Automated pre-column derivatization of amino acids with o-phthalaldehyde by a reagent sandwiching technique

Abstract

A totally automated method for the pre-column derivatization and analysis of the o-phthalaldehyde amino acids has been described. The derivatives are formed by combining the sample and reagent in a mixer in-line with the column and taking the mixture directly to the column after a precisely controlled reaction time. Reagent addition is by the use of a multi-port automated valve. Over-all precision of the method at the 50-100 pmol level is about 1%. The proposed method has several advantages over previously reported automated methods for this analysis: (a) The reaction time is precisely controlled and can be varied reproducibly if needed by altering the initial mobile phase flow-rate. (b) The use of the auto-sampler only for taking the sample should give better reproducibility than when used for taking sample and reagent. The full-loop mode of sampling can be used to improve sampling precision. (c) The auto-sampler trays are used for samples only, not for mixing vials and or reagent vials. As many as 48 samples per day can potentially be analyzed. (d) There is no BASIC program needed to control the sampling, mixing, and injection of the samples. (e) Only the actual volume of sample analyzed is injected, undiluted with reagent and/or quenching solution. (f) The method is easier and less expensive to implement than systems employing robotics, although it is more expensive than procedures which use the auto-sampler for taking the sample and reagent due to the cost of the automated valve for reagent addition.(ABSTRACT TRUNCATED AT 250 WORDS)