Realities of automating OPA HPLC amino acid analyses.
The levels of free amino acids in culture medium can easily be monitored by reversed-phase HPLC chromatography on a C18 column following pre-column derivatization with o-pthaladehyde. The method does not require the prior removal of proteins. A guard column does that. When this method was automated, problems started to occur after about eight injections. Peak resolution rapidly degraded. Unidentified matter was retained by the column and produced both interfering peaks and high back pressures. Better resolution of the closely eluting amino acids occurred when both the pH of the loading buffer was increased and the gradient time was lengthened. Special washes and a carefully controlled time of equilibration with the loading buffer helped to minimize back pressure and interfering substances. Other often skipped routine maintenance and solvent handling procedures were also necessary for optimal performance. These modifications made routine runs of 40 plus samples possible as well as dramatically increased the lifetime of the column.
Krok
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Hygeia Sciences, Newton, MA 02160.
Seaver
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D000596:Amino Acids / Q000302:isolation & purification; D001331:Automation; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D013997:Time Factors; D009764:o-Phthalaldehyde
D000596:Amino Acids; D009764:o-Phthalaldehyde