Quantitative TLC-Image Analysis of Urinary Creatinine Using Iodine Staining and RGB Values

Area of Science:

• Analytical Chemistry
• Chromatography
• Spectroscopy

Background:

• Traditional digital image analysis for thin-layer chromatography (TLC) often requires specialized software and equipment.
• Colorless analytes on TLC plates present challenges for direct visualization and quantification.

Purpose of the Study:

• To develop and demonstrate a low-cost, accessible method for quantitative TLC digital image analysis.
• To validate the use of a universal staining reagent and common office equipment for accurate analyte quantification.

Main Methods:

• Utilized iodine vapor as a universal staining reagent for TLC plates.
• Employed an office scanner and Microsoft Paint software for image acquisition and analysis of RGB values.
• Quantified urinary creatinine as a model analyte in complex biological matrices.

Main Results:

• The "Green" color channel provided the best linear working range (0.0810-0.9260 mg/mL) and precision (2.03% RSD).
• Achieved a detection limit of 0.24 µg per 3 µL spot for urinary creatinine.
• TLC-digital image analysis results for urinary creatinine showed good agreement with high-pressure liquid chromatography (HPLC) findings.

Conclusions:

• This low-cost TLC-digital image analysis method offers a practical and affordable alternative for quantitative analysis.
• The technique is suitable for analyzing complex biological samples, as demonstrated with urinary creatinine.
• The use of readily available tools democratizes advanced analytical techniques.