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To introduce students to the Beer-Lambert Law and its use in basic colorimetric analysis.

1. To determine lmax of a compound.
2. To use colorimetry to determine the concentration of an unknown solution.

This is a “dry” lab since you do not a Spec 20 at home and cannot collect this data without one. Therefore, you will use data collected from an experiment performed at NLC in a campus lab. This data is given in green on your Report Sheet.  Use this data to generate the graphs and fill in the Report Sheet.

Colorimetry involves the use of a device called a colorimeter (or spectrophotometer) to determine the concentration of an unknown sample. The colorimeter is an instrument that measures the fraction of light (of a particular wavelength) absorbed or transmitted by a solution. It consists of a light source (tungsten lamp), a refraction grating, a photodetector, and a display with absorbance and transmittance readings. The solution is inserted into the instrument inside of a cuvette (looks like a test tube but is made of much higher quality glass that does not absorb light.) A water blank is used to set A = 0 (or %T =100.)

At a given wavelength, the absorbance, A, can be calculated using Beer-Lambert’s law [(after August Beer (1825-1863) and Johan Heinrich Lambert (1728-1777)]

A = x l x C

where:

= molar absorptivity (or molar extinction coefficient) of the species

l = pathlength or thickness (cm) of the sample cell

C = concentration (mol/L) of species

Note that the Beer-Lambert law works only for dilute solutions.

The percent transmittance (%T) is the fraction of light transmitted through the sample and can be determined from A:

A = 2 – log(%T)

A = log ( )

A plot of absorbance vs. wavelength will show the wavelength where absorbance is at a maximum. This wavelength is called lmax. Colorimetric analyses are usually done at lmax.

In this experiment, you will:

1. Determine the lmax of a cobalt (II) complex, Co(H2O)62+ .
2. Determine the absorbances of five solutions of known Co(H2O)62+ concentration at lmax.
3. Graph a calibration curve of A vs concentration data.
4. Determine the concentration of an unknown solution of cobalt (II) complex.

Online students will not actually perform this procedure since they do not have access to a spectrophotometer.  Use the information to fill in the Report Sheet and graph the data.

1. Add 2.0, 4.0, 6.0, 8.0, and 10.0 mL portions of a 0.50 M Co(H2O)62+stock solution into 5 clean individual 50 mL beakers. Label each of them as solution A, B, C, D, and E, respectively.
2. Add 18.0, 16.0, 14.0, 12.0, and 10.0 mL of deionized water to content of each beaker, respectively. Note that each beaker has now a total volume of 20.0 mL.
3. Figure out the concentrations of A, B, C, D and E and report these to the correct number of sig figs in the Report Sheet Table in Part III.
4. Add 9.0 mL of your unknown solution into another 50-mL beaker. Add to it 11.0 mL of deionized water. Label this beaker diluted unknown.

PART II:  ABSORPTION SPECTRUM OF Co(H2O)62+

Determination of lmax using Solution C:

1. Read the Absorbance of solution C at the different wavelengths by following the spectrophotometer operating instructions:
1. Set wavelength to 400 nm.
2. With sample compartment empty and the mode set to TRANSMITTANCE, set the display to 0% by adjusting the lower LEFT hand knob. Once this is set, do NOT touch this lower left hand knob again.
3. Switch the mode to ABSORBANCE. Insert clean cuvette that is about half full of deionized water. Adjust the display to 0% by using the lower RIGHT hand knob.
4. Insert the sample and read the absorbance.
2. Reset the wavelength by increasing it 10 nm and repeat Step 1a-d.
3. Once data has been collected for all the wavelengths, plot absorbance versus wavelength. Include this graph in your report sheet.
4. Determine lmax (the wavelength with the maximum absorbance).

PART III: CALIBRATION CURVE

1. Set the wavelength on the spectrophotometer to lmax (determined in part II) using the Wavelength Selector dial.
2. Reset the transmittance and absorbance of the Spectrophotometer as done in Part II Step 1.
3. Measure the absorbances of all 6 solutions (5 knowns and 1 unknown) at lmax .
4. Plot your results of Absorbance vs. concentration. Include this graph in your Report Sheet.
5. From this graph of Absorbance vs. concentration, figure out the concentration of your diluted unknown and enter it into the Report Sheet Table in Part III.
6. From the concentration of the diluted unknown and how you made this dilute solution (step 4 of Part 1), figure out the concentration of the original sample and record this on your Report Sheet Table, Part III.

CALCULATIONS:

(Show work on your Report Sheet for the calculation of Solution C and the unknown solution)

PART I:

Calculate the concentrations of  the 5 samples (A-E) using the dilution formula M1V1 =M2V2.

Sample calculation –  Concentration of Solution A

Mi x Vi = Mf x Vf

Mf   =

Mi  = 0.50 M

Vi = 2.0 mL

Vf = 20.0 mL

Mf =

Mf = 0.050 M

Sample calculation – Concentration of cobalt(II) in the original unknown sample

10.5 mL of  an unknown cobalt(II)  sample was diluted to  30.0 mL.  The concentration of the diluted unknown sample was determined spectrophotometrically as in this experiment and was found to be 6.5 x 10-5 M. What is the concentration of cobalt(II) in the original sample?

Using the dilution formula again:  Mi x Vi = Mf x Vf , you are looking for Mi

Mi =

Mi  = ?

Vi = 10.5 mL

Mf = 6.5 x 10-5 M

Vf = 30.0 mL

Mi =

Mi =  1.9 x 10-4 M

PART II:

1. Plot A vs. wavelength – turn in graph.
2. Determine the wavelength that has the maxium absorbance.

PART III:

1. Plot concentration vs. A – turn in graph.
2. Determine the concentration of the unknown sample from your graph.

OPERATING INSTRUCTIONS

SPECTROPHOTOMETER

Instrument should be warmed up for 15 minutes.

Wipe all cuvettes with Kim Wipes to remove liquid droplets, dust and fingerprints before inserting into the instrument.

1. Set wavelength control to desired wavelength.
2. With sample compartment empty and the mode set to TRANSMITTANCE, set the display to 0% by using the lower LEFT hand knob. Once this is set, do NOT touch this lower left hand knob again.
3. Switch the mode to ABSORBANCE. Insert clean cuvette that is about half full of deionized water. Adjust the display to 0% by using the lower RIGHT hand knob.
4. Insert the sample and read the absorbance.

Colorimetry Report Sheet

 Name: Date:

PART II: Absorbance vs. Wavelength of Solution C

Fill in the data table for the graph of A vs. wavelength.

 Wavelength (nm) Absorbance 400 0.099 410 0.161 420 0.240 430 0.260 440 0.460 450 0.530 460 0.688 470 0.825 480 0.920 490 1.005 500 1.015 510 1.025 520 0.980 530 0.845 540 0.672 550 0.488 560 0.340

λmax =

PART III: Concentration vs. Absorbance at λmax

(Show work for the calculation of Solution C and the unknown solution, fill in the data for the graph of Concentration vs. A, showing concentration of unknown.)

 Solution Concentration (M) Absorbance Blank 0 A 0.317 B 0.626 C 1.035 D 1.220 E 1.280 Unknown ? 0.716

 Concentration of diluted unknown Co(II) complex M Concentration of undiluted Co(II) unknown M