Enhancing the selectivity of polar hydrophilic analytes with a low concentration of barium ions in the mobile phase using geopolymers and silica supports.

Charged analytes such as organic sulfonic acids, sulfates, carboxylates, and phosphates are often analyzed by hydrophilic interaction liquid chromatography (HILIC). In many cases, these analytes do not show any selectivity and elute near the dead time using the conventional acetonitrile-ammonium acetate buffers. In this work, we introduce a powerful selectivity enhancing technique by using a trace amount of Ba2+ ion in the mobile phase as a general approach for HILIC with UV-Vis detection. Silica and a newly developed material called geopolymers are used as stationary phases. Geopolymers are X-ray amorphous aluminosilicate inorganic polymers with cation exchange properties. Barium exchanged geopolymers (Ba-NM-GP) are synthesized from metakaolin based geopolymer. Thorough characterization of Ba-NM-GP is reported using scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), Brunauer-Emmett-Teller (BET) surface area analyzer and laser diffraction particle size distribution analyzer for the determination of their shape, size, porosity, surface area and particle size distribution respectively. It is demonstrated that in the absence of Ba2+, baseline separations of sulfonates, carboxylates, and phosphates is not possible, whereas, in the presence of Ba2+ in the mobile phase, these analytes are easily separated. Barium perchlorate is suggested as an additive for it is UV transparent, and it has practically an unlimited solubility in acetonitrile.

Synthetic aluminosilicate based geopolymers - Second generation geopolymer HPLC stationary phases.

A survey of existing stationary phases classified by the United States Pharmacopeia reveals that 120 groups of chromatographic supports mostly utilize silica-silane chemistry, polymeric materials along with some niche metal oxides. In this work, the synthesis and characterization of transition-metal free geopolymers as a new class of stationary phases for hydrophilic interaction liquid chromatography and normal phase separations is reported. The geopolymers were synthesized by reaction of synthetic aluminosilicate with potassium silicate (fumed silica dissolved in KOH) in a water-in-oil emulsion. For comparative purposes of peak shapes, a geopolymer from natural metakaolin was also synthesized. The geopolymers were examined by X-ray diffraction, energy dispersive spectroscopy, laser diffraction, and N2-adsorption isotherms. This two-step approach gives spherical microparticles with surface area and pore size comparable to silica phases (150 m2/g and 120 Šrespectively). Both synthetic aluminosilicates based and natural metakaolin based geopolymers occupy a unique "spot" in the HILIC selectivity chart when compared to 35 HILIC phases. An additional promising feature of geopolymers is high pH and temperature stability which are used to tune selectivity for small polar analytes. High pH separations are shown with carboxylic acids. Geopolymers also show mixed mode behavior in retention with ion-exchange properties in purely aqueous mobile phases. The separation of derivatized sugars is demonstrated and compared with porous graphitic carbon (Hypercarb™) as another pH-stable stationary phase.

Supercharging and multiple reaction monitoring of high-molecular-weight intact proteins using triple quadrupole mass spectrometry.

RATIONALE: Different supercharging agents were tested to study their effect on the intensity and charge state distributions of high-molecular-weight intact proteins. The goal of this work was to increase chargeability and ionization efficiency for proteins ranging from 66 to 150 kDa, to enable subsequent optimization of multiple reaction monitoring (MRM) mode transitions with a triple quadrupole mass spectrometer for potential top-down quantitative analysis. METHODS: Supercharging agents, such as meta-nitrobenzyl alcohol (m-NBA), dimethylsulfoxide, trifluoroethanol (TFE), and sulfolane were tested in different concentrations in 50/50 acetonitrile/water with 0.5% formic acid to examine the electrospray ionization response for three model proteins: bovine serum albumin (66 kDa), holo-transferrin (78 kDa), and immunoglobulin G (150 kDa). The settings of ionization source temperature and mobile phase flow rate were also examined. MRM transitions were developed for a wide range of precursor ions for each protein, and limits of detection were determined for the proteins in the presence of favorable additive combinations. RESULTS: For most of the proteins, m-NBA (1%) and TFE (5%) worked most effectively, both to shift the charge state and increase intensity. This is the first report of the use of TFE as an effective agent for both increasing protein chargeability and ionization response. TFE increased ionization efficiency between 3- and 14-fold for the model proteins studied. Increases in both source temperature and flow rate reduced the magnitude of the average charge state observed. The MRM transitions of six to eight different precursor ions of the proteins were optimized and limits of detection in the nanogram quantity on column were determined. CONCLUSIONS: The feasibility for top-down quantitative analysis of high-molecular-weight proteins with a triple quadrupole mass spectrometer was demonstrated. Further, additives such as TFE can be highly beneficial for increased chargeability and response of the proteins.