Size Exclusion Mode Research Articles

Molecular Mass Analysis of Antibodies by On-Line SEC-MS

Mass analysis of recombinant protein therapeutics is an important assay for product characterization. Intact mass analysis is used to provide confirmation of proper translation of the DNA sequence and to detect the presence of post-translational modifications such as amino acid processing and glycosylation. We present here a method for the rapid mass analysis of antibodies using a polyhydroxyethyl aspartamide column operated in size-exclusion mode and coupled with ESI-MS. This method allows extremely efficient desalting of proteins under acidic conditions that are optimal for subsequent mass analysis using standard ESI conditions. Furthermore, this technique is significantly faster and more sensitive than rpHPLC methods, typically considered the standard chromatography approach for mass analysis of proteins. This method is flexible and robust, and should prove useful for applications where a combination of speed and sensitivity are required.

Enzyme-catalyzed Production of Oligopeptides from Skim Milk

With the aim of preparing high nutritional dietary supplements containing oligopeptides from skim milk, an Aspergillus protease, papain and pepsin, separately or in association, were used in different enzyme:substract ratios (1%, 2% and 4%). The fractionation of peptides according to their size was used to evaluate the nutritional quality of protein hydrolysates. The hydrolysates were fractionated by high-performance liquid chromatography in size-exclusion mode and the rapid method of Correct Fraction Area was used for the quantification. In general, the isolated action of the enzymes produced better peptide profiles than their simultaneous use, especially in terms of a higher amount of oligopeptides, which reached 56%.

Determination of “free” iron and iron bound in metalloproteins via liquid chromatography separation and inductively coupled plasma-optical emission spectroscopy (LC-ICP-OES) and particle beam/hollow cathode-optical emission spectroscopy (LC-PB/HC-OES) techniques

The abilities of liquid chromatography-particle beam/hollow cathode-optical emission spectrometry (LC-PB/HC-OES) and liquid chromatography-inductively coupled plasma-optical emission spectroscopy (LC-ICP-OES) techniques are demonstrated for the quantitative determination of free iron and bound iron in metalloproteins. Separations were performed in the size exclusion and reversed-phase chromatographic modes. Myoglobin, holo-transferrin and iron(II) chloride were separated by size exclusion chromatography and the eluent species were detected by both PB/HC-OES and ICP-OES through the Fe I 259.9 nm and S I 180.7 nm optical emission. Sulfur optical emission was monitored as means of protein identification through the Fe/S empirical formula differences, with the absence of S emission used to identify the “free” Fe. Both techniques demonstrated detection limits for triplicate injections on the nanogram level for iron (0.9 ng mL−1 ICP-OES and 41.9 ng mL−1 PB/HC-OES) over the concentration range of 0.1 ng mL−1–100 μg mL−1 for iron and iron metalloproteins. The corresponding values for sulfur in the proteins were more similar for the two techniques, being of the order of 20 ng mL−1. The SEC retention times and peak shapes of the three analyte peaks for both techniques are similar to those determined by UV absorbance at 220 nm (Fe-containing metalloproteins) and conductivity (inorganic iron), demonstrating the ability of both techniques to preserve the integrity of the separation. While the LODs in “bulk” sampling were lower with ICP-OES, the signal-to-noise values were comparable for both techniques when sampling chromatographic eluents in real-time. In addition, a reversed-phase separation of the same analyte mixture was carried out with Fe I 259.9 nm and S I 180.7 nm optical emission detection by the PB/HC-OES, demonstrating the compatibility of the PB/HC-OES with a wide range of solvent polarities, its ability to detect non-metals and act as a protein specific detector for liquid chromatography of proteins.

Liquid Chromatography under Limiting Conditions of Enthalpic Interactions: Enthalpic Partition Retention Mechanism

Summary: Applicability of the enthalpic partition (absorption) retention mechanism was evaluated in liquid chromatography under limiting conditions of enthalpic interactions (LC LC). It was shown that the barrier principle of LC LC also held in the case of enthalpic partition retention mechanism. LC columns packed with porous silica C-18 bonded phase in combination with low-polarity PS and PnBMA were employed. The partition-promoting solvent was DMF and the partition-preventing solvent was THF. Pore-permeating molecules of DMF moved slowly along column and acted as a barrier, which hindered fast progression of pore excluded macromolecules. The barrier was either the eluent itself, or a narrow zone of DMF injected immediately before polymer solution. In the former case, the eluent contained a high concentration of DMF and would not allow polymer elution. However, the sample was injected in pure THF and traveled within its zone. Being decelerated by the DMF barrier, both PS and PnBMA eluted independently of their molar mass in total volume of column liquid. They could be efficiently and independently of their molar masses separated from the medium- and high-polarity polymers, which did not exhibit enthalpic partition and eluted in the size exclusion mode. In contrast to recently evaluated adsorption based LC LC procedures, enthalpic partition produced broader, skewed, and often split peaks. This is assumed due to slow establishment of enthalpic partition equilibrium, possible presence of micropores in the C-18 bonded phase, and/or due to deformation of shape of barrier edge. Therefore, limiting conditions of enthalpic partition are to be applied preferably to characterization of polar SEC eluted polymers after their discrimination from the low-polarity macromolecules eluting in the LC LC mode. Fast and efficient separation of polar minor (1%) polymer components from major (99%) polymer component.

Effect of salt on purification of plasmid DNA using size-exclusion chromatography

In the present study, we compared the performances of size-exclusion chromatography for the purification of plasmid DNA when different concentrations (0.5M, 1M, 2M, respectively) of two types of salt (NaCl and (NH(4))(2)SO(4)) are present in running buffers. Our experiment results displayed that it is not only the resolution of RNA but also those of supercoiled plasmid DNA and host's genomic DNA were increased greatly in the presence of high concentration of water-structure salt. We deduce that two separation modes may be involved in the process: The supercoiled plasmid DNA is influenced mainly by compaction effect and eluted in the size-exclusion mode; whereas, RNA and genomic DNA are influenced mainly by hydrophobic effect due to their stretched and loose structures and eluted in the interaction mode. This method led to an improved efficiency of size-exclusion chromatography.

Characterization of EO‐PO Block Copolymers by Liquid Chromatography Under Critical Conditions

AbstractSummary: Block copolymers of ethylene oxide (EO) and propylene oxide (PO) are characterized by liquid chromatography under critical conditions (LCCC) for EO. At the critical adsorption point (CAP) for one structural unit, the non‐critical block can elute in size exclusion (SEC) or adsorption (LAC) mode. Depending on the molar mass and architecture of the polymers, different strategies are applied. For samples with a higher molar mass, the SEC separation is the method of choice, while lower molar masses also allow a LAC separation. Examples for both situations are given, which show, that these approaches yield different information. In the SEC mode, homopolymers and diblocks can be separated from the triblocks. In LAC mode, a baseline resolution of individual oligomers can be achieved, in which homopolymers, diblocks and triblocks with the same number of repeat units of the non‐critical block have the same elution volume.

High-temperature gradient HPLC for the separation of polyethylene–polypropylene blends

Abstract A high-temperature gradient HPLC method has been developed for the analysis of polyethylene–polypropylene blends. For the first time it was possible to separate these polyolefin blends by a chromatographic technique which is operating at 140 °C. Blends of a commercial polypropylene and a medium molar mass linear polyethylene were separated using a mobile phase of ethylene glycol monobutylether (EGMBE) and 1,2,4-trichlorobenzene (TCB) and silica gel as the stationary phase. With the use of n -decanol as sample solvent, a precipitation–redissolution mechanism for polyethylene (PE) was established while polypropylene (PP) is eluted in size exclusion mode.

Liquid chromatography of polymers under limiting conditions of desorption: II. Tandem injection and quantitative molar mass determination

Liquid chromatography under limiting conditions of desorption (LC LCD) is a method which allows molar mass independent elution of various synthetic polymers. A narrow, slowly moving zone of small molecules, which promotes full adsorption of one kind of polymer species within column (an adsorli) acts as an impermeable barrier for the fast moving macromolecules. The latter accumulate on the barrier edge and elute nearly in total volume of liquid within column. At the same time, transport of less adsorptive macromolecules is not hampered so that these are eluted in the size exclusion (SEC) mode. As result, polymers differing in their polarity and adsorptivity can be easily separated without molar mass interference. Three methods of barrier creation are discussed and compared. It is shown that a fraction of sample may elute unretained if the adsorli sample solvent is used as a barrier in connection with a narrow-pore column packing. One part of excluded macromolecules likely breaks-out from the adsorli zone and this results in partial loss of sample and distortion of the LC LCD peaks. This problem can be avoided if the adsorli zone is injected immediately before sample solution. Applicability of the LC LCD method for polymer separation has been demonstrated with a model mixture of poly(methyl methacrylate) (adsorbing polymer) and polystyrene (non adsorbing polymer) using bare silica gel as a column packing with a combination of tetrahydrofuran (a desorption promoting liquid -a desorli) and toluene (adsorli). It has been shown that the LC LCD procedure with tandem injection allows simple and fast discrimination of polymer blend components with good repeatability and high sample recovery. For quantitative determination of molar masses of both LC LCD and SEC eluted polymers, an additional size exclusion chromatographic column can be applied either in a conventional way or in combination with a multi-angle light scattering detector. A single eluent is used in the latter column, which separates the mixed mobile phase, system peaks and the desorli zone from the polymer peaks so that measurements are free from disturbances caused by the changing eluent composition. The resulting LC LCD x SEC procedure has been successfully applied to poly(methyl methacrylate) samples.

Liquid chromatography of polymers under limiting conditions of adsorption: III. Role of experimental variables

LC of polymers under limiting conditions of adsorption (LC LCA) is a novel method based on different mobility of (pore excluded) macromolecules compared to (pore permeating) solvent molecules. Polymer sample is injected in a solvent preventing its adsorption within the column. Eluent promotes sample adsorption. Under these conditions, macromolecules cannot leave its initial solvent and elute from the column independently of their molar mass. In contrast, a less interactive simultaneously injected polymer leaves its initial solvent zone and is eluted in the size exclusion mode. As a result, chemically different polymer species can be discriminated. The effect of selected experimental conditions was studied on the LC LCA behavior of poly(methyl methacrylate)s eluted from bare silica gel columns. The parameters were packing pore diameter, injected sample volume and concentration, as well as column temperature. The size independent elution was only little affected by the above parameters and LC LCA produced well-focused peaks. The LC LCA mechanism was operative even at a very large sample of both volume and concentration. This makes LC LCA a robust and user-friendly method, likely suitable also for characterization of minor components of polymer mixtures.

Retention Behaviors of Block Copolymers in Liquid Chromatography at the Critical Condition

The partitioning of a diblock copolymer (AB) and a triblock copolymer (ABA and BAB) into a pore, at a condition similar to that employed in the experimental liquid chromatography at the critical condition (LCCC), was investigated by lattice Monte Carlo simulations with self-avoiding-walk chains. The B block is set at the predetermined critical condition where the partition coefficient of a homopolymer B has a least dependence on its chain length, and hence becomes chromatographically “invisible”. The A block is set at the size-exclusion mode and is chromatographically “visible”. The partition coefficients of these copolymers were compared with that of a homopolymer A with the same (total) length of the visible A block(s). The partition coefficient of a diblock, KAB, was found to be larger than KA, especially when the A block was short and the B block was long. The difference tends to vanish with an increase in the A block length or a decrease in the B block length. A smaller pore also tends to decrease th...

Dynamic electric field assisted multi-dimensional liquid chromatography of biological samples

Complex biological samples require very high resolution separation strategies. The platform introduced here capitalises on the hyphenation of liquid chromatographic (LC) and electric potential gradient electrochromatographic multi-dimensional separation genres. First-dimension selectivity is provided by simultaneous size exclusion (SEC) and strong cation exchange (SCX) chromatography modes, while the second dimension comprises reversed phase (RP) characteristics in a dynamic (time-variant) electric field. The time-variant potential gradient with reversal of polarity is applied across the second dimension monolithic capillary throughout the duration of the solvent strength gradient elution. Hence, the platform offers comprehensive on-line sample clean-up (matrix depletion, analyte enrichment), fractionation (first dimention LC), and separation (second dimension LC) with the prospect of altering selectivity via polarity reversal dynamic electric field tuning.

Copper binding to prion octarepeat peptides, a combined metal chelate affinity and immunochemical approaches

Based on the hypothetical proposal of Sulkowski [E. Sulkowski, FEBS Lett. 307 (2) (1992) 129] for the implication of transition metal ions in the structural changes/oligomerisation of normal cellular prion protein (PrPc) resulting in the pathological isoform (PrPsc), we focused our study on the octarepat domain of this protein which has been supposed to be the metal binding site. We have studied the copper binding to synthetic prion octarepeat peptides (PHGGGWGQ)n (n=1, 3, 6) using metal chelate and size-exclusion modes of chromatographies. This copper binding induces oligomerisation resulting in multiple aggregates. Moreover, heterogeneity of metal bound octarepeat oligomers by ESI-MS has been demonstrated. In addition, anti prion antibodies specific to the octarepeat region were used to discriminate between metal free and copper, nickel and zinc bound hexamer octarepeat peptide. Differential recognition of Cu(II) and Zn(II) bound complexes has been observed which signify differences in exposed epitopes of aggregated peptides.

Perfil peptídico de hidrolisados enzimáticos de leite em pó desnatado

Sete hidrolisados de leite em po desnatado foram preparados, visando a producao de hidrolisados proteicos, como suplemento dietetico para fenilcetonuricos. Para isso, utilizaram-se uma protease do Aspergillus oryzae (AO), a papaina (PA) e a pepsina (PE), isoladamente ou em associacao, em diferentes relacoes enzima:substrato (E:S), sendo o tempo total de reacao de 5 h e a temperatura de 50 oC. Adotou-se como criterio para a avaliacao nutricional dos hidrolisados, o estudo da distribuicao dos peptideos nas amostras, de acordo com o tamanho da cadeia. Inicialmente, os hidrolisados foram fracionados por cromatografia liquida de alta eficiencia de exclusao molecular (SE-HPLC) e, para a quantificacao dos componentes das fracoes cromatograficas, empregou-se o metodo rapido da Area Corrigida da Fracao (ACF). Os resultados indicaram que, de modo geral, a acao isolada das tres enzimas foi mais vantajosa em relacao ao perfil peptidico em comparacao com as associacoes estudadas. Entre todas as preparacoes testadas, a acao isolada de AO e de PA, e a associacao destas enzimas (relacao E:S de 1% e 2%, respectivamente) produziram hidrolisados com perfis peptidicos semelhantes, mais adequados nutricionalmente, ou seja, com maior teor de di- e tripeptideos e menor proporcao de aminoacidos livres.

Enthalpic Partition‐Assisted Size Exclusion Chromatography: 1. Principle of Method

AbstractA novel high performance liquid chromatographic (HPLC) method viz. “enthalpic partition assisted size exclusion chromatography” deliberately combines entropic and enthalpic partition mechanisms. It enables separation of homopolymers according to their molar mass with increased selectivity, as well as discrimination of polymer species differing in their nature/composition. Enthalpic partition of macromolecules takes place between the mobile phase and the stationary “liquid” of a different chemical nature, which is immobilized within pores of an appropriate carrier (a bonded phase). The extent of enthalpic partition depends on the accessibility of bonded phase for macromolecules and on the difference of polymer solubility in the mobile phase and in the solvated bonded phase. The enthalpic partition in favor of column packing arises from better solubility of polymer solutes in the solvated stationary phase compared to the mobile phase. Macromolecules are “pushed” into the solvated stationary phase and their retention volumes (VR) increase. In the area of high molar masses, the extent of enthalpic partition as rule raises with the increasing size of macromolecules. However, under properly chosen experimental conditions the enthalpic partition may rapidly diminish with the sample molar mass (M), likely due to the solubility changes and/or due to partial exclusion of macromolecules from the pores. As result, the corresponding retention volumes sharply drop within a narrow range of M with the increasing size of macromolecules. This results in the log M vs. VR dependences, which resemble in their form that for size exclusion chromatography but are much more flat indicating highly selective separations of homopolymers according to their molar masses. In this way, enthalpic partition “assists” entropic partition (size exclusion). Polymer species, which do not undergo enthalpic partition, elute from the HPLC column in the conventional size exclusion mode and can be discriminated from the partitioning species. Enthalpic partition assisted size exclusion chromatography can be utilized in separation and characterization of various homopolymers, and polymer blends.

Size-exclusion flow extraction of bisphenol A in human urine for liquid chromatography–mass spectrometry

We report an approach for assessing human exposure to bisphenol A (BPA), which involves measuring the glucuronide in urine sample that were subjected to a novel size-exclusion flow extraction method. The present approach includes the addition of 13C(12)-BPA, enzymatic deconjugation, and the proposed sample preparation method. The sample solution is separated and detected by liquid chromatography-mass spectrometry (LC-MS). The following are used for the LC-MS: a reversed-phase separation column, electrospray ionization (ESI), negative mode, and single ion monitoring (SIM) with m/z 227 for BPA and m/z 239 for 13C(12)-BPA. The detection limit was 0.1 ng ml(-1) and the calibration curves (0.45-90 ng ml(-1)) had correlation coefficients exceeding 0.999. To urine samples requiring deglucuronidation, beta-glucuronidase was added followed by incubation at 37 degrees C for 3 h. After the enzymatic treatment, the samples were subjected to the extraction in the reversed-phase (ODS) and size-exclusion (GPC) modes. It was possible to extract, clean up and concentrate BPA in a single run of 20 min by means of the novel extraction method. The method enables the determination of standards and may be applied to the detection of trace amounts of BPA in human urine samples.

Elution behavior of polyethylene in polar mobile phases on a non-polar sorbent

Linear polyethylene standards in the range of 1-500 kg/mol, dissolved in 1,2,4-trichlorobenzene, were injected into a column packed with oligo(dimethylsiloxane) modified silica gel. Fifteen polar solvents (cyclohexanone, cyclohexylacetate, cyclohexanol, nonylalcohol, dimethylformamide, dimethyl sulfoxide, ethylene- and diethylene glycol monobutyl ether, benzylalcohol, hexylacetate, bis(2-ethyl-hexyl)phthalate, N,N-dimethylacetamide, propylene carbonate, dipropylene glycol and N-methyl-pyrrolidone) were evaluated as mobile phases. Depending on the type of mobile phase evaluated, different elution behaviors are observed for polyethylene: (1) polyethylene was eluted in the size exclusion mode, (2) polyethylene was eluted together with the sample solvent peak at constant elution volume, (3) polyethylene was partially or fully retained on the column. The retained polymer was easily removed from the column by injecting a small volume of trichlorobenzene. The use of ethylene glycol monobutyl ether as the mobile phase enabled separation of the polyethylene from polypropylene. In this case polypropylene is eluted in the size exclusion mode, while polyethylene is eluted at a constant elution volume or remains in the column.

Chromatographic and Spectroscopic Methods in the Analysis of Triacylglycerol Species and Regiospecific Isomers of Oils and Fats

Referee: Dr. Fereidoon Shahidi, Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland, Canada, A1B 3X9The chromatographic, spectrophotometric, and spectroscopic methods usually used for the analysis of oils and fats are reviewed, with special attention to the detection, identification, and quantitation of the triacylglycerol species present in the most common edible vegetable oils. Also, a review of the methods used for the identification of the regiospecific types of the triacylcglycerol species of oils and fats is presented.Methods of high-performance liquid chromatography in the normal, reversed, silver ion, size exclusion, and chiral modes of thin layer chromatography in the normal, reversed, and silver ion modes of supercritical fluid chromatography with capillary glass or stainless steel columns of capillary gas chromatography and of mass spectrometry in different modes are presented and evaluated. The different sample pretreatment methods, the chromatographic columns, the elution systems, and the detectors and detection modes that have been combined with the above methods are also reviewed. Other spectroscopic methods used for the characterization of the status of an oil or fat, such as ultraviolet, fluorescence, chemiluminescence, infrared, and nuclear magnetic resonance are also reported, selectively.The majority of the triacylglycerol species present in an oil or fat could be analyzed and identified by a particular technique of the reviewed methods, but a complete separation in one single run requires a combination of two or more of the existing techniques, especially for samples with high triacylglycerol species variability.

Distribution analysis of cellulose acetate phthalate by ion-exclusion-moderated size exclusion chromatography

Abstract Ion-exclusion is the electrostatic repulsive interaction between a charged polymer and charges of the same sign on the surface of a column packing. Controlled ion-exclusion allows compensation of hydrophobic adsorption in size exclusion chromatography of negatively charged cellulose acetate phthalate (CAP) polymers in acetone/water/LiCl (80/20) as a mobile phase. Properly selected low-ionic-strength conditions provide correct separation in size-exclusion mode also in binary solvent mixtures. Possible interfering effects related to light scattering at low-salt conditions are shown to be negligible if on-line concentration/light scattering detection is used. The absence of these interferences is easily checked by a comparison of experiments at two different low-salt concentrations. Molecular weight averages and distributions identical within the experimental error are obtained when both salt concentrations are properly selected.

Improved performance of protein separation by continuous annular chromatography in the size-exclusion mode

In size-exclusion chromatography (SEC), proteins and peptides are separated according to their molecular size in solution. SEC is especially useful as an effective fractionation step to separate a vast amount of impurities from the components of interest and/or as final step for the separation of purified proteins from their aggregates, in a so-called polishing step. However, the throughput in SEC is low compared to other chromatographic processes as good resolution can be achieved only with a limited feed volume (i.e., maximal approximately 5% of the column volume can be loaded). This limitation opposed widespread application of conventional SEC in industry despite its excellent separation potential. Therefore a continuous separation process (namely preparative continuous annular chromatography) was developed and compared to a conventional SEC system both using Superdex 200 prep grade as sorbent. An immunoglobulin G sample with a high content of aggregates was chosen as a model protein solution. The influence of the feed flow-rate, eluent flow-rate and rotation rate on the separation efficiency was investigated. The height equivalent to a theoretical plate was lower for preparative continuous annular chromatography which could be explained by reduced extra column band broadening. The packing quality was proved to be identical for both systems. The productivity of conventional batch SEC was lower compared to continuous SEC, consequently buffer consumption was higher in batch mode.

Direct analysis of clomipramine in human plasma by microbore high performance liquid chromatography with column-switching

An automated microbore, liquid chromatographic method with column-switching was developed for the determination of clomipramine from human plasma samples. After direct injection of samples (60 μL), plasma proteins and clomipramine were separated in size-exclusion mode using 20% acetonitrile in 20 mM phosphate buffer (pH 7.0) on Capcell Pak MF Ph-1 precolumn (10×4 mm I.D.). By valve switching, a fraction containing clomipramine was directed to an intermediate column for subsequent main separation on a microbore C18 column (250×1.5 mm I.D.) using 50% acetonitrile in 20 mM phosphate buffer (pH 2.5) at 0.1 mL min−1. The method was advantageous for rapidity (total analysis time: 15 min), reproducibility (C.V.<4.8%), and increased sensitivity (1 ng mL−1). The linearity of response was good (r 2≥0.999) over the concentration range 1–250 ng mL−1.