Instrumentation

DEFINITION OF TERMS:

• Energy: entity that this transmitted by electromagnetic waves
• Wavelength: defined as the distance between two successive peaks
• Nanometer: unit expression of wavelength
• Frequency: number of waves that passes a point of observation per one unit of time

SPECTROPHOTOMETRY

• Measures transmitted light in a colored solution
• Measurement is based upon Beer-Lambert-Bouguer Law (Beer’s Law/Beer-Lambert’s Law)

BEER-LAMBERT LAW

• States that concentration of an unknown analyte is directly proportional to the light absorbed and inversely proportional to light transmitted.
• Absorbance is proportional to the inverse log of transmittance

SINGLE-BEAM SPECTROPHOTOMETER

DOUBLE-BEAM SPECTROPHOTOMETER

• Double- beam in time – 1 photodetector
• Double-beam in space – 2 photodetectors (1- sample beam, 2- reference beam)

PARTS OF SPECTROPHOTOMETER

1. LIGHT SOURCE
   • Tungsten: for visible and near infrared region
   • Deuterium: for UV region
   • Xenon discharge lamp: for UV and Visible region

2. ENTRANCE SLIT – minimizes the entry of stray light to the monochromator
3. MONOCHROMATOR – isolates specific wavelength
   • Prisms: light is refracted
   • Diffraction gratings: light is bent; most commonly used
   • Filters: light enters one side and is reflected on the other side.

4. EXIT SLIT – controls bandpass (total range to which wavelengths are transmitted. The narrower the bandpass, the grater the resolution)
5. CUVETTE – contains the solution (known as absorption cell/analytical cell/sample cell)
6. PHOTODETECTOR – aids in the conversion of light transmitted to photoelectric energy
   • Barrier layer cell: simplest. Temperature sensitive. Radiation and visible region.
   • Photodiode: has excellent linearity.
   • Photomultiplier tube: most commonly used. Chemiluminiscence and Fluometry. Measures visible and UV region.
   • Phototube: cathode and anode enclosed in glass case. Fluometry.

7. READ-OUT DEVICE – a monitor that displays the output

ATOMIC ABSORPTION SPECTROPHOTOMETRY

• Measures the amount of light that have been absorbed by a ground state atom
• For measurement of unexcitable metals like calcium and magnesium
• Hollow-cathode lamp: light source
• Atomizer: used for the conversion of ions to atoms
• Chopper: used to modulate amount of light from the hollow-cathode lamp

FLAME EMISSION PHOTOMETRY

• Flame permits the excitation of the electrons; after which, electrons return to a ground state thus radiation is emitted.
• Flame serves as both light source and cuvette.
• Internal standards used: Cesium and Lithium (preferred)
• For measurement of excited ions such as sodium (yellow) and potassium (violet).
• Calcium also shows a colored (brick red) flame

FLUOROMETRY

• Light is absorbed by atoms at a specific wavelength and is emitted at a longer wavelength (with lower energy)
• Light source: xenon lamp or mercury arc
• There are two monochromators
• Primary monochromator: selects wavelength that is best absorbed by solution that is to be measured
• Secondary monochromator: this prevents the incident light from striking the detector
• Disadvantage: Quenching

TURBIDIMETRY

• Measures light blocked by molecules
• Used for immunoglobulins, immune complexes and complement

NEPHELOMETRY

• Measures light scattered by molecules
• Used for measuring amount of antigen-antibody complexes

CHROMATOGRAPHY

• Separation is based upon differences in characteristics (both physical and chemical) of substances
• Used for amino acid determination, drugs and sugars

POTENTIOMETRY

• Measures electric potential
• pH electrode – glass electrode
• pCO₂ electrode
• ion – selective electrode
• Sodium: glass electrode
• Potassium: Valinomycin gel
• Chloride: Tri-N-octyl propyl ammonium chloride decanol

ELECTROPHORESIS

• Separation of proteins is aided by an electric current

• pH of buffer: 6
• support materials:
• Agarose gel – separation by electric charges
• Cellulose acetate – separation by molecular size
• Polyacrylamide gel – separation by charge and molecular size