Fast protein separation and characterization by hydrophobic interaction chromatography (HIC) and low angle laser light scattering photometry (LALLS)

Summary Fast gradient HIC has been interfaced with low angle laser light scattering photometry (LALLS) to separate and characterize proteins. Molecular weights (MWs) have been determined on-line for a set of four proteins. A fast HIC column has been used to elute proteins under fast (5 min) and conventional (10 min) gradients. Certain variables used in the calculation of MW, such as refractive index of the mobile phase (RI) and differential refractive index (dn/dc) have been calculated offline.The MWs for three of the four proteins studied matched literature values at any given gradient time. One of the proteins, -lactoglobulin-A (-LACT), was a dimer when injected. Our results have indicated that fast gradient HIC can be easily interfaced with LALLS in spite of a significant change in RI across the gradient.Dedicated to Professor Leslie S. Ettre on the occasion of his 70th birthday.     

Solution characterization of the novel organometallic polymer poly(ferrocenyldimethylsilane)

A series of four well‐defined poly(ferrocenyldimethylsilane) (PFS) samples spanning a molecular weight range of approximately 10,000–100,000 g mol⁻¹ was synthesized by the living anionic polymerization of dimethyl[1]silaferrocenophane initiated with n‐BuLi. The polymers possessed narrow polydispersities and were used to characterize the solution behavior of PFS in tetrahydrofuran (THF). The weight‐average molecular weights (M_w ) of the polymers were determined by low‐angle laser light scattering (LALLS), conventional gel permeation chromatography (GPC), and GPC equipped with a triple detector (refractive index, light scattering, and viscosity). The molecular weight calculated by conventional GPC, with polystyrene standards, underestimated the true value in comparison with LALLS and GPC with the triple detection system. The Mark–Houwink parameter a for PFS in THF was 0.62 (k = 2.5 × 10⁻⁴), which is indicative of fairly marginal polymer–solvent interactions. The scaling exponent between the radius of gyration and M_w was 0.54, also consistent with marginal polymer–solvent interactions for PFS in THF. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 38: 3032–3041, 2000     

Characterization of Branched Poly (vinyl Acetate) by GPC and Low Angle Laser Light Scattering Photometry

Abstract

Poly (vinyl acetate), PVAC, synthesized by bulk polymerization over a range of initiator concentrations ([AIBN] = 10^?5 to 4 × 10^?3 g-mole/1), temperatures (50°C, 60°C, 70°C, and 80°C) and conversion levels (3 to > 90%) were characterized using low angle laser light scattering (LALLS) photometry to measure M_w of the whole polymers. A number of these samples were characterized using GPC with a differential refractive index (DRI) and LALLS detector to measure the molecular weight distribution (weight fraction versus M_w). M_w for PVAC samples synthesized at suitably low initiator levels at various conversions were found to agree with classical light scattering measurements after Graessley.

An electronic device and a technique which optimizes the sensitivity and the signal-to-noise ratio of the LALLS photometer throughout the molecular weight distribution (MWD) of the GPC chromatogram were devised. These considerably simplify the operation of the LALLS for both offline and online operation with GPC.

Most importantly it was unambiguously shown that the commonly used universal calibration parameter (UCP) with GPC, [n]M_w, is incorrect for polymers with molecules having the same hydrodynamic volume but different molecular weights, i. e., those with only chain branching (LCB), copolymers with compositional drift, and polymer blends. The correct UCP was found to     

Determination of pituitary and recombinant human growth hormone molecular weights by modern high-performance liquid chromatography with low angle laser light scattering detection

The determination of molecular weight for pituitary and recombinant human growth hormone (p-hGH/Crescormon and r-hGH/Protropin) has been performed. This has involved on-line coupling of size-exclusion chromatography (SEC) and gradient elution, reversed-phase high-performance liquid chromatography (RP-HPLC) with low-angle laser light scattering (LALLS) detection. A 5-microns, 300 A, Delta-bond octyl column was used. Traditional specific refractive index increment (dn/dc) and refractive index (n) measurements have been performed in order to derive absolute weight-average molecular weight (Mw) information for p-hGH and r-hGH. Known concentrations of each protein have been separated using reversed-phase gradients utilizing acetonitrile with on-line LALLS determination of excess Rayleigh scattering factors. Accurate Mw data has been obtained for both proteins under conventional RP-HPLC gradient elution conditions. SEC data of both hGHs were found to be concentration, mobile phase, and column dependent for the particular analyses. Both medium- and high-resolution SEC-LALLS studies were performed, and all of these determinations further confirmed our RP-HPLC results. On-line LALLs provides certain advantages in identifying aggregates that may be present, even in medium-resolution SEC, where incomplete resolution occurs. The on-line coupling of modern RP-HPLC for biopolymers with LALLS detection represents a major step forward in the ability of bioanalytical chemists to determine the nature (monomer versus higher-order aggregate) of such materials. Other classes of biopolymers should prove suitable for studies with the same RP-HPLC-LALLS-UV approaches.     

Determination of biopolymer (protein) molecular weights by gradient elution, reversed-phase high-performance liquid chromatography with low-angle laser light scattering detection

The determination of molecular weights for certain proteins has been performed. This has involved the on-line coupling of gradient elution, reversed-phase high-performance liquid chromatography (RP-HPLC) with low-angle laser light scattering (LALLS) detection. A new 1.5-micron, non-porous, Monosphere RP-C8 column has been used in order to perform fast and conventional RP-HPLC gradients (5-45 min). Traditional specific refractive index increment (dn/dc) and refractive index (n) measurements have been performed in order to derive absolute weight-average molecular weight (Mw) information for ribonuclease A, lysozyme, and bovine serum albumin. Standard mixtures of known concentrations of each protein have been separated using reversed-phase gradients utilizing acetonitrile with on-line LALLS determination of excess Rayleigh scattering factors. Accurate Mw data have been obtained for all three proteins, but only under certain, conventional reversed-phase gradient elution conditions. Between 5-10 min of fast gradient elution, each protein appears to exhibit unusual Mw values, suggestive of aggregate formations. Methods have been developed to define the nature of such aggregates. The on-line coupling of modern RP-HPLC for biopolymers with LALLS represents a major step forward in the ability of bioanalytical chemists to determine the nature (monomer versus aggregate) of such materials. Other classes of biopolymers should prove suitable for studies with the same RP-HPLC-LALLS-UV approaches.     

Characterization of proteins separated by displacement chromatography using low angle laser light scattering photometry

Summary -Lactoglobulin A and B (-LACT) were separated by displacement chromatography (DSC) on an ionexchange column using dextran sulfate as the displacer. A LALLS photometer and a UV detector, in series, were used to determine the molecular weight (MW) of the proteins, on-line. The results indicate that both, -LACT A and B, were present as dimers in the buffer used for the mobile phase. The MWs of the proteins were about 6–8% higher than the theoretical MW of a dimer (37,000). Additional control studies have shown the presence of a high molecular weight species in both the proteins, which could possibly be an aggregate. This species was observed in the LALLS signal but was nearly absent in the UV signal. Our work has demonstrated the feasibility of interfacing LALLS with displacement chromatography for detecting impurities or aggregates which may be difficult to detect by conventional detectors used for chromatography.     

Preparation of tea glycoprotein and its application as a calibration standard for the quantification and molecular weight determination of tea glycoprotein in different tea samples by high-performance gel-permeation chromatography

Tea glycoprotein (TGC) was purified by Sephadex G-100 gel filtration, and its purity and molecular weight (MW) were determined by high performance gel permeation chromatography. The gel permeation chromatography (GPC) elution behavior of TGC on an Ultrahydrogel 500 (7.8×300 mm) column was studied using a mobile phase with various concentrations of NaCl. A dextran T system was found to be unsuitable as MW calibration standards in GPC because the MW of TGC was changed with the change of ionic concentration in the mobile phase. Thus, the purified TGC obtained in this study was standardized and used instead as the calibration standards for the determination and comparison of TGCs in tea samples collected from different geographic locations and species varieties. The GPC was run at 35°C on an Ultrahydrogel 500 column (7.8×300 mm) with a refractive index detector. Distilled water was used as the mobile phase at a flow rate of 0.6 ml·min⁻¹. The calibration curve was linear over the TGC concentration range of 0.604–6.04 mg·ml⁻¹, with a correlation coefficient of r=0.9997. The TGC mass recovery ranged from 80.4 to 93.2%, with a relative standard deviation of 5.42%. The assay method developed in this paper was found to be simple, reproducible, and reliable and, thus, ideally suitable for the quality control of TGC-derived products and raw materials.     

Characterization of a series of phenyl‐capped vinyl‐terminated oligomers of styrene

Low molecular weight (MW) polystyrenes were synthesized by radical polymerization in the presence of catalytic chain‐transfer agents. Synthetic conditions are controlled to produce molecules containing one methyl group at one end as well as a double bond at the other end, capped with a phenyl group. Individual oligomers were separated by liquid chromatography, and the properties were analyzed using NMR, ultraviolet–visible (UV–vis) spectroscopy, and size exclusion chromatography with light scattering. The UV–vis spectra, proton NMR spectra, and differential refractive‐index increments exhibit an MW dependence of up to six–eight monomer units. The obtained dependencies can be used for precise characterization of the molecular weight distribution of polystyrene obtained by catalytic chain transfer. The double‐bonded end groups were found to be exclusively in the transconfiguration for all oligomers. © 2001 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 39: 1099–1105, 2001     

Synthesis and Characterization of Monodisperse α,ω-Divinylpolydimethylsiloxane and ω-Vinylpolydimethylsiloxane

Abstract

ω-Vinylpolydimethylsiloxanes (ω-PDMS) and α,ω-divynilpolydimethylsiloxanes (α,ω-PDMS) have been synthesized by anionic polymerization of hexamethylcyclotrisiloxane (D3). The reactions were carried out in n-hexane and tetrahydrofuran (THF), respectively. The initiator was n-butyl-lithium (n-BuLi); tetrahydrofuran was used as an electron donor compound to promote the polymerization of D3. The synthesized polymers have been characterized by infrared (FTIR), proton nuclear magnetic resonance spectroscopy (NMR), light scattering (LALLS), and gel permeation chromatography (GPC). Molecular weights ranging from 20,000 to 100,000 were obtained with relatively narrow molecular weight distributions; Mw/Mn < 1.25.     

Controlled polymerization of 2‐(diethylamino)ethyl methacrylate and its block copolymer with N‐isopropylacrylamide by RAFT polymerization

Poly(2‐(diethylamino)ethyl methacrylate) (PDEAEMA) homopolymers with low polydispersities were synthesized by reversible addition fragmentation chain transfer (RAFT) radical polymerization. The performances of two chain transfer agents, 2‐cyanoprop‐2‐yl dithiobenzoate and 4‐cyanopentanic acid dithiobenzoate (CPADB), were compared. It was found that the polymerization of 2‐(diethylamino) ethyl methylacrylate was under good control in the presence of CPADB with 4,4′‐azobis(4‐cyanopentanoic acid) (ACPA) as initiator in 1,4‐dioxane at 70 °C. The kinetic behaviors were investigated under different CPADB/ACPA molar ratios. A long polymerization inhibition period was observed at high [CPADB]/[ACPA] ratio. The influences of [CPADB]/[ACPA] ratio, monomer/[CPADB] ratio, and temperature were studied with respect to monomer conversion, molecular weight control, and polydispersity index (PDI). The PDI decreased from 1.21 to 1.12, as the CPADB/ACPA molar ratio changed from 2 to 10. The molecular weight of PDEAEMA could be controlled by monomer/CPADB molar ratio. The control over MW and PDI was improved as the temperature increased from 60 to 70 °C; however, an additional increase to 80 °C led to a loss of control. Using PDEAEMA macroRAFT agent, pH/thermo double‐responsive block copolymers of PDEAEMA and poly(N‐isopropylacrylamide) (PDEAEMA‐b‐PNIPAM) with narrow polydispersity (PDI, 1.24) were synthesized. The lower critical solution temperature of PDEAEMA‐b‐PNIPAM block copolymer depended on the environmental pH. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 3294–3305, 2008     

Facile synthesis and characterization of hyperbranched poly(ether amide)s generated from Michael addition polymerization of in situ created AB2 monomers

A new straightforward strategy for synthesis of novel hyperbranched poly (ether amide)s from readily available monomers has been developed. By optimizing the reaction conditions, the AB₂‐type monomers were formed dominantly during the initial reaction stage. Without any purification, the AB₂ intermediate was subjected to further polymerization in the presence (or absence) of an initiator, to prepare the hyperbranched polymer‐bearing multihydroxyl end‐groups. The influence of monomer, initiator, and solvent on polymerization and the molecular weight (MW) of the resultant polymers was studied thoroughly. The MALDI–TOF MS of the polymers indicated that the polymerization proceeded in the proposed way. Analyses of ¹H NMR and ¹³C NMR spectra revealed the branched structures of the polymers obtained. These polymers exhibit high‐moderate MWs and broad MW distributions determined by gel permeation chromatography (GPC) in combination with triple detectors, including refractive index, light scattering, and viscosity detectors. In addition, the examination of the solution behavior of these polymers showed that the values of intrinsic viscosity [η] and the Mark–Houwink exponent α were remarkably lower compared with their linear analogs, because of their branched nature. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 4309–4321, 2007     

A study of aqueous solutions of dextrans by gel permeation chromatography using a multiple detection system

We studied the elution of dextrans in gel permeation chromatography (GPC) using a low-angle laser light-scattering (LALLS) system to monitor the molecular weight and the standard refractive index (RI) detector to measure the concentration. In pure water the dextrans show bimodal chromatograms of the RI response as well as of the LALLS response. The first eluted peak reflects the macroion exclusion due to the repulsion between negatively charged macromolecules and equally charged gel. In spite of the resulting distortion of the chromatograms, correct values of the weight-average molecular weights (M?_w) are obtained from them. If instead of pure water a salt solution (0.1 M KNO₃) is used as eluant, the charge effect is completely suppressed and single-peak chromatograms are found. Again correct values of M?_w are obtained, but number-average molecular weights turn out to be too large owing to axial column dispersion in the coupling arrangement. By introducing a spreading factor, values of M?_n could be corrected. Investigations of the charge effect were also carried out by monitoring the electric conductivity of the eluted dextran zone. In this way, the phenomenon of ion inclusion, evidently provoked by charged dextran molecules, was observed.     

Effect of Topology on the Properties of Poly(N-isopropylacrylamide) in Water and in Bulk

Three macrocyclic poly(N-isopropylacrylamide)s (PNIPAM) with molecular weight (MW) ranging from 6 to 19 kg/mol were synthesized by ‘click’ ring closure of the corresponding α-azido ω-propargyl telechelic linear PNIPAMs, themselves prepared by reversible addition fragmentation chain transfer (RAFT) polymerization of N-isopropylacrylamide. Differential scanning calorimetry (DSC) studies revealed that both the thermal phase separation in water and the glass transition in bulk of PNIPAM were affected by polymer topology. In aqueous solution, the cyclic polymers exhibit a higher phase separation temperature and broader phase transition range than the corresponding linear counterparts. In bulk, the cyclic polymers display a higher glass transition temperature of lesser molecular weight dependence, as compared to their linear precursors.     

Producing bimodal molecular weight distribution polymers through facile one‐pot/one‐step RAFT polymerization

Well‐defined bimodal molecular weight distribution (MWD) polystyrene and polystyrene‐b‐poly(acrylonitrile) were successfully synthesized using a pair of mono/difunctional trithiocarbonate RAFT agents 1 and 2 via one‐pot RAFT polymerization. The kinetics of RAFT polymerization for styrene in bulk with a molar ratio of [St]₀:[AIBN]₀:[ 1 ]₀:[ 2 ]₀ = 1200:1:2.5:2.5 was studied at 75°C. The results indicated that the system showed excellent controllability and “living” characteristics to both higher and lower molecular weight fractions, providing an efficient and facile way to producing bimodal MWD (co)polymers with both controlled molecular weight (MW) and MWD in molecular level, and the plausible mechanism was discussed in this work. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Controlled/living polymerization of 2‐(diethylamino)ethyl methacrylate and its block copolymer with tert‐butyl methacrylate by atom transfer radical polymerization

Polymerization of 2‐(diethylamino)ethyl methacrylate (DEAEMA) via homogeneous atom transfer radical polymerization under various reaction conditions is described. The effects of the initiators and solvents were examined. With 1,1,4,7,10,10‐hexamethyl triethylenetetramine/copper(I) chloride/p‐toluenesulfonyl chloride as the ligand/catalyst/initiator system in methanol, poly(DEAEMA) with a polydispersity index as low as 1.07 was synthesized. Kinetic studies demonstrated the polymerization was very well controlled and exhibited the living characteristic of the process. Well‐defined block copolymers of DEAEMA and tert‐butyl methacrylate (tBMA) were successfully synthesized. The copolymers could be synthesized with equally good results by starting with either p(DEAEMA) or p(tBMA) as the macroinitiators. However, only the macroinitiators terminated with chlorine should be used. The corresponding macroinitiators with bromine as a transferable group did not yield well‐defined copolymers. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 2688–2695, 2003     

Poly(N-acylethylenimine) copolymers containing pendant pentamethyldisiloxanyl groups. I. Synthesis

10-(Pentamethyl disiloxanyl) decyl oxazoline ( Si ) was synthesized. It was copolymerized with either undecyl ( U ) or nonyl ( N ) oxazolines using methyl 4-nitrobenzenesulfonate as initiator. Two series of random poly(N-acylethylenimine) copolymers, U/Si and N/Si , were synthesized over the whole composition range of Si monomer with a total degree of polymerization of about 100. Narrow molecular weight distributions were obtained. At a monomer to initator ratio of about 1060, the final degree of polymerization was 374 with a polydispersity index of 1.93. This shows the effect of chain transfer in this system.     

FTIR-ATR Monitoring and SEC/RI/MALLS Characterization of ATRP Synthesized Hyperbranched Polyacrylates

This work reports the synthesis at 1 L scale of hyperbranched polyacrylates based upon acrylate/diacrylate monomers such as n-butyl acrylate (nBA)/1,6-hexanediol diacrylate (HDDA) and using atom transfer radical polymerization (ATRP). A FTIR-ATR immersion probe was used to monitor the polymerization reaction. The dynamics of the build-up of polymer structure was studied by off-line analysis of samples at different reaction times by size exclusion chromatography (SEC) with detection of refractive index (RI) and multi-angle laser light scattering (MALLS) signals, leading to molecular weight distribution and z-average radius of gyration. Kinetic measurements and observed parameters of the molecular architecture are compared with theoretical predictions which can be used to design new synthesis strategies to improve the homogeneity of hyperbranched polymers. Another goal of this study was elucidating the impact on polymerization of secondary reactions such as intramolecular cyclizations. For comparison purposes, FRP (conventional radical polymerization) of the same monomers is also considered.     

Anionic polymerization of styrenic macromonomers of polyisoprene,polybutadiene, and polystyrene

Anionic polymerization and high‐vacuum techniques were used to prepare a series of well‐defined polyisoprene, polybutadiene, and polystyrene polymacromonomers. The procedure involved (1) the synthesis of styrenic macromonomers in benzene by the selective reaction of the corresponding macroanion with the chlorine of 4‐(chlorodimethylsilyl)styrene (CDMSS) and (2) the in situ anionic polymerization of the macromonomer without previous isolation. The synthesis of the macromonomers [polyisoprene macromonomer: 11 samples, weight‐average molecular weight (M_w) = 1000–18,000; polybutadiene macromonomer: 5 samples, M_w = 2000–4000; and polystyrene macromonomer: 2 samples, M_w = 1300 and 3600] was monitored by size exclusion chromatography with refractive index/ultraviolet detectors. Selectivity studies with CDMSS indicated that polybutadienyllithum had the highest selectivity, and polystryryllithium the lowest. From kinetic studies it was concluded that the polymerization half‐life times were longer but comparable to those of styrene, and they appeared to only slightly depend on the molecular weight of the macromonomer chain (at least for low degrees of polymerization of the polymacromonomer and for M_w < 7000 for the macromonomer side chain). Dependence on the polymerization degree of the polymacromonomer product was also observed. All the prepared polymacromonomers were characterized by size exclusion chromatography with refractive index, ultraviolet and two‐angle laser light scattering detectors, and NMR spectroscopy. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 1038–1048, 2005     

Atom Transfer Radical Polymerization of Styrene Using Multifunctional Iniferter Reagents as Initiators

Summary: Two multifunctional iniferters, 1,4-bis-(α-N,N-diethyldithiocarbamyl-isobutyryloxy)-benzene (BDCIB) and 1,3,5-tris-(α-N,N-diethyldithiocarbamyl-isobutyryloxy)-benzene (TDCIB), were successfully synthesized and used as initiators to initiate the polymerization of styrene in the presence of a CuBr/PMDETA complex. The polymerization results demonstrated that the kinetic plots in all cases were first-order to the monomer, the molecular weight of the polymers increased linearly with the monomer conversion; meanwhile, the molecular weight distribution of the polymer was kept to a very low value (M_w/M_n ≤ 1.35). Furthermore, the measured molecular weights were very close to the calculated values, which indicated the high efficiency of the initiator for the polymerization of styrene. The effect of catalyst concentration and initiator concentration was not obvious and the influence of polymerization temperature was apparent, and the polymerization rate increased with the polymerization temperature. The results of chain-extension and ¹H NMR analysis proved that the polymer obtained was capped with diethylthiocarbamoylthiy (DC) group.     

Large‐scale extrusion processing and characterization of hybrid nylon‐6/SiO2 nanocomposites

Solution impregnations, pulltrusion and film stacking are widely used methods to prepare thermoplastic composite materials. Extruders are used to melt the polymer and to incorporate fibers into the polymer in order to modify physical properties. In this article, the compounding of colloidal silica nanoparticles filled polyamide‐6 (PA‐6) is achieved using a twin‐screw extruder, which has a significant market share due to its low cost and easy maintenance. The experiments were performed at 250 rpm and the bulk throughput was 6 kg h^?1 with a pump pressure of 30 bars. The composites were characterized with nuclear magnetic resonance (NMR), wide angle X‐ray diffraction (WAXD), differential scanning calorimetry (DSC) and transmission electron microscopy (TEM). As determined by WAXD, the PA‐6 showed higher amounts of γ‐phase when compared to other synthesis methods such as in situ polymerization. TEM pictures showed that the silica particles aggregated nevertheless, upon addition of 14% (w/w) silica the E‐modulus increased from 2.7 to 3.9 GPa indicating that an effective mechanical coupling with the polymer was achieved. The behavior, illustrated with dynamic mechanical analysis (DMA) curves, indicated that in general when a filled system is compared to unfilled material, the values of the moduli (E′ and E″) increased and tan δ decreased. Determination of molecular mass distribution of the samples by means of size exclusion chromatography (SEC) coupled to a refractive index (RI), viscosity (DV) and light scattering (LS) detector revealed that the addition of silica did not decrease the average molecular weight of the polymer matrix, which is of importance for composite applications. Copyright © 2004 John Wiley & Sons, Ltd.