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LIGHT ABSORPTION SPECTROSCOPY. colorimetric analysis of nmol samples of macromolecules Prof. Eric Wickstrom.  E.  B. orthogonal electronic (E) and magnetic (B) components of linearly polarized light.   Prism Slit.

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light absorption spectroscopy

LIGHT ABSORPTION SPECTROSCOPY

colorimetric analysis of nmol samples of macromolecules

Prof. Eric Wickstrom

slide2

E

B

orthogonal electronic (E) and magnetic (B) components of linearly polarized light

slide3

 

Prism Slit

slide4

I0 is the current measured by the photomultiplier tube when the light goes through the reference cuvet with buffer but not sample.

  • I is the current measured by the photomultiplier tube when the light goes through the complete sample.
  • The ratio of I to I0 is called the transmittance, T, so T = I/I0 . Therefore T ranges from 0 (total sample absorbance) to 1 (no absorbance at all).
slide5

The absorbance, A, is defined as A= -log T.

  • Because it is a logarithm, absorbance has no units.
  • When the sample absorbs 90% of the light beam, then T = 0.1 = 1/10, and A = -log(1/10) = +log(10) = 1
  • When the sample absorbs 99% of the light beam, then T = 0.01 = 1/100, and A = -log(1/100) = +log(100) = 2
slide6

The most commonly used unit is the absorbance unit, AU, or OD in biochemical slang, an amount of a substance that will give an absorbance of 1.0 at the wavelength of interest in a cuvet with a pathlength of 1.0 cm, when dissolved in 1.0 mL of buffer.

1 mg DNA = 20 A260U

1 mg protein = 1 A280U

slide7

We write the relationship as A= εlc, where l is the pathlength in cm, and c is the concentration in mole/liter.

  • The extinction coefficient, or molar absorptivity, ε, is the theoretical absorbance of a 1.0 M (mole/liter) solution in 1.0 cm cuvet.
  • Because absorbance is unitless, ε has units of liter/mole·cm.
typical initial concentrations
Typical Initial Concentrations
  • Protein Concentration: 0.5 mg/mL
  • Cell Path Length: 10 mm
  • Stabilizers (Metal ions, etc.): minimum
  • Buffer Concentration : 5 mM or as low as possible while maintaining protein stability
  • Contaminants: Unfolded protein, peptides, particulate matter (scattering particles)