| New insights into the capillary electrophoresis (CE) separation of a number of highly hydrophobic porphyrins with biological significance, i.e., Hematoporphyrin D, hematoporphyrin derivative, and porphyrin methyl esters (having 2-8 methyl ester groups), were gained by studying the effects of organic modifiers (e.g., aqueous immiscible/miscible organic solvents, medium chain-length alcohols, or charged/neutral surfactants) present in the sample matrix and/or the running buffer on the separation performance. Also, a novel approach for the biological sample clean-up and concentration in CE, based on the coupling of acetonitrile deproteinization and salting-out extraction with acetonitrile stacking, was demonstrated for the separation and determination of a number of clinically important hydrophobic porphyrins present in urine with high enrichment factors and good precision.; In Chapter 2, an investigation of the basic factors which govern the microemulsion electrokinetic chromatography (MEEKC) and micellar electrokinetic chromatography (MEKC) separation of Hematoporphyrin D and its base hydrolysis product, hematoporphyrin derivative (HpD), was performed. These model compounds contain a complex mixture of porphyrin monomers, dimers and/or oligomers, and were utilized to gain insights into the MEEKC/MEKC separation of samples containing highly lipophilic substances. An interesting and important finding was that the presence of an organic modifier (methanol or acetonitrile at a concentration of 20% or higher) in the sample matrix as well as in the run buffer was essential for the optimal MEEKC or MEKC separation of a number of porphyrin monomers (including hematoporphyrin IX and its acetates, most likely hydroxyacetate, diacetate and vinyl acetate, as well as its dehydration products, hydroxyethylvinyldeuteroporphyrin and protoporphyrin) contained in Hematoporphyrin D.; In Chapter 3, the effects of organic solvents on the CE separation of a number of important biological porphyrin methyl esters---six weakly basic, hydrophobic cyclic tetrapyrroles possessing 2 and 4 to 8 methyl esters groups around the periphery of the porphyrin ring---were investigated in the mode of MEKC, MEEKC and non-aqueous CE. Best separation performances were achieved with non-aqueous CE separations in which the weakly basic porphyrin methyl esters were protonated under strongly acidic conditions (e.g., using 10 mM perchloric acid) in mixed hydro-organic solvents. For example, using a 50:50 mixture of methanol and acetonitrile as the separation medium, baseline separation of all six (positively charged) porphyrin methyl esters can be obtained within 3 min and the average precision (R.S.D., N=13) in terms of migration time and peak area were 0.55% and 2.16%, respectively.; In Chapter 4, a new sample pretreatment method in CE was developed in which acetonitrile was firstly added into the biological samples to allow for protein precipitation and removal (acetonitrile deproteinization), followed by the addition of an appropriate salting-out agent into the deproteinated sample solutions to effect phase separation via salting-out extraction. This approach allowed for concurrent sample clean-up and analyte concentration prior to CE separations. Further enhancement in concentration detection sensitivity was achieved by coupling (off-line) salting-out extraction with an (on-line) CE sample enrichment technique known as "acetonitrile stacking". By optimizing the experiment conditions, a number of model compounds (hydrophobic porphyrins with clinical significances), i.e., zinc-protoporphyrin, protoporphyrin, and coproporphyrin (CP) III and I, can be efficiently extracted from the aqueous sample and a combined enrichment factor of ca.1000 can be obtained. |
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