The Development of On-line Single / Staggered Multi-Step Elution SPE-CE-MS and Heart-cut 2D–CE-MS

• Main Authors: Wei-Han Lee, 李威漢
• Other Authors: Guor-Rong Her
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/36753382916516650744

博士 === 臺灣大學 === 化學研究所 === 98 === A PDMS based two-leveled two cross design interface was proposed for on-line coupling SPE-CE-MS. In this interface, the SPE column and the CE separation column were positioned orthogonally and two crosses were fabricated on the interface. With the two cross design, the operation of SPE could be performed independently without unexpected flow through leakage into the separation column. The performance of the interface was optimized using a peptide mixture. The position of the SPE column related to the CE separation channel was found to be critical to the performance of the system. Under the optimal position, the separation efficiency was similar to a CE-MS experiment without SPE. The peptide signals were enhanced 50 to 100-fold and the repeatability was within 4% RSD for migration time and 10% RSD for peak area. A tryptic digest of cytochrome C was used to demonstrate the feasibility of the interface in protein identification at a level of 1 ng/mL. In a protein mixture analysis, the identification of proteins usually suffers in low sequence coverage in the single run CZE-ESI-MS/MS. An original concept of on-line coupling multistep elution solid phase extraction (SPE) to CZE-MS/MS was proposed to increase sequence coverage of protein mixture analysis. The multistep elution SPE (the first dimension) provides an additional dimension of separation prior to CZE (the second dimension) and extends the separation capacity for protein mixture analysis. Furthermore, a staggered CZE method was described to increase the throughput of each CZE runs in the second dimension separation and thus to reduce entire analysis time. In this study for protein mixture standards, more than 60% of additional peptides were discovered , and more than 50% was improved in sequence coverage by using multistep elution SPE-CE-MS/MS. By using staggered CZE method, half of the entire analysis time could be saved (54%) in comparison with the sequential CZE method used in multistep elution SPE-CE-MS/MS and thus avoiding the time-consuming analytical procedure in comprehensive 2D separation. An interface for heart-cut 2D CE-MS was proposed to increase separation selectivity in mixture analysis. Several concepts were adapted to overcome the limitations of heart-cut 2D-CE designed in the present studies. First, the manipulation of chip-based interface provides an isolated buffer system to connect two sets of capillary electrophoresis. Second, the parallel separation of the two dimensional capillary electrophoresis was detected simultaneously by a pulsed electrospray-based duel-channel CE-MS system. In this study, the system was demonstrated by using capillary zone electrophoresis- micellar electrokinetic chromatography (CZE-MEKC) system to analyze sulfonamide mixtures. Under the consideration of correspondence in EOF for fused silica capillary the PDMS based chip channel, 8 sulfonamide standards can be transferred successfully without loss and peak broadening during the heart-cutting operation. The preliminary feasibility of heart-cut CZE-MEKC with dual-channel CE-MS was studied in sulfonamides analysis. Four sulfonamides(SDZ、SMR、STZ、SMM) were transferred into the MEKC channel by the heart-cut interface after separation in the first dimension of CZE. The migration order of four heart-cut sulfonamides was found similar order in the single-run MEKC.