High-performance liquid chromatography of amino acid peptides and proteins

Summary The retention behaviour of seven globular proteins ranging in molecular weight from 12,000 to 69,000 was investigated using Mono-Q anion-exchange resin as the stationary phase and sodium chloride as the displacer salt. In particular the influence of changes in ionic strength and mobile phase pH on the isocratic retention properties was assessed. Several proteins were found to have significant retention when the pH of the mobile phase was below the reported pl values of the proteins. This behaviour results from the non-uniform charge distribution on the protein surface, which allows interaction with the charged stationary phase even though the protein net charge is equal to or greater than zero. The influence of pH and ionic strength on experimentally observed bandwidths was also investigated. The dependence of the effective reduced plate height on solute capacity factor was found to vary significantly with the mobile phase pH, a behaviour consistent with the interplay of complex multisite binding kinetics. These results provide a basis for further detailed investigations into the mechanism of interaction of proteins not only with charged surfaces associated with adsorptive chromatographic media but also with other macromolecules. For Part LXXXII, see ref. [27].     

Kinetics of the surfactant-free emulsion polymerization of styrene:-The post nucleation stage

The kinetics of the surfactant-free emulsion polymerisation of styrene have been studied. Reactions were sampled at several stages throughout their course, and the samples characterized by electron microscopy, gravimetric weights analysis and gel permeation chromatography, After an initial period of particle nucleation and coagulation, the reaction proceeds at a constant number density. The theories developed for emulsion polymerisation in the presence of surfactant above its critical micelle concentration might be expected, in Interval II, to apply to the surfactant-free system if due regard is taken of the lower number densities and larger particle sizes developed. The results are in best accord with theories invoking a surface phase polymerisation mechanism. None of these theories predict a bimodel molecular weight distribution as found here and which is ascribed to polymerisation in two loci. The activation energy was found to be the same as for surfactant containing emulsion polymerisations.     

Binding of inorganic oxoanions to macrocyclic ligands: effect of the degree of protonation on supramolecular assemblies formed by phosphate and [18]aneN(6)

Five macrocycle-oxoanion adducts have been isolated from aqueous solutions containing 1,4,7,10,13,16-hexaazacyclooctadecane ([18]aneN(6), L) and phosphoric acid whose pH had been adjusted to selected values in the 1-8 range. Four products, (H(6)L)(H(2)PO(4))(6).2H(3)PO(4) (1), (H(6)L)(H(2)PO(4))(6) (2), (H(4)L)(H(2)PO(4))(4).2H(2)O (4), and (H(4)L)(HPO(4))(2).7H(2)O (5) crystallized from aqueous solutions at pH 1, 3, 6, and 8, respectively, while (H(4)L)(H(2)PO(4))(4) (3) crystallized on diffusion of EtOH into an aqueous reaction mixture at pH 6. Single-crystal X-ray structure determinations enabled an examination of supramolecular interactions between protonated forms of [18]aneN(6), phosphoric acid and its conjugate bases, and water of solvation. The macrocycle adopts a variety of conformations in order to accommodate the supramolecular constructs formed by the oxoanions and solvent molecules as the relative proportions of interacting species are altered. At pH 1 and 3, the fully protonated macrocycle, [LH(6)](6+), is found with six H(2)PO(4)(-) anions. At pH 6 and 8, the tetraprotonated macrocycle, [LH(4)](4+), crystallizes with four H(2)PO(4)(-) and two HPO(4)(2)(-), respectively. Variations in the solute of crystallization are evident, with phosphoric acid being present at the lowest pH and water at pH 6 and 8. In 5, the seven unique water molecules form a string-of-pearls motif within which a new heptameric isomer, consisting of a water pentamer that uses a single water to interact with the other two unique water molecules, is found. Structures 1, 2, 4, and 5 exhibit eta-3 H-bonding of ammonium protons to a single oxygen of the guest phosphates located above and below the macrocyclic ring. In 3, two phosphate oxygens of the cavity anion interact with the macrocycle, one of which participates in eta-2 H-bonding with ammonium groups.     

Total selenium and selenomethionine in pharmaceutical yeast tablets: assessment of the state of the art of measurement capabilities through international intercomparison CCQM-P86

Results of an international intercomparison study (CCQM-P86) to assess the analytical capabilities of national metrology institutes (NMIs) and selected expert laboratories worldwide to accurately quantitate the mass fraction of selenomethionine (SeMet) and total Se in pharmaceutical tablets of selenised-yeast supplements (produced by Pharma Nord, Denmark) are presented. The study, jointly coordinated by LGC Ltd., UK, and the Institute for National Measurement Standards, National Research Council of Canada (NRCC), was conducted under the auspices of the Comité Consultatif pour la Quantité de Matière (CCQM) Inorganic Analysis Working Group and involved 15 laboratories (from 12 countries), of which ten were NMIs. Apart from a protocol for determination of moisture content and the provision of the certified reference material (CRM) SELM-1 to be used as the quality control sample, no sample preparation/extraction method was prescribed. A variety of approaches was thus used, including single-step and multiple-step enzymatic hydrolysis, enzymatic probe sonication and hydrolysis with methanesulfonic acid for SeMet, as well as microwave-assisted acid digestion and enzymatic probe sonication for total Se. For total Se, detection techniques included inductively coupled plasma (ICP) mass spectrometry (MS) with external calibration, standard additions or isotope dilution MS (IDMS), inductively coupled plasma optical emission spectrometry , flame atomic absorption spectrometry and instrumental neutron activation analysis. For determination of SeMet in the tablets, five NMIs and three academic/institute laboratories (of a total of five) relied upon measurements using IDMS. For species-specific IDMS measurements, an isotopically enriched standard of SeMet (⁷⁶Se-enriched SeMet) was made available. A novel aspect of this study relies on the approach used to distinguish any errors which arise during analysis of a SeMet calibration solution from those which occur during analysis of the matrix. To help those participants undertaking SeMet analysis to do this, a blind sample in the form of a standard solution of natural abundance SeMet in 0.1 M HCl (with an expected value of 956 mg kg⁻¹ SeMet) was provided. Both high-performance liquid chromatography (HPLC)–ICP-MS or gas chromatography (GC)–ICP-MS and GC-MS techniques were used for quantitation of SeMet. Several advances in analytical methods for determination of SeMet were identified, including the combined use of double IDMS with HPLC-ICP-MS following extraction with methanesulfonic acid and simplified two-step enzymatic hydrolysis with protease/lipase/driselase followed by HPLC-ICP-IDMS, both using a species-specific IDMS approach. Overall, satisfactory agreement amongst participants was achieved; results averaged 337.6 mg kg⁻¹ (n = 13, with a standard deviation of 9.7 mg kg⁻¹) and 561.5 mg kg⁻¹(n = 11, with a standard deviation of 44.3 mg kg⁻¹) with median values of 337.6 and 575.0 mg kg⁻¹ for total Se and SeMet, respectively. Recovery of SeMet from SELM-1 averaged 95.0% (n = 9). The ability of NMIs and expert laboratories worldwide to deliver accurate results for total Se and SeMet in such materials (selensied-yeast tablets containing approximately 300 mg kg⁻¹ Se) with 10% expanded uncertainty was demonstrated. The problems addressed in achieving accurate quantitation of SeMet in this product are representative of those encountered with a wide range of organometallic species in a number of common matrices. Figure Looking into the quantitative speciation of selenium in pharmaceutical supplements Photo courtesy of LGC.     

Examination of the binding behaviour of several proteins with the immobilized copper(II) complexes of o-, m- and p-xylylene bridged bis(1,4,7-triazacyclononane) macrocycles

Three new IMAC chelating systems, incorporating immobilised xylenyl-bridged bis(1,4,7-triaza-cyclonane) ligands, complexed with Cu(2+) ions to form binuclear species, have been prepared. Their binding properties have been investigated with three small globular proteins (hen egg white lysozyme, horse skeletal muscle myoglobin and horse heart cytochrome c). The effects of buffer pH, ionic strength and composition on the binding behaviour of these proteins to these new IMAC sorbents have been examined and compared with those found for the corresponding immobilized mononuclear copper complex of 1,4,7-triazacyclononane (tacn). Higher protein binding affinities were observed with the Cu(2+)-bis(tacn) sorbents compared to the Cu(2+)-tacn system, consistent with the immobilized binuclear copper(II) species undergoing enhanced coordinative interaction with the surface-exposed histidine residues of these proteins. Moreover, the protein binding characteristics of these IMAC sorbents at higher ionic strengths, such as 1M NaCl, also reflect the presence of the aromatic ring in the bis(tacn) ligands, whereby hydrophobic pi/pi stacking interactions can occur with the proteins.     

The synthesis, structure and properties of copper(II) complexes of asymmetrically functionalized derivatives of 1,4,7-triazacyclononane

Reaction of 1-propylamino-4-acetato-1,4,7-triazacyclononane (L1), 1-benzyl-4-acetato-1,4,7-triazacyclononane (L2) and 1-benzyl-4-propylamino-1,4,7-triazacyclononane (L3) with a copper(II) salt gave Na2[CuL1](ClO4)3(1a), [CuL2]Cl (2) and [Cu2L32](ClO4)4.5H2O (3), respectively. [CuL4]ClO4 (4) was formed by reacting 1-formyl-4-ethylacetato-1,4,7-triazacyclononane with cupric chloride in aqueous solution. The X-ray crystal structures of the complexes reveal that the ligands generate distorted square pyramidal or square planar coordination environments about the Cu(II) centre, but in three complexes (1b, 3 and 4) weak interactions to an oxygen atom from a perchlorate anion and, in the case of 4, also to an amide nitrogen leading to tetragonally elongated octahedral Cu(II) geometries. In 4, the formyl group is found to reduce the coordinating ability of the macrocyclic nitrogen to which it is attached, as evidenced by the weak CuN interaction. The formation of five-membered chelate rings on coordination of the ligands further contributes to the distortion from the ideal geometries. The crystal lattices contain a number of novel supramolecular features. 1a contains a negatively charged sodium perchlorate chain of composition [Na2(ClO4)3]x(x-), with a complex series of Na-O-Na bridges flanked by [CuL1]+ units, while 3 contains highly complex hydrogen bonded sheets approximately 20 A thick that stack through van der Waals interactions. One-dimensional chains comprised of copper complexes are found in 2 and 4, and are held together by hydrogen bonds in 2 and acetate bridges between the copper cations in 4. The solution EPR spectra indicate that the copper(II) centres exist in isolated distorted square pyramidal (possibly square planar for 4) environments, while in the solid state there is evidence for the existence of weak exchange and dipole-dipole coupling for some complexes.     

Determination of tributyltin in marine sediment: Comité Consultatif pour la Quantité de Matière (CCQM) pilot study P-18 international intercomparison

The capabilities of National Metrology Institutes (NMIs—those which are members of the Comité Consultatif pour la Quantité de Matière (CCQM)of the CIPM) and selected outside "expert" laboratories to quantitate (C₄H₉)₃Sn⁺ (TBT) in a prepared marine sediment were assessed. This exercise was sanctioned by the 7th CCQM meeting, April 4–6, 2001, as an activity of the Inorganic Analysis Working Group and was jointly piloted by the Institute for National Measurement Standards of the National Research Council of Canada (NRC) and the Laboratory of the Government Chemist (LGC), UK. A total of 11 laboratories submitted results (7 NMIs, and 4 external labs). Two external laboratories utilized a standard calibration approach based on a natural abundance TBT standard, whereas all NMIs relied upon isotope dilution mass spectrometry for quantitation. For this purpose, a species specific ¹¹⁷Sn-enriched TBT standard was supplied by the LGC. No sample preparation methodology was prescribed by the piloting laboratories and, by consequence, a variety of approaches was adopted by the participants, including mechanical shaking, sonication, accelerated solvent extraction, microwave assisted extraction and heating in combination with Grignard derivatization, ethylation and direct sampling. Detection techniques included ICP–MS (with GC and HPLC sample introduction), GC–MS, GC–AED and GC–FPD. Recovery of TBT from a control standard (NRCC CRM PACS-2 marine sediment) averaged 93.5±2.4% (n=14). Results for the pilot material averaged 0.680±0.015 µmol kg^–1 (n=14; 80.7±1.8 µg kg^–1) with a median value of 0.676 µmol kg^–1. Overall, performance was substantially better than state-of-the-art expectations and the satisfactory agreement amongst participants permitted scheduling of a follow-up Key comparison for TBT (K-28), a Pilot intercomparison for DBT (P-43), and certification of the test sediment for TBT content and its release as a new Certified Reference Material (HIPA-1) with a TBT content of 0.679±0.089 µmol kg^–1 (expanded uncertainty, k=2, as Sn) (80.5±10.6 µg kg^–1).Electronic Supplementary Material Supplementary material is available in the online version of this article at .     

Development of a temperature-responsive agarose-based ion-exchange chromatographic resin

A temperature-responsive ion-exchange resin (ItBA) has been prepared by grafting poly(N-isopropylacrylamide-co-acrylic acid-co-tert-butylacrylamide; ItBA) onto cross-linked agarose. A carboxymethylated ion exchanger (CM) of similar charge density was also prepared. Maximum adsorption capacities (B_max) for lactoferrin at 20 °C and 50 °C were determined for both resins by batch adsorption procedures. Dynamic adsorption and desorption characteristics of the CM and ItBA with lactoferrin were established, as well as the ability of ItBA to selectively adsorb and desorb lactoferrin in the presence of other proteins. With the CM-agarose resin there was no significant difference between the B_max values obtained at 20 °C and 50 °C. However, for the agarose-based ItBA resin the B_max value at 50 °C was almost three times higher than the B_max value at 20 °C. Dynamically, lactoferrin adsorbed to the ItBA packed column at 50 °C with a significant proportion of the adsorbed lactoferrin desorbed by reducing the temperature to 20 °C. In addition, anionic proteins did not adsorb to the ItBA packed column, and did not interfere with the dynamic adsorption/desorption behaviour of lactoferrin. These results indicate that this new temperature-responsive agarose-based ItBA resin has potential for the fractionation of whey proteins, with good selectivity for cationic proteins.     

Bioinspired core‐crosslinked micelles from thymine‐functionalized amphiphilic block copolymers: Hydrogen bonding and photo‐crosslinking study

Bioinspired core‐bound polymeric micelles, based on hydrogen bonding and photo‐crosslinking, of thymine have been prepared from poly(vinylbenzylthymine)‐b‐poly(vinylbenzyltriethylammonium chloride). The amphiphilic block copolymer was synthesized by 2,2‐tetramethylpiperidin‐1‐oxyl‐mediated living radical polymerization in water/ethylene glycol solution. Micelle characterization and critical micelle concentration measurements demonstrated that the hydrogen bonding of the attached thymine units stabilizes the micelles. Further, core‐crosslinked polymeric micelles were formed by ultraviolet (UV) radiation showing that the stability of the micelle could be controlled by the UV crosslinking of the attached thymines. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011