The bases of DNA and RNA are good chromophores:īiochemists and molecular biologists often determine the concentration of a DNA sample by assuming an average value of ε = 0.020 ng -1×mL for double-stranded DNA at its λ max of 260 nm (notice that concentration in this application is expressed in mass/volume rather than molarity: ng/mL is often a convenient unit for DNA concentration when doing molecular biology). In our NAD + spectrum we observed A 260 = 1.0, so using equation 4.4 and solving for concentration we find that our sample is 5.6 x 10 -5 M. To use the viewer for absorbance dyes (colorimetric format labels), click the left drop-down menu under 'Current mode. The Spectrum Viewer is set to fluorescent mode by default. As an example, for NAD + the literature value of ε at 260 nm is 18,000 L * mol -1 *cm -1. AAT Bioquest's interactive Spectrum Viewer is a powerful tool for analysis and comparison of the spectra of common compounds used in absorbance and fluorescence formats. If we look up the value of e for our compound at λ max, and we measure absorbance at this wavelength, we can easily calculate the concentration of our sample. In most cases, sample holders are designed so that the path length is equal to 1 cm, so the units for molar absorptivity are L * mol -1 *cm -1. The absorbance will also depend, of course, on the path length - in other words, the distance that the beam of light travels though the sample. If we divide the observed value of A at λ max by the concentration of the sample ( c, in mol/L), we obtain the molar absorptivity, or extinction coefficient ( ε), which is a characteristic value for a given compound. The law is simply an application of the observation that, within certain ranges, the absorbance of a chromophore at a given wavelength varies in a linear fashion with its concentration: the higher the concentration of the molecule, the greater its absorbance. You most likely have performed a Beer – Lambert experiment in a previous chemistry lab. One of the most basic of these applications is the use of the Beer - Lambert Law to determine the concentration of a chromophore. UV-vis spectroscopy has many different applications in organic and biological chemistry. You can see that the absorbance value at 260 nm (A 260) is about 1.0 in this spectrum.Īpplications of UV spectroscopy in organic and biological chemistry To calculate absorbance at a given wavelength, the computer in the spectrophotometer simply takes the intensity of light at that wavelength before it passes through the sample (I 0), divides this value by the intensity of the same wavelength after it passes through the sample (I), then takes the log 10 of that number: This contains the same information as the 'percent transmittance' number used in IR spectroscopy, just expressed in slightly different terms. Here we use a unitless number called absorbance, abbreviated 'A'.

Dna absorbance spectrum