1. Experiment: Extraction of Vitamins A & C Objective: The objective of this experiment is separate vitamins A and C through an extraction and to confirm the separation using NMR. Chemicals Retinol (Vitamin A) Ascorbic acid (Vitamin C) D 2 O (deuterated water) CDCl 3 (deuterated chloroform) Apparatus Thin-tipped spatulas (2) Microcentrifuge tubes (2) Pasteur pipets & bulb CH 113 (M.Hall) Ch11 1 NMR experiment involving vitamins to study intermolecular forces & solubility

2. CH 113 (M.Hall) Ch11 Data & Observations Part I. Extraction of the Vitamins 1.Label a microcentrifuge tube clearly on the top and side of the tube as “Bottom Layer.” Also write your initials and the date. 2.Use a thin-tipped spatula to transfer a small amount of ascorbic acid to the microcentrifuge tube. The solid is very staticky, so it clings to the spatula and tube. Simply dip the spatula into the bottle, and tap whatever sticks to the spatula into the tube. 1.To the same microcentrifuge tube, add a small amount of retinol using a clean spatula. Retinol is very sticky and thick (like earwax), so just scoop some out of the container and scrape it along the inside of the microcentrifuge tube. 2.Use a Pasteur pipet to transfer 0.5-0.7 mL of CDCl 3 to the same tube. You can use the graduations on the tube to guide the volume added. Close the tube firmly and shake it to dissolve the solids as much as possible. 3.Use a fresh Pasteur pipet to add 0.5-0.7 mL D 2 O to the same tube. Mix the contents well, then let the tube sit until the layers separate. 4.Using a Pasteur pipet, decant off the top layer and transfer it to a new microcentrifuge tube labeled with “Top Layer,” your initials, and the date. Experiment: Extraction of Vitamins A & C 2

3. Data & Observations, cont’d Part II. NMR Spectra of the Separated Layers 1.Using a Pasteur pipet, transfer the top layer to a labeled NMR tube. 2.Using a fresh Pasteur pipet, transfer the bottom layer to another labeled NMR tube. 3.Obtain a 1 H NMR spectrum of each sample (using the 300 MHz NMR spectrometer and ICON- NMR). 1.Compare your spectra to the reference spectra on the following pages. Which solvent extracted the ascorbic acid (Vitamin C)? Which solvent extracted the retinol (Vitamin A)? Experiment: Extraction of Vitamins A & C CH 113 (M.Hall) Ch11 3

4. Results Summarize all your results including the identity of the top and bottom layers and the vitamin that was extracted in each layer. Conclusions Describe the results of the experiment and account for the presence of two layers in the extraction as well as the separation of the vitamins into the two solvents. Mention specific intermolecular forces to explain why each vitamin dissolved better in one solvent than in the other. Relate this information to the lifetime of vitamins in the body. Experiment: Extraction of Vitamins A & C CH 113 (M.Hall) Ch11 4

5. Experiment: Extraction of Vitamins A & C Reference 1 H NMR Spectrum Pure Ascorbic Acid (Vit.C) in D 2 O CH 113 (M.Hall) Ch11 full spectrum 5

6. zoom in CH 113 (M.Hall) Ch11 6

7. Experiment: Extraction of Vitamins A & C Reference 1 H NMR Spectrum Pure Retinol (Vit.A) in CDCl 3 CH 113 (M.Hall) Ch11 full spectrum 7

8. zoom ins CH 113 (M.Hall) Ch11 8