6 - Chemical Techniques Part 2

Introduction

• Optical instruments measure light energy using spectrophotometers, flame emission, and atomic absorption techniques.
• Analytical determinations quantify the concentration of atoms or molecules by measuring the absorption or transmission of light energy.
• Electromagnetic energy is transmitted via electromagnetic waves.
• Waves are measured in nanometers (wavelength).

Beer's Law

• States the concentration of a substance is:
  • Directly proportional to the amount of light absorbed.
  • Inversely proportional to the amount of transmitted light.
• Formula for absorbance: Absorbance = 2 log% (Transmittance).
• ROYGBIV relates to increasing wavelengths.
  • Shorter wavelengths equate to stronger radiation.
• Visible light ranges from approximately 400 nm (violet) to 700 nm (red).

Spectrophotometry

• Measures the intensity of color and the light transmitted by a solution to determine a substance’s concentration.
• Converts light into voltage, translating to absorbance value readings.
• Electromagnetic radiation energy is inversely proportional to wavelength. Remember color indicates light bouncing off an object.
• Light travels in waves that vary in lengths and any form of light is radiation.

Components of a Spectrophotometer

• Light Source: Provides incident light for the system
  • Incandescent Tungsten or Tungsten iodide lamp: used for visible and near-infrared spectrums.
  • Deuterium-discharge lamp and Mercury arc lamp: used for the UV spectrum.
  • Silicone carbide: used for the infrared spectrum.
• Monochromator: Isolates specific wavelengths from the light source, utilizing:
  • Interference Filters: Based on constructive interference of waves.
  • Prism: Separates white light into a continuous spectrum allowing for isolating wavelengths.
  • Diffraction Gratings: Bends light and forms wavefronts to either reinforce light or cancel it out.
• Sample Cell: Holds the solution being measured
  • Can consist of a cuvette or analytical cell
  • Soft glass cuvettes are used for the visible range
  • Quartz cuvettes are used for the UV range
• Photo Detector: Converts transmitted radiant energy into electrical energy
  • Photocell (Barrier layer cell): Generates electromotive force (no external voltage).
  • Phototube: Similar to photocell but requires external voltage.
  • Photomultiplier tube: Detects and amplifies radiant energy, highly sensitive.

Flame Emission Spectrophotometry (FES)

• Measures light emitted by excited atoms.
• Measures sodium, potassium, and lithium because they are easily excited.
• A flame, typically using propane, excites atoms to a higher energy state, after which excited atoms return to the ground state by emitting light energy, characteristic for the atom.
  • Sodium emits an intense yellow flame.
  • Potassium emits a violet flame.
  • Calcium emits a brick red flame.
• Components of FES include:
  • Nebulizer: Atomizes and delivers a fine spray of the sample containing the metallic ion to the burner.
  • Burner: Uses a fuel gas (propane) with an oxidizing agent (compressed air) to produce the flame.
  • Monochromator System: Allows only the emitted line spectrum of the specific element to strike the PMT.
  • Photosensitive Detector: Employs a photomultiplier tube

Atomic Absorption Spectrophotometry (AAS)

• Measures light absorbed by ground state atoms and is used to measure the concentration of calcium atoms (not easily excited).
• 100 times more sensitive than FES.
• Components of AAS include:
  • Light Source: Provides incident light for the system
    • Hollow cathode lamp: Consists of an evacuated gas-tight chamber filled with inert gas such as helium or argon.
    • Electrodeless discharge lamp: Consists of a bulb filled with argon and the element to be tested, with a radiofrequency generator to excite the element.
  • Beam Chopper: Modulates the hollow cathode light beam to produce an alternating signal.

Light Emission and Scattering Techniques

• Includes instruments that measure light energy: fluorometry, chemiluminescence, turbidity, nephelometry, and lasers.

Fluorometry

• Measures the concentrations of solutions containing fluorescing molecules.
• Excitation light: Energy emission that occurs when certain compounds absorb electromagnetic radiation, becomes excited, gives off light.
• Principle of fluorescence involves a material absorbing light at one wavelength and emitting light at a different (higher) wavelength.
• Key components in filter fluorometers:
  • Light Source: Gas discharge lamps emitting short-wavelength, high-energy excitation light.
  • Attenuator: Controls light intensity.
  • Primary Filter: Selects the wavelength best absorbed by the sample.
  • Sample Holder: Where the fluorescing sample emits fluorescent light in all directions.
  • Secondary Filter: Passes longer wavelengths of fluorescent light, preventing incident light from striking the photodetector.
  • Photodetector: Employs a photomultiplier tube.

Chemiluminescence

• A portion of chemical energy generates excited intermediates that decay to the ground state by emitting photons.
• The emitted radiation is measured using a photomultiplier tube, and the signal is related to analyte concentration.
• Chemiluminescence arises from one species, not requiring excitation radiation or monochromators, unlike fluorescence.

Turbidimetry

• Uses a spectrophotometer to determine the concentration of particulate matter in a sample by measuring the amount of light blocked by the particle suspension, depending on concentration and size.

Nephelometry

• Similar to turbidimetry, measures light scattered by small particles at an angle to the incident beam on the cuvette.
• Light scattering depends on wavelength and particle size.
  • For macromolecules with sizes close to or larger than the incident lights wavelength, sensitivity improves/increases by measuring forward light scatter.
• Monochromatic light achieves uniform scatter and minimizes sample heating.

Laser

• Based on the interaction of radiant energy with suitably excited atoms or molecules, which leads to stimulated emission of radiation.
• Laser light is polarized and coherent with a narrowness in spectral width, small cross-sectional area, and low divergence.
• Can be a source of incident energy in spectrometry or nephelometry.
• Laser spectrometry aids in determining structure and identification of samples, as well as diagnosis.

Electrophoresis

• Separates molecules based on size and charge, where opposites attract.
  • Negatively charged ions move toward a positive electrodes.
  • Positively charged cations move toward a negative cathode.
• Key components include:
  • Driving force (electrical power).
  • Support medium.
  • Buffer.
  • Sample.
  • Detecting System.

Elctrophoretogram.

• An electropherogram results from electrophoresis, showing separated strands of a macromolecule.

Buffers

• Two buffer properties that affect the charge of ampholytes are pH and ionic strength:
  • If the buffer is more acidic than the isoelectric point (pI), the ampholyte binds H+, becomes positively charged, and migrates toward the cathode.
  • If the buffer is more basic than the pI, the ampholyte loses H+, becomes negatively charged, and migrates toward the anode.
  • A particle without a net charge will not migrate, remaining at the point of application.
• Note*: Isoelectric focusing is the movement of a buffer and solvent relative to their fixed supports.