Consecutive enzymatic reactions of analytes which are affinity bound to immobilized metal ion beads with subsequent direct analysis of the products by matrix-assisted laser desorption/ionization mass spectrometry have been used for detecting phosphorylation sites. The usefulness of this method was demonstrated by analyzing two commercially available phosphoproteins, beta-casein and alpha-casein, as well as one phosphopeptide from a kinase reaction mixture. Agarose loaded with either Fe3+ or Ga3+ was used to isolate phosphopeptides from the protein digest. Results from using either metal ion were complementary. Less overall suppression effect was achieved when Ga3+-loaded agarose was used to isolate phosphopeptides. The selectivity for monophosphorylated peptides, however, was better with Fe3+-loaded agarose. This technique is easy to use and has the ability to analyze extremely complicated phosphopeptide mixtures. Moreover, it eliminates the need for prior high-performance liquid chromatography separation or radiolabeling, thus greatly simplifying the sample preparation.
Micellar electrokinetic capillary chromatography (MECC) is suitable for the separation of mixtures of uncharged and charged solutes. In this paper, the migration behavior of acidic compounds in MECC is quantitatively described in terms of different models. These equations describe the relationships between the two migration parameters in MECC (retention factor and mobility) and the two important experimental parameters (pH and micelle concentration) that have a great influence on the migration behavior and selectivity. Interestingly, the mobility and retention factor of a given solute could behave differently with the variations in pH. This would raise a question of which parameter actually represents the migration behavior of a solute in MECC: retention factor (a chromatographic parameter) or mobility (an electrophoretic parameter). The consequences of micellar-mediated shifts of ionization constants on selectivity and optimization strategies in MECC are discussed. The mathematical models would allow the prediction of migration behavior of solutes based on a limited number of initial experiments. This would greatly facilitate the method development and optimization of separations of ionizable compounds by MECC and, in addition, important physical and chemical characteristics of solutes such as their apparent ionization constants in micellar media and their partition coefficients into micelles (over a wide range pH values) can be determined. The models were verified, as good agreements were observed between the predicted and the experimentally observed migration behavior. Based on the preliminary results, the pH and micelle concentration are likely to be interactive parameters in many situations. As a result, simultaneous optimization of these two parameters would be the most effective strategy to enhance the MECC separation of acidic solutes.
This paper reports successful chiral separations of pharmaceutical racemic amines by nonaqueous capillary electrophoresis (NACE) using beta- and gamma-cyclodextrins (CDs) and various derivatives of beta-CDs. The results in three organic solvents, formamide (FA), N-methylformamide (NMF), and N,N-dimethylformamide (DMF) were compared to those in pure water and in 6 M urea in water systems. The binding constants of trimipramine, mianserin, and thioridazine with beta-CD were determined in the following five solvent systems: water, 6 M urea in water, FA, NMF, and DMF. The binding constants decreased systematically from approximately 10(4) in water to approximately 10 in FA and approximately 10(-2) in DMF. As a result, the optimum CD concentration in the aqueous media is in the high micromolar range, while that in the FA is around 100 mM. In the aqueous media, the occurrence of the optimum at very low concentrations and the rapid changes in enantioselectivity with CD concentration would make it difficult to develop methods based on trial and error. Nevertheless, it is shown that, even under nonoptimum concentrations of the chiral selector, other experimental parameters such as ionic strength, addition of tetraalkylammonium (TAA+), and temperature can be adjusted to achieve acceptable resolutions. This, however, is often achieved at the expense of longer analysis times. In addition, the effects of apparent pH (pH*) and type of cyclodextrin on chiral separations in NACE are studied. The application of negatively charged beta-CD in FA is also reported. Chiral separation of trimipramine was achieved at lower concentration of anionic CD due to the additional Coulombic interactions.
The influence of surfactant type on migration behavior and chemical selectivity in micellar electrokinetic chromatography (MEKC) is investigated through linear solvation energy relationships (LSER) and functional group selectivities. In LSER modeling, solutes' capacity factors are correlated with their structural descriptors such as size, dipolarity, and hydrogen-bonding abilities. Using the LSER methodology, useful information about the nature of solute interactions with different types of surfactant aggregates can be obtained since capacity factor in MEKC is directly related to solute distribution between the bulk aqueous solvent and micelles. High correlations were observed for different LSER models of migration behavior in MEKC for a group of 60 uncharged aromatic compounds of non-hydrogen bonding (NHB), hydrogen-bonding acceptor (HBA) bases, and hydrogen-bonding donor (HBD) acids. In two anionic, hydrocarbon micellar systems of sodium dodecyl sulfate (SDS) and sodium cholate (SC), retention is primarily influenced by the size of molecules and their hydrogen bond accepting basicity. Their dipolarity/polarizability and hydrogen bond donating acidity play minor roles. Capacity factors of solutes in SDS and SC systems increase with their size and decrease for stronger hydrogen bond acceptor bases. These results are similar to those observed for other systems where hydrophobic interactions play a major role, e.g., solute distribution in the 1-octanol-water solvent system or retention in reversed phase LC. In MEKC with an anionic fluorocarbon surfactant, lithium perfluorooctanesulfonate (LiPFOS), however, size and solute HBD acidity are the two predominant factors. The LSER results indicate that compounds find the SDS micellar environments slightly less cohesive (i.e., more apolar) than the SC micelles, while the LiPFOS micelles are the most cohesive among the three surfactant aggregates and 1-octanol provides the least cohesive environment. The fluorocarbon micelles of LiPFOS, on the other hand, are the strongest hydrogen bond donor acids, followed by SDS, SC, and 1-octanol, respectively. The SC micelles have the most hydrogen bond acceptor basic characteristics, followed by 1-octanol, SDS, and LiPFOS micelles. It can be concluded that selectivity differences between these surfactant types in MEKC is primarily due to hydrogen-bonding interactions rather than the dipolar interactions. Comparing the perfluorinated and the hydrocarbon surfactants, even solute size can play a role in selective migration patterns. In addition, information from polar and hydrophobic group selectivities confirm the LSER conclusions about the underlying interactions that control migration behavior and chemical selectivity in MEKC.(ABSTRACT TRUNCATED AT 400 WORDS)
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