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In chromatography, the area of a peak is proportional to the number of moles (n) times some constant of proportionality (k), Area = k×n. The number of moles of compound is equal to the concentration (molarity, M) times the volume, n = MV. From these equations, the following derivation is made:
Two resolved peaks in a chromatogram. The theoretical plate height is given by = where L is the column length and N the number of theoretical plates. [5] The relation between plate number and peak width at the base is given by = ().
Chromatographic response function, often abbreviated to CRF, is a coefficient which measures the quality of the separation in the result of a chromatography.. The CRF concept have been created during the development of separation optimization, to compare the quality of many simulated or real chromatographic separations.
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]
So what does this mean? It means that to increase resolution of two peaks on a chromatogram, one of the three terms of the equation need to be modified. 1) N can be increased by lengthening the column (least effective, as doubling the column will get a 2 1/2 or 1.44x increase in resolution). 2) Increasing k' also helps.
The distribution constant (or partition ratio) (K D) is the equilibrium constant for the distribution of an analyte in two immiscible solvents. [1] [2] [3]In chromatography, for a particular solvent, it is equal to the ratio of its molar concentration in the stationary phase to its molar concentration in the mobile phase, also approximating the ratio of the solubility of the solvent in each phase.
This results in bias towards the unknown concentration. In other words, standard addition will not correct for these backgrounds or other spectral interferences. [5] Analysts also needs to evaluate the precision of the determined unknown concentration by calculating for the standard deviation, .
To normalize the concentration to a 10mm equivalent, the following is done: 0.6 OD X (10/3) * 50 μg/mL=100 μg/mL Most spectrophotometers allow selection of the nucleic acid type and path length such that resultant concentration is normalized to the 10 mm path length which is based on the principles of Beer's law.