The lambda max for Red 40 is 506 nm and the lambda max for Yellow 5 is 421 nm. After analyzing the spectra at these wavelengths, we were
able to determine that Red 40 absorbs independently of Yellow 5, but Yellow 5 does not absorb independently of the Red 40. This allowed for
an exclusion of the term for yellow dye when using Beer’s Law to calculate the unknown concentration of the red dye:
A506 nm= Ered, 506 nm x b x Concred
as opposed to a general formula,
A506nm = Ered, 560 nmx b x Concred +Eyellow, 506 nmx b x Concyellow
Because the spectra for Yellow 5 went to 0 before a wavelength of 506, the term in red was excluded in calculating the concentration of red.
This proved to be a useful tool in eliminating a second unknown variable in order to solve both concentrations.
To solve for the extinction coefficients, Beer’s Law plots were constructed for both dyes at 421 nm, and for Red 40 at 506 nm.
Ered, 506= 0.0487 L/mg*cm
Eyellow, 421=0.042 L/mg*cm
Ered, 421= 0.0135 L/mg*cm
Using the calculated coefficients, solve the unknown concentration of red dye. By substituting this into the combined equation, A421nm = Ered, 421
nmx b x Concred +Eyellow, 421 nmx b x Concyellow, solve for the concentration of yellow dye.
Note: the above Beer’s Law plots should have intercept set to zero. It may be useful to make higher concentrations of Red 40 for obtaining the
extinction coefficient at 421 nm since those absorbance values are low.