Phospholipid–protein coatings for chiral capillary electrochromatography

A phospholipid–bovine serum albumin (BSA) coating was developed for chiral capillary electrochromatographic separation of d- and l-tryptophan. Temperature, liposome composition, and liposome–BSA mixing and extrusion were found to have critical effects on the chiral separation of d- and l-tryptophan in terms of resolution, separation efficiency, and migration times. A solution of 0.5 mM phosphatidylcholine (PC)–1 mg/ml BSA performed better than a solution of 0.5 mM PC/phosphatidylserine (PS) (80:20, mol%)–1 mg/ml BSA as capillary coating; baseline separation of the enantiomers with satisfactory resolution was then achieved. Temperature played a crucial role in the chiral separation, as demonstrated for phospholipid-coated capillaries immobilized with BSA and lysozyme. The d- and l-tryptophans showed a marked difference in separation efficiency on the PC–BSA-coated capillary; the theoretical plate number of l-tryptophan was above 500,000 m−1, whereas that of d-tryptophan was only about 22,000 m−1. Immobilized BSA (pI 4.7) showed better chiral separation selectivity for the enantiomers than did immobilized lysozyme (pI 10.5), α-chymotrypsin (pI 8.1–8.3), or avidin (pI 10.0–10.5); also resolution was better and analysis time was faster. Hydrophobic interactions played an important role in the BSA-immobilized phospholipid-coated capillaries. The importance of protein net charge and molar mass for its immobilization in phospholipid-coated capillaries is discussed.