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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]
Thus, for a fixed path length, UV-Vis spectroscopy can be used to determine the concentration of the absorber in a solution. It is necessary to know how quickly the absorbance changes with concentration. This can be taken from references (tables of molar extinction coefficients), or more accurately, determined from a calibration curve.
A calibration curve is obtained by measuring a series of standard solutions with known concentrations, which can be used to determine the concentration of an unknown sample using linear regression analysis. [10] For example, by comparing the absorbance values of a solution with an unknown concentration to a series of standard solutions with ...
The absorbance of the light is the base 10 logarithm of the ratio of the Transmittance of the pure solvent to the transmittance of the sample, and so the two absorbance and transmittance can be interconverted. [12] Either transmittance or absorbance can therefore be plotted versus concentration using measurements from the Spectronic 20.
Determining the absolute concentration of a compound requires knowledge of the compound's absorption coefficient. The absorption coefficient for some compounds is available from reference sources, and it can also be determined by measuring the spectrum of a calibration standard with a known concentration of the target.
Step 5: Calibration and Quantification:** To determine the actual concentration of the analyte, the instrument is calibrated using standard solutions containing known concentrations of the element. By comparing the measured absorbance of the sample to the calibration curve, the concentration of the analyte in the original sample can be calculated.
In summary, in order to find a standard curve, one must use varying concentrations of BSA (Bovine Serum Albumin) [2] in order to create a standard curve with concentration plotted on the x-axis and absorbance plotted on the y-axis. Only a narrow concentration of BSA is used (2-10 ug/mL) in order to create an accurate standard curve. [23]
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.