Standard Curve

Standard curve

Standard curves are used to determine the concentration of substances. They are obtained by relating a measured quantity to the concentration of the substance of interest in "known" samples, i.e. Standards of known concentration. These standards provide a reference to determine unknown concentrations. Thus amounts chosen of standards need to span the range of concentrations expected to be found in the "unknown" sample concentration.

The quantity (assay measurements as i.e. luminescence, radioactivity, fluorescence, and optical density of various known concentrations of a substance) graphed on y-axis and standard concentrations on x-axis. Data analyzed by fitting a line on curve (see Figure below).

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To determine the unknown concentration of a substance in a sample (with same assay as for standards used), intersect across the assay measurement on y with standard concentration, and down to x. The concentration of substance in unknown sample is the value on x.

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Quantitative estimation of the amount of proteins present in an unknown solution

To determine protein content of an unknown requires:

1- an assay in which measurable quantity is related to concentration

2- standards for comparison

Biuret test: is the method that forms a blue-violet color on reaction of protein with copper ion in alkaline solution. Is a good protein assay.

Spectrophotometer: is an optical instrument that measures the light energy transmitted throughout the continuous band of wavelength in spectromagnetic spectrum. A beam of light is focused by a lens onto an entrance slit, where it is collected by a second lens and refocused on the exit slit after being reflected and dispersed by a diffraction grating (used to select (). After passing the slit , the light goes through the sample being measured and picked up by a phototube. The amount of light absorbed by the sample is read on the dial.

Colorimetry: white light passed through a solution containing...