Enhanced Quantification of Co-eluting Amino Acids Using TLC based Image Analysis and Multivariate Calibration

Articles in Press

Document Type : Original Article

Authors

1 Department of Chemistry, Faculty of Basic Sciences, Semnan University, Semnan, Iran

2 Department of Chemistry, Faculty of Sciences, University of Mohaghegh Ardabili,56199-11367, Ardabil, Iran & Department of Environmental Science, Faculty of Science, University of Zanjan, 45371-38791, Zanjan, Iran

3 Workplace Health Research Center, Neyshabour University of Medical Sciences, Neyshabour, Iran

Abstract

L-Glutamine (Gln) and Glycine (Gly) are two amino acids with overlapping retention times in thin-layer chromatography (TLC). This overlap presents a significant analytical challenge, particularly when both compounds are present in the same mixture, as their spots on the TLC plate may not be fully resolved. Here, we used smartphone-based image analysis combined with PLS regression for semi-separated Gln/Gly quantification in TLC. A smartphone captured TLC plates images under consistent lighting conditions, and custom software processed them to generate chromatographic profiles. Calibration curves showed linear responses, with detection limits of 0.007 M and 0.008 M for Gln and Gly, respectively. Despite mobile-phase optimization, complete spot resolution was unachievable. To address this, partial least squares (PLS) regression deconvoluted the merged signals, enabling accurate quantification. The method was validated using a pharmaceutical product, yielding recovery rates of 100.4 ± 1.4% for Gln and 100.6 ± 1.0% for Gly, demonstrating its reliability for complex samples.

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Applied Chemistry Today

Articles in Press, Accepted Manuscript
Available Online from 26 August 2025

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