基于硅胶整体材料研磨颗粒的多功能聚乙二醇键合固定相的制备及其在高效液相色谱多模式分离中的应用

研究通过对溶胶-凝胶法制备的硅胶整体材料进行研磨、浮选、假晶相转换和水热处理，最终获得了粒径为2~5 μm、孔径为20~60 nm的硅胶颗粒。利用部分含氟的阴离子表面活性剂Capstone FS-66和常用的阳离子表面活性剂十六烷基三甲基溴化铵（CTAB）组成的双胶束模板体系对硅胶基质进行假晶相转换处理；再采用碳酸钠溶液水热处理的方式，进一步扩大孔径。用扫描电镜（SEM）和N₂吸附-解吸等温线测量对扩孔处理前后的硅胶整体材料研磨颗粒进行表征，结果清楚地显示了处理前后的形貌变化和差异。随后将含有长链聚乙二醇（PEG）的硅烷键合到扩孔后的硅胶颗粒表面，分别利用元素分析、红外光谱以及热重分析对固定相进行表征，并对固定相进行色谱性能评价。对键合固定相的元素分析和热重分析数据进行分析表明，硅胶表面键合PEG的含量约为8%。研究揭示了利用假晶相转换法与碳酸钠溶液水热处理和长链PEG硅烷修饰的硅胶整体材料颗粒在尺寸排阻色谱分离蛋白质方面的良好分离效果。同时进一步的高效液相色谱评价结果表明，该键合固定相还可用于疏水作用色谱模式分离核糖核酸酶A和溶菌酶，以及可用于亲水作用色谱模式分离吡啶甲酸、左旋多巴、三聚氰胺和邻苯二酚等极性比较强的化合物。研究显示了PEG键合固定相具有多功能性，及其在多模式高效液相色谱分离中的应用潜力。     

Application of high-performance liquid chromatography to the purification of the putative intestinal peptide transporter

A membrane protein of relative molecular mass (Mr) 127,000 was identified by photoaffinity labelling as (a component of) the uptake system for small peptides and beta-lactam antibiotics in rabbit small intestine. This binding protein is a microheterogeneous glycosylated integral membrane protein which could be solubilized with non-ionic detergents and enriched by lectin affinity chromatography on wheat germ lectin agarose. For the final purification of this protein and separation from aminopeptidase N of Mr 127,000, fast protein liquid chromatography (FPLC) was used. Gel permeation, hydroxyapatite and hydrophobic interaction chromatography were not successful for the purification of the 127,000-dalton binding protein. By anion-exchange chromatography on a Mono Q column with either Triton X-100 or n-octylglucoside as detergent, a partial separation of the 127,000-dalton binding protein from aminopeptidase N was achieved. By cation-exchange chromatography on a Mono S HR 5/5 column at pH 4.5 using Triton X-100 as detergent also only a partial separation from aminopeptidase N could be achieved. If, however, Triton X-100 was replaced with n-octylglucoside, the binding protein for beta-lactam antibiotics and small peptides of Mr 127,000 could be completely separated from aminopeptidase N. These results indicate that Triton X-100 should be avoided for the purification of integral membrane proteins because mixed protein-detergent micelles of high molecular weight prevent a separation into the individual membrane proteins. The putative peptide transport protein was finally purified by rechromatography on Mono S and was obtained more than 95% pure as determined densitometrically after sodium dodecyl sulphate gel electrophoresis. By application of FPLC even microheterogeneous membrane glycoproteins from the intestinal mucosa can be purified to such an extent that a sequence analysis and immunohistochemical localization with antibodies prepared from the purified protein is possible.     

Analysis of polyethyleneoxide macromonomers by liquid chromatography along the critical adsorption line

Polyoxyethylene macromonomers are analyzed by one-dimensional liquid chromatography under different conditions, depending on the required information. These samples may contain polyethylene glycol (PEG) and the corresponding di(meth)acrylate besides the desired mono(meth)acrylate. The molar mass distribution (MMD) of the PEG and the monoester can be obtained by liquid adsorption chromatography (LAC) on a reversed-phase column in acetone–water with a gradient from 10% to 20% acetone. The MMD of the diesters can be obtained with isocratic elution by liquid chromatography at critical conditions (LCCC) on a reversed-phase column in 31% acetone, or using size-exclusion conditions for PEG and LAC conditions for the end groups, which is the case in 40–55% acetone. The absolute amount of the series with different functionality can be obtained by LCCC in ternary mobile phases consisting of acetone, methanol, and water along the critical adsorption line. Under such conditions, all series elute as narrow peaks (regardless their MMD), which can easily be integrated and quantified.     

Determination of polyethylene glycol in low-density polyethylene by large volume injection temperature gradient packed capillary liquid chromatography

Polyethylene glycol (PEG) 20000 in low-density polyethylene has been determined using column switching and inverse temperature programming in reversed-phase packed capillary liquid chromatography with evaporative light scattering detection. PEG 20000 was extracted into water from the polyethylene dissolved in toluene and PEG 35000 was added as an internal standard (I.S.). The samples in aliquots of 100 microl were reconcentrated on the enrichment column using a loading mobile phase of acetonitrile-water (3:97, v/v) at a flow-rate of 75 microl/min for 3 min, then back-flushed and separated on the analytical column with acetonitrile-THF-water (40:5:55, v/v) as mobile phase. The column temperature was reduced from 68 to 55 degrees C with a ramp of -1.5 degrees C/min, held constant for 3 min and then reduced further to 45 degrees C with a -1.5 degrees C/min ramp and kept constant for 1 min. The analysis runtime was 20 min. The recovery of PEG 20 000 was determined to 65.1% with 2.8% RSD and the mass limit of detection of PEG 20 000 was 1.25 microg. The within-assay and between day precision of the retention times of both PEG 20000 and PEG 35000 displayed RSD of less than 1.1% (n = 9), while the overall area ratio RSD of 100 microg/ml PEG 20000 over PEG 35000 was 1.3% (n = 9). The method was linear within the investigated concentration range 25-125 microg/ml (R2 = 0.9983). In addition, a mixture of PEG 4000, 8000, 10000, 20000 and 35000 was analysed on the system to demonstrate the possibility of analysing several PEGs in a sample with the use of temperature gradient elution.     

Tensammetry with accumulation on the hanging mercury drop electrode: Part 2. The behaviour of mixtures of Poly(ethylene glycols) as an Example of Surfactant Mixtures

Model investigations with two-, three- and four-component mixtures of poly(ethylene glycols) (PEG) having different molecular weights (1500–20 000) are described. Three different types of mixture can be distinguished. The first group comprises mixtures of components which have very similar properties and behave additively; such mixtures give only one peak, the height of wich depends linearly on the total concentration of PEG. Examples are mixtures of PEG 9000 with PEG 20 000, and PEG 6000 with PEG 9000 or PEG 20 000. The second group consits of mixtures of components with rather different properties; in such cases, a suitable choice of preconcentration potential enables one component to be determined with adequate precision, even in the presence a 100-fold amount of another component. Examples are mixtures of PEG 4000, PEG 9000 or PEG 20 000 with a 100-fold amount of PEG 1500, and multicomponent mixtures consisting of PEGs 6000, 9000 and 20 000 with PEG 1500 in excess; in the latter case, the three PEGs of higher-molecular-weight behave as a single component. The third group comprises mixtures of components which have similar properties, but which behave nonadditively; their properties are too similar for any component to be eliminated by choosing a suitable preconcentration potential, and two very close peaks of dubious usefulness are obtained. Mixtures of PEG 4000, 6000, 9000 and 20 000, or of PEG 4000, 9000 and 20 000 behave in this way.     

Size exclusion chromatography with Corona charged aerosol detector for the analysis of polyethylene glycol polymer

A technique of using size exclusion chromatography (SEC) with the Corona charged aerosol detector (CAD) was developed and evaluated in comparison with refractive index (RI) and evaporative light scattering detection (ELSD) for fast screening of polyethylene glycol (PEG), a polymer used in preparing pegylated pharmaceutical compounds. These detection techniques were used in the analysis of multiple lots of PEG reagents. CAD was found to provide more accurate impurity and polydispersity profiles of PEG reagents that better differentiate their quality, while RI was not suitable for this application due to its low sensitivity and ELSD led to underestimation of the impurity and polydispersity. The accuracy of polydispersity determination by SEC-CAD was validated against a commercial reference standard of known polydispersity. The SEC-CAD technique and the observed differences between the three detectors can also be applied to polymer analysis in general.     

PEGylation of an artificial O2 and CO receptor: synthesis, characterisation and pharmacokinetic study

A synthetic oxygen (O(2)) and carbon monoxide (CO) receptor (hemoCD) composed of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinatoiron(ii) and a per-O-methylated β-cyclodextrin dimer with a pyridine linker (Py3CD) was functionalised with poly(ethylene glycol) (PEG) to elongate the circulation time of the receptor in the bloodstream. α-PEG monocarboxylic acid (HOOC(CH(2))(3)(CO)O-PEG(mw)-OCH(3); mw = 750 or 5k) or α,ω-PEG dicarboxylic acid (HOOC(CH(2))(3)(CO)O-PEG(mw)-O(CO)(CH(2))(3)COOH; mw = 10k or 20k) was reacted with the amino group of 5-(4-aminophenyl)-10,15,20-tris(4-sulfonatophenyl)porphyrin to afford a porphyrin monomer having a PEG chain or a porphyrin dimer having a PEG linker, respectively. The ferrous complexes of these PEGylated porphyrins (PEG750-, PEG5k-, PEG10k- and PEG20k-hemoCDs) bound O(2) in aqueous solution, P(1/2) values being 6.5-8.1 Torr at pH 7.0 and 25 °C. Each PEG(mw)-hemoCD was infused into the femoral vein of a Wistar male rat. After 6 h of the infusions, 67, 82, 86 and 42% of PEG750-, PEG5k-, PEG10k- and PEG20k-hemoCD were excreted in the urine. PEG750-hemoCD with a hydrodynamic diameter (D(h)) of 3.4 nm seemed to partly leak from the blood vessels (pore size: 2-6 nm) before renal filtration (pore size: 4-14 nm). PEG5k- (D(h) = 6.2 nm) and PEG10k-hemoCDs (9.0 nm) hardly passed through the blood vessels but were fully filtered by the kidney, resulting in high excretion rates. A considerable amount of PEG20k-hemoCD (D(h) = 12.0 nm) was retained in the blood even at 6 h after administration. The present study demonstrates that the behaviour of hemoCD in blood after administration can be controlled by modification of hemoCD with PEG having an appropriate molecular weight.     

Polyethylene glyco-bonded phases for protein separation by high-performance hydrophobic interaction chromatography

Summary This paper further investigates the effects of silica base pore size and the molecular weight of polyethylene glycol (PEG) ligands on the coverage of PEG-bonded phases, as well as the resolution of protein separation in high-performance hydrophobic interaction chromatography (HPHIC). The results demonstrate that among the PEG-bonded phases examined in this study, the bonded phase coupled PEG-1500 on LiChrospher 500 silica exhibited the best resolution in protein separation.     

Theoretical analysis of the effects of reversible dimerization in size exclusion chromatography

Reversible dimer formation in size exclusion chromatography (SEC) can cause peak splitting, merging, tailing, and fronting. Such behavior can be predicted by the association rate and the dissociation rate relative to the convection rate. Slow association and dissociation result in separated monomer and dimer peaks. Fast association and slow dissociation result in one single dimer peak. Slow association and fast dissociation result in one single monomer peak. Intermediate association and dissociation result in a merged, broad peak with either fronting when monomers dominate or tailing when dimers dominate. A diagram based on the two relative rates is generated to predict general peak shape and retention behavior in SEC.     

Purification of human plasma lipid transfer protein using fast protein liquid chromatography

A system for the isolation of human plasma lipid transfer protein (LTP) has been devised using a combination of conventional and high-performance ion-exchange chromatography. Following initial purification by ammonium sulphate precipitation, ultracentrifugation, hydrophobic interaction and cation-exchange chromatography, appropriate fractions were further purified using the Pharmacia fast protein liquid chromatography system. Using this method of purification, human plasma LTP has been purified more rapidly and with greater recovery than with conventional column chromatography. Whereas two forms of LTP were previously reported from the authors' laboratory [LTP-I, molecular mass (Mr) 69,000 and LTP-II, Mr 55,000], with an improved chromatographic system only one form of LTP (LTP-I) has been isolated. This suggests that LTP-II may have been a fragment of LTP-I, produced during the previously used lengthy purification process.     

Applications of Poly(Ethylene)Glycol (PEG) in Separation Science

The wide range of applications of poly(ethylene)glycol (PEG) in primarily chromatography and other closely related analytical methods has been reviewed. PEG has been used as mobile phase modifier in capillary electrophoresis (CE) as well as ion exchange, size exclusion, and hydrophobic interaction liquid chromatography (LC) methods. Generally in the presence of PEG, LC retention of macromolecules is altered and stability of their structure is maintained. PEG was effective in CE as a permanent coating for fused silica capillaries to shield free silanol groups that can cause protein adsorption to the wall resulting in band broadening and low recovery of the separated proteins. In gas chromatography, PEG-based stationary phases were applied for separation of polar analytes. PEG could also serve as an extraction medium in solid phase microextraction and aqueous two phase systems. Selected analytical applications, primarily LC and CE, involving PEG to facilitate the determination of either small molecules or macromolecules such as proteins in their native form are discussed and representative figures provided.

     

Composition analysis of poly(ethylene glycol)-poly(L-lactide) diblock copolymer studied by two-dimensional column chromatography

We used two-dimensional column chromatography to analyze the composition of a sample of presumably a diblock copolymer of poly(ethylene glycol) (PEG) and poly(L-lactide) synthesized from monomethoxy-terminated PEG. The first dimension of the separation is phase fluctuation chromatography to prepare fractions that contain various components of the copolymer in different ratios. The second dimension is size-exclusion chromatography, NMR, and HPLC at the critical condition of PEG. The PEG initiator has small amounts of diol-terminated dimeric components. We found that the copolymer sample contains a triblock copolymer and low-molecular-mass components in addition to the main part of the diblock copolymer. The SEC chromatograms show that the main part consists of two components with distinct peak lengths for the PLLA block. The low-molecular-mass components have a broad distribution in chemical composition. Phase fluctuation chromatography enriched the triblock copolymer and the diblock copolymer with the longer PLLA block in early fractions when the column was packed with carboxymethyl-modified porous silica. When the porous medium was PLLA-grafted silica, size exclusion dominated, but the low-molecular-mass components were separated according to their chemical composition.     

Purification and modification by polyethylene glycol of a new human basic fibroblast growth factor mutant-hbFGF(Ser25,87,92)

The mutant of human basic fibroblast growth factor (hbFGF), hbFGF(Ser25,87,92), which was constructed by replacing the cysteine residues at the positions of the 25th, the 87th and the 92nd with serine residues, was coupled to polyethylene glycol (PEG) with a molecular size of 20 kDa (20K) (PEG(20K)) to obtain hbFGF derivative, PEG(20K)-hbFGF(Ser25,87,92). The optimal modified reaction was conducted at 12 degrees C for 12h with the molar ratio of PEG(20K) to hbFGF(Ser25,87,92) of 30:1. The result of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the modification rate was up to 60%. The PEGylated product retained binding affinity to heparin and could be purified by heparin affinity chromatography. Compared to hbFGF mutant, purified PEG(20K)-hbFGF(Ser25,87,92) retained about 34% of mitogenic activity. Heat-stability assay indicated that the modified product was more stable than the native protein at the temperature of 37 degrees C.     

The application of size exclusion chromatography to the analysis of biopolymers

High performance size exclusion chromatography (HPSEC), employing microparticulate, hydrophilic, surface-modified silicas or hydrophilic, cross-linked, organic polymers and buffered eluents, is an effective means of separating native and denatured proteins of molecular weights in the 10 000–500 000 range. HPSEC can be further used for molecular weight identification, and the fractionation and isolation of proteins.     

Refolding and purification of rhNTA protein by chromatography

RhNTA protein is a new thrombolytic agent which has potential medicinal and commercial value. Protein refolding is a bottleneck for large‐scale production of valuable proteins expressed as inclusion bodies in Escherichia coli. The denatured rhNTA protein was refolded by an improved size‐exclusion chromatography refolding process achieved by combining an increasing arginine gradient and a decreasing urea gradient (two gradients) with a size‐exclusion chromatography refolding system. The refolding of denatured rhNTA protein showed that this method could significantly increase the activity recovery of protein at high protein concentration. The activity recovery of 37% was obtained from the initial rhNTA protein concentration up to 20 mg/mL. After refolding by two‐gradient size‐exclusion chromatography refolding processes, the refolded rhNTA was purified by ion‐exchange and affinity chromatography. The purified rhNTA protein showed one band in SDS‐PAGE and the specific activity of purified rhNTA protein was 110,000 U/mg. Copyright © 2008 John Wiley & Sons, Ltd.     

Purification and identification of PEGlated hemoglobin, a potential blood substitute, by chromatography and capillary electrophoresis

Summary Bovine hemoglobin (Hb) has been chemically modified, by reaction of its lysine residues with the active ester of poly(ethylene glycol) (PEG,M _w=5000), to produce a potential blood substitute for human therapy. Covalent attachment of PEG chain to the protein produced a heterogeneous mixture of Hb from the mixture. This paper describes the use of cation-exchange chromatography (IEC), in flow-through mode, and size-exclusion chromatography (SEC) for purification of the PEG-Hb mixture. The highly modified Hb flowed through the IEC column in the loading buffer without adsorption by the chromatographic medium. SEC was then used for further purification. These two steps were suitable for pilot-scale preparation or for analytical chromatography. The purified product was assessed by high-performance capillary electrophoresis (HPCE), which was also used to optimize the chromatographic parameters.     

Suppression of cell and platelet adhesion to star-shaped 8-armed poly(ethylene glycol)-poly(L-lactide) block copolymer films

To explore the potential of a star-shaped 8-armed poly(ethylene glycol)35K-block-poly(L-lactide)37K (8-armed PEG35K-b-PLLA37K: M(n) of PEG = 35 000, M(n) of PLLA = 37 000) film as a novel bioabsorbable adhesion-prevention membrane, the water structure, surface contact angle, protein adsorption, and cell and platelet anti-adhesion properties of such a hydrated film are investigated. Based on the results, it is found that the 8-armed PEG35K-b-PLLA37K film exhibits a biologically inert surface, which is the result of a large number of PEG chains and a free water layer on the film surface. This leads to a reduction in protein absorption and cell and platelet adhesion onto the film surface. This implies that the star-shaped 8-armed PEG35K-b-PLLA37K film can be utilized as a novel bioabsorbable adhesion-prevention membrane.     

Calibration of chromatographic systems for quantitative prediction of chromatography of homopolymers

A method is proposed for obtaining from the experimental data the main parameters, which determine the chromatographic behavior of homopolymers: a dependence of the adsorption interaction parameter on the mobile phase composition, and the pore volume and pore size values. The interaction parameter can be determined by this method in both adsorption, critical, and SEC-type regimes of chromatography. The method is tested in ‘mathematical experiments’ with theoretical data that mimic experiments, and is applied to calibrate real chromatographic systems. With the obtained calibrations quantitative modeling of chromatograms of PEG samples on two typical ODS columns with different pore size in methanol/water and acetone/water mixed solvents is performed; the simulation results are compared with real chromatograms.     

Simply and effectively preparing high-purity phosvitin using polyethylene glycol and anion-exchange chromatography

An attempt was made to develop a new protocol for preparing phosvitin that could be easy scaled up using polyethylene glycol (PEG6000). Influence of PEG6000 concentration and pH of sample solution on phosvitin isolation was investigated. Phosvitin of high purity (99%) was obtained in good yield (47%) with the optimal condition of pH 4.0 and 3% PEG6000 precipitation. In addition, through evaluating different anion-exchange chromatography methods, the DEAE procedure at pH 7.5 was finally selected as the best procedure to obtain metal-free phosvitin that lost the least protein. Furthermore, it is observed that the purity and characterizations of prepared phosvitin were similar to those of the standard phosvitin from Sigma, and the random coil of phosvitin converted into more compact structure after removing metal ion. In conclusion, the developed method was simple and suitable for scaled up preparation of phosvitin.     

High-performance liquid chromatography of proteins using chemically-modified silica supports

Summary Highly efficient and fast exclusion-chromatographic separations of proteins are possible on chemically-modified, silica stationary phases. By optimizing the pH and the ionic strength of the aqueous eluent secondary interactions of the samples with surface groups can be excluded. Bonded propylamide groups proved to possess optimum properties for exclusion chromatography. With other functional groups adsorption effects cannot be excluded totally. The optimum pore size distribution for protein separation up to relative molecular masses of 500,000 daltnons is between 10nm and 50nm. With these silica-based phases the pore size distribution, the pore volume and the packing characteristics are independent of the eluent, therefore the same column can be used with aqueous as well with organic eluents. It is possible to correlate the elution volume (molecular size) of proteins with those of polystyrene standars. The recovery of the proteins and their biological activity has always been better than 90%. The potentialities of adsorption chromatography of proteins on chemically-bonded stationary plases with different functional groups are demonstrated.