Spectrophotometry & Flame Photometry

Cheatsheet Content

Spectrophotometry Analytical technique measuring the absorption or transmission of light through a sample. 1. Basic Principles Electromagnetic Spectrum: Wavelength ($\lambda$), Frequency ($\nu$), Energy ($E$). $c = \lambda \nu$, $E = h\nu = hc/\lambda$. Absorption: Molecules absorb specific wavelengths of light, promoting electrons to higher energy states. Transmittance ($T$): Ratio of radiant power transmitted ($P$) to incident ($P_0$). $T = P/P_0$. Expressed as %T ($T \times 100$). Absorbance ($A$): Logarithmic inverse of transmittance. $A = -\log_{10} T = \log_{10} (P_0/P)$. Relationship: Higher absorbance means lower transmittance. 2. Beer-Lambert Law Relates absorbance to the concentration of the absorbing species and the path length of the light. Formula: $A = \epsilon b c$ $A$: Absorbance (unitless) $\epsilon$: Molar absorptivity (L mol$^{-1}$ cm$^{-1}$), a constant for a given substance at a specific wavelength. $b$: Path length of the light through the sample (cm), typically 1 cm. $c$: Concentration of the absorbing species (mol L$^{-1}$). Linearity: The law holds true for dilute solutions. Deviations occur at high concentrations due to intermolecular interactions or chemical equilibria. Applications: Quantitative analysis of concentration, reaction kinetics, purity assessment. 3. Spectrophotometer Components A typical spectrophotometer consists of: Light Source: Emits electromagnetic radiation. UV: Deuterium lamp (190-400 nm) Visible: Tungsten-halogen lamp (350-1000 nm) Monochromator: Selects a narrow band of wavelengths. Prism or Diffraction Grating Slits control bandwidth. Sample Holder (Cuvette): Holds the sample. Glass for visible light. Quartz for UV light. Detector: Converts light signal into an electrical signal. Photodiode, Photomultiplier Tube (PMT). Readout Device: Displays absorbance or transmittance. 4. Types of Spectrophotometry UV-Vis Spectrophotometry: Most common, uses UV and visible light. Measures concentrations of colored and colorless compounds. Infrared (IR) Spectrophotometry: Uses infrared light to identify functional groups in molecules based on vibrational modes. Atomic Absorption Spectrophotometry (AAS): Measures the absorption of specific wavelengths by free atoms in a flame, used for trace metal analysis. 5. Practical Considerations Calibration Curve: Plot of absorbance vs. concentration for known standards to determine unknown concentrations. Blank: Sample containing all reagents except the analyte, used to zero the instrument and correct for background absorbance. Wavelength Selection: Choose $\lambda_{max}$ (wavelength of maximum absorbance) for highest sensitivity and adherence to Beer-Lambert law. Cuvette Care: Clean, scratch-free, orientation. Flame Photometry Atomic emission technique used for the quantitative determination of certain metal ions (e.g., Na, K, Li, Ca) in solution. 1. Basic Principles Atomization: Sample is introduced into a flame, where solvent evaporates, and metal salts decompose into free atoms. Excitation: Thermal energy from the flame excites valence electrons of these atoms to higher energy levels. Emission: Excited electrons are unstable and quickly return to their ground state, emitting light of characteristic wavelengths. Detection: The emitted light is isolated by filters and measured by a detector. The intensity of emitted light is proportional to the concentration of the metal in the sample. 2. Flame Photometer Components Nebulizer/Atomizer: Draws sample into the flame as a fine spray. Typically pneumatic (air or gas flow). Burner: Provides a stable flame (e.g., air-acetylene, air-propane, air-natural gas). Temperature must be consistent. Optical System: Lens/Mirror: Collects emitted light. Filter: Selects specific wavelength characteristic of the analyte (e.g., 589 nm for Na, 766 nm for K). Detector: Photocell or photomultiplier tube converts light intensity into an electrical signal. Readout Device: Displays concentration or intensity. 3. Working Mechanism Sample Introduction: Liquid sample is drawn into the nebulizer. Aerosol Formation: Nebulizer creates a fine mist (aerosol). Flame Atomization: Aerosol enters the flame, solvent evaporates, and analyte salts dissociate into free atoms. Excitation & Emission: Thermal energy excites atoms, which then emit characteristic photons as they return to ground state. Wavelength Isolation: Optical filters isolate the specific wavelength for the target element. Signal Measurement: Detector measures the intensity of the emitted light. 4. Applications Clinical analysis (Na, K, Li in blood serum, urine). Environmental analysis (metals in water). Agricultural analysis (soil and plant samples). Industrial quality control (cement, glass, ceramics). 5. Advantages & Limitations Advantages: Relatively inexpensive and simple to operate. High sensitivity for alkali and alkaline earth metals. Fast analysis time. Limitations: Limited to easily excitable metals (low ionization energy). Spectral interference from overlapping emission lines. Ionization interference (high flame temperatures can ionize atoms). Matrix effects (other components in the sample affecting signal). 6. Interferences Spectral: Emission from other elements or molecular species overlapping with the analyte's emission. Ionization: At high temperatures, atoms can ionize, reducing the number of neutral atoms available for excitation and emission. Suppressed by adding an ionization buffer (e.g., Cs or K). Chemical: Formation of stable compounds in the flame that don't dissociate into free atoms (e.g., phosphates with Ca). Can be overcome by releasing agents. Matrix: Viscosity or surface tension of the sample affecting nebulization efficiency.