Kinetic Detection of Overlapped Amino Acids in Thin-Layer Chromatography with a Direct Trilinear Decomposition Method

Kinetic fluorescence detection (KFD) was employed to determine the concentrations of two overlapped components after a thin-layer chromatographic (TLC) separation. Two amino acids, glycine and glutamine, were used as model analytes. These species exhibited very similar retardation factors (R f ) under our experimental conditions. A reaction that produced a fluorescent product was performed subsequent to the separation. The reaction can be described by the following scheme : amino acid + CPA → fluorescent product → nonfluorescent product. Here OPA stands for o-phthalaldehyde. The kinetic profile of the reaction was monitored with a charge-coupled device camera by taking sequential images of the separation medium after the reaction starts. A direct trilinear decomposition (TLD) method was used to analyze the resulting third-order data that consist of fluorescence intensities as a function of elution distance, reaction time, and sample number. This approach was used to determine the initial concentrations of the two overlapped components based on the different kinetics and retention exhibited by these species. This paper discusses the kinetic approach and the applicability and limitations of the direct trilinear decomposition (TLD) method using both synthetic and experimental data. Efforts to optimize the experimental conditions are also reported. The major focus of this work is to explore the application of this novel kinetic fluorescence detection method for TLC separations (TLC-KFD) combined with the TLD data analysis method.