UV-Visible Spectroscopy PDF

Analytical Chemistry SCACA3-44 WEEK 3: UV-VISIBLE SPECTROSCOPY 1. Electromagnetic Radiation (EMR): EMR is radiant energy released through electromagnetic processes, covering a range of frequencies called the electromagnetic spectrum. UV/VIS spectroscopy u...

Analytical Chemistry SCACA3-44 WEEK 3: UV-VISIBLE SPECTROSCOPY 1. Electromagnetic Radiation (EMR): EMR is radiant energy released through electromagnetic processes, covering a range of frequencies called the electromagnetic spectrum. UV/VIS spectroscopy uses specific parts of this spectrum (UV: 200-400 nm, Visible: 400-700 nm) to analyze matter. 2. Spectroscopy vs. Spectrometry: Spectroscopy studies the interaction between matter and radiated energy. Spectrometry is the quantitative measurement of the spectrum (light intensity vs. wavelength). Spectrophotometry measures how much light is reflected or transmitted by a sample at diPerent wavelengths. PRINCIPLE OF UV/VIS SPECTROSCOPY UV/VIS spectroscopy: measures the absorbance of ultraviolet (UV, 185-400 nm) and visible (VIS, 400-700 nm) light by a sample. When light passes through the sample, some wavelengths are absorbed by the sample, and the remaining light is transmitted. Each compound absorbs light at a specific wavelength (the analytical wavelength), which provides information about its concentration and composition. The absorbance is due to the excitation of electrons in chromophores (molecular regions that absorb light) from a lower to a higher energy state. The absorbance (A) follows the Beer-Lambert law. Beer-Lambert law: absorbance is proportional to the concentration of absorbing species in the sample.  Analytical Chemistry SCACA3-44 HOW IT WORKS (WORKING PRINCIPLE) Radiation is passed through a solution of a compound (radiation at wavelength range between 200 and 700 nm) and the bonding electrons become excited. They then occupy a higher quantum state by absorbing some of the energy passing through the solution. The more loosely held the electrons are (within the bonds of the molecule) the lower the energy of the radiation absorbed and the longer the wavelength at which it absorbs energy. Transmitted light is measured and unabsorbed light that passes through sample solution INTERACTIONS OF UV/VIS RADIATION WITH CHROMOPHORES Chromophores: functional groups within a molecule responsible for light absorption, they are molecular structures where the energy gap between orbitals aligns with visible light These structures, often involving alternating double bonds, are responsible for the colour of substances by absorbing specific wavelengths and transmitting others. Isolated chromophores: absorb at specific wavelengths. When chromophores are conjugated, the absorbance shifts towards longer wavelengths (bathochromic shift) and becomes more intense (hyperchromic ePect). Examples of chromophores: the amine group (-NH₂) at 195 nm and the nitro group (-NO₂) at 210 nm. This interaction allows the determination of molecular structure or quantification of compounds. GENERAL APPLICATIONS Quantitative Analysis: UV/VIS is widely used to determine the concentration of compounds in solution by applying the Beer-Lambert law. Qualitative Analysis: Identifies compounds based on their absorption spectrum. Monitoring Reactions: Tracks changes in absorbance over time to study reaction kinetics. Purity Testing: Detects impurities in pharmaceuticals, chemicals, and biological samples STRENGTHS AND LIMITATIONS STRENGTHS Non-destructive and simple to use. Quick, providing fast results. Can be used for both qualitative and quantitative analysis. Inexpensive compared to more advanced techniques like NMR or MS. LIMITATIONS Limited structural information—can only determine the presence of chromophores, not the entire structure. Overlapping spectra of compounds in mixtures can complicate analysis. Requires samples to have chromophores for light absorption  Analytical Chemistry SCACA3-44 SCHEMATIC DIAGRAM OF THE INSTRUMENT COMPONENTS INSTRUMENT USED : UV/VIS spectrophotometer o UV/VIS spectrophotometer: an analytical instrument that measures the amount of ultraviolet (UV) and visible light that is absorbed by a sample COMPONENTS Light Source: Usually a deuterium lamp for UV (160-400 nm) and a tungsten lamp for visible light (400-700 nm). Xenon arc lamps cover both UV and visible spectra Monochromator: Disperses light into its component wavelengths using gratings. Common designs include Czerny-Turner configurations, which use concave mirrors and gratings to direct the light beam Slits: Control the beam width, impacting the resolution. Smaller slit widths allow higher resolution but lower light intensity Optics: Lenses or mirrors (often achromatic) are used to focus light onto the sample and then the detector Sample Cells: Typically made of quartz for UV measurements (since glass absorbs UV light) and glass for visible measurements. Cuvettes are the most common form Detector: Photomultiplier tubes (PMTs) or photodiode arrays (PDAs) are used to convert light into an electrical signal. PDAs are preferred for rapid, multi-wavelength detection  Analytical Chemistry SCACA3-44 LIGHT INTENSITY AND BEER-LAMBERT LAW Transmission (T): The ratio of transmitted light intensity (I) to the incident light intensity (I0) Absorbance (A): The logarithmic measure of the amount of light absorbed by the sample Beer-Lambert Law: Relates absorbance to the concentration of the analyte: Where: o A: absorbance, o ε: molar absorptivity o c: concentration o l: the path length of the sample cell o The law allows for the determination of concentration from absorbance ABSORBTION (A) VS TRANSMISSION (T) Absorption (A): Represents how much light a sample absorbs, with higher absorbance indicating more light is absorbed. Transmission (T): Represents the amount of light that passes through the sample. Relationship: A =−logT, so a higher transmission corresponds to lower absorption CALCULATING A AND T FOR RADIATION Given an intensity I before the sample and I0 after, you can calculate: And from transmission: Using these equations, you can determine both transmission and absorbance based on the measured intensities of light before and after it passes through the sample  Analytical Chemistry SCACA3-44 WEEK 3: ATOMIC SPECTROSCOPY PRINCIPLE Understanding the Principle of Atomic Spectroscopy Atomic spectroscopy is used to determine the elemental composition of a sample by analyzing the electromagnetic radiation emitted or absorbed by the atoms of an element. Each element has a unique electromagnetic radiation signature due to diPerences in electronic structure, which allows for highly accurate detection of specific elements even in small quantities. The spectroscopy relies on transitions between electronic energy levels: atoms absorb energy to move to an excited state and release energy (in the form of photons) as they return to their ground state. HOW AAS AND FES WORK ATOMIC ABSORPTION SPECTROSCOPY (AAS) The sample solution is introduced into a flame or heated to form free atoms in a gaseous state. Atoms in their ground state absorb energy from a light source, causing their electrons to be excited to a higher energy level. The amount of light absorbed (at a specific wavelength) is measured, which correlates to the concentration of the element in the sample. AAS measures absorption of energy by ground-state electrons. FLAME EMISSION SPECTROSCOPY (FES) Atoms are thermally excited in a flame (typically around 2000–3000°C), causing electrons to jump to a higher energy level. When the electrons return to the ground state, they emit photons (light) at characteristic wavelengths. The intensity of the emitted light is measured to determine the element's concentration. FES focuses on the emission of energy by excited electrons returning to the ground state. GENERAL APPLICATIONS Atomic spectroscopy (both AAS and FES) is widely used in environmental analysis, clinical chemistry, pharmaceuticals, and metallurgy to detect and quantify elements. Common applications include: o Water quality testing (e.g., detecting trace metals like lead or mercury) o Food safety (e.g., measuring mineral content) o Pharmaceutical analysis (e.g., monitoring metal impurities) o Geological surveys (e.g., determining mineral compositions)  Analytical Chemistry SCACA3-44 STRENGTHS AND LIMITATIONS STRENGTHS AAS: Highly sensitive for detecting trace amounts of elements. Provides precise quantification due to narrow spectral lines. Can be used for a wide range of elements, including metals and some non-metals. FES: Simple and cost-e\ective for elements that emit visible light (alkaline earth metals). Rapid analysis and straightforward setup for routine applications. LIMITATIONS AAS: Limited to quantitative analysis (does not provide information on molecular forms or chemical states). Requires a light source specific to the element being analyzed, making multi- element analysis slower. FES: Less sensitive than AAS, particularly for elements that do not emit strongly visible light. More prone to interference from the flame’s background emissions or sample matrix. INSTRUMENT COMPONENTS 1. Light Source (Hollow Cathode Lamp) Purpose: Emits light at specific wavelengths corresponding to the element being analyzed. Description: Contains a cathode made of the element of interest (e.g., lead), with a fill gas (argon or neon). The cathode is ionized, releasing energy in the form of light. Alternatives: Electrodeless Discharge Lamps (EDL), which oPer higher intensity but less stability. 2. Sample Introduction (Nebulization) Purpose: Transforms the liquid sample into a fine mist to be introduced into the flame. Process: The sample is drawn into the burner as a fine mist using a Venturi e\ect, mixed with air, and combusted with a fuel gas. Types: o Flame Nebulization: Uses a burner and flame to atomize the sample. o Graphite Furnace Atomization: Replaces flame with a graphite tube that can reach up to 3000 K, improving sensitivity for trace detection.  Analytical Chemistry SCACA3-44 3. Flame and Burner Purpose: Provides the heat needed to atomize the sample, converting it into atomic gas. Description: The flame (usually air/acetylene) heats the sample, causing it to reach the atomic state. The burner is aligned with the optical path of the instrument. Alternative Flames: Higher temperatures can be achieved with a nitrous oxide flame. 4. Monochromator Purpose: Selects the specific wavelength of light that corresponds to the element being measured. Description: It isolates a narrow band of wavelengths, filtering out unwanted light and allowing only the light absorbed by the sample to reach the detector. 5. Detector Purpose: Measures the intensity of light after it passes through the flame. Function: If the element is present, the detector will measure a reduced light intensity compared to the original light source. This reduction is correlated with the concentration of the element in the sample. Types: Often uses photomultiplier tubes due to their sensitivity in detecting low light intensities.  Analytical Chemistry SCACA3-44 INTERFERANCE Ionization Cause: High temperatures in flames can cause partial ionization of elements, decreasing free atom concentration. E\ect: Reduced sensitivity of measurements. Treatment: Add ionization suppressors (e.g., potassium salts) or an ionization buPer (e.g., sodium or potassium salt) to prevent ionization. Viscosity Cause: High viscosity of the sample, particularly in graphite furnaces, leading to incomplete atomization. E\ect: Background absorption and interference due to unburned particles or organic molecules. Treatment: Correct with advanced instruments like double-beam spectrometers or background correction methods (deuterium lamp, Zeeman ePect, or Smith– Hieftje method). Anionic Interference Cause: Presence of anions in the sample (e.g., phosphate ions). E\ect: Complexes with the analyte, making it harder to detect. Treatment: Add liberating agents (e.g., strontium or lanthanum chloride) to free the analyte. Spectral Interference Cause: Overlapping absorption or emission lines, especially from graphite furnace walls or matrix compounds. E\ect: Incorrect measurements due to confusion between analyte and interference signals. Treatment: Use diPerent wavelengths or pulsed HCL voltage to separate absorption from emission.  Analytical Chemistry SCACA3-44 WEEK 3: INDUCTIVELY COUPLED PLASMA (IPC) PRINCIPLE ICP Optical Emission Spectroscopy (ICP-OES): uses an argon plasma to excite atoms and ions. Excited electrons emit radiation at specific wavelengths characteristic of the elements, allowing for their identification and quantification. The emission intensity is proportional to the concentration of the element in the sample. INSTRUMENTATION Nebulizer: Introduces a liquid sample, which is vaporized and atomized. Torch: Converts argon gas into an electron-rich plasma using high radio frequency to generate an electromagnetic field, causing excitation of electrons in the atoms. Polychromator: Disperses emitted radiation into diPerent directions to isolate various wavelengths. It allows for the simultaneous detection of multiple wavelengths, unlike a monochromator, which isolates one beam at a time. Detector: Measures the light intensity from diPerent wavelengths, typically using photomultiplier tubes. APPLICATIONS Industrial: Monitoring metal content in oils to assess machinery wear. Environmental: Analyzing soil, water, and crops. Forensic: Metal poisoning detection. Clinical Medicine: Analysis of tissue and biological fluids. Mining: Detecting metal deposits in ores.  Analytical Chemistry SCACA3-44 STRENGTHS AND LIMITATIONS STRENGTHS High sensitivity with the ability to detect elements in ppm, ppb, and ppt. Rapid analysis of over 75 elements in as little as 3 seconds. Applicable to a wide range of elements, including non-metals, from 5 to 250 atomic mass units (amu). LIMITATIONS Requires skilled technicians for operation. Expensive instrumentation.