Through optimization of the printing processand orientation, a suitably developed surface area has beenrealized upon a 3D printed polymer substrate to facilitatechromatographic separations in a planar configuration. Usingan Objet Eden 260VS 3D printer, polymer thin layerchromatography platforms were directly fabricated withoutany additional surface functionalization and successfullyapplied to the separation of various dye and protein mixtures.The print material was characterized using gas chromatographycoupled to mass spectrometry and spectroscopic techniquessuch as infrared and Raman. Preliminary studies included theseparation of colored dyes, whereby the separation performancecould be visualized optically. Subsequent separations wereachieved using fluorescent dyes and fluorescently taggedproteins. The separation of proteins was affected by differences in the isoelectric point (pI) and the ion exchange propertiesof the printed substrate. The simple chromatographic separations are the first achieved using an unmodified 3D printedstationary phase.