LIGHT ABSORPTION SPECTROSCOPY colorimetric analysis of nmol samples of macromolecules Prof. Eric Wickstrom

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

 Prism Slit

I 0 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 I 0 is called the transmittance, T, so T = I/I 0. Therefore T ranges from 0 (total sample absorbance) to 1 (no absorbance at all).

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

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 A 260 U 1 mg protein = 1 A 280 U

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 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)