Characterization Of Functionalized Polyethylene Glycol By High Performance Liquid Chromatography

Polyethylene glycol (PEG) and end-group functional PEGs have been widely used in biomedical and pharmaceutical field due to the good biocompatibility and hydrophilicity. However, the PEGylation and further targeting conjugation at the other end-group of PEG both require clear end-group information for PEG and functional PEG derivatives. In this work, several examples of functional PEG derivatives for biomedical application were given showing challenges and opportunities in separation and characterization of biomedical polymers by HPLC.In Chapter 1, the biomedical applications of functional PEG derivatives and the separation and characterization of polymers were introduced. The principle and research progress of separation and characterization of polymers by HPLC were introduced.In Chapter 2, the separation and characterization of PEGs and amino-substituted derivatives on common silica-based reversed-phase packing columns using isocratic elution is described. This separation is achieved by liquid chromatography under critical conditions (LCCC), based on the number of amino functional end-groups without obvious effect of molar mass for PEGs. The mobile phase is acetonitrile in water with an optimal ammonium acetate buffer. The separation mechanism of PEG and amino-substituted PEG under the near LCCC on silica-based packing columns is confirmed to be ion-exchange interaction. Under the LCCC of PEG backbone, with fine tune of buffer concentration, the retention factor ratios for benzylamine and phenol in buffered mobile phases, a(benzylamine/phenol)-values, were used to assess the ion-exchange capacity on silica-based reversed-phase packing columns. This is the first report on separation of amino-functional PEGs independent of the molar mass by isocratic elution using common C18 or phenyl reversed-phase packing columns.In Chapter 3, in order to further separate and characterize other functional PEGs by HPLC, linear PEGs and 3-arm PEG were modified with maleimide and benzaldehyde. The maleimide-substituted PEGs and benzaldehyde-substituted PEGs with different molar masses and functional degrees were synthesized. The functionalization of 3-arm PEG with maleimide and benzaldehyde resulted in tri-functionalized maleimide PEGs and tri-functionalized benzaldehyde PEGs, respectively. The chemical structures of thes obtained functional PEGs were confirmed preliminarily by 1H NMR, which could be used for the HPLC analysis described in the next two chapters.In Chapter 4, the separation of PEGs and maleimide-substituted PEGs prepared in Chapter 3 based on the number of maleimide end-groups under LCCC has been investigated on a reversed-phase column. The LCCC for non-functional PEGs and bi-functional maleimide-substituted PEGs were determined to be identical at about 40% acetonitrile in water on a reversed-phase octadecyl carbon chain-bonded silica column using mixtures of acetonitrile and water of varying composition as the mobile phase at 25?. The maleimide-functionalized PEGs were successfully separated according to maleimide functionality (with zero, one, two, or three maleimide end-groups, respectively) under the LCCC without obvious effect of molar mass. The separation was mainly due to the hydrophobic interaction between the maleimide end-groups and the column packing. Off-line (matrix-assisted laser desorption/ionization time of flight mass spectrometry) MALDI-TOF-MS was used to identify the repeating units and, especially, the end-groups of the maleimide-substituted PEG. LCCC could provide useful information to optimize the synthesis of functional PEGs. This is the first report of the baseline separation of maleimide-functionalized PEGs based on the functionality independent of the molar mass without derivatization by isocratic elution.The distributions of non-, mono-and bi-functional benzaldehyde-substituted PEGs need to be accurately determined for biomedical applications. In Chapter 5, the baseline separation of PEGs and their derivatives based on the number of functional end-group without obvious effect of molar mass is achieved by HPLC and ultra-performance liquid chromatography (UPLC) using reversed-phase packing columns under the LCCC. The effects of organic solvent percentage, column temperature and buffer concentration on the retention times of PEGs and benzaldehyde-substituted PEGs have been investigated. The separation was mainly due to the hydrophobic interaction between the benzaldehyde end-groups and the column packing. The LCCC analysis can provide useful information to optimize the synthesis of functional PEGs. This is the first report on separation of benzaldehyde-functional PEGs based on the functionality independent of the molar mass by isocratic elution.