At any particular wavelength the absorption of light when visible light passes through a solution depends on two factors: * The length of the light path * The concentration of the colored path
The connection of these two variables is known as the Beer-Lambert Law:
Absorbance (A) = ε c l
Where ε is the absorption coefficient
C is the concentration of the compound
And l is the length of light usually 1cm
When I is constant, this proves a linear relationship between absorbance and concentration. This means it can be rearranged to find the find the optimum concentration for maximum absorbance.
C = A/ ε
The two reagents used are Bromophenol blue and Methyly orange.
1 Set up the colorimeter at 400nm, measure the absorbance of both reagents against distilled water, this will be used as a blank
(make sure to zero the colorimeter after each interval)
2 Repeat this procedure at 20nm from 420nm to 700nm
3 After getting a range of results make further readings in smaller intervals to precisely locate the wavelength for λmax
1 Using the protocol provided prepare a series of dilutions in testubes for each compound for the range of concentrations from 1 to 10mg/l.
2 Using the λmax for each reagent from Experiement 1 set the colorimeter against distilled water and measure the absorption
3 Repeat for other dilution series
Experiment 1: Bromophenol Blue | | Further readings to determine λ max | | Wavelength (nm) | Absorbance | Wavelength (nm) | Absorbance | 400 | 0.093 | 595 | 0.98 | 420 | 0.113 | 590 | 0.942 | 440 | 0.035 | 610 | 0.672 | 460 | 0.048 | | | 480 | 0.069 | | | 500 | 0.111 | | | 520 | 0.154 | | | 540 | 0.301 | | | 560 | 0.451 | | | 580 | 0.728 | | | 600 | 0.976 | | | 620 | 0.347 | | | 640 | 0.066 | | | 660 | 0.011 | | | 680 | 0.002 | | | 700 | 0 | | |
Methyl orange | | Further
References: http://www.chemguide.co.uk/analysis/uvvisible/beerlambert.html Accessed: 25/11/12