Effect of temperature and pH on 31P nuclear magnetic resonances of phospholipids in cholate micelles

Accurate and precise determination of phospholipid composition by ³¹P NMR spectroscopy requires correct assignments and adequate spectral resolution. Because temperature and pH may affect chemical shifts (δ), our first aim was to establish the temperature coefficient (Δδ/ΔT) of common phospholipid classes when using sodium cholate as detergent. This parameter can then be used to aid in resonance assignments. The second goal was to investigate the pH dependence of δ so that, in addition to temperature, pH control can be used to minimize spectral overlap. For phosphatidylcholine, sphingomyelin, dihydrosphingomyelin and phosphatidylglycerol, δ values were invariant with pH and temperature. Whereas the Δδ/ΔT for phosphatidylinositol was 4 × 10^?3 ppm/°C, regardless of pH, these coefficients were highly pH-dependent for phosphatidic acid, phosphatidylethanolamine and phosphatidylserine, exhibiting maximal variations with the deprotonation of the headgroup, particularly for phosphatidic acid. These trends indicate the importance of H-bonding on δ and Δδ/ΔT for phospholipid resonances.