Redetermination of 2-[4-(2-hydroxyethyl)piperazin-1-ium-1-yl]ethanesulfonate at 100 K

The crystal structure of the title compound (common name HEPES), C8H18N2O4S, has been redetermined at 100 K in order to properly elucidate the protonation state of the HEPES molecule. The piperazine ring has a chair conformation and one of the N atoms in the ring is protonated, which was not previously reported [Gao, Yin, Yang, & Xue (2004). Acta Cryst. E60, o1328–o1329]. The change of protonation state of the nitrogen atom significantly affects the intermolecular interactions in the HEPES crystal. The structure is stabilized by N—H⋯O and O—H⋯O hydrogen bonds and ionic interactions, as the title compound in solid state is a zwitterion. HEPES molecules pack in layers that are held together by ionic and weak interactions, while a hydrogen-bonded network connects the layers.

The crystal structure of the title compound (common name HEPES), C 8 H 18 N 2 O 4 S, has been redetermined at 100 K in order to properly elucidate the protonation state of the HEPES molecule. The piperazine ring has a chair conformation and one of the N atoms in the ring is protonated, which was not previously reported [Gao, Yin, Yang, & Xue (2004). Acta Cryst. E60, o1328-o1329]. The change of protonation state of the nitrogen atom significantly affects the intermolecular interactions in the HEPES crystal. The structure is stabilized by N-HÁ Á ÁO and O-HÁ Á ÁO hydrogen bonds and ionic interactions, as the title compound in solid state is a zwitterion. HEPES molecules pack in layers that are held together by ionic and weak interactions, while a hydrogenbonded network connects the layers.

Comment
The title compound, (I), known commonly as HEPES, is a sulfonic compound used as a zwitterionic buffer. HEPES and analogous compounds (known as the Good buffers) are used during the study of biological processes (Good et al., 1966;Good & Izawa, 1972;Ferguson et al., 1980), and very often during crystallization of macromolecules.
The crystal structures of HEPES crystallized from methanol (determined at 293 K -Wouters et al., 1996) and from water In order to localize all hydrogen atoms, the structure determination was performed at 100 K. The crystal structure of the title compound reported here is isomorphous to the structure reported by Gao et al., but a more detailed analysis revealed that the HEPES molecules are zwitterionic and the nitrogen atom (N1) is protonated (Fig. 1). Change of the localization of the hydrogen atom in comparison with the previously reported structure (Gao et al., 2004) significantly affects the hydrogen bond network (Fig. 2, Table 1), which we believed was previously incorrectly determined. To confirm our finding, we also performed a structural analysis of HEPES crystals (crystallized from water or taken directly from the bottle provided by SIGMA) at 293 K. In both cases (100 K and 293 K) the structures had the same protonation state, which excluded the possibility of temperature dependent changes of protonation.  supplementary materials sup-2 Experimental HEPES was purchased from SIGMA (99.5% purity, lot 036 K5461). The crystal of (I), used for X-ray diffraction study, was obtained by slow evaporation of HEPES solution in water.

Refinement
All hydrogen atoms were localized using the difference density Fourier map. Their positions and isotropic displacement parameters were refined.
Figures Fig. 1. The molecular structures of the title compound. Displacement ellipsoids are drawn at the 75% probability level and hydrogen atoms are drawn as spheres of an arbitrary radius.

2-[4-(2-Hydroxyethyl)piperazin-1-ium-1-yl]ethanesulfonate
Crystal data Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating Rfactors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.