The separation of intact microbes by capillary electrophoresis

“Microbes play a vital role in the lives of every human being. Different species of microbes are helpful in the production of food and medicine and many other areas. Microbes are also important in health care and are either directly or indirectly responsible for many types of diseases which range from mildly irritating to deadly. Despite the importance of microbes, little attention has been devoted to the development of modern analytical techniques for the separation and quantitation of intact microbes. This dissertation describes the development of a fast, efficient analytical technique for the separation and quantitation of intact mixtures of microbes. This method utilizes capillary electrophoresis (CE) with a dissolved polymer in the running buffer. In addition to being able to separate mixtures of intact microbes, this CE technique can be used in the diagnosis of urinary tract infections (UTIs) and as an assay for commercial products which contain bacteria”--Abstract, page iii

Separating Microbes in the Manner of Molecules. 1. Capillary Electrokinetic Approaches

Selective, high-efficiency separations of intact bacteria may, in some cases, allow them to be identified and quantified in much the same way that molecules are done today. Two different capillary electrokinetic approaches were utilized. The first approach used a dissolved polymer-based CE separation that may be affected by size and shape considerations. Another approach uses capillary isoelectric focusing to separate bacteria by their surface charge or isoelectric point. Good peak shapes and extremely high efficiencies are observed (up to ~ 1 600 000 theoretical plates/m). Careful sample preparation and separation runs are essential in order to obtain reproducible separations. Expansion of these types of rapid, efficient microbial separations could have profound effects on many branches of science and technology

High Efficiency Separation of Microbial Aggregates Using Capillary Electrophoresis

Recent advances in the technique of capillary electrophoresis have demonstrated fast, highly efficient separation of mixtures of intact microbes. This paper describes the application of this technique for the separation of microbial aggregates of Micrococcus luteus, Saccharomyces cerevisiae, or Alcaligenes faecalis. The aggregates of these microbes were resolved into several highly efficient peaks with analysis times under 10 min and efficiencies approaching 1 000 000 plates m-1 in some cases. A reproducible relationship was found between the electrophoretic mobility and the aggregation number or size of the cluster under a given set of experimental conditions. Often, cellular aggregation was reversible with brief immersion in an ultrasound bath. This reversibility was confirmed by visual microscopy and electrophoretic data

High binding capacity surface grafted monolithic columns for cation exchange chromatography of proteins and peptides

Poly(glycidyl methacrylate-co-ethylene methacrylate) monoliths have been prepared in 100 μm i.d. capillaries and their epoxy groups hydrolyzed to obtain poly(2,3-dihydroxypropyl methacrylate-co-ethylene methacrylate) matrix. These polymers were then photografted in a single step with 2-acrylamido-2-methyl-1-propanesulfonic acid and acrylic acid to afford stationary phases for a strong and a weak cation exchange chromatography, respectively. Alternatively, poly(ethylene glycol) methacrylate was used for grafting in the first step in order to enhance hydrophilicity of the support followed by photografting with 2-acrylamido-2-methyl-1-propanesulfonic acid or acrylic acid in the second step. These new columns were used for the separation of proteins and peptides. A mixture of ovalbumin, α-chymotrypsinogen, cytochrome c, ribonuclease A and lysozyme was used to assess the chromatographic performance for large molecules while a cytochrome c digest served as a model mixture of peptides. All tested columns featured excellent mass transfer as demonstrated with very steep breakthrough curves. The highest binding capacities were found for columns prepared using the two step functionalization. Columns with sulfonic acid functionalities adsorbed up to 21.5 mg/mL lysozyme while the capacity of the weak cation exchange column functionalized with acrylic acid was 29.2 mg/mL. © 2009 Elsevier B.V. All rights reserved