Experiment with Intermolecular Forces

1. Begin with 3.0 g egg white in a small glass beaker (~50 mL beaker works well).
2. Use a p1000 micropipette to take 0.5 mL of 6 M HCl, being careful of drips or other hazards that can come from pipetting strong acid.
   • If you don’t have a micropipette, you can use a glass Pasteur pipette with a rubber bulb.
3. Add the acid DROPWISE into the egg white, swirling slightly to observe the chemical changes happening over time.
   • The slow addition of the acid is important for visualizing change.
4. Continue to observe the beaker over the next half hour, and eventually you can poke gently with a pipette tip to study the final texture of your egg white.

1. Measure 1 C of flour and mix it with 1⁄2 C of water. Mix the flour and water together until a ball of dough begins to form, adding more water if needed. The ball should not feel sticky—if it does, add more flour.
   
2. Knead the ball of dough until it becomes more elastic and shiny on the outside, up to 5 minutes.
3. Let the ball sit for at least 10 minutes, which will encourage a higher gluten content.
4. Begin to wash away the water soluble carbohydrate component of the dough by putting the dough ball under a gentle stream of water at the tap, or by gently kneading it in a bucket of fresh water. If you are using a bucket of water, change the water as it becomes milky to continue to efficiently remove the carbohydrates.
5. Once the water no longer looks milky, you have removed all of the carbohydrates.

Study the properties of your ball of protein.  What conclusions can you draw about the effects of their intermolecular interactions?

6. If you’d like, you can bake your ball at this stage to see how the heat affects the gluten in the absence of all the other bread-making ingredients.

What conclusions can you draw about how might heat affect gluten protein structure?

1. To each tube of foodstuffs, add 1 mL of chloroform:methanol (2:1, v/v) solution.
2. Secure the cap on the tube and vigorously shake the samples for 30 seconds.
3. Place the tubes back into a rack, and remove the caps.
   • At this point, you have created a monophasic system whereby components are dissolving in the solution.
4. Add 267 μL of the 0.73% NaCl solution to each tube to make a chloroform: methanol: water mixture (2:1:0.8, v/v/v).
5. Secure the caps back onto the tubes and again shake vigorously for 30 seconds.
   • The addition of the aqueous salt solution transforms the mixture into a biphasic system, and helps to separate the hydrophobic lipid components from water-soluble (hydrophilic) components.
   • Because chloroform is more dense than methanol and water, the chloroform-lipid phase will move toward the bottom of the tube, and on top of this will sit the less dense methanol-salt phase (thereby creating a biphasic system).
6. Spin conical tubes in a benchtop centrifuge at 2,500 rpm for 2 minutes.
7. Carefully remove the tubes from the centrifuge, being sure not to disturb the phase separation.
   • If you do not have a benchtop centrifuge, you can allow the tubes to sit, undisturbed, for 30-60 minutes, which will allow the phases to completely separate.
8. Transfer the top, methanol-water phase, to a waste container labeled for methanol disposal.
   • If you have interest and ability, this phase can be used to assess non-lipid components of the food materials.
9. Label a fresh glass tube with the appropriate food label.
10. Using a glass pipette, transfer the bottom, chloroform-lipid phase to this freshly labeled glass tube.
    • There will still be a small amount of biomass in the tube, above the chloroform-lipid phase — try to get to the chloroform-lipid phase without disturbing the biomass too much. you can just quickly “push” your glass pipette tip through the biomass to get to the bottom.

What observations can you make about the lipid samples?

10. The lipid is now ready to be analyzed using TLC.

1. Using 2 small pieces of tape, attach the TLC plate to the hotplate such that the bottom of the plate is touching the hotplate.
2. Mark the origin (bottom) of your plate with a pencil and label each lane.
3. Slowly spot the standard in the first lane in increments of 5 μL, allowing the spot to dry.
4. Repeat 3 times for a total of 15 μL standard.
5. Next, slowly spot the lipid extract in the designated lanes in increments of 5 μL, allowing the spots to dry.
6. Repeat 5 times for a total of 25 μL sample.
   • If you do not have micropipettes, you can use a glass Pasteur pipette to add your lipid extract to the plate in a dropwise fashion, being sure that you allow the “spot” to dry in between each drop.
7. Place the plate into the charged TLC chamber, in front of the wick.
8. Secure the lid to the chamber and allow the plate to develop.
9. Once the solvent front nears the top of the plate, remove the plate from the TLC chamber, quickly mark the solvent front with a pencil, and allow it to dry.
   • It typically takes 30-60 minutes for the solvent front to reach near the top of the plate.
10. Place the plate into the iodine chamber for visualization and secure the lid.
11. Once adequately stained (about 3-5 minutes), remove and lightly outline the spots and/or take a picture.

What conclusions can you draw about the polarity of the substances?

Discuss one purpose of this technique in research or industry.