Chemical characterisation of different separation media based on agarose by static time-of-flight secondary ion mass spectrometry

In this paper, the novel application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) for qualitative and semi-quantitative investigation of the surface chemistry of separation media based on beaded agarose is reported. Five different media were studied: DEAE Sepharose Fast Flow, Q Sepharose Fast Flow, SP Sepharose Fast Flow, Phenyl Sepharose Fast Flow at ligand densities between 7 and 33% (w/w) and the base matrix Sepharose 6 Fast Flow. The obtained TOF-SIMS spectra reveal significant chemical information regarding the ligands (DEAE, Q, SP and Phenyl) which are covalently attached to the agarose-based matrix Sepharose 6 Fast Flow. For the anion-exchange media (DEAE and Q Sepharose Fast Flow), the positive TOF-SIMS spectra yielded several strong characteristic fragment peaks from the amine ligands. Structural information was obtained, e.g. from the peak at m/z 173.20, originating from the ion structure [(C2H5)2NCH2CH2NH(C2H5)2l+, which shows that the ligand in DEAE Sepharose Fast Flow is composed of both tertiary and quaternary amines. The positive spectrum of Phenyl Sepharose Fast Flow contained major fragments both from the base matrix and the ligand. The cation-exchanger (SP Sepharose Fast Flow) gave rise to a positive spectrum resembling that of the base matrix (Sepharose 6 Fast Flow) but with a different intensity pattern of the matrix fragments. In addition, peaks with low intensity at m/z 109.94, 125.94 and 139.95 corresponding to Na2SO2+, Na2SO3+ and Na2SO3CH2+, respectively, were observed. The positive TOF-SIMS spectrum of Sepharose 6 Fast Flow contains a large number of fragments in the mass range up to m/z 200 identified as CxHyOz and CxHy structures. The results clearly show that positive TOF-SIMS spectra of different media based on Sepharose 6 Fast Flow are strongly influenced by the ligand coupled to the matrix. The negative TOF-SIMS spectra contained several ligand-specific, characteristic peaks for the cation-exchanger, having sulphonate as the ion-exchange group. Negative fragments such as S-, SO-, SO2-, SO3-, C2H3SO3-, C3H5SO3- and OC3H5SO3- were observed. Phenyl Sepharose Fast Flow, which has an uncharged group (Phenyl) coupled to the agarose matrix yielded one ligand-related peak corresponding to the C6H5O- fragment. DEAE and Q ligands could only be identified by the appearance of the fragments CN- and CNO- in the negative spectrum. However, a strong peak corresponding to the counter ion (Cl-) was observed. TOF-SIMS analysis can also be used for the investigation of residues from the coupling procedure that bonds the ligands to the matrix. One example is the observation of bromine peaks in the negative spectrum of Q Sepharose Fast Flow. Furthermore, it has also been shown that different ligand concentrations of Phenyl Sepharose Fast Flow can easily be detected by TOF-SIMS analysis. Information regarding the difference between the ligand density on the surface of the beads and in the bulk can also be obtained. However, spectra registered on the outermost surface and on the pore surface (crushed beads) of DEAE Sepharose Fast Flow clearly show that the agarose and the DEAE groups are homogeneously distributed in the beads.     

Poly(tetrafluoroethylene) separation capillaries for capillary electrophoresis. Properties and applications

Poly(tetrafluoroethylene) (PTFE) is a material widely known for its inertness and excellent electrical properties. It is also transparent in the UV region and has a reasonable thermal conductivity. These properties make PTFE a suitable material for the separation capillary in capillary electrophoresis. Differences in the chemistry of the capillary wall compared to fused silica (FS) can make PTFE an interesting alternative to FS for some special applications. In this work, properties of a commercial PTFE capillary of approx. 100 microm i.d. were investigated, including the dependence of electroosmotic flow (EOF) on pH for unmodified and dynamically modified PTFE, optical properties, and practical aspects of use. The main problems encountered for the particular PTFE capillary used in this study were that it was mechanically too soft for routine usage and the crystallinity of the PTFE caused light scattering, leading to high background absorbance values in the low UV region. The profile of the EOF versus pH for bare PTFE surprisingly showed significantly negative EOF values at pH < 4.2, with an EOF of -30 x 10(-9) m2 V(-1) s(-1) being observed at pH 2.5. This is likely to be caused by either impurities or additives of basic character in the PTFE, so that after their protonation at acidic pH they establish a positive charge on the capillary wall and create a negative EOF. A stable cationic semi-permanent coating of poly(diallyldimethylammonium chloride) (PDDAC) could be established on the PTFE capillary and led to very similar magnitudes of EOF to those observed with FS. A hexadecanesulfonate coating produced a cathodic EOF of extremely high magnitude ranging between +90 and +110 x 10(-9) m2 s(-1) V(-1), which are values high enough to allow counter-EOF separation of high mobility inorganic anions. In addition, pH-independent micellar electrokinetic capillary chromatography (MEKC) separations could be easily realised due to hydrophobic adsorption of sodium dodecylsulfate (used to form the micelles) on the wall of the PTFE capillary. The use of polymers that would be mechanically more robust and optically transparent in the low-UV region should make such CE capillaries an interesting alternative to fused silica.     

Unusual Gruneisen and Bridgman parameters of low-density amorphous ice and their implications on pressure induced amorphization

The low-temperature limiting value of the Grüneisen parameter for low-frequency phonons and the density dependence of the thermal conductivity (Bridgman parameter) of low-density amorphous (LDA) ice, high-density amorphous (HDA) ice, hexagonal ice Ih, and cubic ice Ic were calculated from high-pressure sound velocity and thermal conductivity measurements, yielding negative values for all states except HDA ice. LDA ice is the first amorphous state to exhibit a negative Bridgman parameter, and negative Grüneisen parameters are relatively unusual. Since Ih, Ic, and LDA ice all transform to HDA upon pressurization at low temperatures and share the unusual feature of negative Grüneisen parameters, this seems to be a prerequisite for pressure induced amorphization. We estimate that the Grüneisen parameter increases at the ice Ih to XI transition, and may become positive in ice XI, which indicates that proton-ordered ice XI does not amorphize like ice Ih on pressurization.     

Speciation in the aqueous H+/H2VO4-/H2O2/phosphate system

The speciation in the aqueous H(+)/H(2)VO(4)(-)/phosphate (dihydrogen phosphate, P(-)) and H(+)/H(2)VO(4)(-)/H(2)O(2)/P(-) systems has been determined in the physiological medium of 0.150 M Na(Cl) at 25 degrees C. A combination of multinuclear NMR integral and chemical shift (Bruker AMX500) as well as potentiometric data (glass electrode) have been collected and treated simultaneously by the computer program LAKE. The pK(a)-values for phosphoric acid have been determined by potentiometric and (31)P NMR chemical shift data, and have been found to be 1.85 +/- 0.02, 6.69 +/- 0.02 and 11.58 +/- 0.07. The errors given are 3sigma. Altogether nine vanadate-phosphate species have been found in the ternary H(+)/H(2)VO(4)(-)/P(-) system in the pH region 1-11, with the following compositions: VP, VP(2) and V(14)P. Equilibrium is very slow in acidic solutions, requiring more than 3 months for the formation of V(14)P species. On the other hand, less than 15 min are needed for equilibration at neutral and alkaline pH. In the quaternary H(+)/H(2)VO(4)(-)/H(2)O(2)/P(-) system, four new species have been found in addition to all binary and ternary complexes. They are of VXP and VX(2)P compositions, where X denotes the peroxo ligand. (51)V and (31)P NMR chemical shifts, compositions and formation constants are given, and equilibrium conditions are illustrated in distribution diagrams as well as the fit of the model to the experimental data. Biological and medical relevance of the species is also discussed and physiological conditions are modelled.     

An organoiron approach to thyroid hormone analogues

An approach to thyroid hormone analogues was proposed involving sequential substitution of cationic cyclopentadienyl(1,4-dichlorobenzene)iron(II) complexes with phenoxide/thiophenoxide and hydroxide/amine, followed by decomplexation. Although the selectivity for monosubstitution with phenolates and thiophenolates was poorer than previously observed, it was often possible to control the reaction with sterically less demanding phenolates of intermediate nucleophilicity. The subsequent introduction of a polar substituent into the monosubstituted product was successful with amine nucleophiles. A modified approach, based on the reverse order of substitution was also attempted. Whereas clean monosubstitution with hydroxide/hydroxide equivalents was unsuccessful, cyclopentadienyl(N-alkyl-1-chloro-4-aminobenzene)iron(II) complexes could be prepared in fair yields and further substituted with nucleophiles such as thiophenolates.     

On the degree of weighted polynomial approximation of holomorphic functions

[4] , —. , , , [2] [7].     

Spontaneous transformation of water's high-density amorph and a two-stage crystallization to ice VI at 1 GPa: a dielectric study

Dielectric relaxation spectra of a metastable crystal phase formed on implosive and exothermic transformation of pressure-amorphized hexagonal ice have been measured in situ at 0.97 GPa pressure over a range of temperature. The metastable phase showed no relaxation peak at 130 K and 0.97 GPa. When heated at a fixed pressure of 0.97 GPa, it began to transform at approximately 145 K exothermally to a phase whose relaxation rate and equilibrium dielectric permittivity increased. A second, but slower exothermic transformation also occurred at approximately 175 K. After keeping at 213 K, the relaxation rate and equilibrium permittivity reached the known values of these two quantities for ice VI. Thus the metastable phase transformed to ice VI in two stages. It is conjectured that the intermediate phase in this transformation could be ice XII. The rate of transformation is not determined by the reorientational relaxation rate of water molecules in the ices.     

Immobilized metal ion affinity chromatography of synthetic peptides. Binding via the alpha-amino group.

Peptides synthesized by the solid-phase method can be efficiently purified in a single immobilized metal affinity chromatography step based on interaction with the alpha-amino group if, after coupling of each amino acid residue, unreacted amino groups are irreversibly blocked by acetylation and if no strongly metal-binding amino acids (His, Trp, Cys) are present in the sequence. A difference in basicity for alpha- and epsilon-amino functions of ca. 2 pH units is sufficiently large to allow selective binding of peptides to immobilized metal ions via the unprotonated alpha-amino group. The binding is pH-dependent: on Cu(2+)- and Ni(2+)-loaded supports most peptides are maximally retarded at pH values around 7.5 and 8.5, respectively. The decreased binding strength at lower pH values is due to protonation of the alpha-amino function, whereas the reduced affinity at higher pH is caused by metal ion transfer from the matrix to the peptide. The metal ion is captured in a multidentate chelate where, in addition to the alpha-amino group, up to three adjacent deprotonated amide nitrogens are coordinated to the metal. If the pH is raised further, additional metal ions may be bound in biuret-like structures. Immobilized Ni2+, owing to its higher selectivity and affinity, is the preferred chromatographic support if slightly basic conditions can be tolerated.