Molar Absorptivity

scantor145

I need help in devising a procedure to determine the molar absorptivity of an iron-dye complex in two solutions at a wavelength of 660nm. I have a UV-VIS spectrophotometer.

Solution 1 is an acidic buffer (R1) containing the iron-binding dye Chromazurol B and ferric chloride. Some amount of an iron standard will be added to this solution, hopefully forming a colored complex with the dye.

Solution 2 is Solution 1 with the addition of a neutral buffer (R2) raising the pH. I believe the molar absorptivity will change under varying pH. Accounting for dilution of Solution 1 would have to be taken into account.

The ratios of the volumes of iron standard to R1 to R2 are 16:200:60.

Yanick

You need to know the concentration of your chromophore precisely (level of precision depends on needs/goals of course). Get a baseline corrected absorbance measurement at the λ_max for a range of concentrations. Plot absorbance as a function of concentration. Do a linear least squares fit. According to the Beer-Lambert Law, the slope of this line is the extinction coefficient (molar absorptivity) mulitplied by the path length.

A very simple procedure. The most difficult part most times is being able to know the concentration of the analyte very precisely.

Also why would you add Iron to solution 1 if you already have ferric chloride? Do you need to add Fe(II) for the complex to form?

Also why not just make a new solution 2 instead of diluting with buffer? If the extinction coefficient is not so high, you may dilute the analyte below a measurable absorbance.

Typically you want to measure absorbances between 0.1-1.0 AU (and your calibration curve should cover atleast that much range), but this depends on the instrument you are using.

I just thought of another thing, if you are indeed using Fe(II) in aqueous solutions, you need to keep the pH pretty acidic. Fe(II) is not very stable above ~pH 4 (IIRC), quickly oxidizes to Fe(III) and precipitates out as rust. Chelators help to stop this but pH affects chelation depending on what you are using. Just a few things off the top of my head for you to keep in mind.