Application of the TLC image analysis technique for the simultaneous quantitative determination of L-proline and L-lysine in dietary supplement

Abstract

A simple and sensitive thin-layer chromatography (TLC) method coupled with an image analysis technique was developed for the simultaneous quantitative determination of L-proline and L-lysine in dietary supplement with good precision and accuracy. Separation was performed on silica gel plates using ethanol‒toluene (2:3, V/V) as the mobile phase. The visualization of chromatograms was based on iodine–azide reaction; therefore, pre-chromatographic derivatization reaction of amino acids with phenyl isothiocyanate was performed. Digital images of TLC plate chromatograms were converted into peak chromatograms, and quantitative analysis was conducted using TLSee software.

Image Analysis as a Tool for Understanding Quantitative Analysis in Chromatography

“…The image analysis (IA) technique requires only flatbed scanner ,,, or digital camera ,, and appropriate software. The scanner (digital camera) allows us to obtain an image of the TLC plate.…”

“…The scanner (digital camera) allows us to obtain an image of the TLC plate. The conversion of spots to peaks and its analysis can be performed using commercially available (TLSee, AlfaTech, Italy; JustTLC, Sweday, Sweden) or freeware license (ImageJ, NIH, USA) software. ,− Recently, some new protocols have been presented. In these protocols, a smartphone with installed app (free of charge) has been used to get the TLC plate’s image and its analysis. ,− Eliminating the need for expensive equipment means that the cost of analysis drops drastically, allowing TLC experiments to be implemented in student exercises.…”

“…1 However, during the past few years quantification based on digital image analysis of a TLC chromatogram has often been used as an alternative to densitometry. 2−11 The image analysis (IA) technique requires only flatbed scanner 3,5,9,10 or digital camera 4,6,8 and appropriate software. The scanner (digital camera) allows us to obtain an image of the TLC plate.…”

“…However, during the past few years quantification based on digital image analysis of a TLC chromatogram has often been used as an alternative to densitometry. − …”