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Correct! Octanol has a lower density (0.83 g/cm3) than water (1.00 g/cm3), so it will be the upper layer.
Correct! Octanol has a lower density (0.83 g/cm3) than water (1.00 g/cm3), so it will be the upper layer.
Preparation of atrazine extraction.
Separating the layers and measuring the absorbance of the aqueous layer
Separating the layers and measuring the absorbance of the aqueous layer
Materials needed: 10.00 mL volumetric pipet, 3 x 15 mL centrifuge tubes with caps, graduated pipet, octanol solution (pre-saturated with water), centrifuge, 2 plastic pipets, UV cuvet
Materials needed: 10.00 mL volumetric pipet, 3 x 15 mL centrifuge tubes with caps, graduated pipet, octanol solution (pre-saturated with water), centrifuge, 2 plastic pipets, UV cuvet
Use a 10.00 mL volumetric pipet to add 10.00 mL of atrazine stock solution to a 15 mL centrifuge tube.
Use a graduated pipet to add 0.20 mL of the octanol solution to the centrifuge tube.
Repeat steps 1 & 2 for a total of three centrifuge tubes filled with 10.00 mL of atrazine solution and 0.20 mL of octanol.
Cap the three centrifuge tubes and invert them gently end over end continuously for 5 minutes. Note: Do not shake the tubes, as this will cause an emulsion to form!
Place the tubes in a centrifuge so that they are placed opposite each other (add the centrifuge tubes of another group if needed, in order to balance the centrifuge). Centrifuge the tubes for 3 minutes.
Remove the tubes from the centrifuge (make sure not to agitate them too much!). Use a plastic pipette to remove the top layer from each of the three tubes.
Use a plastic transfer pipet to add approximately 2 mL of the aqueous layer from one centrifuge tube into a cuvet and cap the cuvet. Repeat this for each centrifuge tube for a total of three cuvets.
Use a capped cuvet filled with water to blank the UV/Vis spectrophotometer. Make sure to use the same spectrometer that you used in Part 1!
Measure and record the absorbance of each solution at the λmax. Dilute your solutions if needed in order to keep the absorbance readings within the range of your previously made callibration curve.
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Think and discuss!
Think and discuss!
1. Why are you using an octanol solution that is pre-saturated with water?
1. Why are you using an octanol solution that is pre-saturated with water?
2. Why are you removing the octanol layer first, instead of directly pipetting the aqueous layer out?
2. Why are you removing the octanol layer first, instead of directly pipetting the aqueous layer out?
2. What is preferrable: removing as much of the octanol layer as possible first without removing any of the water layer OR removing some of the water layer as well in order to ensure all of the octanol layer has been removed?
2. What is preferrable: removing as much of the octanol layer as possible first without removing any of the water layer OR removing some of the water layer as well in order to ensure all of the octanol layer has been removed?
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