Separation of phenylthiohydantoin amino acids by capillary electrochromatography.
Capillary electrochromatography (CEC) was employed as a rapid and high-efficiency method for the isocratic separation of all 20 important phenylthiohydantoin (PTH) amino acids, the end products of Edman degradation during N-terminal protein sequencing. For this purpose, 75 microm ID fused-silica capillaries were packed with standard 3 microm Hypersil octadecyl silica (ODS) particles using a two-step column fabrication process, which represents a fast, reliable and efficient means of producing long-term stable columns. The influence of solvent composition, pH, type of buffer cation, buffer concentration, and temperature on retention behavior of PTH amino acids was investigated. Same-day and day-to-day reproducibility of the retention times (over a period of two months) were found to be better than 3%. When comparing this new technique with traditional reversed phase-high performance liquid chromatography (RP-HPLC) methods applied in automated protein sequenators, CEC shows essentially shorter separation times and superior resolution.
Institute of Medical Chemistry and Biochemistry, University of Innsbruck, Innsbruck, Austria.
Thomas
T
Mueller
Ulrike C
UC
Posch
Herbert H
HH
Lindner
D000097:Acetonitriles / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry / Q000302:isolation & purification*; D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D005663:Furans / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D010669:Phenylthiohydantoin / Q000737:chemistry / Q000302:isolation & purification*; D011506:Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D012822:Silicon Dioxide; D013696:Temperature; D013995:Time
D000097:Acetonitriles; D000596:Amino Acids; D002021:Buffers; D005663:Furans; D011506:Proteins; D010669:Phenylthiohydantoin; C018674:tetrahydrofuran; D012822:Silicon Dioxide; C032159:acetonitrile