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Instructions
Take four clean test tubes and measure 100 microliters of ethyl acetate into the first with your pipette. Add 50 microliters to the second, 25 to the third and 5 to the fourth.
Change pipette tips and add deionized water to each tube. To the first tube add 1.8 mL, then 1.9 mL to the second tube, 1.95 mL to the third tube and 1.6 mL to the fourth. Stir with the pipette tip to mix, being sure to change pipette tips between tubes.
Transfer 1 mL of the mixture in each tube to four clean cuvettes. Again, be sure to change pipette tips between tubes.
Insert the first of the four cuvettes into a blanked, calibrated spectrophotometer and measure absorbency at 215 nm. If you aren't sure how to change the wavelength and/or blank your spectrophotometer, consult the manufacturer's instructions; the procedure may vary somewhat depending on the device. Record the value you measure and take measurements for the contents of the other three cuvettes.
Take a clean separatory funnel and make sure the stopcock or drain at the end is closed. Remove the stopper at the top.
Add 2 mL of the ethyl acetate, followed by 15 mL of water and 15 mL of octanol. If necessary, you can support your separatory funnel with a ring on a ring stand while you do so.
Remove the glass funnel and replace the stopper. Hold the separatory funnel in both hands with one hand positioned over the stopper to make sure it does not come out. Shake the funnel back and forth a couple times. Then tilt it backwards and open the stopcock to relieve pressure. Repeat this procedure until you have shaken the contents for a total of 30 seconds or so. Make sure you always shake it once or twice and then relieve pressure. If you allow pressure to build up inside, it could cause the stopper to come out and release all the contents onto your lab bench.
Place the separatory funnel on a ring attached to a ring stand. Allow the contents to separate into two layers. The upper layer is octanol; the lower layer is water. Collect the water phase and measure its absorbency in the UV region of the spectrum.
Place a small (~25 mL) flask beneath the separatory funnel and drain a few mL of the water layer into the flask. Remove 1 mL of the contents of the flask with your micropipette and transfer it to a clean cuvette.
Insert the cuvette into the spectrophotometer and measure the absorbency at 215 nm.
Take your absorbance data and enter it into a spreadsheet program like Open Office or Excel. For the four ethyl acetate samples you prepared at the beginning, enter the molarity in one column and the absorbency in a neighboring column. The molarity for the four samples is as follows: 100 microliter sample: 0.5091 molar; 50 microliter sample: 0.2545 molar; 25 microliter sample: 0.1273 molar; and 5 microliter sample: 0.02545 molar.
Use your spreadsheet program to prepare a scatter-plot graph of these four data points with molarities or concentrations on the x-axis and absorbency on the y. If you are not sure how to do this, refer to the user's guide or help menu for your spreadsheet program. Using the spreadsheet program, fit a linear equation or line of best fit of the form y = mx + b to this data. The x-variable in this equation is the concentration, while the y-variable is the absorbency.
Take the absorbency of your 2 mL ethyl acetate sample from the separatory funnel and divide it by the m-value from the equation your spreadsheet program calculated and subtract the b-value. If the m-value is 1.2, for example, and the b-value is 0.2 while the absorbency is 0.42, subtract 0.2 from 0.42 and divide the result by 0.5 to obtain a concentration of 0.183 moles per liter.
Multiply your concentration value by 0.015 liters since you had 10 mL of water in your separatory funnel. If your concentration was 0.183, for example, you would multiply by 0.015 to obtain 2.745 x 10^-3 moles of ethyl acetate present.
Subtract the result from Step 14 from 0.02036 moles of ethyl acetate, which was the amount you originally added. If you had 2.745 x 10^-3 moles in the water layer, for example, subtract this number from 0.02036 moles to obtain 0.01762 moles present in the octanol layer.
Divide the amount present in the octanol layer by the amount present in the water layer, and this gives you the partition coefficient of your liquid. This example uses ethyl acetate, but you can use other liquids as long as you adjust the concentrations and values for molarity appropriately.