Gradient Separation of PTH-Amino Acids Employing Supercritical CO2 and Modifiers

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Abstract
Twenty-four PTH-amino acids are rapidly and efficiently separated on a packed cyanopropyl Zorbax column by gradient elution of supercritical CO2 and tetramethyl-ammonium hydroxide-modified methanol. Complete or partial resolution of 22 derivatives is observed with only valine coeluting with norleucine and lysine coeluting with asparagine. A wide variety of stationary phases and modifiers are investigated with supercritical CO2 in attempting to achieve the separation in less than 15 min. Critical to achieving a rapid and efficient separation is the control of modifier flow in to the CO2. No modifier is required for elution of neutral PTH-amino acids. The addition of base plays a major role in the elution of acidic and basic PTH-amino acids. Peak tailing is minimized and the elution order of several peaks is altered upon incorporation of reagent into the mobile phase.

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