Single-isomer sulfated cyclodextrins (SISCDs) have proven to be reliable, effective, robust means for separation of enantiomers by capillary electrophoresis (CE). SISCD derivatives used as chiral resolving agents in CE can carry the sulfo groups either at the C2, C3 or C6 positions of the glucopyranose subunits which provides varied intermolecular interactions to bring about favorable enantioselectivities. The first single-isomer, sulfated β-CD that carries the sulfo group at the C2 position, the sodium salt of heptakis(2-O-sulfo-3-O-methyl-6-Oacetyl) cyclomaltoheptaose (HAMS) has been synthesized. The purity of each synthetic intermediate and of the final product was determined by HILIC and reversed phase HPLC. The structural identity of each intermediate and the final product was verified by 1D, and 2D NMR, and MALDI-TOF mass spectrometry. HAMS has been used as chiral resolving agent for the CE separation of a set of nonionic, weak base and strong acid enantiomers in pH 2.5 background electrolytes. Rapid separations with satisfactory peak resolution values were obtained for the enantiomers of most of the nonionic and weak base analytes. Typically, low concentrations of HAMS were required to effect good enantiomer resolution. The trends in the effective mobilities and separation selectivities as a function of HAMS concentrations followed the predictions of the ionic strength-corrected charged resolving agent migration model (CHARM model). HAMS showed poor complexation with the anionic strong electrolyte enantiomers for which no peak resolution was observed. The separation patterns observed with HAMS as chiral resolving agent were compared with those of other β-cyclodextrin analogues, including heptakis(2-O-methyl- 3-O-acetyl-6-O-sulfo)-b-cyclodextrin (HMAS), heptakis(2-O-methyl-3,6-di-O-sulfo)-b- cyclodextrin (HMdiSu), heptakis(2,3-di-O-acetyl-6-O-sulfo)-b-cyclodextrin (HDAS) and heptakis(2,3-di-O-methyl-6-O-sulfo)-b-cyclodextrin (HDMS).