Poly (

N

-isopropylacrylamide) (PNIPAAm) grafted onto silica

which may be used for reverse phase chromatography(RPC)

was simulated and synthesized for protein separation with temperature-triggered adsorption and desorption.Molecular dynamics simulation at an all-atom level was performed to illustrate the adsorption/desorption behavior of cytochrome c

the model protein

on PNIPAAm-grafted-silica

a temperature responsive adsorbent.At a temperature above the lower critical solution temperature(LCST)

the PNIPAAm chains aggregate on the silica surface

forming a hydrophobic surface that is favorable for the hydrophobic adsorption of cytochrome c

which has a high exposure of hydrophobic patches.At temperatures below the LCST

the PNIPAAm chains stretch

forming hydrophilic surface due to hydrogen bonding between PNIPAAm and surrounding water.Desorption of cytochrome c on the PNIPAAm-grafted-silica surface occurs as a result of competition with water

which forms hydrogen bonds with the protein.The conformational transitions of both cytochrome c and PNIPAAm are monitored

providing molecular insight into this temperature-responsive RPC tec

hnique.PNIPAAm-grafted-silica beads were synthesized and used for the adsorption and desorption of cytochrome c at approximately 313 K and 290 K

respectively.The experimental results validate the molecular dynamics simulation.In comparison to conventional RPC

using temperature as a driving force for RPC reduces the risk of protein denaturation caused by exposure to chaotropic solvents.Moreover

it simplifies the separation process by avoiding the buffer exchange operations between the steps.