Isoelectric trapping (IET) has become an accepted preparative-scale electrophoretic
separation technique. However, there are still a number of shortcomings that limit its
utility. The performance of the current preparative-scale IET systems is limited by the
serial arrangement of the separation compartments, the difficulties in the selection of the
appropriate buffering membranes, the effect of Joule heating that may alter separation
selectivity and a lack of methods for the determination of the true, operational pH value
inside the buffering membranes. In order to bolster the current membrane pH
determination methods which rely on the separation of complex ampholytic mixtures, a
fluorescent carrier ampholyte mixture was synthesized. The use of a fluorescent mixture
allows for a reduced load of carrier ampholytes, thereby reducing a possible source of
error in the pH determinations. A mixture of carrier ampholytes tagged with an
alkoxypyrenetrisulfonate fluorophore was shown to have suitable fluorescence and
ampholytic properties and used to accurately determine the pH of high pH buffering
membranes under actual IET conditions. In a more elegant solution to the difficulties
associated with pH determinations, a method utilizing commercial UV-transparent carrier ampholytes as the ampholyte mixture to be separated was developed. By using
commercial carrier ampholytes and eliminating the need to synthesize, purify, and blend
fluorescently tagged ampholytes, the new method greatly simplified the determination of
the operational pH value of the buffering membranes. In order to address the remaining
limitations, a new system has been developed that relies on (i) parallel arrangement of
the electrodes and the collection compartments, (ii) a directionally-controlled convection
system for the delivery of analytes, (iii) short anode-to-cathode distances, (iv) short
intermembrane distances, and (v) an external cooling system. This system has been
tested in four operational modes and used for the separation of small molecule
ampholytic mixtures, for the separation of protein isoforms, and direct purification of a
target pI marker from a crude reaction mixture.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2009-05-424 |
| Date | 2009 May 1900 |
| Creators | North, Robert Yates |
| Contributors | Vigh, Gyula |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | thesis, text |
| Format | application/pdf |
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