I just wanted some quick help with a lab I'm doing, based on absorption spectroscopy. I'm given an aqueous stock solution of Yellow Dye #5 with a concentration of 54.5 micro moles. I'm asked to create a standard curve. It gives me the diluted volume as 50 mL, and has a data table with the volume of stock solutions and absorbance values for each. It asks me to create a standard curve (absorbance versus concentration) in excel for the Yellow Dye #5. First off, would I just use the constant concentration of 54.5 micro moles for this standard curve graph and the absorbance values in the data table? I'm not familiar with what a standard curve is, or if the volume of stock solution affects the concentration value in the graph. If anyone replies I'll probably comment on the right answer with a couple more, thanks for the help! It means a lot!
Supposed that Yellow Dye #5 refers to the FD&C numbering, this is a food colorant more known as tartrazine outside the U.S.
A standard curve correlates absorbance at a particular wavelength with the concentration of the dye.
Supposed that no distortion (e.g. due to aggregation effects) is present, the relation is given by the Lambert-Beer law: $$E_\lambda = \epsilon_\lambda \cdot c \cdot d$$
where $E_\lambda$ is the absorbance, $\epsilon_\lambda$ is the molar absorption coefficient (specific for a particular dye at a particular wavelength $\lambda$ in a certain solvent), $c$ is the concentration of the dye and $d$ is the path length of the cuvette. Please pay attention to the units in your calculations!
In order to create a standard curve,
Record the uv spectrum of the stock solution. If you have two-channel spectrometer, put a second cuvette with the neat solvent in that channel.
Get a couple of volumetric flasks and dilute the stock solution.
- Record the uv spectra for each of these solutions.
- Plot absorbance (probably at around 420 nm) vs concentration.