Abstract: Accordance to various embodiments, there are methods of separating molecules, devices, and method of making the devices. The method of separating molecules can include providing a nanofluidic device including a plurality of nanochannels on a top surface of a substrate, wherein each of the plurality of nanochannels has a first end and a second end and extends from the top surface into the substrate. The nanofluidic device can also include a dielectric layer disposed over each of the plurality of nanochannels, an inlet at the first end of the plurality of nanochannnels, an outlet at the second end of the plurality of nanochannels, and an optically transparent cover disposed over the plurality of nanochannels to form a seal. The method of separating molecules can further include providing a solution in the plurality of nanochannels through the inlet and creating a longitudinal pH gradient along each of the plurality of nanochannels.