Column Chromatography of Dyes: Microanalysis with a Thin Layer Chromatography Scanner Works to Beat the Bands

Quantitative analysis students have performed the following experiment in our laboratories for 15 yr with generally disappointing results: column chromatographic separation of a dye mixture on alumina followed by spectrophotometric analysis of the eluates and quantification of the dyes using Beer's law. Previous workers claimed that these dyes, Victoria Blue R, methylene blue, and fluorescein, are separable under the chromatography conditions, but we have always had loss of dye on the column or poor separation or both. We have formulated a replacement dye mixture (Oil Red O, Victoria Blue R, fluorescein) that separates cleanly with inexpensive solvents. For preliminary studies we used TLC plates with novel sample application discs and a TLC scanner to measure λ maxvalues for microgram quantities of the dyes. The dyes mentioned can be recovered nearly quantitatively from alumina columns. The mud-colored dye mixture becomes aesthetically pleasing as the dyes move down the column and separate into brightly colored bands, making this experiment useful for classroom demonstrations of the column chromatographic technique.