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At a wavelength of 260 nm, the average extinction coefficient for double-stranded DNA is 0.020 (μg/mL) −1 cm −1, for single-stranded DNA it is 0.027 (μg/mL) −1 cm −1, for single-stranded RNA it is 0.025 (μg/mL) −1 cm −1 and for short single-stranded oligonucleotides it is dependent on the length and base composition.
In biochemistry, the molar absorption coefficient of a protein at 280 nm depends almost exclusively on the number of aromatic residues, particularly tryptophan, and can be predicted from the sequence of amino acids. [6] Similarly, the molar absorption coefficient of nucleic acids at 260 nm can be predicted given the nucleotide sequence.
Once a reference signal is acquired, it is compared to the transmitted light Is of the measured sample. In order to calculate the associated extinction coefficient μ t, Beer's law can be applied as follows: I s = I o exp(-μ t d), where d is the sample thickness. The extinction coefficient is therefore: μ t = -ln(I s /I o)/d.
Extinction coefficient refers to several different measures of the absorption of light in a medium: Attenuation coefficient , sometimes called "extinction coefficient" in meteorology or climatology Mass extinction coefficient , how strongly a substance absorbs light at a given wavelength, per mass density
These two different types of hemoglobin exhibit different absorption spectra that are normally represented in terms of molar extinction coefficients, as shown in Figure 1. The molar extinction coefficient of Hb has its highest absorption peak at 420 nm and a second peak at 580 nm. Its spectrum then gradually decreases as light wavelength increases.
Variable pathlength absorption spectroscopy uses a determined slope to calculate concentration. As stated above this is a product of the molar absorptivity and the concentration. Since the actual absorbance value is taken at many data points at equal intervals, background subtraction is generally unnecessary.
When an isosbestic plot is constructed by the superposition of the absorption spectra of two species (whether by using molar absorptivity for the representation, or by using absorbance and keeping the same molar concentration for both species), the isosbestic point corresponds to a wavelength at which these spectra cross each other.
Several formulas are used to calculate T m values. [10] [11] Some formulas are more accurate in predicting melting temperatures of DNA duplexes. [12] For DNA oligonucleotides, i.e. short sequences of DNA, the thermodynamics of hybridization can be accurately described as a two-state process.