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The secondary benefit of using spectrophotometric analysis for nucleic acid quantitation is the ability to determine sample purity using the 260 nm:280 nm calculation. The ratio of the absorbance at 260 and 280 nm (A 260/280) is used to assess the purity of nucleic acids.
The absorbance of a material that has only one absorbing species also depends on the pathlength and the concentration of the species, according to the Beer–Lambert law =, where ε is the molar absorption coefficient of that material; c is the molar concentration of those species; ℓ is the path length.
Absorbance within range of 0.2 to 0.5 is ideal to maintain linearity in the Beer–Lambert law. If the radiation is especially intense, nonlinear optical processes can also cause variances. The main reason, however, is that the concentration dependence is in general non-linear and Beer's law is valid only under certain conditions as shown by ...
The spectra of basic, acid and intermediate pH solutions are shown. The analytical concentration of the dye is the same in all solutions. In spectroscopy, an isosbestic point is a specific wavelength, wavenumber or frequency at which the total absorbance of a sample does not change during a chemical reaction or a physical change of the sample ...
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.
In particular, the pH of a solution can be predicted when the analytical concentration and pK a values of all acids and bases are known; conversely, it is possible to calculate the equilibrium concentration of the acids and bases in solution when the pH is known. These calculations find application in many different areas of chemistry, biology ...
A calibration curve plot showing limit of detection (LOD), limit of quantification (LOQ), dynamic range, and limit of linearity (LOL).. In analytical chemistry, a calibration curve, also known as a standard curve, is a general method for determining the concentration of a substance in an unknown sample by comparing the unknown to a set of standard samples of known concentration. [1]
The pH of a solution is defined as the negative logarithm of the concentration of H+, and the pOH is defined as the negative logarithm of the concentration of OH-. For example, the pH of a 0.01M solution of hydrochloric acid (HCl) is equal to 2 (pH = −log 10 (0.01)), while the pOH of a 0.01M solution of sodium hydroxide (NaOH) is equal to 2 ...