Columns switch recycling size exclusion chromatography for high resolution protein separation

Columns switch recycling size exclusion chromatography (csrSEC) was proposed to achieve high resolution protein separation with good biocompatibility. Proteins were firstly separated by two serially coupled SEC columns, and fractions were in sequence switched back to the first column by two-position valves for further separation in terms of close-loop recycling until satisfactory resolution was achieved. Compared to SEC, the separation window was broadened by increasing column length via cycling without further increase on back pressure. Compared to recycling SEC (rSEC), the overtaking of later eluted components by early eluted ones after several cycles could be avoided for complex sample analysis, by parking fractions in the second SEC column before transferred in turn back to the first one for cycling ordinally. In our experiments, the baseline separation of five proteins with molecular weight ranging from 10 kDa to 80 kDa was achieved by csrSEC. Furthermore, a multidimensional csrSEC–RPLC platform was constructed, and peak capacity up to 3600 was obtained for protein separation. All these results demonstrated that csrSEC is a promising protein separation mode with good biocompatibility, broadened separation window and improved resolution.     

High-performance size exclusion chromatography as a rapid method for the separation of steroid hormone receptors

Triazine dyes, bound to polyethylene glycol, have been used to influence the partition of some enzymes within a dextran-polyethylene glycol-water two-phase system. The enzymes, present in a protein extract from baker's yeast, included glucose 6-phosphate dehydrogenase, glyceraldehyde phosphate dehydrogenase, 3-phospho-glycerate kinase and alcohol dehydrogenase. The partition coefficients of the enzymes could be changed by a factor of 10-500 in favour of the polyethylene glycol-rich phase, while the partition of bulk protein was much less affected. The influence of the concentration of polymer-bound dye and phase-forming polymers, temperature, pH, kind and concentration of salt and the presence of nucleotides on this affinity partitioning effect was studied. The extraction was effective even at high concentrations of dye and protein (40 g/l). A partial purification (32-fold) of glucose 6-phosphate dehydrogenase was carried out by an extraction in five steps.     

Dichloropentafluoropropanes as solvents for size exclusion chromatography

We have found that HCFC225s (HCFC225ca: 3,3-dichloro-1,1,1,2,2-pentafluoropropane, HCFC225cb: 1,3-dichloro-1,1,2,2,3-pentafluoropropane) are superior mobile phases for size exclusion chromatography (SEC). As alternatives of CFC113, they have been shown to possess a number of excellent properties, such as low flammability, low viscosity, low cost, high purity, and environmental and operational friendliness. In addition, they have distinct advantages for the SEC measurement, because they solubilize some kinds of acrylate such as poly(methyl methacrylate) (PMMA) and commercial monodispersed PMMA can be used to prepare calibration curves necessary for the measurement of equivalent molecular weight of some polymers. Furthermore, we propose an HCFC225/1,1,1,3,3,3-hexafluoroisopropanol mixed solvent for use in the SEC of poly(ethylene terephthalate) (PET) and polyamides. Poly(2-(perfluorooctyl)ethyl acrylate), whose PMMA equivalent weight average molecular weight was 118,100 Da, was evaluated by a multi-angle laser light scattering (MALLS) detector to have an absolute molecular weight of 439,000 Da. The difference can be attributed to the molecular size of the polyfluorinated polymer compared to the non-fluorinated one. The possible application of this novel mobile phase system for molecular size and molecular weight characterization of perfluoropolyethers, PET, nylon 6 and nylon 6,6 are also discussed.     

Prototype for integrated two-dimensional gel electrophoresis for protein separation

Two-dimensional gel electrophoresis practitioners have long waited for a fully automated system. This article presents an integrated platform that is capable of complete automation from sample introduction to spots detection. The strip gel for the first dimensional separation is fixed on the edge of a discrete planar stage before separation. A pair of platinum pin electrodes for isoelectric focusing (IEF) makes contact from underneath the stage. IEF is performed directly after rehydration and protein loading. After the first dimensional separation, sodium dodecyl sulfate (SDS) equilibration is done on the same stage without moving the gel. The IEF stage is then moved horizontally to couple with a precast second dimensional gel. The <0.5 mm gap between the two gels is filled with poly (ethylene oxide) solution. After SDS-polyacrylamide gel electrohporesis separation, a charge-coupled device camera is used to detect spots via protein native fluorescence excited by a Hg (Xe) lamp with the gel inside the running cell. Potential for full automation is demonstrated with 0.5 microg of Escherichia coli proteins on this miniaturized platform. More than 240 spots are detected in a total experiment time of <2.5 h.     

Off-line comprehensive two-dimensional high-performance liquid chromatography system with size exclusion column and reverse phase column for separation of complex traditional Chinese medicine Qingkailing injection

A comprehensive two-dimensional liquid chromatography system was constructed on the combination of size exclusion chromatography (SEC) and reverse phase liquid chromatography (RPLC). The first dimension used a SEC column with 300 mm x 8mm i.d., packed with Toyopearl HW-40S. The column was eluted with 0.05 mol/l Tris-HCl (pH 6.9) at a flow rate of 0.4 ml/min. The second dimension used a RPLC column with 100 mm x 4.6mm i.d., which was operated in gradient form at a flow rate of 1.0 ml/min. An automatic switching valve was used to collect the effluent of the SEC column every 3 min, and the effluent was automatically injected into the RPLC column. Mass spectrometer was used for peak identification. This system was used to separate Qingkailing injection, a traditional Chinese medicine (TCM). The result showed that the total peak capacity of this system could reach 1134 and the qualitative analysis of seven chemical components of the Qingkailing injection was accomplished by this system. The results show that comprehensive two-dimensional liquid chromatography system is of great importance and high value in the separation of complex TCM.     

Hexafluoroisopropanol as size exclusion chromatography mobile phase for polyamide 6

The present study deals with the use of hexafluoroisopropanol (HFIP) as size exclusion chromatography (SEC) mobile phase for polyamide 6 (PA6). Contradictory conclusions relating to the use of HFIP as SEC mobile phase for polyamides are found in the literature. By using a multi-detector SEC apparatus equipped with on-line viscometer and multi-angle light scattering we have studied the chromatographic artifacts and the validity of the universal calibration (UC) in HFIP for different PA6 samples (hydrolytic and anionic, monofunctional or bifunctional activator). Appropriate SEC columns and optimized experimental conditions allow most of the chromatographic artifacts to be avoided, even in neat HFIP. The use of a salt in the mobile phase, namely 0.01 M tetraethylammonium nitrate (TEAN), slightly increases the elution volume for both PA6 and PMMA polymers. Under the right conditions, the UC substantially holds for PA6. The validity of the UC is not linked to the presence of TEAN in the mobile phase. With some PA6 samples, namely those anionically synthesized using a bifunctional activator, aggregation becomes a problem and the molar mass in neat HFIP is overestimated. Addition of TEAN prevents any aggregation of the above anionically synthesized PA6. In contrast, the use of a different salt, namely potassium trifluoroacetate, increases the extent of aggregation.     

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。     

Recent progress in carbohydrate separation by high-performance liquid chromatography based on size exclusion

Since the introduction of stationary phases based on microparticulate porous silica and polymeric sorbents, rigid and semi-rigid, size-exclusion chromatography (SEC) has become established as a form of high-performance liquid chromatography. In recent years, there have beeen revolutionary developments in detection systems for high-performance SEC, which have placed the use of the method for the determination of molecular-size and molecular-weight distributions of polymers on a sound theoretical basis andincreased the range of information on molecular characteristics that can be retrieved from SEC data. This review surveys these changes in SEC systems and their application to the separation and molecular-weight distribution analysis of carbohydrates.     

Phenomena of insulin peak fronting in size exclusion chromatography and strategies to reduce fronting

Insulin peak fronting in size exclusion chromatography (SEC) results in more than 10% yield loss in the production of insulin. The goal of this study is to understand the mechanisms of peak fronting and to develop strategies to reduce fronting and increase insulin yield. Chromatography experiments ruled out pressure surge, viscous fingering, and adsorption as the cause for peak fronting. Theoretical analysis based on a general rate model indicated that reversible dimerization is the major cause for peak fronting and reducing the dimerization equilibrium constant is the most effective method for reducing fronting. Two strategies were developed and tested to reduce the degree of dimer formation. The first strategy was to use 0.1N acetic acid as the presaturant and eluent. The second strategy was to use 0.8 or 2.8N acetic acid in 20vol.% denatured ethanol as the mobile phase. The experimental results showed that both strategies can reduce insulin peak fronting in SEC, maintain desired product purity, and increase insulin yield to higher than 98%.     

Composite dextran gels as size exclusion chromatography stationary phases

Composite dextran gels have been prepared using epichlorohydrin as the covalent cross-linker and a modifying agent, typically a polyvalent metal compound creating complexes with the residual OH groups of the polymer matrix. Two series of gels were prepared and investigated by a comparison of their swellabilities in water as well as the separation characteristics when used as size exclusion chromatography (SEC) stationary phases. The gels were in situ modified with sulfates of Mg(II), Zn(II), Co(II), Ni(II), Cu(II), Mn(II), Al(III), Fe(III) and tetraethylorthotitanate or with the oxides: MgO, ZnO, Al₂O₃, TiO₂ and SiO₂. Large differences in the swellabilities and SEC calibration dependences for poly(ethylene glycol) standards in an aqueous eluent have been found and discussed.     

Novel polystyryl resins for size exclusion chromatography

New size exclusion chromatography (SEC) resins based on a crosslinker having independent vinyl groups have been produced and compared with SEC resins based on divinylbenzene-55 (DVB). 1,2-Bis(p-vinylphenyl)ethane (p,p-BVPE) and its meta-isomers were suspension-copolymerized with p-methylstyrene in the presence of different porogens to give particles of about 5 μm average diameter. The porous particles were slurry-packed into stainless steel columns for SEC evaluation. Calibration curves were obtained using narrow disperse polystyrene standards with molecular weights ranging from 580 to 3,040,000. The calibration curves for the new BVPE resins covered wider useful molecular weight ranges than those for comparable divinylbenzene resins. Particle size, surface morphology and the properties of pores were studied using a Coulter Multisizer II, scanning electron microscopy, nitrogen adsorption, and mercury porosimetry. © 1994 John Wiley & Sons, Inc.     

Silica nanoparticles as pseudostationary phase for protein separation

This paper investigated the potential use of silica nanoparticles (SNPs) as pseudostationary phase (PSP) for protein separation. The wall adsorption of SNPs as well as the influences of SNPs and poly(ethylene oxide) (PEO) were studied. The SNPs showed selectivity toward the proteins, and the concentration ratio of SNPs to PEO influenced the separation. The proteins that could not be baseline-resolved under conventional CZE mode were separated in a buffer consisting of 30 mM phosphoric acid, 0.05% PEO, and 0.05% SNPs at pH 2.37, with detection limits ranging from 2 to 45.5 ppm. The intraday and interday RSDs of the migration times were in the ranges of 2.1-2.8% (n = 5) and 2.5-3.4% (three days, n = 3 x 5 = 15), respectively.     

Relations between the separation coefficient,longitudinal displacement and peak broadening in size exclusion chromatography of macromolecules

The separation of a polymer by size exclusion chromatography is described as a series of interactions, i.e. consecutive establishments of equilibria between polymer fractions in the mobile and stationary phases followed by displacements of mobile phase containing the polymer. The elution curve is derived as the longitudinal concentration profile in the column observed in one position in space during the time of the analysis. The mean value of elution volume of a particular polymer species turns out to be the interstitial volume of the separation system divided by the mean fraction of polymer in the mobile phase. The number of the displacement-equilibrium steps can be estimated from the limiting values of the variance of the spreading function.     

Comparison of in-gel protein separation techniques commonly used for fractionation in mass spectrometry-based proteomic profiling

Fractionation of complex samples at the cellular, subcellular, protein, or peptide level is an indispensable strategy to improve the sensitivity in mass spectrometry-based proteomic profiling. This study revisits, evaluates, and compares the most common gel-based protein separation techniques i.e. 1D SDS-PAGE, 1D preparative SDS-PAGE, IEF-IPG, and 2D-PAGE in their performance as fractionation approaches in nano LC-ESI-MS/MS analysis of a mixture of protein standards and mitochondrial extracts isolated from rat liver. This work demonstrates that all the above techniques provide complementary protein identification results, but 1D SDS-PAGE and IEF-IPG had the highest number of identifications. The IEF-IPG technique resulted in the highest average number of detected peptides per protein. The 2D-PAGE was evaluated as a protein fractionation approach. This work shows that the recovery of proteins and resulting proteolytic digests is highly dependent on the total volume of the gel matrix. The performed comparison of the fractionation techniques demonstrates the potential of a combination of orthogonal 1D SDS-PAGE and IEF-IPG for the improved sensitivity of profiling without significant decrease in throughput.     

Inverse size exclusion chromatography and universal calibration

The mechanism of size-exclusion chromatography (SEC) is reciprocal in respect to the properties of the porous material and of the solutes immersed. The porosimetric interpretation (inverse SEC) yields information about the morphology of porous solids. It depends on a number of assumptions and in particular on the steric properties of the probing macromolecules whose ‘Universal Calibration’ remains disputed to this day. The central question concerns the equilibrium distribution of molecules in confined spaces of complex geometry, and is, as such, not limited to the particulars of chromatographic technique. This survey hopes to help focus future research activities in this field of analytical chemistry and material science.     

Optimization strategies for off-line two-dimensional liquid chromatography

A step by step strategy of optimization of comprehensive off-line two-dimensional liquid chromatography (2D-LC) separations is proposed. The goal of an optimization process in the separation sciences is either to achieve a given resolution (a target peak capacity in 2D-LC) within as short a time as possible or to reach the highest possible resolution in a given analysis time. The proposed method takes into account the characteristics of the columns used in the first and the second dimension and the number of fractions of the first dimension eluent that should be collected. The effect of the time spent during the analysis on the second dimension column to carry out necessary tasks that are not the separation itself (called the additional time) on the maximum peak capacity that is achievable was carefully investigated. It was shown that (1) an increase in the peak capacity of the first dimension column combined with the collection of larger volume fractions permits a significant reduction of the time needed to achieve the desired peak capacity; and (2) there is an optimum fraction collection ratio (or number of collected fractions per peak) which yields the target peak capacity in the minimum time. The proposed strategy was used for the optimization of the separation of samples of BSA tryptic digest by an off-line 2D-LC using an SCX⊗RP$\text{SCX}\otimes \text{RP}$-HPLC method. As a result of this optimization, a peak capacity of 4000 could be achieved in about 5 h with the two columns available. The time needed for the optimized analysis was less than two thirds of the analysis time that would have been needed had the conventional rule of thumb of sample collection in comprehensive on-line 2D-LC (4 samples/peak) been followed.     

Recent advances in proteolysis and peptide/protein separation by chromatographic strategies

This review gives a broad glance on the progress of recent advances on proteolysis and peptide/protein separation by chroma-tographic strategies in the past ten years, covering the main research in these areas especially in China. The reviewed research focused on enzymatic micro-reactors and peptide separation in bottom-up approaches, and protein and peptide separation in top-down approaches. The new enzymatic micro-reactor is able to accelerate proteolytic reaction rate from conventionally a couple of hour...     

A new organic carbon detector for size exclusion chromatography

A novel organic carbon detector (OCD) for size exclusion chromatography (SEC), and its application to the characterisation of aquatic natural organic matter (NOM) in natural and treated potable water samples, is described. The instrument uses a conventional UV-persulfate oxidation technique to convert organic carbon to CO(2). The novelty of the technique is detection of the evolved CO(2) using a sensitive Fourier transform infrared (FTIR) spectroscopy 'lightpipe' detector originally designed for detection of analytes after gas chromatographic separation. With the exception of the lightpipe, the OCD system was constructed using simple, inexpensive, readily available components. The system was designed to minimise deadvolume, allowing for use of smaller sample sizes and smaller columns, substantially shortening analysis time, while maintaining chromatographic integrity through the OCD system. Downscaling resulted in some loss of separation but it was shown that this was caused by the lower separation efficiency of the smaller capacity column, rather than from sample dispersion within the OCD system.