Biotechnology: Fundamentals of Spectrophotometry

Fundamentals of Spectrophotometry

• Spectrophotometry involves the measurement of light absorption by substances, revealing key properties of materials in biotechnology.
• Light energy (E) inversely relates to its wavelength (λ), with increased frequency (ν) leading to higher energy.

Properties of Light

• The electromagnetic spectrum encompasses various types of light.
• The colors of visible light correspond to wavelengths absorbed by different chemicals, revealing complementary color relationships.

Absorption of Light

• The concentration of a substance is directly proportional to the light absorbed; greater absorption indicates higher concentration.
• DNA analysis relies on both qualitative (size and quality) and quantitative (purity and yield) measures, utilizing techniques like agarose gel electrophoresis and spectrophotometry.

Common Absorbing Biochemicals

• Key nucleobases absorb light at specific wavelengths, which are critical for quantitative analysis of nucleic acids:
  • Guanine: λmax = 275 nm, ε = 8.0 mM⁻¹cm⁻¹
  • Adenine: λmax = 260 nm, ε = 12.9 mM⁻¹cm⁻¹
  • Cytosine: λmax = 265 nm, ε = 5.8 mM⁻¹cm⁻¹
  • Thymine: λmax = 258 nm, ε = 8.0 mM⁻¹cm⁻¹

% Transmittance Relationships

• The relationship between % transmittance, light path length, and concentration is fundamental:
  • %T can range from 0 to 100, where a lower %T indicates increased absorption.
  • The equation for transmittance is: ( T = \frac{I_T}{I_0} = e^{-\alpha c l} )
    • Where α is the extinction constant, c is the concentration, and l is the light path length.

Spectrophotometer Design

• A spectrophotometer measures absorbance or transmittance by directing light through a sample.
• Two main types of light sources are utilized:
  • Tungsten Lamp: emits visible light (320-2500 nm) through black body radiation principles.
  • Deuterium Lamp: generates ultraviolet light (160-375 nm) using D2 or H2 gas, which disassociates in an electric arc to produce light.

Explore the fundamental concepts of spectrophotometry and its applications in biotechnology. This quiz covers key properties of light, different types of light, and the electromagnetic spectrum. Test your knowledge on how these elements interact and are utilized in clinical laboratory settings.