Exclusion Mode Research Articles

A study of the relationship between the thermodynamic quality of mobile phases and their strength for the critical conditions of liquid chromatography of polystyrenes

The relationship between the thermodynamic quality of mobile phases and their strength e was studied for the critical conditions of liquid chromatography. The chromatography of reference polystyrene samples was carried out in the adsorption and exclusion modes, and also under critical conditions, with binary mobile phases and a silica gel column used. The thermodynamic quality of solvent mixtures with respect to one another was evaluated by the intrinsic viscosity [η].

Liquid Chromatography Under Limiting Conditions of Insolubility, II

AbstractSummary:Liquid chromatography under limiting conditions of insolubility (LC LCI) is a novel member of the group of liquid chromatographic methods working under limiting conditions of enthalpic interactions (LC LC). These employ differences in the velocity of small, pore permeating molecules (eluent components, auxilliary substances) and large, excluded macromolecules (sample components) in the course of their transport along an LC column packed with porous particles. A “barrier” of slowly moving small molecules of an appropriate nature selectively decelerates certain kinds of macromolecules due to their enthalpic interactions either with the column packing mediated by the barrier or with the barrier itself. The retained macromolecules accumulate on the barrier edge and elute from the column independently of their molar mass. Other kind(s) of macromolecules may be inert towards the barrier effect and elute in the exclusion mode. In this way, polymer species of different natures can be easily and efficiently separated. In LC LCI, a barrier impermeable to macromolecules consists of a narrow zone of non‐solvent, which is injected into the column immediately before the sample solution. The effects of basic experimental variables on the LC LCI elution of selected model polymer species were studied in the present work. These included both the polarity and pore size of the column packing, the nature and composition of the eluent, sample concentration and volume and the non‐solvent barrier width, as well as temperature. The general LC LCI behavior of macromolecules was confirmed. It was only slightly affected by the experimental conditions mentioned above, as long as the precipitation power of the non‐solvent barrier remained high enough. It has been demonstrated that LC LCI operates over a very broad polymer molar mass range, from a few thousand g · mol−1up to several million g · mol−1. Under well chosen experimental conditions, LC LCI produces well focused polymer peaks, even for extremely large sample volumes reaching 16% of the total column volume. Narrow pore column packings are preferred because wide pore materials may produce skewed or even split LC LCI peaks. Chromatographically strong eluents and barrier components prevent adsorption of the sample on the column packing so that the mixed retention mechanism is avoided and the elution behavior of macromolecules is controlled exclusively by their solubility. In conclusion, LC LCI is a robust, user‐friendly technique which can help solve numerous separation problems in polymer science. For example, LC LCI enables fast and efficient separation of polymer species, which differ in their chemical composition.Fast and efficient separation of the LC LCI eluted polystyrene from the SEC eluted poly(methyl methacrylate) of similar molar mass.magnified imageFast and efficient separation of the LC LCI eluted polystyrene from the SEC eluted poly(methyl methacrylate) of similar molar mass.

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.

Looking inside the pores of a chromatographic column: I. Variation of the pore volume with mobile phase composition

The dependence of void volume and pore volume on mobile phase composition was studied on a reversed phase column based on poly(divinyl benzene) in mobile phases consisting of 100% tetrahydrofuran (THF) and 40–90% THF–water. Void volumes V 0 are obtained from a series of polymers with different molar mass by extrapolation of elution volume to M = 0, the interstitial volume V i from the elution volume of polymers with a sufficiently high molar mass. The pore volume V p is obtained as the difference ( V 0 − V i). While V i does not depend on mobile phase composition, a dramatic decrease of the pore volume is observed, when the mobile phase is changed from THF to THF–water. This effect of the mobile phase composition on the pore volume is observed using PEG and PPG as test macromolecules, regardless the retention mechanism: at 40–50% THF both PEG (in exclusion mode) and PPG (in adsorption mode) experience the same reduced pore volume, which is considerably lower than that in 100% THF.

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

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 × 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.

Characterization of amphiphilic polymers: Independent analysis of blocks in poloxamers by liquid chromatography under critical conditions

Block copolymers of ethylene oxide (EO) and propylene oxide (PO) are characterized by liquid chromatography under critical conditions (LCCC) for EO on a Diol column in acetone water 78:12 (w/w) and for PO on a poly(divinyl benzene) column in acetone water 92:8 (w/w) or on a silica based poly(ethylene glycol) phase in 45:55 (w/w). Under these conditions, the other (non-critical) block elutes in exclusion mode, which allows an independent determination of the molar mass distribution of the individual blocks. These systems allow also the identification of homopolymers. The results thus obtained are compared to those from size exclusion chromatography (SEC) with coupled density and refractive index (RI) detection, which allows the determination of the entire MMD and the chemical composition along the MMD.

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.

Liquid chromatography of polymers under limiting conditions of adsorption: IV. Sample recovery

The high performance liquid chromatography of polymers under limiting conditions of adsorption (LC LCA) separates macromolecules, either according to their chemical structure or physical architecture, while molar mass effect is suppressed. A polymer sample is injected into an adsorption-active column flushed with an adsorption promoting eluent. The sample solvent is a strong solvent which prevents sample adsorption. As a result, macromolecules of sample elute within the zone of their original solvent to be discriminated from other, non-adsorbing polymer species, which elute in the exclusion mode. LC LCA sample recovery has been studied in detail for poly (methyl methacrylate)s using a bare silica gel column and an eluent comprised toluene (adsorli) and tetrahydrofuran (desorli). Sample solvent was tetrahydrofuran. It was found that a large part of injected sample may be fully retained within the LC LCA columns. The amount of retained polymer increases with decreasing packing pore size and with higher sample molar masses and, likely, also with the column diameter. The extent of full retention of sample does not depend of sample volume. An additional portion of the injected desorli sample solvent (a tandem injection) does not fully eliminate full retention of the sample fraction and the reduced recovery associated with it. The injected sample is retained along the entire LC LCA column. The reduced sample recovery restricts applicability of many LC LCA systems to oligomers and to discrimination of the non-adsorbing minor macromolecular components of complex polymer mixtures from the adsorbing major component(s). The full retention of sample molecules within columns may also complicate the application of other liquid chromatographic methods, which combine entropic and enthalpic retention mechanisms for separation of macromolecules.

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.

Un-cooled infrared (8–12 μm) emitters of positive and negative contrast

We report studies of infrared emitters with positive and negative contrast for the 8–12 μm spectral range. The operating principle is based on modulation of semiconductor thermal emission in charge carrier injection and exclusion modes. The sources, made of Ge, have semitransparent n–p or p +–p contacts on the radiating face. This makes it possible to realise vertical output of radiation and uniform emission from the working area of emitter. The results of investigations of the principal characteristics of the sources are presented, and practical aspects of their application in infrared engineering are discussed.

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.

Theory of liquid chromatography of mono- and difunctional macromolecules: I. Studies in the critical interaction mode

The theory of liquid chromatography of mono- and difunctional polymers based on the model of ideal polymer chain in wide slit-like pores is presented. Analytical equations describing chromatographic behavior of functional macromolecules in both adsorption, exclusion and critical modes are derived and compared with experiments. The focus of this experimental study was on the verification of the theory in chromatography at critical conditions. Chromatographic behavior of low molar mass end-functionalized polyethylene glycols was found to be in a very good qualitative and in a reasonable quantitative agreement with the theory.

効率的な発現プロファイリングのためのプロテオーム解析システムの開発 疾患標的蛋白質の迅速な同定と定量に向けて

In the post-genome era, there is an immediate requirement for the analysis of proteome, which gives a great impact on the methodology of disease diagnosis and drug discovery in medicine and pharmaceutical sciences. Mass spectrometry (MS) is essential for high throughput and accurate detection/identification of potential drug target proteins in low abundance localized to intrinsic functions at the extremely early stage of disease progression. Conventional proteome analysis strategy with two-dimensional electrophoresis (2-DE) techniques is not compatible for the drug discovery process and MS analysis, mainly due to poor solubility of many hydrophobic proteins in the current 2-DE sample buffers and its drawbacks including being low reproducibility, time consuming and very labor intensive. Higher throughput identification of proteins, including membrane-binding ones, was accomplished by introducing one-dimensional (1D) sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein fractionation coupled with nano-liquid chromatography (nanoLC)/nano-electrospray ionization (nanoESI)/ion trap (IT)-MS/MS. Toward fully automated proteome analysis, a five-dimensional (5D)-LC-based system was constructed, consisting of two-dimensional LC for protein fractionation, followed by proteolytic fragmentation of each protein fraction, and two-dimensional nanoLC for peptide fractionation directly linked with nanoESI/IT-MS/MS. A combination of the isotope-coded affinity tags (ICAT) assay with 1D-SDS-PAGE fractionation or the 5D-LC proteome workstation followed by nanoESI/IT-MS/MS data-dependent measurements under the dynamic exclusion mode will bring a promising approach for identification and quantification of proteins expressed in a disease progression. This strategy leads to the high throughput discovery of key target components related to disease progression or origin.

Simultaneous determination of neutral, anionic and cationic compounds within one chromatographic run using an inductively coupled plasma mass spectrometer as element‐specific detector

AbstractTwo simple methods for the simultaneous separation of anionic and cationic arsenic species in a single injection were developed. One method was a dual column system connected with anion and cation exchange columns; the other was a dual mode system. The dual mode system was a combination of ion exclusion and cation exchange modes. The cation exchange resin, functionalized by dicarboxylic acid, was used for the dual mode column. Separation conditions for the two separation methods were optimized for eight arsenic species. The eight arsenic standards were completely separated within 40 min in both methods. On the dual column system, a good reproducibility (peak area reproducibility was 5% and under) for arsenic species was obtained by applying the column switching method, although the retentions of anionic arsenic species did fluctuate by interference of strong adsorptive anions. Inductively coupled plasma mass spectrometry as an element‐selective detector was used. Detection limits for the eight arsenic compounds ranged from 0.1 to 0.7µgAsdm−3, and reproducibility (relative standard deviation, n = 8) ranged from 2.4 to 8.0% at 0.01 mg As dm−3. The simultaneous separation methods developed were applied to the determination of arsenic species in human and rat urine. Good agreement on the quantified values by the two separation methods was obtained for all arsenic species in the urine samples. Copyright © 2001 John Wiley & Sons, Ltd.

MIXED-MODE GPC USED TO CONTROL THE REACTION BETWEEN N,N-DIMETHYL-PROPYLENE-1,3-DIAMINE AND TRIGLYCERIDES

Gel permeation chromatography (GPC) was utilised to control the progress of the reaction between N,N-dimethylpropylene-1,3-diamine (DMPDA) and triglycerides (fats). As per “ideal size exclusion mode”, the sample is separated into triglycerides (TGC), diglycerides (DGC), monoglycerides (MGC), and glycerine (GC), as the component groups with sufficiently different molecular weights. Moreover, the separation was obtained for MGC and amidoamines (AA), i.e. the reaction products, despite the fact that their molecular weights are the same in practice (Mw=348 and 358, respectively). The increased retention time for amidoamines was assumed to be accounted for by the additional adsorption effects in “adsorption mode”. Resulting from the overlapping of two types of chromatographic separation mechanisms, an analytical method was obtained which could be called “mixed mode” conditions. This method allows for the control of the amidolysis reaction of fats at its intermediate stages. The method is applicable for the respectively narrow distribution of molecular weights of triglycerides, which is the case in lard, sunflower oil and/or rape-seed oil.

Effects of electrostatic and hydrophobic interactions on the chromatographic behavior of biopolymers in HPLC columns

AbstractThe effect of electrostatic and hydrophobic interactions on the chromatographic behavior of biopolymers with the use of chemically bonded silica‐based HPLC columns and aqueous buffered mobile phases containing neutral salts in a wide range of concentration is discussed. Two columns packed with stationary phases appositely designed for biopolymer HPLC in size exclusion and anion exchange mode, respectively, are examined. Experimental data are evaluated by plotting the measured isocratic elution volumes of several standard proteins of different isoelectric point against the salt concentration in the mobile phase. Depending on the concentration and nature of salt, both columns exhibit different domains where either sieving effect or electrostatic or hydrophobic interactions are predominant. At sufficiently low salt concentrations electrostatic interactions are predominant leading to either increasing or decreasing elution volumes depending on the sign of the charges on the stationary phase and the protein, respectively. On the other hand, at high salt concentrations of a salt with sufficiently high molal surface tension increment proteins may be retained by hydrophobic interactions.

The Decomposition of a Commercial Perfluoropolyalkylether on Alpha and Gamma Catalytic Aluminas

The decomposition of a commercial perfluoropolyalkylether liquid lubricant (PFPE Z) was studied using a thermal gravimetric and differential scanning calorimetry unit. Small porous alumina pellets (alpha and gamma forms) were charged with approximately 5 mg of PFPE Z, and the charged pellets were then placed inside the thermal units where decomposition started to occur over the. narrow temperature range of 198° to 210°C under a nitrogen atmosphere. At the end of a test, 1,1,2-trichlorotrifluoroethane solvent was used to extract PFPE Z and decomposition products from the pellets. This extract was analyzed using high-pressure liquid chromatography in the size exclusion mode. The alumina pellets were examined using X-ray photoelectron spectroscopy. Analysis of the thermal, chromatography, and X-ray photoelectron spectroscopy data support evidence that there are two different decomposition mechanisms for PFPE Z. The data indicated, that active surface sites, other than Lewis acid sites, were responsible for PFPE Z decomposition.