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Method optimization in capillary zone electrophoretic analysis of hGH tryptic digest fragments
1Eli Lilly and Company, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285.
Journal of Chromatography
|September 7, 1990
Summary
Optimizing capillary zone electrophoresis mobile phase composition enhanced the separation of human growth hormone tryptic fragments. Tricine buffer concentration proved most critical for resolution, with morpholine aiding separation of closely eluting species.
Area of Science:
- Analytical Chemistry
- Biochemistry
Background:
- Capillary zone electrophoresis (CZE) is a powerful separation technique.
- Human growth hormone (hGH) analysis requires robust separation methods for its tryptic fragments.
Purpose of the Study:
- To optimize mobile phase composition for separating tryptic digest fragments of human growth hormone using CZE.
- To investigate the impact of pH, buffer, ionic strength, and mobile phase additives on separation performance.
Main Methods:
- Systematic variation of pH (2.4, 6.1, 8.1, 10.4) to determine optimal conditions.
- Optimization of tricine buffer, sodium chloride, and morpholine concentrations at selected pH.
- Evaluation of effects on electroosmotic flow, current, and chromatographic resolution.
Main Results:
- pH 8.1 was identified as optimal for selectivity.
- Tricine concentration significantly impacted resolution, with 0.1 M being crucial.
- Morpholine (0.02 M) improved the separation of closely eluting peaks.
- Sodium chloride was found to be unnecessary for optimal separation.
Conclusions:
- The optimized CZE method provides effective separation of hGH tryptic fragments.
- Mobile phase composition, particularly tricine concentration and pH, is key to achieving high resolution.
- This optimized method can be applied to the analysis of protein digests.