We demonstrate a new procedure for separation of single-stranded DNA (ssDNA) fragments that are anchored to the surface of a gold electrode by end hybridization. The new separation procedure takes advantage of the strong yet evolving nonuniform electric field near the gold surface in contact with a buffer solution gradually being diluted with deionized water. Separation of short ssDNA fragments is demonstrated by monitoring the DNA at the gold surface with in situ fluorescence measurement. The experimental results can be rationalized with a simple theoretical model of electric double layer that relates the strength of the surface pulling force to the ionic concentration of the changing buffer solution.