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Capillary isoelectric focusing with UV-induced fluorescence detection
1Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Brno.
Journal of Chromatography. A
|May 31, 2001
Summary
New fluorescent compounds serve as effective isoelectric point (pI) markers for isoelectric focusing (IEF) analysis. These markers enable precise pI determination and protein separation using UV-based detection methods.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Separation Science
Background:
- Isoelectric focusing (IEF) is a key technique for protein separation based on isoelectric point (pI).
- Accurate pI markers are crucial for calibrating and validating IEF separations.
- Existing markers may have limitations in detection or applicability with certain methods.
Purpose of the Study:
- To develop and characterize novel low-molecular-mass fluorescent compounds as pI markers for IEF.
- To evaluate these markers for UV-based detection, including fluorometric detection.
- To demonstrate their utility in capillary IEF separations of proteins.
Main Methods:
- Synthesis and characterization of new fluorescent pI markers.
- Determination of pI values for 18 markers using non-IEF methods.
- Implementation of capillary IEF with UV photometric and fluorometric detection.
- Separation of derivatized proteins using the developed pI markers.
Main Results:
- 18 new UV-excitable fluorescent pI markers were synthesized and characterized.
- The pI values of these markers span a wide range from 2.1 to 10.3.
- Successful application of these markers in capillary IEF for protein separation with both photometric and fluorometric detection was demonstrated.
Conclusions:
- Novel fluorescent pI markers offer a valuable tool for isoelectric focusing.
- These markers are suitable for UV-based detection, enhancing analytical capabilities.
- The developed system facilitates accurate pI determination and protein analysis in complex samples.