Monolithic polymer layer with gradient of hydrophobicity for separation of peptides using two-dimensional thin layer chromatography and MALDI-TOF-MS detection.

Superhydrophobic monolithic porous polymer layers supported onto glass plates with a gradient of hydrophobicity have been prepared and used for 2-D thin layer chromatography of peptides. The 50 μm-thin poly(glycidyl methacrylate-co-ethylene dimethacrylate) layers prepared using UV-initiated polymerization in a simple mold were first hydrolyzed using dilute sulfuric acid and then hydrophilized via two-step grafting of poly(ethylene glycol) methacrylate to obtain superhydrophilic plates. The hydrophobicity was then formed by photografting of lauryl methacrylate. The exposure to UV light that initiates photografting was spatially controlled using moving shutter that enabled forming of the diagonal gradient of hydrophobicity. This new concept enables the solutes to encounter the gradient for each of the two sequential developments. Practical application of our novel plates was demonstrated with a rapid 2-D separation of a mixture of model peptides gly-tyr, val-tyr-val, leucine enkephalin, and oxytocin in dual reversed-phase mode using different mobile phases in each direction. Detection of fluorescent-labeled peptides was achieved through UV light visualization while separation of native leucine enkephalin and oxytocin was monitored directly using MALDI mass spectrometry.