Double beam Advantages Disadvantages a. Compensate for fluctuation a. Expensive in I & λ b. If reference beam does b. Useful when λmax is not not pass through a blank known then the I is not corrected. c. λmax easily obtained pages 710-713 9/19/2024 56 Prepared By: Analytical Chemistry Team 2022  2.9. UV-vis spectroscopy Monochromators Disperse light into its component wavelengths and selects a narrow band of wavelengths to pass on to the sample or detector Filter Prism Grating pages 710-713 9/19/2024 57 Prepared By: Analytical Chemistry Team 2022  2.9. UV-vis spectroscopy Sample compartment Cuvette: The cell which contain samples. Most common cuvettes have 1.00 cm pathlength Made of fused silica, quartz, and glass UV Visible Glass absorbs ultraviolet radiation pages 710-713 9/19/2024 58 Prepared By: Analytical Chemistry Team 2022  2.9. UV-vis spectroscopy Detector – Phototube, Photomultiplier tube, photodiodes converts radiation energy into an electrical signal for measurement PMT Photo-multiplier tube photodiodes 220px-Pmside a semiconductor detector consisting of multiple individual diodes typically constructed from silicon or germanium pages 710-713 9/19/2024 59 59 Prepared By: Analytical Chemistry Team 2022  2.10. Advantages of UV-vis spectroscopy Advantages of UV-VIS spectroscopy 1. Applicable to organic, inorganic & biological samples. 2. High sensitivity , detection limit (10-5 M to 10-7 M) 3. High selectivity 4.High accuracy, 95-99% pages 710-713 5. Easy automation 9/19/2024 60 Prepared By: Analytical Chemistry Team 2022  2.11. Interferences and solutions in UV-vis spectroscopy Problems Solutions liquid-liquid extraction/ solid phase extraction Interferences add it to sample and standards Deviation from Beer-Lambert’s A>1 Dilution of law standards & unknown pages 710-713 9/19/2024 61 Prepared By: Analytical Chemistry Team 2022 References Skoog, D.A., West, D.M., Holler, F.J., and Crouch, S.R. (2014) Fundamentals of Analytical Chemistry, 9th ed. USA: Brooks/Cole, Cencage Learning. Christian, G. D. (2003). Analytical Chemistry, 6th ed. USA: John Wiley & Sons. Harris, D.C. (2007) Quantitative Chemical Analysis, 7th ed. USA: W.H. Freeman and Company Dean, J.R., Jones. A.M., Homes, D., Reed, R., Wyers, J. and Jones, A. (2002) Practical Skills in Chemistry. Great Britain: Ashford Colour Press Gosport, Hants. 62 Prepared By: Analytical Chemistry Team 2022