Molecular weight characterization of soluble high performance polyimides. 1. Polymer-solvent-stationary phase interactions in size exclusion chromatography

Soluble, fully cyclized m-amino phenyl acetylene terminated polyimides based on several anhydride/diamine monomers were prepared in N-methylpyrrolidine (NMP) and cyclized by solution imidization to controlled molecular weight. The polyimides and a polyamic acid precursor were successfully analyzed by size exclusion chromatography (SEC) utilizing online parallel coupled refractive index and differential viscometer detectors. The calculated M _nvalues were varied from 3,000 to 20,000 daltons. N-methylpyrrolidone (NMP), tetrahydrofuran (THF), and chloroform served as mobile phases for the cross-linked polystyrene gel packings. Normal retention behavior of the polyimides was observed in chloroform, THF, and NMP containing LiBr, or in NMP stirred over P₂O₅ before use. Values of Mark-Houwink-Sakurada exponents for narrow distribution linear polystyrene indicate that pure NMP and NMP with 0.06 M LiBr are good solvents for polystyrene standards at 60°C. In contrast, SEC behavior of polyimides in pure NMP leads to splitting of the peaks with the major portion observed to pass through the columns at the exclusion limit. In contrast to strong polymeric chain expansion of the polyamic acid in dilute solution, presumably due to a polyelectrolyte effect, no increase of intrinsic viscosity of polyimide samples in pure NMP was observed. This exclusion effect of polyimides analyzed in NMP is discussed in terms of possible ion-exclusion from pores of the stationary phase. Differences in polystyrene calibration in NMP with or without additives and the temperature dependence of calibration curves in these mobile phases is discussed as well. ©1995 John Wiley & Sons, Inc.     

Molecular weight characterization of soluble high performance polyimides. 2. Validity of universal SEC calibration and absolute molecular weight calculation

Six different soluble high-performance aromatic polyimides, each prepared by solution imidization to three controlled average molecular weights, were analyzed by size exclusion chromatography (SEC) using on-line parallel coupled refractometric and viscometric detectors. N-methylpyrrolidone (NMP) with 0.06 M LiBr and NMP stirred over P₂O₅ were used as mobile phase for four of the polyimides; NMP with 0.06 M LiBr and NMP stirred over P₂O₅ were used as mobile phases for four of the polyimides; NMP with 0.06 M LiBr tetrahydrofuran (THF) and chloroform served as mobile phases for the other two polyimides. For all the samples the stationary phase in the SEC columns was cross-linked polystyrene beads. Molecular weight averages of the polyimides were calculated using universal SEC calibration with polystyrene standards in each solvent. The agreement of the calculated molecular weight averages in the different solvents confirms that the universal SEC calibrations are valid for these semiflexible polymers. There was good agreement with weightaverage molecular weights obtained by low-angle laser light scattering (LALLS) performed in pure NMP. Intrinsic viscosity and molecular weight data for a series of nine samples of one polyimide covering a M_w = 20,000–70,000 g mol^–1 interval were treated to obtain Mark-Houwink-Sakurada constants. Unperturbed chain dimensions of this polyimide were obtained by application of the Stockmayer-Fixman extrapolation procedure to these data. ©1995 John Wiley & Sons, Inc.     

Rapid determination of molecular parameters of synthetic polymers by precipitation/redissolution high‐performance liquid chromatography using “molded” monolithic column

Rapid high‐performance liquid chromatography (HPLC) of polystyrenes, poly(methyl methacrylates), poly(vinyl acetates), and polybutadienes using a monolithic 50 × 4.6 mm i.d. poly(styrene‐co‐divinylbenzene) column have been carried out. The separation process involves precipitation of the macromolecules on the macroporous monolithic column followed by progressive elution utilizing a gradient of the mobile phase. Depending on the character of the separated polymer, solvent gradients were composed of a poor solvent such as water, methanol, or hexane and increasing amounts of a good solvent such as THF or dichloromethane. Monolithic columns are ideally suited for this technique because convection through the large pores of the monolith enhances the mass transport of large polymer molecules and accelerates the separation process. Separation conditions including the selection of a specific pair of solvent and precipitant, flow rate, and gradient steepness were optimized for the rapid HPLC separations of various polymers that differed broadly in their molecular weights. Excellent separations were obtained demonstrating that the precipitation‐redissolution technique is a suitable alternative to size‐exclusion chromatography (SEC). The molecular weight parameters calculated from the HPLC data match well those obtained by SEC. However, compared to SEC, the determination of molecular parameters using gradient elution could be achieved at comparable flow rates in a much shorter period of time, typically in about 1 min. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 2767–2778, 2000     

Industrial application of green chromatography--I. Separation and analysis of niacinamide in skincare creams using pure water as the mobile phase

In this work, chromatographic separation of niacin and niacinamide using pure water as the sole component in the mobile phase has been investigated. The separation and analysis of niacinamide have been optimized using three columns at different temperatures and various flow rates. Our results clearly demonstrate that separation and analysis of niacinamide from skincare products can be achieved using pure water as the eluent at 60 °C on a Waters XTerra MS C18 column, a Waters XBridge C18 column, or at 80 °C on a Hamilton PRP-1 column. The separation efficiency, quantification quality, and analysis time of this new method are at least comparable with those of the traditional HPLC methods. Compared with traditional HPLC, the major advantage of this newly developed green chromatography technique is the elimination of organic solvents required in the HPLC mobile phase. In addition, the pure water chromatography separations described in this work can be directly applied in industrial plant settings without further modification of the existing HPLC equipment.     

Two-dimensional preparative liquid chromatography system for preparative separation of minor amount components from complicated natural products

An on-line comprehensive two-dimensional preparative liquid chromatography system was developed for preparative separation of minor amount components from complicated natural products. Medium-pressure liquid chromatograph (MPLC) was applied as the first dimension and preparative HPLC as the second one, in conjunction with trapping column and makeup pump. The performance of the trapping column was evaluated, in terms of column size, dilution ratio and diameter-height ratio, as well as system pressure from the view of medium pressure liquid chromatograph. Satisfactory trapping efficiency can be achieved using a commercially available 15 mm × 30 mm i.d. ODS pre-column. The instrument operation and the performance of this MPLC × preparative HPLC system were illustrated by gram-scale isolation of crude macro-porous resin enriched water extract of Rheum hotaoense. Automated multi-step preparative separation of 25 compounds, whose structures were identified by MS, ¹H NMR and even by less-sensitive ¹³C NMR, could be achieved in a short period of time using this system, exhibiting great advantages in analytical efficiency and sample treatment capacity compared with conventional methods.     

Size exclusion chromatography of plasmid DNA isoforms

There is considerable interest in using size exclusion chromatography (SEC) to analyze and purify specific plasmid isoforms, but there is currently no fundamental understanding of the effects of plasmid size and morphology on plasmid behavior in SEC. Experiments were performed for plasmids from 3.0 to 17.0 kbp in size. The linear and open-circular isoforms were generated from the supercoiled plasmid by appropriate enzymatic digestion. SEC retention data were obtained using a Sephacryl S-1000 SF resin packed column and an Agilent HPLC system over a range of flow rates using buffers of different ionic strength and composition. The plasmid partition coefficients, K_P, were evaluated from the first statistical moment of the chromatographic peak. The partition coefficient decreased with increasing plasmid size as expected; K_P varied from 0.299 to 0.045 for supercoiled plasmids of 3.0 to 17.0 kbp. The partition coefficient also increased with increasing ionic strength due to the compaction of the DNA associated with the shielding of the intramolecular electrostatic interactions. For any plasmid size, the supercoiled isoform had the highest K_P followed by the open-circular and then the linear isoform, consistent with independent estimates of the plasmid radius of gyration as determined by static light scattering. The experimental data were analyzed using available theoretical models for the partitioning of linear and cyclic polymer chains in well-defined pore geometries. These results provide important insights into the behavior of different plasmid isoforms in size exclusion chromatography.     

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.     

The size exclusion chromatography calibration of ‘Mixed-A’ and ‘Mixed-D’ columns using various polymers and compounds: An application to coal-derived materials

This work attempts to obtain the calibration curves of two different size exclusion chromatography (SEC) columns operating with 1-methyl-2-pyrrolidinone (NMP) as eluent by using various standards. Polystyrene (PS) and polymethylmethacrylate (PMMA) standards were used for obtaining calibration curves, and checked against polysaccharide (PSAC) standards, some small aromatic polycyclic standards and miscellaneous polymers. Polystyrenes and polymethylmethacrylates gave identical calibrations while polysaccharides and miscellaneous polymers lay within 1 or 2 min of the polystyrene calibration. Small molecules of mass less than 1000 units lay on or near to the polystyrene calibration lines, with a shift to late elution for the smallest molecules. This shift may be caused by the interaction with the column packing. A sample has been examined by analytical size exclusion chromatography, which was calibrated using polystyrene and polymethylmethacrylate standards. Molecular mass (MM) distributions of the sample have been examined in terms of these calibrations.     

1,3,5,7-Tetramethyl-8-aminozide-difluoroboradiaza-s-indacene as a new fluorescent labeling reagent for the determination of aliphatic aldehydes in serum with high performance liquid chromatography

A BODIPY-based fluorescent derivatization reagent with a hydrazine moiety, 1,3,5,7-tetramethyl-8-aminozide-difluoroboradiaza-s-indacene (BODIPY-aminozide), has been designed for aldehyde labeling. An increased fluorescence quantum yield was observed from 0.38 to 0.94 in acetonitrile when it reacted with aldehydes. Twelve aliphatic aldehydes from formaldehyde to lauraldehyde were used to evaluate the analytical potential of this reagent by high performance liquid chromatography (HPLC) on C₁₈ column with fluorescence detection. The derivatization reaction of BODIPY-aminozide with aldehydes proceeded at 60 °C for 30 min to form stable corresponding BODIPY hydrazone derivatives in the presence of phosphoric acid as a catalyst. The maximum excitation (495 nm) and emission (505 nm) wavelengths were almost the same for all the aldehyde derivatives. A baseline separation of all the 12 aliphatic aldehydes (except formaldehyde and acetaldehyde) is achieved in 20 min with acetonitrile–tetrahydrofuran (THF)–water as mobile phase. The detection limits were obtained in the range from 0.43 to 0.69 nM (signal-to-noise = 3), which are better than or comparable with those obtained by the existing methods based on aldehyde labeling. This reagent has been applied to the precolumn derivatization followed with HPLC determination of trace aliphatic aldehydes in human serum samples without complex pretreatment or enrichment method.     

Solvent effects on jet evolution during electrospinning of semi-dilute polystyrene solutions

Linear polystyrene with a weight average molecular weight of 393,400 g/mol was used with various solvents including tetrahydrofuran (THF), chloroform, carbon disulfide (CS₂), 1-methyl-2-pyrrolidinone (NMP), and N,N-dimethylformamide (DMF) to produce solutions, corresponding to a Berry number of about 9. The jet breakdown behavior of each of these solutions was studied with a high speed camera (2000 frames/s). The structure of the electrospun polymer was examined with a scanning electron microscope. The results indicate that jet breakdown with THF and chloroform entailed significant extensional flow, followed by the onset of instabilities, leading to the formation of numerous secondary jets under steady-state conditions. By comparison, the solution jets with DMF and NMP exhibit extensive whipping and splaying to produce a cloud of jets. In this case, few secondary jets were observed under steady-state conditions. A highly refined structure was observed in the electrospun polymer for NMP and DMF, in accordance with the extensive instabilities observed during jet breakdown. Limited jet instability observed with CS₂ solution suggests the significant effect of solvent evaporation. Typical primary jet velocities were measured to be on the order of 2-5 m/s.     

高效液相色谱表征高聚物*

最常用的测试高聚物的分子量和分子量分布的体积排除色谱(SEC)是高效液相色谱 (HPLC)的一个重要分支,HPLC的另一个重要分支是相互作用液相色谱, 它是20世纪90年代开始用于高分子分离和表征的研究领域。相互作用液相色谱可以根据高分子的化学结构（如共混物组成、共聚物组成、端基）来分离,它比SEC 有更高的分离效率。本文介绍了高聚物液相色谱的分离模式,并就高聚物体积排除色谱、相互作用液相色谱、临界液相色谱和全二维液相色谱用于分离和表征高聚物的研究进展进行了较系统的综述,并对该技术目前存在的问题和今后可能的发展前景进行了探讨。     

Semi-micro size-exclusion chromatography: Molecular-weight measurement of poly (ethylene terephthalate) and separation of oligomers and prepolymers

Summary Several polystyrene gels of different pore sizes were packed into a 500 mm×2.1 mm I.D. column. Semi-micro size-exclusion chromatography (SEC) using these columns was carried out with a system consisting of a triple piston pump, a micro loop injector and a flow cell with 1.0-μl cell volume constructed for semi-micro HPLC, because the dead volume of the injector and the cell volume of flow cell for conventional HPLC caused a significant loss in column efficiency. The effects of sample amount, injection volume and mobile phase flow rate on column efficiency and retention volume were examined and the optimized operational variables of the sample amount (below 500 μg), the injection volume (less than 15 μl) and the flow rate range (30–70 μl/min) determiend for semi-micro SEC. Oligostyrene, epoxy resin, phenol-formaldehyde resin and phthalates were analyzed by the optimized semi-micro SEC system under the given conditions. In addition, molecular weight distribution of four different poly(ethylene terephthalate) films was successfully measured by using a mixture of chloroform and hexafluoroisopropanol as the eluent.     

A green and fast chromatographic method for determining organic compound mobility in soils

Soil leaching column chromatography (SLCC) employing totally aqueous mobile phases has been used for the estimation of retardation factors (R) of heterocyclic compounds and heteroatom-substituted aryl derivatives Aniline, Aniside, Cresol, Hydroquinone, p-Nitroaniline, Phenol, Phenylenediamine, Piridine, Sulfanilamide, Sulfathiazole, Sulfamethoxazole, Metolachlor and Toluene in soils with low fraction of organic carbon. Small columns (0.39 cm × 10 cm i.d.) were packed with soils of different compositions (alluvial sediment, coarse sand and standard Eurosoil4 mixed with quartz). The theory of linear chromatography was employed to correlate statistical peak moments to R. Rs estimated through SLCC were compared with those derived from water–organic carbon partitioning coefficient reported in the literature. The adsorption isotherm of Metolachlor was measured by frontal analysis on a small column packed with alluvial sediment. The approach proposed for measuring the adsorption isotherm requires small amounts of chemicals and soils and does not need fraction collection nor detector calibration. The information obtained by SLCC experiments was used to predict the elution of Metolachlor and Phenol on a large column (8.9 cm × 100 cm i.d.) packed with alluvial sediment. The method used in this work, not making use of any chemical solvents, respects the fundamental concepts of green chemistry.     

Use of itaconic acid-based polymers for solid-phase extraction of deoxynivalenol and application to pasta analysis

Molecular modelling and computational design were used to identify itaconic acid (IA) as a functional monomer with high affinity towards deoxynivalenol (DON), a Fusarium-toxin frequently occurring in cereals. IA-based polymers were photochemically synthesised in dimethyl formamide (porogen) using ethylenglycol dimethacrylate as cross-linker and 1,1′-azo-bis(cyclohexane carbonitrile) as initiator, and the relevant binding interactions with DON in solvents with different polarity were investigated. The performances of the non-imprinted IA-based polymer (blank polymer, BP) and the corresponding molecularly imprinted polymer (MIP) were compared using DON as a template. Both BP and MIP were able to bind about 90% DON either in toluene, water or water containing 5% polyethylene glycol. Non-imprinted polymers with different molar ratios of IA to cross-linker were evaluated as adsorbents for solid-phase extraction (SPE) clean-up and pre-concentration of DON from wheat and pasta samples prior to HPLC analysis. Samples were extracted with PBS/0.1 M EDTA solution and cleaned up through a cartridge containing blank IA-based polymer. The column was washed with PBS (pH 9.2) and the toxin was eluted with methanol and quantified by reversed-phase HPLC with UV detector (λ = 220 nm), using methanol:water:acetic acid (15:85:0.1, v/v/v) as the mobile phase. Effective removal of matrix interferences was observed only for pasta with DON recoveries higher than 70% (RSD < 7%, n = 3) at levels close to or higher than EU regulatory limit.     

On-line coupling of counter-current chromatography and macroporous resin chromatography for continuous isolation of arctiin from the fruit of Arctium lappa L.

In this work, we have developed a novel hybrid two-dimensional counter-current chromatography and liquid chromatography (2D CCC × LC) system for the continuous purification of arctiin from crude extract of Arctium lappa. The first dimensional CCC column has been designed to fractionalize crude complex extract into pure arctiin effluent using a one-component organic/salt-containing system, and the second dimensional LC column has been packed with macroporous resin for on-line adsorption, desalination and desorption of arctiin which was effluent purified from the first CCC dimension. Thus, the crude arctiin mixture has been purified efficiently and conveniently by on-line CCC × LC in spite of the use of a salt-containing solvent system in CCC separation. As a result, high purity (more than 97%) of arctiin has been isolated by repeated injections both using the ethyl acetate–8% sodium chloride aqueous solution and butanol–1% sodium chloride aqueous solution. By contrast with the traditional CCC processes using multi-component organic/aqueous solvent systems, the present on-line CCC × LC process only used a one-component organic solvent and thus the solvent is easier to recover and regenerate. All of used solvents such as ethyl acetate, n-butanol and NaCl aqueous solution are low toxicity and environment-friendly. Moreover, the lower phase of salt-containing aqueous solution used as mobile phase, only contained minor organic solvent, which will save much organic solvent in continuous separation. In summary, our results indicated that the on-line hybrid 2D CCC × LC system using one-component organic/salt-containing aqueous solution is very promising and powerful tool for high-throughput purification of arctiin from fruits of A. lappa.     

Preparation and characterization of a thermoresponsive gigaporous medium for high-speed protein chromatography

A high-speed thermoresponsive medium was developed by grafting poly(N-isopropylacrylamide-co-butyl methacrylate) (P(NIPAM-co-BMA)) brushes onto gigaporous polystyrene (PS) microspheres via surface-initiated atom transfer radical polymerization (ATRP) technique, which has strong mechanical strength, good chemical stability and high mass transfer rate for biomacromolecules. The gigaporous structure, surface chemical composition, static protein adsorption, and thermoresponsive chromatographic properties of prepared medium (PS–P(NIPAM-co-BMA)) were characterized in detail. Results showed that the PS microspheres were successfully grafted with P(NIPAM-co-BMA) brushes and that the gigaporous structure was robustly maintained. After grafting, the nonspecific adsorption of proteins on PS microspheres was greatly reduced. A column packed with PS–P(NIPAM-co-BMA) exhibited low backpressure and significant thermo-responsibility. By simply changing the column temperature, it was able to separate three model proteins at the mobile phase velocity up to 2167 cm h⁻¹. In conclusion, the thermoresponsive polymer brushes grafted gigaporous PS microspheres prepared by ATRP are very promising in ‘green’ high-speed preparative protein chromatography.     

A novel dual-function molecularly imprinted polymer on CdTe/ZnS quantum dots for highly selective and sensitive determination of ractopamine

A novel dual-function material was synthesized by anchoring a molecularly imprinted polymer (MIP) layer on CdTe/ZnS quantum dots (QDs) using a sol–gel with surface imprinting. The material exhibited highly selective and sensitive determination of ractopamine (RAC) through spectrofluorometry and solid-phase extraction (SPE) coupled with high performance liquid chromatography (HPLC). A series of adsorption experiments revealed that the material showed high selectivity, good adsorption capacity and a fast mass transfer rate. Fluorescence from the MIP-coated QDs was more strongly quenched by RAC than that of the non-imprinted polymer, which indicated that the MIP-coated QDs acted as a fluorescence sensing material could recognize RAC. In addition, the MIP-coated QDs as a sorbent was also shown to be promising for SPE coupled with HPLC for the determination of trace RAC in feeding stuffs and pork samples. Under optimal conditions, the spectrofluorometry and SPE-HPLC methods using the MIP-coated QDs had linear ranges of 5.00 × 10⁻¹⁰–3.55 × 10⁻⁷ and 1.50 × 10⁻¹⁰–8.90 × 10⁻⁸ mol L⁻¹, respectively, with limits of detection of 1.47 × 10⁻¹⁰ and 8.30 × 10⁻¹¹ mol L⁻¹, the relative standard deviations for six repeat experiments of RAC (2.90 × 10⁻⁹ mol L⁻¹) were below 2.83% and 7.11%.     

Separation of α-cyclohexylmandelic acid enantiomers using biphasic chiral recognition high-speed counter-current chromatography

This work concentrates on a chiral separation technology named biphasic recognition applied to resolution of α-cyclohexylmandelic acid enantiomers by high-speed counter-current chromatography (HSCCC). The biphasic chiral recognition HSCCC was performed by adding lipophilic (−)-2-ethylhexyl tartrate in the organic stationary phase and hydrophilic hydroxypropyl-β-cyclodextrin in the aqueous mobile phase, which preferentially recognized the (−)-enantiomer and (+)-enantiomer, respectively. The two-phase solvent system composed of n-hexane-methyl tert-butyl ether–water (9:1:10, v/v/v) with the above chiral selectors was selected according to the partition coefficient and separation factor of the target enantiomers. Important parameters involved in the chiral separation were investigated, namely the types of the chiral selectors (CS); the concentration of each chiral selector; pH of the mobile phase and the separation temperature. The mechanism involved in this biphasic recognition chiral separation by HSCCC was discussed. Langmuirian isotherm was employed to estimate the loading limits for a given value of chiral selectors. Under optimum separation conditions, 3.5–22.0 mg of α-cyclohexylmandelic acid racemate were separated using the analytical apparatus and 440 mg of racemate was separated using the preparative one. The purities of both of the fractions including (+)-enantiomer and (−)-enantiomer from the preparative CCC separation were over 99.5% determined by HPLC and enantiomeric excess reached 100% for the (±)-enantiomers. Recovery for the target compounds from the CCC fractions reached 85–88% yielding 186 mg of (+)-enantiomer and 190 mg of (−)-enantiomer. The overall experimental results show that the HSCCC separation of enantiomer based on biphasic recognition, in which only if the CSs involved will show affinity for opposite enantiomers of the analyte, is much more efficient than the traditional monophasic recognition chiral separation, since it utilizes the cooperation of both of lipophilic and hydrophilic chiral selectors.     

Determination of cefuroxime axetil in tablets and biological fluids using liquid chromatography and flow injection analysis

In the present work, the separations of calixarene derivatives have been investigated using both high-performance liquid chromatography (HPLC) and nonaqueous capillary electrophoresis (NACE) techniques. HPLC-1 method with LC-318 (pore size = 300 Å) column and MeCN mobile phase was optimized for the separation of calixarenes. At the flow-rate of 1 ml/min p-nitrocalix[6]arene, calix[4]arene and calix[6]arene could be well baseline and symmetrically separated within 5 min. For the separation of p-tert-butylcalix[n]arenes (n = 4, 6, 8), HPLC-2 and NACE methods have been optimized. The optimal conditions in HPLC-2 method included NH₂ column and MeCN mobile phase, and p-tert-butylcalix[n]arenes (n = 4, 6, 8) were baseline separated within 10 min at 0.8 min/min. The optimal conditions for NACE method employed MeCN-H₂O (8:2, v/v) as the nonaqueous medium and 120 mM Tris/HCl (pH 9.0) as the buffer, and p-tert-butylcalix[n]arenes (n = 4, 6, 8) were successfully baseline resolved within 16 min. With the detection at 280 nm, the calibration lines were linear in the ranges of 1-200 μg/ml for calixarene derivatives by HPLC-1 and HPLC-2 methods, and of 2.5-200 μg/ml for p-tert-butylcalix[n]arenes (n = 4, 6, 8) by NACE method, respectively. The detection limits (S/N = 3) and recoveries ranged from 0.5 to 1.4 μg/ml and from 98.1 to 102.4% by both HPLC-1 and HPLC-2 methods, and from 1.3 to 2.0 μg/ml and from 97.9 to 105.1% by NACE method, respectively. The intra-day reproducibility of the methods was determined with satisfactory results. The proposed HPLC and NACE methods were accurate and reproducible, and could be utilized to separate and determine calixarene derivatives.     

New Chromatographic and Hyphenated Techniques for Hydrophilic Copolymers

Most synthetic polymers are distributed in more than one parameter of molecular heterogeneity. For hydrophobic copolymers there are different chromatographic techniques available to analyse these distributions. As a result of the increasing interest in hydrophilic polymers and copolymers new chromatographic techniques are developed for the characterization of these polymers as well. However, very frequently these polymers contain highly polar or charged functional groups making them soluble only in aqueous mobile phases. There are several problems related to the use of aqueous mobile phases in polymer chromatography. Even the SEC analysis of such copolymers is not straightforward. As for HPLC in aqueous mobile phases, there are only a few applications in the literature so far. In addition to the fact that only a very limited number of stationary phases is available for aqueous HPLC of polymers, the interactions of polyelectrolytes in such chromatographic systems are not well understood. The present paper addresses the problems related to the application of SEC and HPLC in aqueous mobile phases. For graft copolymers with a polyethylene oxide backbone, e.g. PEG-g-polymethacrylic acid and PEG-g-polyvinyl alcohol, it will be shown that methods can be developed that give accurate molar mass and chemical composition information. Two-dimensional chromatography where aqueous HPLC and SEC are coupled on-line will be shown to be the most powerful analysis tool for the analysis of such copolymers. The hyphenation of the chromatographic separation techniques with spectroscopic detection techniques provides further insight into the molecular complexity of these copolymers.