High performance microcolumn exclusion chromatography of proteins and peptides

A new method for the determination of the molecular weight of proteins and peptides has been developed. It is based on microcolumn exclusion chromatography in trifluoroacetic acid on silica gel sorbents of different porosities with a linear molecular-weight calibration dependence in the range of 5 × 10² - 7 × 10⁴ Da. It was shown that in this eluent proteins and peptides adopt the random-coil conformation and do not undergo hydrolysis for 2–3 days at room temperature.     

分子排阻色谱硅质填料的制备及其对蛋白质的分离机理

利用自制的四种不同粒径的硅溶胶,通过堆积法来制备分子排阻色谱多孔硅质填料,该填料在进行化学键合改性后,形成二醇固定相。利用二醇固定相对蛋白质进行分离分析方面的研究。此填料粒径小,有利于蛋白质生物大分子的高效快速分离分析。     

Anion-exchange and size-exclusion chromatography of proteins in mouse ascites fluid: Changes in the protein pattern during tumour growthIn vivo

Summary During the transition of Ehrlich mouse ascites tumour cells from the proliferating to the resting state of growth a large loss of purine and pyrimidine compounds occurs. This decrease is accompanied by a change in the amount of protein in the supernate of ascites fluid, which is known from the study of the ATP-consumption during protein synthesis. The ascites fluid was investigated by anion-exchange and size-exclusion chromatography (SEC). SDS-PAGE (sodium, dodecyl sulfate-polyacrylamide gel electrophoresis) data were compared with SEC data. The total amount of protein increased by 50% between day 5 and 12 of growth. At least 5 new peaks are observed in the chromatograms of an ion-exchange separation of Ehrlich ascites fluid at day 12 postinoculation. The amounts of transferrin, albumin and IgG (immunoglobulin G) were increased to 132, 134, 157%, respectively duringin vivo growth.     

Error introduced into determination of pore size distribution by size exclusion chromatography

Simultaneous use of large standard molecules and small particles of the product examined gives rise to errors in pore size determination by size exclusion chromatography. This error is calculated for packings of spherical particles, thus making corrections possible.     

Oscillatory transverse electric field enhances protein resolution and capacity of size-exclusion chromatography

Protein separations by a novel size-exclusion electrochromatography (SEEC) are presented. The present SEEC, denoted as pSEEC, was established with an oscillatory low-voltage electric field perpendicular to the mobile-phase streamline. Retention experiments with different proteins indicated that the influence of electric field strength on the partition coefficient is different for different proteins as well as for the same protein under different mobile-phase conditions. These results of protein retention led to the experimental design of protein separations with binary mixtures of BSA and immunoglobulin G (IgG), myoglobin (Myo) and lysozyme (Lys), as well as ovalbumin (Oval) and Myo. The separation results for the binary protein systems sufficiently exhibited the applicability of the pSEEC for various separations in terms of their molecular weights (MWs) as well as pIs. For example, it was possible to separate the gel-excluded proteins (BSA/IgG) as well as gel-permeable and similar-molecular-weight proteins (Myo/Lys) by the pSEEC. Moreover, in the cases of Oval/ Myo, which could be partially separated by size-exclusion chromatography, the use of the pSEEC greatly improved the resolution and the separation became possible at high sample loading. The results indicate that the pSEEC technology is promising for preparative protein separations.     

Indirect fluorimetric detection of proteins via postcolumn mixing with fluorescence probe in size-exclusion chromatography

Summary Proteins were visualized by postcolumn mixing with 2-p-toluidinyl-6-naphthalene sulfonate or 1-anilino-8-naphthalene sulfonate in size-exclusion chromatography. The indirect detection is based on fluorescence enhancement of the fluorescence probe owing to hydrophobic interaction with proteins. Bovine serum albumin gave the highest signal intensity among the proteins examined.     

Separation of pyranoanthocyanins from red wine by column chromatography

With the aim of monitoring the formation of anthocyanin-derived pigments and contributing to the study of their chromatic properties, stability and relative contribution to the colour of red wines, a method for fractionation of the colouring material was set up. The method was based on the distinct reactivity of the different pigment families towards bisulfite (hydrogen sulfite). The wine, acidified and bleached with NaHSO₃, was placed in a Toyopearl^® HW-40(s) gel column and submitted to elution with ethanol. Two fractions with different pigment compositions were collected and analysed by liquid chromatographay diode array detection-mass spectrometry. Compounds present in each fraction were identified according to their UV-visible and MSⁿ mass spectra, showing that the first one was mostly constituted of pyranoanthocyanins, whereas the second basically contained anthocyanins and anthocyanin-flavanol condensation products. A large variety of new pigments were detected, some of which had not been previously reported in red wines, as far as we know. Characteristic MS² and MS³ fragmentation patterns were observed within each family of compounds, which could be further applied for characterisation of unknown pigments in other wines.     

Size‐exclusion chromatography for the determination of the boiling point distribution of high‐boiling petroleum fractions

The paper describes a new procedure for the determination of boiling point distribution of high‐boiling petroleum fractions using size‐exclusion chromatography with refractive index detection. Thus far, the determination of boiling range distribution by chromatography has been accomplished using simulated distillation with gas chromatography with flame ionization detection. This study revealed that in spite of substantial differences in the separation mechanism and the detection mode, the size‐exclusion chromatography technique yields similar results for the determination of boiling point distribution compared with simulated distillation and novel empty column gas chromatography. The developed procedure using size‐exclusion chromatography has a substantial applicability, especially for the determination of exact final boiling point values for high‐boiling mixtures, for which a standard high‐temperature simulated distillation would have to be used. In this case, the precision of final boiling point determination is low due to the high final temperatures of the gas chromatograph oven and an insufficient thermal stability of both the gas chromatography stationary phase and the sample. Additionally, the use of high‐performance liquid chromatography detectors more sensitive than refractive index detection allows a lower detection limit for high‐molar‐mass aromatic compounds, and thus increases the sensitivity of final boiling point determination.     

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.     

Interlaced size exclusion liquid chromatography of monoclonal antibodies

Size exclusion chromatography is a widely performed analysis of monoclonal antibodies, primarily used to monitor the levels of higher weight molecular species such as aggregates. Owing to the subtleties of these separation mechanisms and frequently observed partial resolutions of components in these separations, many common methods for increasing the method throughput are not practical as they trade off resolution for speed. Short columns, high flow rates and smaller particles are examples of these approaches. In this paper a practical method is demonstrated for injecting samples onto the column in rapid succession and gating the detection window to monitor the elution of each sample individually. At any given instant approximately two samples are eluting through the column. By co-ordinating the injection and detection time windows the samples can be kept discrete and significant throughput enhancements achieved, up to nearly 2-fold improvements are demonstrated. A rudimentary theory is development to show that the throughput improvements can be predicted to approximation by simple column characteristics. Experimental results for a series of monoclonal antibodies demonstrate the equivalency of the method to a conventional injection approach, the throughput increase, and the robustness of the method.     

Size exclusion chromatography on porous fractals

Summary Some porous packings used in chromatography have been claimed to be fractals with a scale of sizes a<l<L, where a is a molecular size and L is the size of the largest pores. For a fractal porous packing, the excluded volume for molecules in solution in the vicinity of the packing surface is directly related to D_f, the fractal dimension of the pore surface (2<D_f<3). Since retention in size exclusion chromatography is itself directly related to this excluded volume, the fractal nature of the packing provides a model of retention in this technique. According to this model there is a linear relationship between log R_s and log(1-K_d), where R_s is the hydrodynamic radius of the solute macromolecules and K_d the distribution coefficient. The fractal dimension is derived from the slope of this plot. Size exclusion chromatographic retention data have been analyzed according to the model. It is found that some HPLC packings are fractals with fractal dimensions ranging from about 2.15 to 2.6, depending on the material. Such a large range of D_f values indicates large variations in the selectivities and domains of applications of the different packings. For some classical gel filtration chromatographic gels, the fractal retention model does not seem to apply.Presented at the 17th International Symposium on Chromatography, September 25–30, 1988, Vienna, Austria.     

Size-exclusion separation of proteins using a biocompatible polymeric monolithic capillary column with mesoporosity

Biocompatible poly(ethylene glycol methyl ether acrylate-co-polyethylene glycol diacrylate) monoliths were prepared for size exclusion chromatography (SEC) of proteins in the capillary format using Brij 58P in a mixture of hexanes and dodecanol as porogens. The monolithic columns provided size separation of four proteins in 20 mM sodium phosphate buffer (pH 7.0) containing 0.15 M NaCl, and there was a linear relationship between the retention times and the logarithmic values of the molecular weights. Compared to SEC monoliths previously synthesized using a triblock copolymer of polyethylene oxide and polypropylene oxide, an increase in mesoporosity was confirmed by inverse size exclusion chromatography. As a result, improved protein separation in the high molecular weight range and reduced column back-pressure were observed.     

Chromatographic analysis of allergens

Summary Extracts of insect, mite, fungal and mammalian origin, known to provoke an allergic response in man, have been examined and fractionated by high-performance size exclusion chromatography. The mild conditions employed for separation have permitted direct immunological assessment of the fractions obtained, and recognition of the biologically important components. These are being isolated for further study. This chromatographic procedure has proved to be of value for assessing different extracts from the same source material as potential reference preparations. It can also be employed as a sensitive method for identification of extracts derived from a variety of sources.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Characterization of complex hydrocarbon mixtures using on-line coupling of size-exclusion chromatography and normal-phase liquid chromatography to high-resolution gas chromatography

An on-line coupling of size-exclusion Chromatography (SEC), normal-phase liquid Chromatography (NPLC), and gas Chromatography (GC) for the characterization of complex hydrocarbon mixtures is described. The hyphenated system separates according to size, polarity, and boiling point. The use of size exclusion as the first separation step allows for the direct injection of complex (“dirty”) samples withont prior clean-up. SEC-NPLC coupling was realized using an on-line solvent evaporator based on fully concurrent solvent evaporation (FCSE) using a modified loop-type interface, vapor exit and co-solvent trapping. Complete reconcentration of the analytes was realized by the introduction of a cryogenic cold trap. For the subsequent hydrocarbon group-type separation an ammo-silica column with n-heptane as eluent was used. The NPLC-GC coupling was based on an on-column interface using partially concurrent solvent evaporation (PCSE) and an early vapor exit. Initial results obtained on the analysis of a residue from the atmospheric crude-oil distillation (a so-called long residue) are presented as an example of the enormous separation power of the SEC-NPLC-GC system. The application of the system for quantitative analysis has not yet been studied.     

Semi‐micro reversed‐phase liquid chromatography for the separation of alkyl benzenes and proteins exploiting methacrylate‐ and polystyrene‐based monolithic columns

Monolithic columns were synthesized inside 1.02 mm internal diameter fused‐silica lined stainless‐steel tubing. Styrene and butyl, hexyl, lauryl, and glycidyl methacrylates were the functional monomers. Ethylene glycol dimethacrylate and divinylbenzene were the crosslinkers. The glycidyl methacrylate polymer was modified with gold nanoparticles and dodecanethiol (C₁₂). The separation of alkylbenzenes was investigated by isocratic elution in 60:40 v/v acetonitrile/water. The columns based on polystyrene‐co‐divinylbenzene and poly(glycidyl methacrylate)‐co‐ethylene glycol dimethacrylate modified with dodecanethiol did not provide any separation of alkyl benzenes. The poly(hexyl methacrylate)‐co‐ethylene glycol dimethacrylate and poly(lauryl methacrylate)‐co‐ethylene glycol dimethacrylate columns separated the alkyl benzenes with plate heights between 30 and 60 μm (50 μL min^?1 and 60°C). Similar efficiency was achieved in the poly(butyl methacrylate)‐co‐ethylene glycol dimethacrylate column, but only at 10 μL min^?1 (0.22 mm s^?1). Backpressures varied from 0.38 MPa in the hexyl methacrylate to 13.4 MPa in lauryl methacrylate columns (50 μL min^?1 and 60°C). Separation of proteins was achieved in all columns with different efficiencies. At 100 μL min^?1 and 60°C, the lauryl methacrylate columns provided the best separation, but their low permeability prevented high flow rates. Flow rates up to 500 μL min^?1 were possible in the styrene, butyl and hexyl methacrylate columns.     

Effect of method of solution on observed molecular weight distribution of barley starch and starch derivatives in size exclusion chromatography

Summary Samples of native barley starch and six starch derivatives were suspended (0.1% sample concentration) in four different solvents: the eluent (pH 11 buffer), dimethylsulfoxide, 0.1 M NaOH or 0.5 M NaOH and kept in a boilling water bath for 5 to 60 minutes or shaken for 60 minutes. The average molecular weight values , and the polydispersity value were determined with a TSK PW-type column using narrow standard calibration. Only a small part of the samples dissolved in the eluent. The dissolution of sample in dimethylsulfoxide was dependent on sample type. Of the NaOH solutions, 0.5 M NaOH was the only one that dissolved all the samples. Therefore, 0.5 M NaOH appears to be the solvent of choice for starch molecules.