Spectrophotometry Study Notes

Spectrophotometry Notes

Page 1: Spectrophotometry Components
  • Components:
    • Lamp
    • Detector
    • Sample Meter
    • Slits
    • Grating
  • Application:
    • JAMP WFCC Concentration Measurement
Page 2: Introduction to Organic Structural Determination
  1. Start with Pure Substance:
    • Synthesis or Isolation: Ensure the product is pure.
    • Techniques: Crystallization, Distillation (simple, fractional, vacuum, steam), Sublimation, Extraction, Chromatography, Electrophoresis, etc.
  2. Physical Characterization:
    • Organoleptic properties (odour, colour):
      • Melting Point (solid)
      • Freezing Point
      • Boiling Point (liquid)
      • Density
      • Molecular Weight
      • Refractive Index
      • Optical Rotation (natural products)
  3. Chemical Characterization:
    • Qualitative and quantitative elemental analysis
    • Solubility Classification
    • Acid-Base Determination
    • Functional Group Identification
    • Derivative Preparation
    • Bibliographical Consultation
    • Spectroscopic Techniques
Page 3: Nature of Light
  • Nature: Light exhibits both particle and wave properties.
    • Wave properties: Reflection, diffraction, transmission, etc.
    • Electromagnetic Properties: Vectors are mutually perpendicular.
Page 4: Characteristics of Light Radiation
  • Each light radiation is characterized by:
    • Frequency ($
      u$) which is independent of the medium.
    • Wavelength ($ ext{λ}$) which depends on the medium.
    • Relationship: <br/>ν=cλ<br />\nu = \frac{c}{λ}
    • Unit of Frequency: Hertz (s$^{-1}$)
    • Light can be monochromatic (single wavelength) or polychromatic (a range of wavelengths).
Page 5: Electromagnetic Spectrum
  • Consists of all discovered wavelengths, divided into several regions or ranges.
  • Energy associated with a wave: E=hνE = h \nu
    • Where: $h$ = Planck's constant.
Page 6: Absorption of Radiation
  • Various types of radiation affect molecules:
    • X-rays & Cosmic Rays: Molecular ionization
    • UV-Visible: Electron transitions between atomic and molecular orbitals
    • Infrared: Chemical bond deformation
    • Microwaves: Bond rotations
    • Radiofrequencies: Electronic or nuclear spin transitions
  • Chromophores: Atoms/molecules absorbing radiation.
  • Auxochromes: Groups that modify the absorption characteristics of chromophores.
Page 7: Spectroscopic Techniques
  • X-ray Absorption: Total molecular structure and stereochemistry.
  • UV-Visible: Identifies chromophores/conjugation from observed absorptions.
  • Infrared: Functional groups identified from absorbance.
  • Mass Spectrometry: Determines molecular formula and substructures from observed ions.
  • Nuclear Magnetic Resonance (NMR): Provides data on functional groups, substructures, connectivities, stereochemistry.
Page 8: Visible Spectrophotometry
  • Visible Range: Wavelengths between 400 nm and 700 nm.
Page 9: Complementary Colors in Absorption
  • Color Absorption and corresponding observation:
    • 420-430 nm: Absorbs Green-Yellow (appears Yellow)
    • 500-520 nm: Absorbs Red (appears Red)
    • Total Absorption: Appearance is Black.
    • Total Reflection: Appearance is White.
  • Colored solutions absorb complementary colors:
    • Color vs. Wavelength:
    • Violet: Absorbs Green-Yellow (570-580 nm)
    • Blue: Absorbs Yellow-Orange (570-600 nm)
    • Green: Absorbs Orange-Red (600-800 nm)
    • Yellow: Absorbs Blue (450-470 nm)
    • Orange: Absorbs Blue-Green (460-480 nm)
    • Red: Absorbs Green (490-590 nm)
Page 10: Laws of Absorption of Electromagnetic Radiation
  • Light absorption depends on incident radiation power ($P_0$):
    • Higher incident power increases transmitted power ($P_t$).
  • Transmittance (T): T=P<em>tP</em>0T = \frac{P<em>t}{P</em>0} or ext{%T} = rac{Pt}{P0} imes 100
  • Absorbance (A): A = 2 - ext{log} ext{%T}
  • Transmittance is expressed relative to 1 or 100.
Page 11: Beer-Lambert Law
  • Monochromatic radiation absorption defined by:
    A=extεimesbimesCA = ext{ε} imes b imes C
  • Where:
    • A = Absorbance (unitless)
    • ε = Molar absorptivity (cm$^{-1}$ L/mol)
    • b = Light path length (cm)
    • C = Concentration (mol/L)
  • Absorbance is dimensionless.
Page 12: Spectral Curves
  • Absorptivity variations create a spectral curve, showing absorption characteristics related to wavelength.
  • Often plotted as Absorbance vs. Wavelength (absorption spectrum) or Transmittance vs. Wavelength (transmission spectrum).
Page 13: Practice Problems
  • Exercise 1:
    • Solution of Ni(NO3)2
    • Given: 1000 ppm, 20% transmittance at 395 nm
    • a) Calculate absorbance for 2 cm cell
    • b) Calculate %T for 0.5 cm cell.
  • Exercise 2: 50 mg of substance X in 200 mL, 40% transmittance, 2 cm at 300 nm
    • a) Calculate specific absorptivity of X
    • b) Find molar absorptivity if MW = 250 g/mol.
Page 14: Additional Practice Problems
  • Convert transmittance to absorbance: 0.42%, 50%, 36.8%, 0.8%, 10%.
  • Convert absorbance to transmittance: 0.434, 0.76, 2.0, 0.1, 0.4, 0.00.
  • Given: Transmission 40% in 1 cm cell, find values for 1.5 cm and 2 cm cells.
Page 15: Problem Solving Steps
  • Exercise 6: 20 mg% organic compound (MW 150 g/mol) has 12% transmittance in a 0.5 cm cell.
    • Calculate: Specific absorptivity, molar absorptivity, and concentration for 0.434 absorbance in a 1 cm cell.
  • Exercise 7: 6000 ppm cobalt ion with 38% transmittance at 510 nm in 1 cm cell.
    a) Absorbance in 2 cm cell?
    b) Concentration for same absorbance in a 2 cm cell?
    c) Concentration for initial transmittance in a 2 cm cell?
Page 16: Instrumentation
  • Colorimeter: Selects wavelength using optical filters.
  • Spectrophotometer: Uses monochromators to select wavelengths.
Page 17: Light Sources
  • Excitation Source: Emits polychromatic light for interaction with samples.
Page 18: Monochromator Function
  • Separates polychromatic rays into monochromatic rays using:
    • Lenses, Mirrors, Diffraction Gratings, and/or Prisms.
Page 19: Sample Compartment
  • Where the cuvette holding the sample is placed.
    • Material transparency:
    • UV: Quartz
    • VIS: Glass, quartz, or clear plastic
    • IR: NaCl, KBr, CsI.
Page 20: Detector Function
  • Device receiving radiation, producing a signal upon absorption.
  • Ideal characteristics: sensitive, linear response, short response time, stability.
  • Common detectors: Phototubes made of alkali metal oxides (200-1000 nm, UV, VIS, NIR).
Page 25: Method Standardization
  • To check if a solution satisfies Beer’s Law, determine the wavelength of maximum absorption at constant concentration to create a spectral curve.
  • Graph absorbance vs concentration.
  • If linear, the system follows Beer-Bourger-Lambert Law.
  • Equations:
    • A<em>1/C</em>1=A<em>2/C</em>2A<em>1/C</em>1 = A<em>2/C</em>2 (Standard Method)
    • C<em>x=fimesA</em>xC<em>x = f imes A</em>x (Factor Method)
    • F=1mF = \frac{1}{m} where m=A<em>2−A</em>1C<em>2−C</em>1m = \frac{A<em>2 - A</em>1}{C<em>2 - C</em>1}.
Page 26: Optimal Concentration Range
  • Beer’s Law applies only within a certain concentration range.
  • Determine by a Ringbom curve: Absorbance (100 - %T) vs Log C.
Page 27: Simultaneous Determinations
  • Multiple absorbing species can be analyzed without prior separations, based on different spectral characteristics.
  • Apply additivity property of absorbance: Total measured absorbance is the sum of individual component absorbances, influenced by molar absorptivity and concentration.
Page 30: Analysis Example
  • Given %T for M and N at specific wavelengths, calculate concentrations in mg/mL considering molecular weights of 157 and 320 g/mol.
Page 40: Stoichiometry Determination of Complexes
  • Allows measures without disturbing equilibria.
  • Continuous Variation Method: Mix varying concentrations of components but maintain constant total volume.
Page 42: Final Steps
  • To confirm stoichiometry, plot absorbance against molar ratios.
  • Graph comparisons with complexation reactions.