Resolution Of Oligomers Research Articles

Vibrational Spectra of Nucleotides in the Presence of the Au Cluster Enhancer in MD Simulation of a SERS Sensor

Surface-enhanced Raman scattering (SERS) nanoprobes have shown tremendous potential in in vivo imaging. The development of single oligomer resolution in the SERS promotes experiments on DNA and protein identification using SERS as a nanobiosensor. As Raman scanners rely on a multiple spectrum acquisition, faster imaging in real-time is required. SERS weak signal requires averaging of the acquired spectra that erases information on conformation and interaction. To build spectral libraries, the simulation of measurement conditions and conformational variations for the nucleotides relative to enhancer nanostructures would be desirable. In the molecular dynamic (MD) model of a sensing system, we simulate vibrational spectra of the cytosine nucleotide in FF2/FF3 potential in the dynamic interaction with the Au20 nanoparticles (NP) (EAM potential). Fourier transfer of the density of states (DOS) was performed to obtain the spectra of bonds in reaction coordinates for nucleotides at a resolution of 20 to 40 cm−1. The Au20 was optimized by ab initio density functional theory with generalized gradient approximation (DFT GGA) and relaxed by MD. The optimal localization of nucleotide vs. NP was defined and the spectral modes of both components vs. interaction studied. Bond-dependent spectral maps of nucleotide and NP have shown response to interaction. The marker frequencies of the Au20—nucleotide interaction have been evaluated.

X-ray Crystallographic Structures of Oligomers of Peptides Derived from β2-Microglobulin.

Amyloid diseases such as Alzheimer's disease, Parkinson's disease, and type II diabetes share common features of toxic soluble protein oligomers. There are no structures at atomic resolution of oligomers formed by full-length amyloidogenic peptides and proteins, and only a few structures of oligomers formed by peptide fragments. The paucity of structural information provides a fundamental roadblock to understanding the pathology of amyloid diseases and developing preventions or therapies. Here, we present the X-ray crystallographic structures of three families of oligomers formed by macrocyclic peptides containing a heptapeptide sequence derived from the amyloidogenic E chain of β2-microglobulin (β2m). Each macrocyclic peptide contains the heptapeptide sequence β2m63-69 and a second heptapeptide sequence containing an N-methyl amino acid. These peptides form β-sheets that further associate into hexamers, octamers, and dodecamers: the hexamers are trimers of dimers; the octamers are tetramers of dimers; and the dodecamers contain two trimer subunits surrounded by three pairs of β-sheets. These structures illustrate a common theme in which dimer and trimer subunits further associate to form a hydrophobic core. The seven X-ray crystallographic structures not only illustrate a range of oligomers that a single amyloidogenic peptide sequence can form, but also how mutation can alter the size and topology of the oligomers. A cocrystallization experiment in which a dodecamer-forming peptide recruits a hexamer-forming peptide to form mixed dodecamers demonstrates that one species can dictate the oligomerization of another. These findings should also be relevant to the formation of oligomers of full-length peptides and proteins in amyloid diseases.

Analysis and purification of peptide nucleic acids by denaturing PAGE

A flexible and convenient protocol for the analysis and purification of peptide nucleic acid (PNA) oligomers and PNA-peptide chimeras by denaturing PAGE is described. Vertical slab gel electrophoresis, 26% in polyacrylamide and 8 M urea at pH 3, was suitable for analysis of oligomers ranging in size from tetramers (4-mers) to tetradodecamers (24-mers). Single-base resolution of oligomers was achieved and separations are generally superior to those given by standard RP-HPLC techniques. The separation of a related series of PNA oligomers showed the distance migrated was linearly dependent on the logarithm of the molecular weight. The migration of oligomers through the gel is dependent on the number of basic functional groups present, such as amino groups, and the A and C content of the oligomer. PNAs are amenable to detection by UV-shadowing technique illuminated at 260 nm or Coomassie blue staining, both with similar, sub-microgram per band detection limits.

Organic and inorganic di‐cations for capillary silica coating and EOF modulation in CE: Example of application in PEG analysis

EOF measurements, by using 1,4-di-(4-aza-1-azonia-bicyclo[2.2.2]octane)butane diiodide, barium and strontium tetraborate as silica wall modifiers, are reported and, as an example of application, analysis of PEG (PEG 400-2000) polydisperse preparations in free solution CZE is shown. PEGs have been derivatized with phthalic anhydride so as to form singly or doubly charged derivatives with strong UV absorbance at 214 nm. Whereas separations in plain tetraborate buffer, pH 9.0, without any EOF control, did not lead to good resolution of all-size oligomers and suffered from long analysis times, excellent resolution of all oligomers up to 40 ethylene oxide (EO) units could be obtained under EOF control. Such EOF modulation was engendered by addition of 1 mM M7C4M7, a doubly charged organic cation able to stick tenaciously to the silica wall. Further modulation of EOF and silica surface modification could be achieved also by addition of inorganic cations, notably those of group II, whereas monovalent cations did not seem to affect much the EOF flux. Among the doubly charged cations investigated, Ca++, Mg++, Sr++ and Ba++, the latter did seem to offer best EOF control and reproducible runs. A judicious blend of M7C4M7 (0.33-1 mM range) with barium (10-20 mM range) allowed baseline resolution of all PEG oligomers investigated up to PEG 2000 and >40 EO units in length. In this last case, best results in terms of reproducibility and separation efficiency of the more heavy homologues were obtained using Li+ salt in small amounts.

In vivo resolution of oligomers with fluorescence photobleaching recovery histograms

Simple independent enzyme-catalyzed reactions distributed homogeneously throughout an aqueous environment cannot adequately explain the regulation of metabolic and other cellular processes in vivo. Such an unstructured system results in unacceptably slow substrate turnover rates and consumes inordinate amounts of cellular energy. Current approaches to resolving compartmentalization in living cells requires the partitioning of the molecular species in question such that its localization can be resolved with fluorescence microscopy. Standard imaging approaches will not resolve localization of protein activity for proteins that are ubiquitously distributed, but whose function requires a change in state of the protein. The small heat shock protein sHSP27 exists as both dimers and large multimers and is distributed homogeneously throughout the cytoplasm. A fusion of the green fluorescent protein variant S65T and sHSP27 is used to assess the ability of diffusion rate histograms to resolve compartmentalization of the 2 dominant oligomeric species of sHSP27. Diffusion rates were measured by multiphoton fluorescence photobleaching recovery. Under physiologic conditions, diffusion rate histograms resolved at least 2 diffusive transport rates within a living cell potentially corresponding to the large and small oligomers of sHSP27. Given that oligomerization is often a means of regulation, compartmentalization of different oligomer species could provide a means for efficient regulation and localization of sHsp27 activity.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of polymers

Matrix-assisted laser desorption/ionization mass spectrometry has been demonstrated to be a powerful analytical technique for the analysis of polymeric materials. The advantages of this technique for such analyses include low sample consumption, ease of sample preparation, short analysis times, and soft ionization which leads to negligible or no fragmentation of analytes. It provides absolute, fast and accurate molecular masses for polymers with narrow polydispersity as opposed to relative masses provided by other techniques. It provides masses for the entire polymer distribution instead of the average value, hence providing molecular mass information which can be used to obtain the mass of the end-groups, mass of the repeat unit (monomer), and chemical modifications on the polymer if oligomer resolution is attained. This review concentrates on the developments in methodology that have allowed for the increased use of this technique for polymer analysis.

Liquid exclusion–adsorption chromatography, a new technique for isocratic separation of nonionic surfactants: IV. Two-dimensional separation of fatty alcohol ethoxylates with focusing of fractions

Fatty alcohol ethoxylates can be analyzed using a combination of liquid chromatography under critical conditions as the first dimension and liquid exclusion–adsorption chromatography as the second dimension. Transfer of fractions from the first to the second dimension is achieved using the full adsorption–desorption (FAD) technique. The peaks of interest in the first dimension are trapped on a short precolumn before injecting them into the second dimension. Full adsorption is achieved by increasing the water content in the mobile phase before the FAD column. When the fractions are desorbed by switching to the mobile phase of the second dimension, they are focused and reconcentrated. In this way, a full resolution of oligomers is achieved. As both dimensions are run in isocratic mode, density and refractive index detection can be applied, which allows an accurate quantitation.

NMR Oligomer Resolution by Intramolecular Complexation in a Poly(ethylene glycol) with an Electron-Acceptor End Group

NMR Oligomer Resolution by Intramolecular Complexation in a Poly(ethylene glycol) with an Electron-Acceptor End Group

Retention Behavior of Oligomers and Cooligomers in Reversed-phase and in Normal-phase Interactive Liquid Chromatographic Systems

Models describing the retention of small molecules combined with the additivity rules for the contributions to the retention can be used for prediction of the retention and resolution of oligomers, small synthetic polymers and copolymers in interactive liquid chromatography with dependency on the molar mass distribution and on the composition of the mobile phase, both in reversed-phase and in normal-phase chromatographic systems, for isocratic or gradient elution. Synthetic copolymers or cooligomers with two (or more) repeat monomer units show not only molar mass distribution, but also composition and sequence distribution of the individual repeat units. By combination of adequately selected separation conditions in normal-phase and in reversed-phase systems, the separation conditions can be adjusted to enhance or to suppress the chromatographic resolution according to each of the two distribution modes and to achieve the separation following the distribution of any of the two repeating groups under “critical conditions” of interactive HPLC. The sequence distribution, i.e., the distribution of the position of the blocks was found to affect significantly the retention behavior of the individual species in the block cooligomers. Based on the retention mechanism suggested, optimization of the conditions for isocratic and gradient-elution separations of cooligomers is possible.

Improved separation of polyethylene glycols widely differing in molecular weight range by reversed-phase high performance liquid chromatography and evaporative light scattering detection

Polyethylene glycols (PEGs) of nominal molecular weight (M) 200, 400, 600, 1000, 1500, 3000, 4000 and 6000 were chosen as model compounds and subjected to reversed-phase high performance liquid chromatography (RP-HPLC) on an octadecasilyl silica gel (C18) stationary phase using a binary gradient composed of acetonitrile and water and evaporative light scattering detection (ELSD). Satisfactory resolution of oligomers up to M of 3000 was accomplished; the higher M samples PEG-4000 and PEG-6000 could not be further resolved into the constituent oligomers and therefore, M=4000 marks the upper limit of oligomer resolution. Despite some peak overlapping as a consequence of the more or less broad oligomer distribution, individual types of PEG samples can be distinguished from each other by their characteristic chromatographic fingerprint patterns, as shown with a mixture consisting of PEG-400, PEG-1000, PEG-3000, PEG-4000 and PEG-6000. For this reason, the method is well-suited for characterization of samples containing PEGs widely differing in M. In addition, matrix assisted laser desorption ionization time of flight mass spectroscopy (MALDI-TOF/MS) performed with PEG-600, PEG-1000 and PEG-3000 revealed that the optimum degree of oligomer resolution has been achieved by use of the present method.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the analysis of polydienes

An analytical method based on matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry has been developed to provide information on oligomer structure, average molecular weight, and molecular weight distributions of polydienes (e.g., polybutadiene and polyisoprene), an important class of industrial polymers. This MALDI method involves the use of all-trans-retinoic acid as the matrix, copper (II) nitrate as the cationization reagent, and tetrahydrofuran as the solvent. The incorporation of this copper salt generates Cu+ adducts with the polymer chains. It also improves the signal strength and extends the upper mass range when used with all-trans-retinoic acid, as compared to silver nitrate. With this formulation, it is demonstrated that polybutadienes of narrow polydispersity with masses up to 300,000 u and polyisoprenes of narrow polydispersity with masses up to 150,000 u can be analyzed. The upper molecular weight limit is set by the requirement of using higher matrix-to-polymer ratios with increasing polymer molecular weight, to the point where the instrument can no longer detect the small quantity of polymer present in the matrix host. It is also shown that this sample preparation generates previously unreported adduction behavior. The practical implications of this adduction behavior on polymer structural analysis, accuracy of molecular weight determination, and the upper molecular weight limit of oligomer resolution are discussed. It is illustrated that, in a linear time-lag focusing MALDI instrument, oligomer resolution can be obtained for polydienes with molecular weights up to 24,000, providing structural confirmation of the end-groups and the repeat unit. The average molecular weights of a number of polydienes of narrow polydispersity determined by MALDI are compared to those obtained by gel permeation chromatography, and discrepancies are noted.

Separation of polyester oligomers by gradient high-performance liquid chromatography

A highly efficient method for the separation of commercially available polyesters composed of a multitude of individual oligomers by gradient reversed-phase high-performance liquid chromatography (gRP-HPLC) was developed. Oligomers up to a molecular mass of more than 10 000 could be sufficiently resolved on an octadecylsilyl silica (C 18) stationary phase using a ternary gradient consisting of acetonitrile, tetrahydrofuran (THF) and aqueous acetic acid. Detection was performed by measurement of signal responses from either UV detection at 230 nm or evaporative light scattering detection (ELSD). Addition of THF to the mobile phase is essential and omission of the co-solvent provided elution of only a few oligomers, the residual amount being trapped on the highly hydrophobic stationary phase. Due to the substantial UV mismatch invoked by THF exhibiting marked absorbance at the chosen wavelength, ELSD, which is not associated with baseline drift phenomena, is much more suited for identification of individual polyester samples on the basis of the chromatographic fingerprint. In one case about sixty sufficiently resolved peaks each attributable to a single oligomer could be observed. A concentration of about 10 mg/ml of sample was required for an unequivocal distinction of the individual polyesters. The use of a C 18 stationary phase is an ultimate prerequisite for efficient oligomer resolution as impressively evidenced by an almost complete lack of separation into individual oligomers on C 8 or C Phenyl matrices. In the latter cases only few low-molecular-mass oligomers are preceding the broad and unresolved bulk peak envelope of sample constituents.

Time-lag focusing MALDI time-of-flight mass spectrometry for polymer characterization: oligomer resolution, mass accuracy, and average weight information.

We report a polymer characterization study by matrix-assisted laser description/ionization (MALDI) on a linear time-of-flight instrument equipped with pulsed ion extraction for time-lag focusing. It is demonstrated that time-lag focusing MALDI provides improved mass resolution and mass accuracy over continuous extraction instruments. Oligomer resolution is extended to a much higher mass range than that observed even by continuous extraction reflectron systems. This allows new opportunities to study the chemical composition and determine the molecular weight of individual components in a mixture of higher molecular weight polymers. It is shown that oligomer resolution can be obtained for poly(ethylene glycol) (repeat unit mass of 44) of mass up to 25,000 u and poly(styrene) (repeat unit mass of 104) up to 55,000 u. Mass measurement accuracy of 80 ppm or better is demonstrated, and the relevance to end-group analysis is shown for two derivative of poly(ethylene glycol) used as slow-release drugs. The analysis of the molecular weight distribution was investigated at several extraction pulse potentials to determine if there was an effect on the relative peak area. We found that the values of the number-average molecular weight (Mn and the weight-average molecular weight (Mw) do not change significantly for a poly(styrene) blend with oligomer masses between 2,000 and 15,000 u and a polydispersity of 1.155. The values are within the 1.6% standard deviation observed for repeat analyses at the same extraction pulse.

Analysis of Structurally Complex Polymers by Time-Lag Focusing Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

The analytical utility of matrix-assisted laser desorption ionization (MALDI) mass spectrometry for the characterization of structurally complex polymers is demonstrated. The advantages and limitations of the MALDI method are discussed through the analysis of several low molecular weight copolymers. Oligomer resolution is achieved by using a MALDI time-of-flight mass spectrometer equipped with pulsed ion extraction for time-lag focusing. Accurate mass analysis of the oligomers provides data sufficient for the confirmation of the repeat unit and end group structures for alternating copolymers. It is shown that for alternating copolymers poly[(o-cresyl glycidyl ether)-co-formaldehyde] and glycidyl end-capped poly[(bisphenol A)-co-epichlorohydrin], the molecular weight as well as detailed structural and compositional information can be obtained from a single spectrum of each. It is found to be more difficult to obtain compositional information on block copolymers or random copolymers. Through the analysis of poly[(propylene glycol)-b-(ethylene glycol)-b-(propylene glycol)]bis(2-aminopropyl ether) and Dow polyol A and polyol B, it is shown that prior knowledge of the end group and monomer masses is required to determine information on the monomer composition. Loss of unit mass resolution complicates the analysis for monomer composition. The issue of obtaining the necessary quantitative information for complete characterization of the above polymer systems is also addressed.

High resolution slab gel electrophoresis of 8-amino-1,3, 6-pyrenetrisulfonic acid (APTS) tagged oligosaccharides using a DNA sequencer.

A novel electrophoretic method for the analysis of oligosaccharides using DNA sequencer technology is illustrated using malto-oligosaccharide distributions obtained following isoamylase digestion of glycogen, wheat starch and potato starch. The debranched starches were derivatized at the reducing and with the charged fluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS). This highly reproducible method provides baseline resolution of oligomers from chain lengths of 3 to more than 80 glucose units, and exhibits high sensitivity with detection thresholds of one femtomole per resolved band. In addition, the reductive amination procedure attaches a single fluorophore per oligosaccharide, allowing calculation of the results on either a mass or a molar basis. The efficacy of the method is illustrated through the determination of the profile of individual oligosaccharides of chain length with a degree of polymerization (DP) < 80, derived from loading less than 15 ng per analysis of glycogen, wheat and potato starches. While the results obtained were superior in resolution and sensitivity to previously reported observations using a range of techniques, they were nonetheless consistent with the overall differences between these polysaccharides. The resolution, sensitivity, reproducibility and high throughput of the method provides substantial advantages over existing methods for the analysis of linear oligosaccharide chain length distributions.

Analysis of Ethoxylated Nonylphenol Surfactants by High Performance Size Exclusion Chromatography (HPSEC)

Abstract Since Size Exclusion Chromatography does not provide a complete oligomer resolution, it has not been generally considered as a choice method for analyzing ethoxylated surfactant. However, this situation might be changing. In effect, the use of new column technology together with non aqueous carrier solvent, and some operational optimization, can result in fairly good distribution data on the number of ethylene oxide groups per molecule (EON). It is shown that HPSEC provides an excellent EON average estimate over a wider and higher EON range than the the up-to-date HPLC techniques.

Mechanism of Asymmetric Polymerization of Triphenylmethyl Methacrylate, Separation and Optical Resolution of Oligomers

Abstract Asymmetric oligomerization of triphenylmethyl methacrylate (TrMA) was carried out with (−)-sparteine-fluorenyllithium complex in toluene at −78 °C and the oligomers of methyl methacrylate (DP=2 and 3) derived therefrom were separated into diastereomers and optical isomers on silica gel and chiral HPLC columns, respectively. On the basis of the obtained data, the mechanism of asymmetric polymerization of TrMA was discussed.

Thin-layer and gel permeation chromatographic separation of low molecular weight polyethylene glycol oligomers

We describe a gel permeation and a thin-layer Chromatographic technique for the complete resolution of oligomers of ethylene glycol up to a chain length of fourteen ethylene oxide [OCH 2CH 2] units. We employed columns of Bio-Gel P-2 with 0.02 mol/l ammonium acetate as the eluant to prepare milligram quantities of each of the individual oligomers. Thin-layer chromatography on silica gel G plates with an ethyl acetate/methanol/water solvent provides a sensitive and simple method for monitoring multiple specimens. We determined the flame ionization detector response of purified individual ethylene glycol oligomers relative to tetraethylene glycol. These data permit the accurate quantification of the oligomeric profile of commercially available mixtures of low molecular weight polyethylene glycols (PEG). When PEG 400 is administered orally to normal subjects, aged three months through adulthood, they excrete in their urine a mixture of unmetabolized oligomers with a mean molecular weight of 360 ± 14 daltons. The ability to measure absolute, rather than relative, amounts of ethylene glycol oligomers will permit more accurate studies of passive intestinal permeability in human subjects.

Effects of Column Length, Particle Size, Flow Rate, and Pressure Programming Rate on Resolution in Pressure-Programmed Supercritical Fluid Chromatography

In pressure-programmed supercritical fluid chromatography, particle diameters in the range of 122 ..mu..m to 205 ..mu..m were found to yield maximum resolution of oligomers of 800 molecular weight polystyrene when 5% isopropanol in pentane was employed as a mobile phase at 240/sup 0/C. The ability of a given column to achieve maximum resolution was found to depend on the ratio of the pressure-programming rate to the average flow rate of the mobile phase. Resolution could be increased while analysis time decreased, when a fast pressure-programming rate was used in conjunction with a high flow rate. In addition, due to the unusual thermophysical properties of the super-critical mobile phase, operation at linear velocities that are generally considered to be in the region of turbulent flow were found to be necessary for maximum resolution.

Low Pressure Gel Chromatographic Separation of Oligosaccharides

AbstractA method is described for the separation and analysis of oligosaccharides by gel chromatography, using Bio‐Gel P‐2, 200–400 mesh and Bio‐Gel P‐4, 200–400 mesh. Resolution of malto‐oligosaccharides containing up to 13 glucose residues was obtained on both the P‐2 and the P‐4 columns. This resolution is slightly lower than that obtainable using the finer minus 400 mesh gel. However, when the two columns were connected in series, resolution of oligomers containing up to 15 glucose residues was obtained. This resolution is comparable to that obtainable on a single P‐2 minus 400 mesh column. Improved resolution of lower oligomers was obtained by recycle gel chromatography on the P‐2 column. Some of the practical difficulties associated with the operation of a minus 400 mesh gel column were avoided as high pressure is not required to operate the 200–400 mesh gel columns.