Development of gradient programmer and its application to automated amino acid analyzer based on reversed phase ion pair chromatography

Although the gradient elution technique is very useful for HPLC separation of multi-component sample, a major bottleneck of its use especially in routine analysis might be the poor reproducibility. To overcome this problem, a new one-chamber continuous solvent programmer for HPLC was constructed using electromagnetic valves, a mixing chamber made of glass, a silicon rabber stopper, a magnetic stirrer, a vacuum pump, a programable controller etc. This system controlled by the programable controller permitted to prepare not only simple exponential curves but also more complex shaped gradient curves. The programmer also realized reproducible separations by the use of different types of reciprocating pumps with constant flow rate. The device was applied to an automated amino acid analyzer based on reversed phase ion pair chromatography. The separation was carried out with a stainless steel column (250×4mm i.d.) packed with NS-Gel C8 (5μm). Sodium lauryl sulfate was used as a counter ion. For the monitoring of amino acids eluted from the column, a fluorescence detection system using o- phathalaldehyde and 2-mercaptoethanol was used.