Exclusion Mode Research Articles

Liquid chromatography of polymer mixtures applying combination of exclusion and full adsorption mechanisms. I. Analysis of polystyrene in its mixture with polymethylmethacrylate single column/single eluent approach

AbstractComponents of polymer mixtures with different chemical composition can be separated by liquid chromatography by applying appropriate conditions (column packing, eluent, temperature, and pressure) in which just one type of macromolecule is eluted in the size exclusion mode while the other component is fully retained within on column packing. The feasibility of this idea has been demonstrated using the mixtures of polystyrene (PS) plus polymethylmethacrylate (PMMA) with silica gel as the column packing and toluene as an eluent. The PMMA was retained by the silica get at ambient temperature and moderate pressures of several MPa while the PS was eluted by a typical SEC mecahanism. Furthermore, the molar mass distribution of the PS could be determined in one step with the same precision as if injected alone. Surprisingly large amounts of PMMA were trapped within the column prior to its SEc characteristics being influenced. After a series of analyses the column has to be regenerated with a brief flushing with a desorbing liquid, tetrahydrofuran in the system studied. The re‐equilibrization of the column with the initial eluent, toluene, was fast and a new series of analyses could be started soon after the characterization step had seen finished. © 1995 John Wiley & Sons, Inc.

Critical Retention Behavior of Homopolymers

Abstract The isocratic retention behaviour of a homopolymer at a specific temperature and varying solvent compositions can be divided into three modes: exclusion mode, transition mode (critical conditions) and adsorption mode. Under critical conditions (a specific non-solvent/solvent composition at a specific temperature) the retention of a homopolymer is independent of the molar mass. The critical conditions depend upon the temperature, the type of polymer and the type of non-solvent/solvent mixture. A difference in the critical conditions between polystyrene standards and polybutadiene standards was found. No significant influence of different types of column packing on the critical solvent composition was noticed. The exact mechanism of the retention of a polymer under critical condition is not clear yet. In order to determine the critical conditions, a new method has been developed. The method can be easily performed on existing equipment. The method contains a different setup of the data acquired by isocratic HPLC than methods described in literature. Therefore, less experiments need to be performed to determine the critical conditions compared to the current method. The newly developed method is more efficient than the methods described in the literature.

Chromatographic investigations of macromolecules in the critical range of liquid chromatography: 4. Analysis of poly(styrene- b-methyl methacrylate)

Poly(styrene- b-methyl methacrylate)s were analysed by liquid chromatography at the critical point of adsorption. By operating at chromatographic conditions corresponding to the critical mode for poly(methyl methacrylate) and the size exclusion mode for polystyrene, the molar mass and the polydispersity of the polystyrene block in the block copolymers may be determined. The data were found to be in excellent agreement with the corresponding parameters of the polystyrene precursor homopolymers.

Chromatographic investigations of macromolecules in the critical range of liquid chromatography: 7. Analysis of the poly(methyl methacrylate) block in poly(styrene- block-methyl methacrylate)

Poly(styrene- block-methyl methacrylate)s were analysed by liquid chromatography at the critical point of adsorption. Operating at chromatographic conditions, corresponding to the critical mode of polystyrene and a size exclusion mode for poly(methyl methacrylate), the molar mass and the polydispersity of the poly(methyl methacrylate) block in the block copolymers was determined. The chemical composition of the block copolymers was obtained using size exclusion chromatography with coupled density-refractive index detection, and compared to data calculated from critical chromatography experiments. The agreement of the data obtained by the different methods was found to be very good.

Combination of Size Exclusion and Adsorption Phenomena on a Hydrophilic Polymer Gel Column Using Organic Solvents

Abstract Elution behavior of alkylbenzenes, phthalate esters, oligostyrenes, and prepolymers of epoxy resins and methylated melamine-formaldehyde resins has been investigated on a column packed with hydrophilic polyhydroxyethyl methacrylate gels using tetrahydrofuran (THF), methanol, and mixtures of THF and methanol or n-hexane as the mobile phases. Size-exclusion effect was predominant when THF was used as the mobile phase and adsorption interactions between solutes and the gels superimposed the size-exclusion effect when methanol or mixtures of THF and methanol or n-hexane was the mobile phase. The gel-THF system was assumed to be SEC. The addition of methanol or n-hexane to THF in the mobile phase retarded the elution of solutes. The combination of size exclusion and adsorption modes in the same column was possible by changing composition of the mobile phase and resolution of some peaks was improved than by SEC alone. The peak identification by the present system is also improved than by SEC.

Determination of functional type distribution of oligocaprolactonediols by liquid chromatography under critical conditions

Functional type separation of oligocaprolactonediols by liquid chromatography of macromolecules under critical conditions was investigated. The analysis was performed with the aid of a novel variant of HPLC in the critical region on the boundary of the exclusion and adsorption separation modes. This method is characterized by practically complete disappereance of separation by molecular masses thus giving only information on the type of functionality.

Assessment of progestin receptor polymorphism by various synthetic ligands using HPLC

Tritiated R5020 and [ 3H]ORG-2058 were utilized to investigate apparent polymorphism of progestin receptors by vertical-tube gradient centrifugation and HPLC in size exclusion (HPSEC), ion-exchange (HPIEC) and chromatofocusing (HPCF) modes. Rapid centrifugation (3 h) following molybdate stabilization (1 h) showed mainly 8–9S receptor species with 90–96% recovery. [ 3H]R5020 appeared to associate with a receptor isoform sedimenting faster than that bound to [ 3H]ORG-2058. Excess unlabeled R5020 did not eliminate all of the [ 3H]R5020 binding by the 8–9S component suggesting some nonspecific association while excess unlabeled ORG-2058 suppressed this binding by either ligand. Separate labeling of cytosol with each ligand and mixing prior to gradient separation showed at least two receptor species isoforms sedimenting in the 8–9S region with mol. wt of 190,000 and 173,000 D. Sephacryl S-300 chromatography revealed two radioactive peaks with either ligand but with slight molecular weight differences. HPSEC confirmed the presence of isoforms with different molecular size and shape as a function of the radioactive ligand employed. HPIEC showed the presence of two labeled receptor species irrespective of the ligand used. The first peak appeared at the void volume of the column (10 mM), co-eluted with free ligand, indicating the possibility of ligand stripping by the column. The second peak bound both steroids specifically and eluted with 100 mM phosphate. HPCF identified a single specific receptor eluting at a pH of 5.6–6.1, but with free steroid in the void volume irrespective of the ligand used. [ 3H]ORG-2058 appeared to be less prone to the stripping phenomenon than was [ 3H]R5020. These data suggest these ligands either bind to different progestin receptor species or they modify receptor characteristics in a fashion that allows separation based upon size and shape.

Functional type separation of oligocarbonates by liquid chromatography under critical conditions

The separation of linear oligocarbonates has been carried out by the type of functionality, namely the number and nature of terminal chloroanhydride and hydroxyl groups. The analysis was performed with the aid of a novel variant of HPLC — “separation in a critical region” (SCR), on the boundary of the exclusion and adsorption separation modes. The SCR method is characterised by practically complete disappearance of separation by molecular masses (sizes) while chromatograms obtained in the critical region give information only on the types of functionality of oligomers under study. The critical point for the oligocarbonates has been found on silicas “Silasorb 600” and “Lichrosphere Si 500” in a binary solvent carbon tetrachloride-chloroform. An effect of pore size on the retention volumes of macromolecules of different functionality under critical conditions is discussed.

Determination of the tanning capacity of tannic acids by high-performance liquid chromatography

The complexation of several tannic acids (purified tannins) with soluble and insoluble proteins has been studied by high-performance liquid chromatography, Tannins are analysed in both the straight phase and the reversed-phase mode. Bovine serum albumin (BSA) and beer proteins were chromatographed in the size exclusion mode. Soluble proteins seem to complex preferentially with the higher molecular weight tannin polygalloyl glucose components, whereas insoluble standardized hide powder binds preferentially to lower molecular weight tannin components. The tanning capacity of different tannic acids can be expressed in milligrams of BSA bonded per milligram of tannin under standard conditions.

A system for coupled multiple-column separation of proteins

Purification of proteins is commonly a multiple-step process involving size exclusion, ion exchange, affinity, hydrophobic, and other modes of chromatography. In an effort to circumvent the laborious process of collecting the solutes from each column and reintroducing them onto a second column, a valving system is described that directs the samples eluted from a high-performance liquid chromatographic column through a detector with a high-pressure cell into either a second column or into storage loops of a multiloop value. This multiloop value is referred to as a high-pressure fraction collector. After development of the first column is complete, a second solvent can be directed to the second column or high-pressure fraction collector to elute the solutes back through the detector and onto any other column in the system. The process of eluting a sample from a column through a single detector and directing it to the high-pressure fraction collector or any other column in the system may be repeated a number of times. Such valving systems make it possible to chromatograph a single protein component on two or three columns in a short time.

Propagation of elastic waves in DNA

The mathematical analyses of longitudinal and torsional elastic waves transmitted along DNA molecule undergoing Brownian motion in solution are presented. Longitudinal vibrations in DNA are shown to be responsible for drug intercalation and breathing. The near neighbor exclusion mode of drug intercalation is explained. Torsional oscillations in DNA are shown to be responsible for conformation transitions from a right handed to a left handed form, depending on sequence specificity in high salt concentration.

Band dispersion in high-performance exclusion chromatography

The dispersion of polymer samples eluted in the exclusion mode from columns of Hypersil and SG60F (a highly porous silica gel) has been measured for a range of polymer standards over a range of elution velocities and from columns of different lengths. From experiments with columns of different lengths operated at the same velocity, the polydispersities of the polymer standards were obtained and ranged from 1.007 to 1.052. From experiments at different linear velocities, mass transfer coefficients were obtained from which the ratio of the diffusion rates of polymers in bulk eluent to the diffusion rates within the silica gel matrix could be found. For Hypersil these ranged from ca. 6 for PS2K and PS4K (MW 2000 and 4000 respectively) to ca. 17 for PS33K (MW 33,000) indicating fairly severe restrictions to diffusion within the pores of the matrix especially for nearly excluded materials. Measurements with a pure oligomer of polystyrene (MW ≈ 3000) confirmed the validity of the above method of separating spreading effects of polydispersity and kinetics and gave a reduced plate height—velocity curve overlapping that for benzene. Measurements of spreading of excluded polymer samples indicated anomalous behaviour at low flow-rates and with long columns. Measurements of the loading capacity in exclusion chromatography showed that samples should not exceed ca. 1 μg per cm 3 of column packing if overloading is to be avoided. It is concluded that the dispersion of bands of polymers eluted in the exclusion mode from silica gels obeys the same kinetic relationships as the dispersion of bands in retentive chromatography. The study confirms, however, that the major contributor to the dispersion of polymer samples eluted in the exclusion mode is their polydispersity.

Combination of size exclusion and normal-phase partition modes in high performance liquid chromatography

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCombination of size exclusion and normal-phase partition modes in high performance liquid chromatographySadao. Mori and Akira. YamakawaCite this: Anal. Chem. 1979, 51, 3, 382–384Publication Date (Print):March 1, 1979Publication History Published online1 May 2002Published inissue 1 March 1979https://doi.org/10.1021/ac50039a015RIGHTS & PERMISSIONSArticle Views62Altmetric-Citations8LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (378 KB) Get e-Alerts Get e-Alerts

Structural and Energetic Considerations of Wave Propagation in DNA

An important dynamic aspect of DNA in solution is the presence of coupled motions in its structure that involve bending, stretching, unwinding, and shearing components. It is postulated that these motions arise from wave propagation in the polymer, excited through impulses generated by the random and continuous bombardment of DNA by solvent molecules along its length. This gives rise to travelling structural distortions in DNA that result in DNA breathing and in drug intercalation. These distortions can be thought of as premelting conformational changes in DNA, and, in this respect, could have important implications in understanding the structure of DNA active in RNA transcription, DNA replication, and genetic recombination. The nature of these distortions is further documented and their relationship to DNA breathing and to drug intercalation is discussed.A key intermediate in these processes is the multiply kinked structure, ..beta.. kinked DNA. This structure has both B and A characteristics, and it is postulated that this structure is a conformational intermediated in the B ..-->.. A polymorphic transition. In addition, a class of intercalators known as the bis functional intercalators, molecules that have two intercalative chromophores separated by about 10.2 A is discussed. These bifunctional intercalators appear to bind DNAmore » in a neighbor exclusion mode, and could, therefore, be probes to detect the migratory ..beta.. kinked DNA structure that has been postulated. The nature of the excitation force due to Brownian motion of solvent molecules that gives rise to wave motion in DNA is examined. This force is temperature dependent and one would therefore expect the average energy density along the DNA molecule to reflect this. As one raises the temperature of DNA in solution, additional localized regions of DNA premelting could arise. Events such as these could have important implications in understanding the mechanism of DNA melting.« less

Determination of molecular-weght distribution of chitosan by high-performance liquid chromatography

Optimal conditions for using high-performance liquid chromatography (HPLC) in the size exclusion mode have been determined for measuring the molecular-weight (MW) distribution of chitosan samples. Physical separation according to molecular size was accomplished on the stationary phase of glass supports having controlled pore sizes ranging from 2500 to 40 Å. Selection of column combinations was based on the requirements to resolve the higher MW fraction of chitosan and to give a linear calibration curve within the required MW range. The best combination of glass pore sizes and column lengths in two foot sections joined sequentially was: 2500 Å (2 ft.), 1500 Å (4 ft.), 550 Å (6 ft.), 250 Å (2 ft.), 100 Å (2 ft.), and 40 Å (2 ft.). a loading study showed that an injection load of 500 μg, i.e. 100 μl at 5 g/l or 50 μl at 10 g/l (w/v), was the optimal load to give reproducible elution volumes, precision in quantitation, and minimum viscosity effects. The best calibration curve using defined dextran standards was obtained from the geometric mean of M w (weight average MW) and M n (number average MW) values and peak elution volumes. Precision in determining MW distributions of chitosan as well as dextran standards was better than 5% relative standard deviation, and the differences between these results and the manufacturer's data on the dextran standards were t 60 −17%. The MW distribution of a selected chitosan sample in 2% acetic acid thus determined was M w = 2,055,000, M n = 936,000, dispersity = 2.16, and the most abundant species was around 1,103,000. Analysis time for the HPLC separation was less than 20 min per sample. Chitosan is an effective coagulating agent for the treatment of food processing wastes and activated sludge from biological treatment systems. It is manufactured from chitin in shrimp and crab wastes. The rapid methods developed here for determining the MW distribution of chitosan preparations will be used to optimize the manufacturing process and guide the selection of more effective chitosan products.