Tudies applied continuous-wave (CW) EPR spectroscopy to observe motional (tumbling) effects around the TEMPOL line shape, from T variation-induced dynamical transitions within the surrounding solvent. This led to a calibrated Tg for pure water of 136 K,15 in agreement with the reported value obtained by extrapolation of calorimetric data.13 Calibrated Tg’ values for the mesodomain within the heterogeneous frozen glycerol-water method at distinct glycerol concentrations were also determined.14 These research recommend that application of high-resolution EPR spectroscopic tactics will reveal detailed microscopic information regarding sugar-water mesodomain structure. Here, we apply many CW- and pulsed-EPR approaches to samples containing a constant, trace (0.two mM, 0.0034 ) concentration of TEMPOL spin probe, to characterization of microscopic parameters of the sucrose-water mesodomain, such as solute density and volume, in each heterogeneous liquid and solid states, more than a wide array of added sucrose concentrations [0 ?75 (w/v)].Buy2-(3,4,5-Trimethoxyphenyl)acetonitrile The following approaches had been employed to probe the solute concentration, volume, and microscopic structure in the mesodomain: (a) Continuous-waveNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptLangmuir. Author manuscript; available in PMC 2014 April 02.Chen et al.PageEPR spectroscopy of TEMPOL mobility (tumbling) was employed to figure out the dynamical transition (related for the powerful glass transition temperature, Tg’) inside the mesodomain over 200-270 K. (b) Electron spin echo envelope modulation (ESEEM) spectroscopy at six K was employed to detect the hyperfine interaction of TEMPOL with 2H-labeled sucrose, as a probe in the relative volume in the mesodomain. (c) Electron spin echo (ESE) etected longitudinal, or spin-lattice relaxation times (T1) at 6 K had been utilised to ascertain the concentration of TEMPOL within the mesodomain, and thus, the volume fraction from the mesophase. (d) The ESE phase memory time (TM; correlated with all the transverse relaxation time, T2) at six K was also used as an indicator of the relative concentration of TEMPOL within the mesodomain.α-(Bromomethyl)-2-pyrazinemethanol structure Integrated interpretation on the outcomes results in a novel microscopic model of a heterogeneous mesodomain structure, in which maximally freeze-concentrated sucrose1 types an ordered sucrose hydrate phase, that excludes solutes, as well as a disordered, amorphous sucrose-water glass phase.PMID:23903683 The volume fractions with the ordered and disordered phases are exquisitely sensitive to the initial, added sucrose concentration.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptEXPERIMENTALSample preparation Sucrose (99.5 , Sigma-Aldrich, St. Louis, MO) and deuterated sucrose ([6,6-2H2fru]sucrose (Omicron Biochemicals, Inc., South Bend, IN) had been made use of without having further purification. Water was purified to a specific impedance of 13.six M-cm (Nanopure, Siemens). Samples with distinct concentrations of sucrose [1-75 (w/v)] were prepared by mixing organic abundance or deuterated sucrose and water. The final concentration of TEMPOL (4-Hydroxy-TEMPO, Sigma-Aldrich, St. Louis, MO) spin probe in all samples was 0.two mM [0.0034 (w/v)]. All samples have been cautiously degassed by freeze-thaw cycles prior to loading into standard quartz 4 mm EPR tubes (Wilmad-LabGlass, Buena, NJ). Samples had been initial frozen within a 252 K freezer and maintained at this temperature for 12 hours. Samples had been subsequently transferred to liquid nitrogen (LN2 ) for storage. CW-EPR spectroscopy CW-EPR exp.