Study on Capillaries Covalently Bound with Phospholipid Vesicles for Open-Tubular Capillary Electrochromatography

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Julkaisun nimi: Study on Capillaries Covalently Bound with Phospholipid Vesicles for Open-Tubular Capillary Electrochromatography
Tekijä: Tiala, Heidi
Muu tekijä: Helsingin yliopisto, Matemaattis-luonnontieteellinen tiedekunta, Kemian laitos
Opinnäytteen taso: Lisensiaatintyö
Tiivistelmä: The starting point for the work was to develop a method for covalent binding of phospholipids on fused silica capillaries for capillary electrochromatography (CEC). The method consists of three steps. First, the silanol groups in the fused silica capillary were aminopropylsilylated with 3-(aminopropyl)-triethoxy silane (APTES). Secondly, the product of aminopropylsilylation was allowed to react with glutaraldehyde giving an imidoaldehyde, which further reacted with the primary amino group of phospholipids. The stability of the coating was verified by measuring the electroosmotic flow (EOF) mobility after aminopropylsilylation with APTES and liposome coating. The highest stability of the EOF was obtained using coatings made of liposomes comprising 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS). The EOF remained stable even after injection of a large number of analytes. The tested phospholipids were selected to mimic biological membranes. In the coating procedure the following phospholipids were used: POPC, 1,2-didodecanoyl-sn-glycero-3-phosphate (DLPA), 1,2-didodecanoyl-sn-glycero-3-phosphoethanolamine (DLPE), and POPS. Primary amino groups were needed for stable covalent binding of liposomes to the fused silica capillary wall. The phospholipids POPC and POPS contained primary amino groups. The highest stability of the covalent binding was achieved using liposome dispersions of 3 mM POPC/POPS 80/20 mol-% but the retention factors for aldosterone, testosterone, and progesterone were higher using a 2 mM POPC/POPS 80/20 mol-% dispersion. The thickness of the liposome layers was measured with quartz crystal microbalance (QCM). The mass of the adsorbed liposomes was higher for the 2 mM POPC/POPS 80/20 mol-% dispersion than for the 3 mM POPC/POPS 80/20 mol-% dispersion, which explained the higher retention factors for the analytes studied. The developed coating method was used to the separation of model steroids by on-line open-tubular CEC mass spectrometry (OT-CEC-MS). The steroids, which are poorly visible or non-visible by UV-detection, could be separated and detected by on-line OT-CEC-MS. Six different type of steroids were separated and detected by this method. There was no ion suppression in the MS due to lipid leakage. The effect of reducing the formed Schiff’s base with sodium borohydride and the impact of liposome composition on the stability of the coating were investigated. NaBH4 treatment did not improve the stability of the EOF, but rather decreased it. The effect of NaBH4 reduction on 40/40/20 mol % POPC/POPS/cholesterol-coated capillaries was also investigated, and the results were similar to those obtained with the 80/20 mol % POPC/POPS-coated capillaries: the stability of the coating was better without NaBH4 treatment. The effect of cholesterol in the phospholipid coating was studied and the results show that cholesterol can well be included in phospholipid coatings covalently attached to silica. 2 mM 40/40/20 mol% POPC/POPS/cholesterol showed greater stability than 2 mM 80/20 mol% POPC/POPS. However, much higher retention factors of aldosterone, testosterone, and progesterone were obtained with 2 mM 80/20 mol % POPC/POPS liposome coatings than with 2 mM 40/40/20 mol% POPC/POPS/cholesterol coatings. This may be due to the fact that cholesterol is likely to increase the rigidity of lipid membranes. Also the stability and robustness of the capillary column was tested by individual injections of a large amount of analytes into the capillary: almost 400 injections were made into the 40/40/20 mol% POPC/POPS/cholesterol coated capillary (different type of drugs). The EOF was stable for over 200 injections.
Päiväys: 2017-01-24
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