Question

Explain how do we construct a standard curve in the UV/Vis spectrometer lab,what do we use the standard curve for?

Answer 1

Spectrophotometry is a technique that uses the absorbance of light by an analyte (the substance to be analyzed) at a certain wavelength to determine the analyte concentration. UV/VIS (ultra violet/visible) spectrophotometry uses light in UV and visible part of the electromagnetic spectrum.

The absorbance (A) of such solutions can be plotted against the quantity or concentration (c) of the test substance producing the color. This graph is known as the calibration curve (or standard curve). Since it is often possible to produce comparatively high absorbances with relatively small amounts of material

The amount of light absorbed by the dye, A, can be used to determine the amount of dye in the sample by comparing the absorbance data of the sample with those of carefully prepared solutions of blue dye of known concentrations called standard solutions using a mathematical relationship called BeerLambert’s Law:

where A is the absorbance, c is the concentration of standard or sample solutions, b is the length at which light passes through and ε is a constant called molar absorptivity . The value of ε depends on the wavelength used for analysis and the nature of species in solution. To make the standard curve plot of absorbance, A, on the y-axis against concentration of standard solutions, c, on the x-axis will yield a straight line with a slope equal to (εb). Such plot is called a standard curve, which is obtained by measuring the absorbance signal from a series of standard solutions of known concentration. The standard curve is then used to determine the concentration of dye (cunk) in the sample solutions by locating the absorbance measurement of the unknown on the yaxis (Aunk) and following a line until it intersects the standard curve. The corresponding value on the xaxis is the concentration of substance in the unknown sample