Separation of 3'-sialyllactose (SL) and lactose is an essential final step for the production of the next generation of infant formulas containing sialyllated prebiotics. Due to the difference in molecular weight (MW) between SL and lactose and the charge density of SL, nanofiltration could provide a rapid, inexpensive alternative for the separation of SL and lactose compared to traditional chromatography. The performance of four commercial nanofiltration membranes (NF45, DSS-ETNAO1PP, NTR-7540 and NP010) for the separation of SL and lactose was assessed at various pH. The difference in retention between SL and lactose was only significant in the NP010 and NTR-7450 membranes, whereas the NF45 and DSS ETNA01PP membranes exhibited either too high lactose retention (i.e. insufficient separation) or too low SL retention (i.e. losing the target SL compound), respectively. Operation at increased pH did not affect SL retention significantly. The expected increase in retention levels of SL at high pH - due to repulsion between the negative charge of the membrane and the charged SL - was apparently offset by pore swelling of the NF membranes at high pH. The water permeability was measured before and after a membrane was used for filtration of a mixture of lactose and SL. For the NP010 and DSS-ETNA membranes, the decline in water permeability was lower when the experiments were conducted at high pH, which is ascribed to the electrostatic repulsion of SL by the membrane. Further improvements in the ratio of retention of SL and lactose were achieved by changing the operational pressure. The best suited membrane was used in a final 10-rounds diafiltration, which enabled total separation of SL and lactose. The study also reveals that while charge differences between solutes can be utilized during nanofiltration, the trade-off between electrostatic repulsion and pore swelling must be addressed when optimizing the nanofiltration process. (C) 2014 Elsevier B.V. All rights reserved.