OD₂₆₀ Concentration

OD₂₆₀ basics

Optical density 260 (OD₂₆₀) is a measurement of how much light is absorbed by a molecule suspended in solution. The heterocyclic ring structures found in the nucleotide base structures absorb the maximum amount of light at 260nm and therefore, the absorbance of light emitted at 260nm is an accurate readout for the concentration of nucleotides in solution. The OD₂₆₀ of a solution can be measured using a spectrophotometer, which is a common piece of equipment found in most laboratories. Factors such as pH, buffer type, guanine content, and non-specific contaminating nucleotides can affect the OD₂₆₀ readout and should be taken into consideration when calculating concentrations.

Beer-Lambert Law

The Beer-Lambert Law relates the reduction of light to the materials which it travels through and is expressed using the equation below, where A = absorbance, ε = molar absorptivity, l = length of light path, and c = concentration. By applying this law with a spectrophotometer, you can find the concentration of RNA and DNA by measuring how much light the sample absorbs at a specific wavelength. A spectrophotometer works by shining a light beam through the sample and measuring the light that comes out on the other side. The change in light intensity, from before to after it passes through the sample, gives the absorbance value.

Figure 1. The equation for the Beer Lambert Law which describes the relationship between
the attenuation of light and the concentration of a solute in solution.

Molar absorptivity

Molar absorptivity, also known as the molar extinction coefficient, is a measure of how well a substance absorbs light at a specific wavelength, and it is expressed in units of L/(mol*cm). It is determined experimentally by measuring the absorbance of a series of known concentrations of the substance using a spectrophotometer and applying the Beer-Lambert Law. If the length of the light path is fixed and the molar absorptivity of a molecule is known, the absorbance can be used to directly calculate the concentration of DNA and RNA in water. The average molar extinction coefficients for single-stranded DNA, double-stranded DNA, and single-stranded RNA are listed below. These coefficients are used in the equation above to derive equations which can be used to directly calculate the concentration of an oligo in solution if the OD₂₆₀ is measured by a spectrophotometer.