Separation of Mixtures via Precipitation

1. Precipitation of CaCO₃

1. Prepare 5 mL of 1 M CaCl₂.
2. Prepare 5 mL 1 M Na₂CO₃.
3. In a small centrifuge tube (1.5 mL), add 750 µL of CaCl₂ and 750 µL of Na₂CO₃.
4. Wait 2 min for the reaction to occur. The solution should turn cloudy.
5. Centrifuge the mixture at 10,000 × g for 5 min.
6. Decant the supernatant.
7. Add 1 mL of cold water to the pellet.
8. Resuspend the pellet by mixing in a vortex mixer for 10 s.
9. Centrifuge the mixture at 10,000 × g for 5 min.
10. Decant the supernatant.

2. Precipitation of Milk Proteins

1. Pour the milk in a beaker and add a stir bar.
2. Warm milk gently until 40 °C in a stirring hot plate. Do not heat over 40 °C.
3. Prepare a 15% (v/v) of acetic acid by mixing 7.5 mL of acetic acid and diluting in enough water to reach 50 mL.
4. Immerse the electrode of a pH meter in warm milk, and monitor the pH.
5. Add the acetic acid drop-wise to the milk until a pH of 4.6 is reached.
6. Filtration of Milk
   1. Flute a piece of filter paper and place it in a funnel.
   2. Place the funnel in an flask, and pour the acidified milk solution into the funnel.
   3. As the solution is poured, the filter paper may get clogged. Using a stirring rod, agitate the solution and filter paper occasionally to unclog. If it does not improve the passage of solution, change the filter paper.
   4. Place a new filter paper on the bench top and transfer as much of the wet solid to the new filter paper. This should absorb more water from the solid.
   5. If the new filter paper gets too wet, continue to change it until there is minimal amount of wetness on the filter paper. Press it lightly to absorb more water if needed.
   6. Take the dried solid and re-suspend in about 70% ethanol. Filter the solid again following steps 2.6.1 to 2.6.5.
7. Centrifugation of Milk (as an alternative to filtration)
   1. Transfer 50 mL portions of the mixture to 50-mL centrifuge tubes.
   2. Centrifuge at 4,500 × g for 10 min, then decant the supernatant.
   3. Add 50 mL 70% ethanol to the pellet.
   4. Using a stirring rod, resuspend the pellet in the ethanol.
   5. Centrifuge this suspension following step 2.7.2.
8. Resuspend the pellet in buffer for further analysis such as SDS-PAGE, otherwise store it at 4°C.

3. Recrystallization of KCl

1. Weigh 50 g of KCl in an Erlenmeyer flask, and add 100 mL of water
2. Heat the mixture until water boils. Make sure all KCl powder is dissolved. Some impurities may not dissolve in water.
3. Heat another (empty) Erlenmeyer flask along with the mixture and keep it very warm.
4. Place a funnel with filter paper in the warm, empty flask.
5. Pour the solution through the filter paper to remove undissolved impurities. The receiving flask is kept warm to make sure no temperature changes occur during the filtration, otherwise a crude precipitate will form. If that happens, re-heat the mixture until all the precipitate dissolves.
6. Remove the flask with the solution from heat.
7. Keep it in a cool place in the room and let it cool down slowly for about 30 min, or until it is no longer warm to touch.
8. Once cooled down to room temperature, place the flask in an ice bath to further lower the temperature. Alternatively, one can leave the flask inside the fridge or a temperature-controlled room at 4 °C.
9. Crystals can be harvested by filtering as in steps 3.4–3.5 (use a flask and funnel at room temperature).