title: Quantitative TLC-Image Analysis of Urinary Creatinine Using Iodine Staining and RGB Values.
abstract: Digital image analysis of the separation results of colorless analytes on thin-layer chromatography (TLC) plates usually involves using specially tailored software to analyze the images generated from either a UV scanner or UV lamp station with a digital camera or a densitometer. Here, a low-cost alternative setup for quantitative TLC-digital image analysis is demonstrated using a universal staining reagent (iodine vapor), an office scanner and a commonly available software (Microsoft Paint) for analysis of red, green and blue colors (RGB values). Urinary creatinine is used as a model analyte to represent a sample in complicated biological matrices. Separation was carried out on a silica gel plate using a butanol-NH4OH-H2O (40 : 10 : 50, v/v) mobile phase with a 6-cm solvent front. It is important that the TLC plate be stained evenly and with sufficient staining time. Staining the TLC plate in a 23.4 × 18.8 × 6.8 cm chamber containing about 70 g iodine crystals yielded comparable results for the staining times of 30-60 min. The Green value offered the best results in the linear working range (0.0810-0.9260 mg/mL) and precision (2.03% RSD, n = 10). The detection limit was found to be 0.24 µg per 3 µL spot. Urinary creatinine concentrations determined by TLC-digital image analysis using the green value calibration graph agree well with results obtained from high-pressure liquid chromatography (HPLC).
authors.affiliation: Department of Chemistry, Xavier University, 3800 Victory Parkway, Cincinnati, OH 45207, USA.
authors.forename: Emily
authors.initials: E
authors.lastname: Kerr
authors.affiliation: Department of Chemistry, Xavier University, 3800 Victory Parkway, Cincinnati, OH 45207, USA.
authors.forename: Caroline
authors.initials: C
authors.lastname: West
authors.affiliation: Department of Chemistry, Xavier University, 3800 Victory Parkway, Cincinnati, OH 45207, USA kradtas@yahoo.com.
authors.forename: Supaporn
authors.initials: S
authors.lastname: Kradtap Hartwell
mesh_terms: D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer / Q000379:methods*; D003404:Creatinine / Q000652:urine*; D006801:Humans; D007455:Iodine / Q000737:chemistry*; D015203:Reproducibility of Results; D013194:Staining and Labeling; D014466:Ultraviolet Rays
chemical_list: D007455:Iodine; D003404:Creatinine