Semester

Spring

Date of Graduation

2010

Document Type

Dissertation

Degree Type

PhD

College

Eberly College of Arts and Sciences

Department

Chemistry

Committee Chair

Lisa A Holland

Abstract

Phospholipids were used as an additive in capillary electrophoresis to enhance the separation of glycans derived from alpha1-acid glycoprotein, fetuin, and ribonuclease B. The properties of phospholipid preparations were dependent upon composition, hydration, and temperature. Separation performance was evaluated as a function of these variables. A preparation of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-snglycero-3-phosphocholine (DHPC), with [DMPC]/[DHPC] = 2.5, in 10% lipid/aqueous buffer at 25 °C provided the best separation efficiency at an electric field strength of 400 V/cm. Resolution was enhanced with the additive as well.;Phospholipids were also investigated as an additive for capillary electrophoresis separation of DNA molecules. Lipid preparations with [DMPC]/[DHPC] = 2.5 at hydrations of 6%, 8%, 10%, 12% and 15% were used to separate a 50 base pair double stranded DNA ladder. The experiments demonstrated that separation of DNA in phospholipids occurred via a sieving process. Electric field as low as 100 V/cm was suitable for the separation of DNA fragments larger than 450 base pairs. For double stranded DNA smaller than 450 base pairs the electric field strength ranging from 100 to 300 V/cm is suitable. The best resolution of small DNA fragments was obtained at high phospholipid concentration, while dilute phospholipid solution was favorable for the separation of larger fragments. The pore size of phospholipid was estimated by plotting the absolute mobility into a standard equation obtained from a Ferguson plot. The calculated value was approximately 29 nm, which is close to the pore size (27 nm) of the polymer solution employed for the separation of a similar DNA ladder.

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