Direction of temperature gradient for normal-phase temperature gradient interaction chromatography in polystyrene fractionation

Temperature gradient interaction chromatography (TGIC) is a powerful technique for molecular weight fractionation of polymers, in which the interaction strength is controlled by varying the column temperature. In the present paper, the effects of the sign of the temperature dependence of the retention and the direction of the temperature gradient (raising or lowering) on TGIC in the normal-phase mode were studied for the molecular weight fractionation of polystyrene samples in organic mobile phases. It was found that a positive temperature gradient was effective in the system consisting of amino-modified silica (NH(2)) column and the eluent mixture of tetrahydrofuran and n-hexane where retention decreased with increasing temperature. A negative temperature gradient was effective for the systems consisting of a bare-silica column//chloroform/n-hexane and NH(2)-column//chloroform/n-hexane, where retention increased with increasing temperature. Increasing retention with increasing temperature has been found, so far, only for a water-soluble polymer (PEO) in an aqueous mobile phase in RP-TGIC.     

Characterization of polydisperse poly(vinyl chloride) by temperature gradient interaction chromatography

Temperature gradient interaction chromatography (TGIC) was employed to fractionate a commodity polymer, poly(vinyl chloride) (PVC) with wide molecular weight distribution (MWD). The TGIC fractionation was carried out with C18 bonded silica and dimethylformamide (DMF) as the stationary and mobile phase, respectively. TGIC exhibited a high resolution to fractionate the PVC into the fractions with a narrow MWD comparable to the anionically polymerized standards. In combination with light scattering detection, TGIC is able to characterize the polymers with wide MWD and shows a good potential to be further developed as a new preparative fractionation method of synthetic polymers.     

On-chip temperature gradient interaction chromatography

This paper reports the first integrated microelectromechanical system (MEMS) HPLC chip that consists of a parylene high-pressure LC column, an electrochemical sensor, a resistive heater and a thermal-isolation structure for on-chip temperature gradient interaction chromatography application. The separation column was 8 mm long, 100 microm wide, 25 microm high and was packed with 5 microm sized, C18-coated beads using conventional slurry-packing technique. A novel parylene-enhanced, air-gap thermal isolation technology was used to reduce heater power consumption by 58% and to reduce temperature rise in the off-column area by 67%. The fabricated chip consumed 400 mW when operated at 100 degrees C. To test the chromatography performance of the fabricated system, a mixture of derivatized amino acids was chosen for separation. A temporal temperature gradient scanning from 25 to 65 degrees C with a ramping rate of 3.6 degrees C/min was applied to the column during separation. Successful chromatographic separation of derivatized amino acids was carried out using our chip. Compared with conventional temperature gradient HPLC system which incorporates "macro oven" to generate temporal temperature gradient on the column, our chip's thermal performance, i.e., power consumption and thermal response, is greatly improved without sacrificing chromatography quality.     

Rapid molecular weight analysis of polymers by temperature gradient interaction chromatography

Temperature gradient interaction chromatography (TGIC) has been established as a high-resolution technique for the characterization of synthetic polymers. So far, most of the TGIC investigations focused on the high-resolution analysis and little effort has been made on the reduction of the analysis time. In this study, we examined the effect of the column heating rate, the eluent flow rate, and the column length on the TGIC analysis time. We found that the heating rate is the most important experimental parameter to control the TGIC retention time. With a C18 silica column (50 mm x 4.6mm I.D.), a set of PS standards of wide molecular weight range (5 - 648 kg/mol) could be separated within 4 min at a heating rate of 8 degrees C/min.     

Characterization of alkyl polyglycosides by both reversed-phase and normal-phase modes of high-performance liquid chromatography

Alkyl polyglycosides today represent the most important sugar surfactant. Nonionic sugar surfactants produced via different synthetic routes are mixtures of alkyl homologues, oligomers, anomers and isomers. Alkyl homologues and oligomers of alkyl mono- and diglucosides were separated by reversed-phase high-performance liquid chromatography (HPLC) with methanol-water as the mobile phase using a gradient elution. The gradient was optimized in respect to a simultaneous separation of alkyl glycosides according to their alkyl chain length and alkyl polyoxyethylene glucosides with regard to their length of the polyoxyethylene spacer. The separation of alkyl glycosides into alpha- and beta-anomers was carried out by normal-phase HPLC with isooctane-ethyl acetate (60:40, v/v)-2-propanol in the gradient mode. Light scattering detection was used. Matrix-assisted laser desorption ionization time-of-flight mass spectra of alkyl glucosides and dodecyl glucosides with oxyethylene spacer groups are presented.     

Effect of pore size of packing materials on molecular-weight separation by temperature gradient interaction chromatography

Temperature gradient interaction chromatography (TGIC) is an interactive polymer chromatography technique varying the column temperature during the elution in a programmed manner to control the solute retention. In the present paper, the effect of the pore size of packing materials on the molecular-weight separation of polystyrene and poly(methyl methacrylate) standard samples by TGIC was studied by using the columns (octadecyl modified silica) with different pore size (100, 300 and 1000 Å) and eluent mixture of CH₂Cl₂/CH₃CN. By rising temperature gradient, both polymers were separated by molecular weight from lower to higher. It became clear that each sample elutes out earlier as the pore size is larger. These experimental results could be explained by the theory based on the scaling concept of Gorbunov and Skvortsov.     

Turbidimetric titration and gradient high-performance liquid chromatography of polystyrene samples

Summary Polystyrene samples of narrow molecular-weight distribution have been eluted according to their molecular weight from columns packed with bare silica Si50, phenyl, or C18 bonded phase by gradients of methanol and tetrahydrofuran (THF) or ofiso-octane and THF. Among the six combinations investigated,iso-octane/THF with a silica column formed a proper normal-phase system whereas methanol/THF with a C18 column formed a proper reversed-phase system. The combinations of C18 column andiso-octane/THF or of Si50 column and methanol/THF gradient did not correspond to the approved polarity rules in high-performance liquid chromatography but were nevertheless effective in separating polystyrene mixtures by molecular weight. Methanol andiso-octane are nonsolvents for polystyrene whereas THF is a solvent. The solubility of polystyrene as a function of molecular weight and concentration was determined by means of turbidimetric titration of solutions in THF with the nonsolvents used in the gradients. The solubility and elution characteristics were almost identical on C18 columns or in methanol/THF combinations. The elution from phenyl bonded phase and Si50 columns usingiso-octane/THF gradients required more THF than the solubility experiments. Information is also given on the occurrence of multimodal elution patterns.     

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

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

Analysis of polystyrene-b-polyisoprene copolymers by coupling of liquid chromatography at critical conditions to NMR at critical conditions of polystyrene and polyisoprene

For the investigation of the molecular heterogeneity of polystyrene-b-polyisoprene block copolymers, a chromatographic separation method, namely liquid chromatography at critical conditions was developed. This method was coupled on-line with (1)H-NMR(where NMR stands for nuclear magnetic resonance) for the comprehensive analysis of the polystyrene-b-polyisoprene copolymers. The copolymers were synthesized by two different methods: sequential living anionic polymerization and coupling of living precursor blocks. While (1)H-NMR allows just for the analysis of the bulk chemical composition of the block copolymers, the coupling with liquid chromatography at critical conditions provides selective molar mass information on the polystyrene and polyisoprene blocks within the copolymers. The polyisoprene block molar mass is determined by operating at chromatographic conditions corresponding to the critical point of adsorption of polystyrene and size exclusion chromatography mode for polyisoprene. The molar mass of the polystyrene block is determined by operating at the critical conditions of polyisoprene. In addition to the molar mass of each block of the copolymers, the chemical composition distribution of the block copolymers was determined. By using the coupling of liquid chromatography at critical conditions to (1)H-NMR, one can also detect the homopolymers formed during synthesis. Finally the microstructure of the polyisoprene block in the copolymers was evaluated as a function of molar mass.     

Characterization of the extremely high molecular mass polystyrene by gel permeation chromatography

Summary The extremely high mol. mass (EHMM) polystyrene (PS) samples prepared by emulsion polymerization with an initiator forming heterogeneous phase were characterized by gel permeation chromatography (GPC). A bicomponent eluent methyl ethyl ketone (MEK) and methanol (McOH) in a ratio slightly different from the composition of theta solvent for PS was applied in GPC measurements. The GPC apparatus was calibrated with a series of PS reference materials. The course of calibration curves in the EHMM region was mathematically or graphically simulated by the third and second degree polynomial respectively. Correction of GPC data for the broadening effect was made mathematically by solving the Tung integral equation by the iterative method ofChang andHuang. The values of the EHMM PS samples varied between 1.07 × 10⁷ and (>4 × 10⁷). The values of were higher than 1.5. Besides distribution analysis by GPC also light scattering (LS) was used for measurement of of EHMM PS samples and a sample was partially characterized also by the method of sedimentation in ultracentrifuge (UC).

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Zusammenfassung Polystyrol-Proben mit extrem hohen Molekulargewichten (EHMM-PS) wurden durch Emulsions-Polymerisation mit einem eine heterogene Phase bildenden Initiator hergestellt. Sie wurden mittels GPC charakterisiert unter Verwendung einer MEK-MeOH-Mischung, deren Zusammensetzung etwas vom -Lösungsmittel abweicht. Die Eichung erfolgte mit mehreren Referenz-Polystyrolen. Der Verlauf der Kalibrierungs-Kurven im EHMM-PS-Bereich wurde mathematisch oder graphisch unter Zugrundelegung eines Polynoms 2. und 3. Grades simuliert. Die GPC-Daten wurden unter Berücksichtigung des Verbreiterungs-Effektes unter Verwendung von in der Literatur angegebenen Gleichungen korrigiert.M _w von EHMM-PS wurde von 1,07 · 10⁷ bis > 4 · 10⁷ variiert;M _w /M _n war > 1,5. Ferner wurdeM _w mittels Lichtstreuung und für eine Probe durch Sedimentations-Messungen bestimmt.

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With 4 figures 2 tables

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Dedicated to Prof. Dr. G. Rehage on the occasion of his 60th birthday.     

Characterization of a 4-miktoarm star copolymer of the (PS-b-PI)3 PS type by temperature gradient interaction chromatography

Temperature gradient interaction chromatography (TGIC) was applied for the separation of a complex miktoarm star copolymer which has one polystyrene (PS) arm and three polystyrene-b-polyisoprene (PS-b-PI) diblock copolymer arms. Such miktoarm star polymers are much more difficult to characterize than branched homopolymers since the byproduct, typically polymers with missing arm(s) or coupled products, have not only different molecular weights but also different compositions. TGIC was able to fully separate the byproducts, and the composition of the molecular species corresponding to the different separated elution peaks was determined by two methods, fractionation/NMR and multiple detection (UV and RI). A reasonable agreement between the results of the two methods was obtained. By using the composition found, the corresponding molecular weights were determined by multi-angle light scattering detection. Based on the composition and the molecular weight we were able to identify the structure of the different molecular species.     

Axial temperature gradient and mobile phase gradient in microcolumn high-performance liquid chromatography

The effect of axial temperature gradient (ATG) along a microcolumn on the separation performance at both isocratic and gradient elution mode was investigated. A thermostat system was designed to form an ATG along the packed column. Polycyclic aromatic hydrocarbons (PAHs) were separated on a 0.53 mm  × 150 mm i.d. 5 μm C₁₈ microcolumn, with water and acetonitrile as mobile phase. The separation results obtained at mobile phase gradient (MPG) and ATG in microcolumn HPLC were compared with the results performed at ambient conditions. Extrapolated curves of peak width at half height (wh)versus lnk showed that wh is narrower at the same retention time when ATG was applied in addition to MPG. The column efficiency was enhanced 20-30% and the resolution was slightly reduced because of reduction of selectivity at elevated temperature at ATG condition. The RSD of retention time in ATG mode was less than 2.5%.     

Prediction of peptide retention times in normal-phase liquid chromatography with only a single gradient run.

Previous studies of peptide separation by normal-phase liquid chromatography have shown a linear relationship between the logarithm of the capacity factor and the logarithm of the volume fraction of modifier in the mobile phase. This permitted the use of a model to predict isocratic and gradient retention times based on data obtained by two initial gradient runs. In the present study, chromatographic behavior of 25 peptides in normal-phase liquid chromatography with isocratic elution have been studied and a linear relationship between the slope (S) and intercept [log k(0)] was obtained. This relationship was combined with the algorithm of prediction reported in the previous paper. The prediction of peptide retention times with only a single experimental gradient retention data was investigated.     

Phase separation in polyisoprene/polystyrene blends by a systematically coarse-grained model

The authors have successfully developed a structurally coarse-grained 1,4-cis-polyisoprene-atactic polystyrene blend model by systematic mapping between a detailed atomistic model and a mesoscale model. This is to their best knowledge the first time that a chemically specific polymer blend model has been used to study the phase separation morphology and kinetics in a blend. A structurally optimized force-field model has many advantages over simple bead-spring models in terms of representing the chain microstructure. It keeps the identity of the polymers, particularly the structure through radial distribution functions. Starting from randomly mixed initial configurations, the blends show a clear phase separation for chain lengths around 10 monomers and this separation becomes more pronounced with the increase of chain length. The ensuing morphology is lamellar at equiweight concentrations and cylindrical or spherical at unbalanced concentrations. Morphologies are validated to be stable under increasing system sizes and further characterized quantitatively by density profiles.     

Size exclusion chromatography with dual-beam refractive index gradient detection of polystyrene samples

Non-aqueous size exclusion chromatography (SEC) of polystyrenes (as model analytes) is examined using the microscale molar mass sensor (μ-MMS) for detection. The μ-MMS is combined with SEC to demonstrate this simultaneously universal and molar mass selective detection method for polymer characterization. The μ-MMS is based on measuring the refractive index gradient (RIG) at two positions (upstream and downstream) within a T-shaped microfluidic channel. The RIG is produced from a sample stream (eluting analytes in the mobile phase) merging with a mobile phase stream (mobile phase only). The magnitude of the RIG is measured as a probe beam deflection angle and is related to analyte diffusion coefficient, the time allowed for analyte diffusion from the sample stream toward the mobile phase stream, and the bulk phase analyte refractive index difference relative to the mobile phase. Thus, two deflection angles are measured simultaneously, the upstream angle and the downstream angle. An angle ratio is calculated by dividing the downstream angle by the upstream angle. The μ-MMS was found to extend the useful molar mass calibration range of the SEC system (nominally limited by the total exclusion and total permeation regions from ∼100,000 g/mol to ∼800 g/mol), to a range of 3,114,000-162 g/mol. The injected concentration LOD (based on 3 s statistics) was 2 ppm for the upstream detection position. The point-by-point time-dependent ratio, termed a ‘ratiogram’, is demonstrated for resolved and overlapped peaks. Within detector band broadening produces some anomalies in the ratiogram shapes, but with highly overlapped distributions of peaks this problem is diminished. Ratiogram plots are converted to molar mass as a function of time, demonstrating the utility of SEC/μ-MMS to examine a complex polymer mixture.     

Surfactant-mediated hydrophobic interaction chromatography of proteins: gradient elution

Addition of 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulphonate (CHAPS) to mobile phases in gradient elution hydrophobic interaction chromatography (HIC) on SynChropak Propyl causes changes in observed elution times for nine globular proteins. The nine proteins showed different percentage reductions in capacity factor, k', demonstrating the ability of CHAPS to change the selectivity of the separations. Three basic types of gradient experiments have been explored for surfactant-mediated gradient elution HIC. Type I gradients are conducted with constant salt and variable surfactant concentration. Type II gradients with variable salt and constant surfactant concentration, and Type III gradients with variable salt and surfactant concentrations. By the criterion of a linear relationship between gradient time and retention time the linear solvent strength condition applies to Type II and Type III gradients. Type III gradients, with the fastest re-equilibration time, are preferable for repetitive analyses. Type I gradients are relatively ineffective in making use of the solvent strength of CHAPS, and Types I and II gradients require long equilibration times due to large changes in surface concentration of CHAPS which occur during elution. The presence of CHAPS had a negligible effect on peak shapes of the proteins examined, except for bovine serum albumin which yielded a narrower, less distorted peak in the presence of CHAPS.     

Analysis of purines and pyrimidines by mixed partition-adsorption normal-phase high-performance liquid chromatography

This paper summarizes the results in the development of mixed partition-adsorption (MPA) normal-phase high-performance liquid chromatography published in the last 10 years. The MPA normal-phase systems are an alternative approach not only to the adsorption normal-phase mode but also to the most widely used reversed-phase mode in the separation area of purine and pyrimidine derivatives. It is shown that the MPA systems are applicable in analytical practice.     

Comparison of provitamin A determination by normal-phase gravity-flow column chromatography and reversed-phase high performance liquid chromatography

Summary The provitamin A content of some food samples was determined by methods involving MgO: Hyflosupercel gravityflow column chromatography (GFCC) and reversed phase high performance liquid chromatography (HPLC), the quantitation being done by external standardization (HPLC-ES) or internal standardization (HPLC-IS) with Sudan. The results obtained with - and -carotene in carrots, -carotene and -cryptoxanthin in papaya and -carotene in tomato and kale agreed well, showing that any of the these techniques can be used, provided the analysis is done under optimum conditions. Good separation of the different provitamins using GFCC depends on the analyst's skill and visual acuity. HPLC-ES required a constant supply of provitamin standards, thus the varying purity of commercially available standards and the high instability of these compounds could pose grave problems. Due to the stability of Sudan, HPLC-IS appeared to be the method of choice although passage of the extract through a MgO: Hyflosupercel minicolumn was required prior to injection to separate chlorophylls, dihydroxy- and polyoxycarotenoids which would otherwise elute with Sudan. Nonconformity of the Sudan structure to those of the provitamins did not effect the quantitative results. The chromatographic separation, identity and quantification of the provitamins could be more easily established by using HPLC-IS, complemented with GFCC.