How Do I Protonate An Acidic Lipid?

Theory

Protonate the phospholipid using acid. Wash the excess acid and salt from the product.

Equipment

• Separatory funnel (teflon stopcock) or suitable glass vessel
• Rotary evaporator or nitrogen/argon stream
• pH indicating paper

Reagents

• Chloroform
• Methanol
• HCl
• Deionized water

Procedure

1. Dissolve the lipid in a volume of chloroform: methanol (2:1 v/v) such that the lipid con centration is 10-50 mg/ml. (e.g. 9.0 ml)
2. Add deionized water to the solution. (0.2 times the volume of the chloroform: methanol: e.g. 1.8 ml)
3. Shake the solution and let the phases separate.
4. Adjust the water phase to < pH 3.0 using acid. (pKa of the phosphate is approximately 3.5.)
5. Shake the acidified solution and allow the phases to separate.
6. Isolate the bottom phase. (This phase contains the acidified phospholipid.)
7. Remove the chloroform by rotary evaporation or nitrogen/argon stream under a hood.
8. Dissolve the residue in a suitable organic solvent.