Modified Procedure

### Experiment 1: Absorption Spectroscopy

Beer-Lambert's Law. Spectrometric determination of iron

Modified Procedure (March '97)

This procedure supersedes the one on page 51 in the laboratory manual.

Part One

A. Examining the Visible Spectrum

Same as in Lab Manual.

B. Determination of the wavelength of maximal absorbance of [Fe(SCN)]2+

Goes into Part Two

Part Two

1. Obtain about 10 mL of the 0.005M Fe(III) stock solution in a clean test tube. Label it Fe(III).

2. Using a graduated pipette transfer exactly 5.0 mL of the stock solution into a 250mL Erlenmeyer flask. Measure exactly 95.0 mL of distilled water in a 100mL graduated cylinder and add it to the 250mL Erlenmeyer flask. Swirl to ensure that the solutions are thoroughly mixed. You have just prepared a 1:20 dilution of the Fe(III) solution. Label it Fe(III) diluted.

3. Prepare six solutions of the [Fe(SCN)]2+ complex by setting up six large, clean test tubes on the test tube rack. Pipette into each tube the volumes of diluted Fe(III) solution, 8% NH4SCN, and distilled water specified in the following chart:

Tube # | Vol. diluted Fe(III) (mL) | Vol. 8% NH4SCN (mL) | Vol. distilled H2O (mL)
1 | 0.0 | 1.0 | 19.0
2 | 1.0 | 1.0 | 18.0
3 | 3.0 | 1.0 | 16.0
4 | 5.0 | 1.0 | 14.0
5 | 7.0 | 1.0 | 12.0
6 | 9.0 | 1.0 | 10.0

Using Parafilm, gently mix the solutions by inverting the test tubes three times. This will give you a range of concentrations of Fe(III) (present as the complex cation [Fe(SCN)]2+) from 1.25x10-5 M to 11.25x10-5 M.

4. Fill a cuvette about two-thirds full with solution #1. This is your blank for the entire experiment. Fill another cuvette with solution #6. Read and record the absorbance of this solution for the wavelengths 390 nm through 600 nm at 10 nm intervals. Use the 0-1A scale mode in the LKB. Remember at each wavelength setting to first set the LKB to 0.000 Absorbance with the blank. If the spectrometer's digital reading fluctuates, reset it to 0.000 with the blank and try again. Determine the wavelength of maximal absorbance by examining your data.

5. Set the spectrophotometer at the wavelength of maximal absorbance and set the absorbance to 0.000 with your blank. Record the absorbance of all the samples in order of increasing concentration (solutions #2 through #6), starting with the most diluted one (solution #2). Follow this procedure for all five dilutions. Rinse the cuvette with the solution you are about to use each time. Remember to set the absorbance to 0.000 with the blank before reading each sample.

Part Three

Iron Determination

Same as in Lab manual, with the following modifications:

- In step 1: Weigh about 0.040 g of vitamin (instead of 0.025 g).

- In step 2: Pour about 10 mL of 0.01M HCl (instead of 5 mL).

- In step 9: Find the absorbance (instead of the % transmittance).

- Step 10, that says: "The above procedure should be repeated twice,...", means: Repeat steps 6, 7, 8, and 9.

WRITE-UP

I. Calibration Curve

a) Calculate the concentrations of Fe(III) in the six the solutions tested in Part Two by using the dilution equation:

> > Vi Ci = Vf Cf

b) Plot the visible spectrum of the [Fe(SCN)]2+ complex expressed as Absorbance (y axis) vs. Wavelength in nm (x axis). Refer to the section on Graphing in the Data Analysis Guide (p.19).

c) Plot the calibration curve of [Fe(SCN)]2+ solution as Absorbance vs. Concentration. Do Least Squares Analysis (refer to section on LSA in the Data Analysis Guide, p. 21) of the data to determine the slope of the curve. The slope of the curve is the extinction coefficient (e) times l, the pathlength. In our case the cuvettes employed have a nominal pathlength of 1.00 cm, so the slope is effectively equal to e. Using a calculator or a computer to perform LSA is highly recommended.

II. Iron Determination

Refer to Lab Manual

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