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where l is the optical path length, ε is a molar absorbance at unit path length and c is a concentration. More than one of the species may contribute to the absorbance. In principle absorbance may be measured at one wavelength only, but in present-day practice it is common to record complete spectra.
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]
In addition, the total concentration of the two binding partners, the pH and ionic strength of the solution must all be maintained at fixed values throughout the experiment. Finally, there must be only one complex in solution which predominates over all others under the conditions of the experiment.
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 ...
The linear graph acquired from the assay (absorbance versus protein concentration in μg/mL) can be easily extrapolated to determine the concentration of proteins by using the slope of the line. It is a sensitive technique. It is also very simple: measuring the OD at 595 nm after 5 minutes of incubation.
Conversely, when pH = pK a, the concentration of HA is equal to the concentration of A −. The buffer region extends over the approximate range pK a ± 2. Buffering is weak outside the range pK a ± 1. At pH ≤ pK a − 2 the substance is said to be fully protonated and at pH ≥ pK a + 2 it is fully dissociated (deprotonated).
A titration curve is a curve in graph the x-coordinate of which represents the volume of titrant added since the beginning of the titration, and the y-coordinate of which represents the concentration of the analyte at the corresponding stage of the titration (in an acid–base titration, the y-coordinate usually represents the pH of the solution).
For example, if the concentration of the conjugate base is 10 times greater than the concentration of the acid, their ratio is 10:1, and consequently the pH is pK a + 1 or pK b + 1. Conversely, if a 10-fold excess of the acid occurs with respect to the base, the ratio is 1:10 and the pH is p K a − 1 or p K b − 1.