Spectrophotometry and Colorimetry

Light's Dance - Absorbance Rules

• Spectrophotometry & Colorimetry: Techniques measuring substance concentration based on light absorption/transmission.
  • Spectrophotometry: Uses specific wavelengths.
  • Colorimetry: Uses visible light; often for colored solutions.
• Principle: Interaction of light with matter. Light absorbed by a solution is proportional to the concentration of the absorbing substance.
• Beer-Lambert Law: $A = \epsilon c l$
  • $A$: Absorbance (no units)
  • $\epsilon$: Molar absorptivity (L·mol⁻¹·cm⁻¹)
  • $c$: Concentration (mol/L)
  • $l$: Path length (cm)
• Absorbance & Transmittance: $A = -\log T$ or $A = \log(1/T)$, where $T$ is transmittance.
• Limitations: Deviations at high concentrations, chemical reactions, stray light, non-monochromatic light.

⭐ Beer-Lambert Law is the cornerstone of quantitative analysis using spectrophotometry, directly relating absorbance to concentration.

The Machine - Peeking Inside

• Spectrophotometer Components:
  • Light Source: UV (Deuterium lamp), Visible (Tungsten lamp).
  • Monochromator: Prism or Grating; selects specific wavelength ($\lambda$).
  • Sample Holder: Cuvette (path length usually 1 cm).
  • Detector: Photomultiplier Tube (PMT) or Photodiode; measures transmitted light intensity.
  • Readout Device: Displays absorbance/transmittance.
• Colorimeter Components:
  • Light Source: LED or Tungsten lamp.
  • Filter: Selects a specific range of wavelengths.
  • Sample Holder: Cuvette.
  • Detector: Photocell or Photodiode.
  • Readout Device.
• Cuvette Types:
  • Quartz: For UV light (<340 nm).
  • Glass/Plastic: For visible light.
  • 📌 Mnemonic: "Quite Useful Glass Visible" (Quartz-UV, Glass-Visible).
• Key Difference: Spectrophotometer uses a monochromator (precise $\lambda$); Colorimeter uses a filter (broader $\lambda$ range).

⭐ Quartz cuvettes are essential for UV spectrophotometry as glass absorbs UV light significantly, especially <340 nm.

Lab Detective - Finding Clues

• Quantitative Analysis (Concentration):
  • Proteins: Biuret, Lowry methods.
  • Nucleic Acids: Absorbance at $A_{260}$.
  • Metabolites: Glucose (GOD-POD), cholesterol.
  • Hemoglobin: Cyanmethemoglobin method ($A_{540}$).
• Qualitative Analysis (Identification):
  • Absorption spectra identify substances by unique peak absorbance wavelengths.
• Enzyme Kinetics:
  • Measure reaction rates by monitoring changes in absorbance of substrate/product over time.
• Purity Assessment (Nucleic Acids):
  • $A_{260}/A_{280}$ ratio:
    • Pure DNA ≈ 1.8.
    • Pure RNA ≈ 2.0.
  • Ratios < 1.8 (DNA) or < 2.0 (RNA) suggest protein/phenol contamination.

⭐ The $A_{260}/A_{280}$ ratio is a widely used, quick method to assess the purity of nucleic acid preparations.

• Clinical Examples:
  • Hemoglobin estimation: Drabkin’s (cyanmethemoglobin) method.
  • Glucose estimation: GOD-POD (Glucose Oxidase-Peroxidase) method.

Color Power - Simpler & Specific

• Colorimetry:
  • Simpler technique; uses filters for broad wavelength bands (visible range).
  • Principle: Measures colored solution intensity.
  • Follows Beer-Lambert Law: $A = \epsilon c l$.
• Calibration Curve:
  • Plot Absorbance vs. known concentrations of standards.
  • Essential for quantification.
• Blanks:
  • Purpose: Zero instrument; correct for solvent/reagent absorbance.
  • Types: Reagent blank, sample blank.

⭐ Colorimeters use filters to select a range of wavelengths, making them simpler and cheaper but less specific than spectrophotometers which use monochromators.

High‑Yield Points - ⚡ Biggest Takeaways

• Beer-Lambert Law governs: Absorbance proportional to concentration & path length.
• Spectrophotometry measures substance concentration via light absorbance/transmittance at specific wavelengths.
• Colorimetry is a spectrophotometry type limited to the visible spectrum.
• Cuvette choice crucial: quartz for UV, glass/plastic for visible light.
• A blank solution is essential for accuracy, correcting background absorbance.
• Key uses: Quantifying DNA, RNA, proteins; monitoring enzyme kinetics.
• High concentrations can cause deviation from Beer-Lambert's linearity.