# Standard curve of absorption and concentration

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!

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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,

1. 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.

2. Get a couple of volumetric flasks and dilute the stock solution.

3. Record the uv spectra for each of these solutions.
4. Plot absorbance (probably at around 420 nm) vs concentration.
• Perfect thank you! And just to verify, the concentration of the tartrazine given is a single value, meaning when I plot the standard curve of absorbance vs concentration I will have a straight, flat line since concentration doesn't change? – Matt Goodrich Feb 4 '15 at 9:53
• @soccer66 When you dilute by taking 1 mL of the stock solution and fill it up to 10 mL, 25 mL or 100 mL in volumetric flasks, the concentration does change! That's the idea behind the dilution ;) For each of these solutions, you'll measure a different absorbance. Plotting these data vs concentration will give a straight line, but not a flat one. – Klaus-Dieter Warzecha Feb 4 '15 at 9:58
• Oh! That makes sense! Should I just use the idea of initial concentration times initial volume equals final concentration times final volume to calculate the concentration, where the final volume is the amount of dye in the dilute mixture? And thanks again for giving me all of this extra help! – Matt Goodrich Feb 4 '15 at 10:06
• Think practical ;) If the assignment does not require a particular concentration range, measure the stock solution first and decide how low you can go with the concentration before the uv spectra start to look crappy. For the lower concentration range, remember that diluted solutions can be diluted again ;) Don't use all of the stock to prepare samples of very low concentrations, you cannot "undilute" them ;) – Klaus-Dieter Warzecha Feb 4 '15 at 10:25
• And, as a rule of thumb, try to get your absorbance to be between 0.1 and 1. – cbeleites supports Monica Feb 4 '15 at 16:54