• Spectrophotometry is a method used to estimate the level of an analyte in solution. It relies on the principle that materials absorb light of a certain wavelength as it passes through the solution.
  • Beer’s Law states that the amount of light of a particular wavelength absorbed by a substance across a constant distance (lightpath) is proportional to the concentration of that substance.
ie. An = l x c
where An is the absorbance at n nm
l is the light path (cm)
c is the concentration
  •  A spectrophotometer is a device which measures the absorbance of a solution as light of a specified wavelength is passed through it.

Principle of spectrophotometry

  • If we measure the absorbance of a solution containing a known concentration of an analyte, we can use this value to estimate the concentration of the analyte in an unknown solution by comparing the two absorbance values
  • The range over which absorbance is proportional to concentration varies according to the analyte and the wavelength of light used. To ensure that there is a direct relationship between absorbance and concentration, we must prepare a standard curve. Despite its name, the part of the standard curve that gives a proportional relationship is a straight line.
  • To prepare a standard curve, we prepare a series of dilutions of a standard solution of our analyte of known concentration. The first tube always contains none of the analyte (eg. a concentration of 0g/L) and we call this a blank. We use this to calibrate the spectrophotometer to take into account the natural absorbance of the diluents. Each of the following tubes contain increasing concentrations of the analyte. The absorbances of each of the standards are read using the spectrophotometer.

Setting up a standard curve

  • To create the standard curve, we plot a line graph of Absorbance (Y axis) vs Concentration (X Axis) for each of the standards. A line of best fit is then drawn through the points.

Plotting the standards on a standard curve

  • To estimate the concentration of an unknown solution of the analyte, we read the absorbance, and then use the standard curve :

Using a standard curve to find the concentration of an unknown