PHAR201_UV_Fluori_Sep_Chrom_Revision__1__unlocked
UV-Visible Spectroscopy Overview
Course Information
Course: PHAR 201 NGU
Instructor: Marwa Fouad, PhD
Contact: marwa.fouad@ngu.edu.eg
UV and Visible Light Range
Ultraviolet (UV): 200 - 400 nm
Visible Light: 400 - 900 nm
Types of Electrons in Electronic Transitions
Excited State vs Ground State
Sigma (σ) Electrons:
Bonding electrons of sigma covalent bonds (single bonds)
Lowest energy, most stable
Pi (π) Electrons:
Bonding electrons of pi covalent bonds (double bonds)
Higher energy than sigma electrons
Non-bonding (n) Electrons:
Electrons that do not participate in bonding
Highest energy level of the ground state
Electronic Transitions
σ - σ*: Requires high energy, associated with shorter wavelengths (UV)
n - σ*: Also requires high energy, associated with short wavelengths (UV)
n - π and π - π**: Induced transitions by UV or visible light
Chromophores
Definition: Groups of atoms and electrons in organic compounds that interact with light to produce color.
Requirements for Absorption: Molecules must contain conjugated double bonds.
Conjugation Effect: More conjugated = longer absorption wavelength.
Beer-Lambert Law and Absorbance
Absorbance (A): Measured light intensity at specific wavelengths.
Transmittance (T): Ratio of light transmitted through a sample.
Formula: T = (Io/I) expressed as a percentage.
Molar Absorption Coefficient (ε): Indicates the ability of a substance to absorb light, measured in L mol⁻¹ cm⁻¹; typical values: 10,000 - 30,000 L mol⁻¹ cm⁻¹.
Specific Absorbance
Definition: Absorbance of 1% m/v solution in 1 cm path length. Used in analytical work, common values provided by the British Pharmacopoeia.
Calculations
Molar Absorption Coefficient Example:
Given: Concentration = 2.305 × 10⁻⁵ mol L⁻¹, Absorbance (A) = 0.497, Path length (l) = 0.5 cm.
Calculation: A = ε * c * l ⇒ ε = A / (c * l) = 43,100 L mol⁻¹ cm⁻¹.
Specific Absorbance Example:
Given: Absorbance = 0.715, Concentration = 0.00140% m/v.
Calculation: A(1%, 1 cm) = 511.
UV/Vis Spectrophotometer Components
Light Sources: Tungsten lamp (for visible range), Deuterium lamp (for UV range).
Monochromators: Filters, prisms, gratings.
Sample Compartment: Cuvette.
Detectors: Photocells, photomultiplier tubes.
Determining Concentration Using Absorbance
Methods
Calibration Curve: Graphical method for relating absorbance to concentration.
Beer-Lambert Law: A = εcl.
Example Calculation using Beer-Lambert Law:
Methyltestosterone Example: Given absorbance and ε, solve for c. Final Concentration: c = 0.00165% m/v.
Simultaneous Determination of Compounds
Method: Choose wavelength where only the analyte absorbs.
Equations: At wavelengths λ1 and λ2, set up equations using absorption coefficients to solve for concentrations of a and b.
Quality Control in Pharmaceuticals
Examples of Assaying Brufen® Tablets:
Titration Method: Calculate ibuprofen content from NaOH volume.
UV Spectrophotometric Assay: Measure absorbance at 264 nm to calculate percentage purity.
UV-Visible Spectroscopy Overview: 80/20 Rule Explanation
Key Concepts (20% of content):
Beer-Lambert Law: This law is fundamental as it relates the absorbance of light by a sample to its concentration. Understanding this law allows for the estimation of concentrations in practice, which is crucial for any analytical work involving UV-Vis spectroscopy.
Formula: A = εcl
Chromophores: Essential to know about chromophores because they are key groups in organic compounds that produce color when interacting with light. This is where the absorption begins, making this concept pivotal in any analysis.
Molar Absorption Coefficient (ε): Understanding how this coefficient indicates a substance's light absorption ability is vital for properly applying the Beer-Lambert Law in calculations and experimental setups.
Supporting Details (80% of content):
Types of Electrons: While details on sigma, pi, and non-bonding electrons provide depth, knowing the basics about how these electrons relate to energy states isn't essential for every user.
Electronic Transitions: Various transitions (e.g., σ-σ*, n-π) offer insights into theoretical underpinnings but focusing on practical applications like the Beer-Lambert Law is more impactful.
UV-Vis Spectrophotometer Components: Components such as light sources, detectors, and monochromators are important to know for practical setup but are secondary after understanding core principles.
Practical Application: Focus primarily on mastering the Beer-Lambert Law and the concept of chromophores to analyze and interpret UV-Vis spectroscopy results effectively. Once these foundational elements are understood, delve into the other aspects as needed for deeper applications or troubleshooting.
UV-Visible Spectroscopy: Pages 12 to 22 Overview
Advanced Concepts in UV-Visible Spectroscopy
Factors Affecting UV-Vis Absorption:
Solvent Effects: The choice of solvent can influence absorption spectra due to solvent-solute interactions.
pH Changes: The ionization state of compounds can change with pH, affecting their absorbance.
Temperature: Higher temperatures can increase molecular motion, altering absorption characteristics.
Instrumentation for UV-Vis Spectrophotometry:
Cuvettes: Material choices (glass, quartz, plastic) based on the wavelength range.
Wavelength Calibration: Importance of calibrating instruments for accurate measurements.
Baseline Correction: Ensuring a baseline is established to compare sample absorbance against a blank.
Data Analysis Techniques:
Spectral Data Interpretation: Understanding peaks and their significance in identifying compounds.
Quantitative Analysis: Utilizing absorbance values for determining concentrations via the Beer-Lambert Law.
Applications of UV-Visible Spectroscopy
Pharmaceutical Analysis: Quality control and quantitative analysis of drugs.
Environmental Monitoring: Measuring pollutants and analyzing water quality.
Food Industry: Analyzing food additives and contaminants.
Biochemical Assays: Monitoring enzyme activities and protein concentrations.
Example Applications**:
Antibiotic Assay: Use of UV-Vis to determine concentration in a sample.
Colorimetric Methods: Utilizing color changes in reactions to analyze concentrations.
Kinetics Studies: Monitoring reaction progress through absorbance changes over time.
Summary of Key Points
Focus on the impact of various external factors on UV-Vis absorption for accurate analysis.
Understand the importance of proper instrumentation and data analysis for reliable results.
Explore diverse applications in pharmaceuticals, environmental science, food safety, and biochemistry.
Summary of Key Points
Key Focus: Understand the influence of external factors like solvent choice, pH, and temperature on UV-Vis absorption to ensure accurate analyses.
Instrumentation Importance: Recognize the significance of using proper instruments and techniques for reliable data collection and analysis results.
Diverse Applications: Familiarize yourself with how UV-Visible spectroscopy is employed across various fields, including pharmaceuticals, environmental monitoring, food safety, and biochemistry for practical applications.