Methods and systems for injecting a sample during electrophoresis, that generally comprise: loading a sieving matrix through a first end of a separation channel; having the an end of the sieving matrix at a set distance from the intersection of the separation channel and a loading channel; pressure loading a sample through the loading channel and filling an empty portion of the separation channel; applying an electric field across the separation channel while flowing a washing buffer through the loading channel; and injecting a portion of the sample into the separation channel, wherein the portion of the sample injected is of a size that is determined by a distance between the end of the sieving matrix and the intersection of the loading and separation channels.