BY452 Revision Notes

BY452 Revision Notes

Learning Outcomes

• Define the electromagnetic spectrum.

• Understand concepts of light absorbance.

• Apply Beer-Lambert law.

• Learn about applications of spectroscopy in biology.

• Calculate Relative Centrifugal Field (RCF).

• Understand the different types of centrifugation used to separate different particles.

• Understand the theory of chromatography as applied to:

  • Gel Filtration Chromatography.

  • Ion-Exchange Chromatography.

  • Affinity Chromatography.

Electromagnetic Spectrum

• Definition: The entire range of electromagnetic radiation, varying in wavelength from very long, lower-energy waves to very short, higher-energy waves.

• Wavelength (λ) & Frequency (ν):

  • Wavelength increases from high-energy gamma rays, through X-rays, visible light, infrared, microwaves, to radio waves.

  • Visible Light Range: 400 nm (violet) to 700 nm (red).

  • Example of Frequencies and wavelength of electromagnetic waves:

  • Radio Waves: $3 imes 10^{10} Hz$

  • Microwaves: $3 imes 10^{12} Hz$

  • Ultraviolet: $3 imes 10^{16} Hz$

  • Visible: $6 imes 10^{14} Hz$

Properties of Light

• Light as a Wave:

  • Wavelength (λ): Peak-to-peak distance between waves.

  • Amplitude: Related to power.

  • Frequency (ν): Number of oscillations per second (units: $s^{-1}$ or Hz).

  • Relationship: $c = ν imes λ$, where c = speed of light ( approximately $2.998 imes 10^8 m/s$ in a vacuum).

Photon Energy

• Each Photon Energy (E): $E = hν$, where:

  • h = Planck’s constant = $6.626 imes 10^{-34} Js$.

• Energy Relationships:

  • Energy is inversely proportional to wavelength.

  • Energy is directly proportional to frequency:
    $E ext{ is proportional to } ν$

Spectroscopy Principles

• When a molecule absorbs a photon, it gains energy and is promoted to an excited state. Conversely, emitting a photon lowers its energy.

• Ground State: The lowest energy state of a molecule.

• Qualitative Analysis: Specific wavelengths of absorption help identify substances.

• Quantitative Analysis:

  • Light absorption decreases the radiant power (irradiance, P) of light passing through the sample.

  • Transmittance (T) is the fraction of original light passing through: $T = rac{P_{ ext{transmitted}}}{P_{ ext{incident}}}$

  • Absorbance (A) is defined as: $A = - ext{log}(T)$

Beer-Lambert Law

• Definition: Absorbance is directly proportional to concentration: $A = ε imes b imes c$ Where:

  • $ε$ = molar absorptivity (M$^{-1}$cm$^{-1}$), specific to a substance at a given wavelength.

  • $b$ = pathlength in cm.

  • $c$ = concentration in M (moles/liter).

• Conditions for Validity:

  • Monochromatic light is used.

  • Sample must be dilute; collisional effects in concentrated solutions can alter molar absorptivity.

• Limitations:

  • Applies if the analyte does not undergo chemical changes during measurement.

Centrifugation

• Purpose: Separates particles in a liquid based on differences in size, shape, and density by speeding up sedimentation.

• Factors Influencing Sedimentation:

  • Applied centrifugal force.

  • Density difference between particle and liquid.

  • Size and shape lead to varying settling velocities.

  • Viscosity of the medium.

• Measurements:

  • RPM (Revolutions per Minute): Speed of the centrifuge.

  • RCF (Relative Centrifugal Force):
    $RCF = 1.12 imes R imes rac{( ext{RPM})^{2}}{1000^{2}}$
    Where R = radius in mm.

  • RCF expressed as multiples of gravitational force (g = 9.807 m/s²).

Types of Centrifugation

• Differential Centrifugation: Separates particles based on sedimentation rates; effective for subcellular fractionation.

• Density Gradient Centrifugation: Utilizes differences in buoyant density for separation:

  • Rate Zonal—ex: sucrose density gradient.

  • Isopycnic—particles stop migrating when they reach neutral buoyancy density.

Chromatography

• Definition: The separation process of molecules between stationary and mobile phases.

• Phases:

  • Stationary Phase:

  • Fixed and acts as adsorbent.

  • Characteristics: selectivity, capacity, stability, cost.

  • Mobile Phase:

  • Carrier solution (liquid or gas) moving through stationary phase.

• Types of Chromatography:

  • Gel Filtration Chromatography (GF): Separates based on molecular size; large molecules elute first.

  • Exclusion Limit: Pore size limitation in stationary phase beads.

  • Ion-Exchange Chromatography (IEX): Uses charge properties to separate molecules based on ionic interactions.

  • Affinity Chromatography (AC): Uses specific binding interactions to isolate a target molecule from a mixture.

Gel Filtration Chromatography (GF)

• Operational Principle: Small molecules enter beads; larger molecules cannot, causing different elution times.

• Important Constants:

  • Void Volume (V₀): Volume excluded from stationary phase.

  • Total Bed Volume (Vₐ): Total volume including stationary and mobile phases.

  • Pore Volume (Vₗ): Volume accessible to solutes.

  • Resolution: Defined using distribution coefficients (K_d):
    $K_d = rac{C_{ ext{stationary}}}{C_{ ext{mobile}}}$.

Ion-Exchange Chromatography (IEX)

• Types of Exchangers:

  • Cation Exchangers—bind positively charged analytes.

  • Anion Exchangers—bind negatively charged analytes.

• Elution Conditions: Change in pH or ionic strength to release bound analytes based on competitive binding.

Affinity Chromatography (AC)

• Mechanism: Separates molecules based on specific interactions using ligands attached to a stationary phase.

• Types of Elution Methods:

  • Buffer composition changes.

  • pH extremes or high concentrations of chaotropic agents (caution on stability).

  • Specific competition in binding.

• Applications: Useful for assays such as HbA1c correlating to specific ligands (e.g., phenylboronic acid).