On-line Coupling Of Size Exclusion Chromatography Research Articles

Quantitative Studies of Block Copolymers and Their Containing Homopolymer Components by Diffusion Ordered Spectroscopy

AbstractSeveral polystyrene‐b‐poly(methyl methacrylate) (PS‐b‐PMMA) and polyisoprene‐b‐PMMA (PI‐b‐PMMA) block copolymers are studied with high‐resolution diffusion ordered spectroscopy (DOSY) in order to quantify the amounts and molar masses of the copolymers and the containing homopolymers. These studies are particularly challenging because these homopolymers and one block of the copolymers consist of the same monomer type with different molar masses and compositions and cause a total overlap of these polymer components in the NMR spectra. However, DOSY can be proven as a powerful tool for separation and quantification of these moieties. Whereas exponential fittings only deliver averaged diffusion data, biexponential fittings of the individual magnetization curves can separate the overlapping polymer parts. This DOSY approach can be successfully tested for different blends of PS as well as PS and PS‐b‐PMMA of different molar masses and weighed compositions. The DOSY separation is also compared to size exclusion chromatography (SEC). In the case of the PS‐b‐PMMA and PI‐b‐PMMA block copolymers, DOSY identifies the contaminations with homopolymers, quantifies their individual amounts as well as determines the molar masses of both the copolymers and homopolymers. Two mathematical evaluations are used for quantifying these homopolymers with DOSY. These results are compared with the online coupling of SEC and NMR.

Elemental Mass Size Distribution for Characterization, Quantification and Identification of Trace Nanoparticles in Serum and Environmental Waters.

Accurate characterization, quantification, and identification of nanoparticles (NPs) are essential to fully understand the environmental processes and effects of NPs. Herein, the elemental mass size distribution (EMSD), which measures particle size, mass, and composition, is proposed for the direct size characterization, mass quantification, and composition identification of trace NPs in complex matrixes. A one-step method for the rapid measurement of EMSDs in 8 min was developed through the online coupling of size-exclusion chromatography (SEC) with inductively coupled plasma mass spectrometry (ICP-MS). The use of a mobile phase with a relatively high ionic strength (a mixture of 2% FL-70 and 2 mM Na2S2O3) ensured the complete elution of different-sized NPs from the column and, therefore, a size-independent response. After application of a correction for instrumental broadening by a method developed in this study, the size distribution of NPs by EMSD determination agreed closely with that obtained from transmission electron microscopy (TEM) analysis. Compared with TEM, EMSD allows a more rapid determination with a higher mass sensitivity (1 pg for gold and silver NPs) and comparable size discrimination (0.27 nm). The proposed EMSD-based method was capable of identifying trace Ag2S NPs and core-shell nanocomposite Au@Ag, as well as quantitatively tracking the dissolution and size transformation of silver nanoparticles in serum and environmental waters.

Determining the solution conformational entropy of oligosaccharides by SEC with on-line viscometry detection

Introduced here is a method for determining the solution conformational entropy of oligosaccharides (−ΔS) that relies on the on-line coupling of size-exclusion chromatography (SEC), an entropically-controlled separation technique, and differential viscometry (VISC). Results from this SEC/VISC method were compared, for the same injections of the same sample dissolutions and under identical solvent/temperature conditions, to results from a benchmark SEC/differential refractometry (SEC/DRI) method which has been applied successfully over the last decade to determining −ΔS of a variety of mono-, di-, and oligosaccharides. The accuracy (as compared to SEC/DRI) and precision of SEC/VISC were found to be excellent, as was the sensitivity of the viscometer in the oligomeric region. The experiments presented here contrast three sets of (1→4)-β-d-oligosaccharides, namely manno-, cello-, and N-acetylchitooligosaccharides of degree of polymerization (DP) 2 through 6. For each series, the dependence of −ΔS on DP was found to be monotonic while, between series, differences at each DP could be ascribed to either the additional degrees of freedom imparted by large, multi-atomic substituent groups, or to the presence or absence of additional intramolecular hydrogen bonds, depending on the axial versus equatorial arrangement of particular hydroxyl groups. An hypothesis is advanced to explain the unexpectedly high sensitivity of viscometric detection for low-molar-mass analytes. The method presented can be extended to the analysis of oligosaccharides other than those studied here.

Molar Mass and Microstructure Analysis of PI‐b‐PMMA Copolymers by SEC‐NMR

AbstractThe online coupling of size‐exclusion chromatography and NMR is used to characterize block copolymers consisting of polyisoprene (PI) and poly(methyl methacrylate) (PMMA) regarding their distributions of molar mass (MMD) and chemical composition (CCD). Using the CCD, $\overline{M}_{\rm n}$ and $\overline{M}_{\rm w}$ are calculated on the basis of PI and PMMA homopolymer calibrations. The microstructure distribution of PMMA and the distribution of isomeric units of PI in dependence of molar mass is also demonstrated. Furthermore, a simulation analysis is presented for a bimodal eluting sample. It allows for full separation, quantification and molar mass determination of the coeluting co‐ and homopolymer fractions. The quantification of the fractions is verified by liquid chromatography at critical conditions.

On-line coupling of size-exclusion chromatography and capillary zone electrophoresis via a reversed-phase C18 trapping column for the determination of peptides in biological samples.

Since biologically active peptides usually exhibit their effects in low concentrations, the development of sensitive analytical methods has become a challenge. In this paper, a multidimensional system is presented, consisting of a size-exclusion chromatographic (SEC) separation followed by a trapping procedure on a 4 mm x 3 mm ID reversed-phase C18 (RP18) column with subsequent elution of the trapped fraction and separation by capillary zone electrophoresis (CZE). The system has been tested with mixtures of six enkephalins and albumin, mimicking biological matrices such as plasma and cerebrospinal fluid. After separation of albumin from the enkephalins in the SEC dimension a heart-cut of 200 micro L, containing the enkephalin peak, is taken, concentrated on the RP18 microcolumn and, after elution with a 20 micro L plug of 80% acetonitrile, electrokinetically injected into the CZE system, where stacking and separation is achieved. While validation shows generally good linearity and reproducibility, the quantitation limit with UV detection is acceptable (2.5 micro g/ mL with an injection volume of 50 micro L).

On-line coupling of SEC and RP–LC for the determination of structurally related enkephalins in cerebrospinal fluid

On-line coupled analytical techniques can be advantageous in the assay of smaller peptides in complex biological matrices such as plasma, cerebro-spinal fluid (CSF) and tissues. The present study shows the feasibility of the recently reported on-line coupled size exclusion chromatography (SEC) and reversed phase liquid chromatography (RP–LC) separation system for the quantitation of structural related peptides in biological matrices, as demonstrated for a number of enkephalins in CSF. The degradation of the peptides, caused by endogenous peptidases in the matrix, could sufficiently be inhibited with imipramine HCL to allow an assay with satisfactory linearity and intraday (0.70–4.9%) precision. The sensitivity of the method, with a concentration limit of quantitation (LOQ) of 2 μg/ml is comparable with other kinds of assays for peptides and sufficient for the quantitation of peptide drugs with higher therapeutic ranges in biological matrices. However, for the assay of low concentrations of peptides, such as endogenous components of a biological matrix, the sensitivity may need improvement. The LOQ cannot be improved by increasing the sample amount, because of interference of other endogenous components of the CSF. This indicates that a larger selectivity is desired. The LOQ may be improved by using more sensitive and selective detection methods such as mass spectrometry or fluorescence after post-column derivatization. Miniaturization of the system, combined with on-line trapping may also contribute to a better sensitivity.

A method for studying optical anisotropy of polymers as a function of molar mass.

Optical properties of polymers are extremely important in many end-use applications, as is the ability of anisotropic polymers to depolarize incident radiation. To date, most light-scattering studies of the optical anisotropy of macromolecules have dealt with the bulk state or measured ensemble properties of dilute solutions. Here, we introduce a method to determine the optical anisotropy as a continuous function of molar mass. By direct, on-line coupling of size-exclusion chromatography and depolarization multiangle light scattering (SEC/D-MALS), molar mass averages, polydispersities, molar mass distributions, and the distribution of the optical anisotropy as a function of molar mass may all be determined. To quantify the anisotropy, it has been expressed in terms of the Cabannes factor, thus permitting the Rayleigh ratio necessary for light-scattering calculations to be corrected for anisotropy. The effects of tacticity, heavy atom substitution on the main chain, and chain helicity on the depolarization behavior of polymers have been studied using atactic and isotactic PMMA; atactic and brominated PS; and the semiflexible polypeptide PBLG, which maintains an extended structure in solution. An introduction to the theory of SEC/D-MALS is given.

Transformation of Iodide in Natural and Wastewater Systems by Fixation on Humic Substances

Separation of different fractions of humic substances (HS) by their molecular weight was carried out by size exclusion chromatography (SEC). The fixation of inorganic iodide by HS of these fractions was determined by on-line coupling of SEC with inductively coupled plasma mass spectrometry (ICP-MS). Possible changes of HS during this transformation process of HS/iodine species could be followed by UV absorption. A 129I-labeled sodium iodide solution was used to determine the transformation of iodide into iodine fixed on humic substances. In the case of natural samples, iodide was exclusively fixed by HS fractions where natural HS/iodine species have also been observed in the original sample. In wastewater samples from sewage disposal plants organoiodine compounds were also identified which were not affected by the transfer of iodide. A strong influence of the transformation process was seen on the microbiological activity of the sample and a complicated transfer mechanism by the formation of different intermediate HS/iodine species was observed. In general, the results contribute to a better understanding of the biogeochemical cycle of the important trace element iodine, but they are also important for the possible fixation of radioactive iodine, e.g. emitted from nuclear plants, in natural aquatic systems. On the other hand, it must be assumed that substantial amounts of iodine are removed as HS/iodine species during water processing, where humic substances are usually separated. This reduces the iodine content in drinking and mineral waters with respect to the natural level of this essential trace element.

Determination of pituitary and recombinant human growth hormone molecular weights by modern high-performance liquid chromatography with low angle laser light scattering detection

The determination of molecular weight for pituitary and recombinant human growth hormone (p-hGH/Crescormon and r-hGH/Protropin) has been performed. This has involved on-line coupling of size-exclusion chromatography (SEC) and gradient elution, reversed-phase high-performance liquid chromatography (RP-HPLC) with low-angle laser light scattering (LALLS) detection. A 5-μm, 300 Å, Delta-bond octyl column was used. Traditional specific refractive index increment (d n/d c) and refractive index ( n) measurements have been performed in order to derive absolute weight-average molecular weight ( M w) information for p-hGH and r-hGH. Known concentrations of each protein have been separated using reversed-phase gradients utilizing acetonitrile with on-line LALLS determination of excess Rayleigh scattering factors. Accurate M w data has been obtained for both proteins under conventional RP-HPLC gradient elution conditions. SEC data of both hGHs were found to be concentration, mobile phase, and column dependent for the particular analyses. Both medium- and high-resolution SEC—LALLS studies were performed, and all of these determinations further confirmed our RP-HPLC results. On-line LALLS provides certain advantages in identifying aggregates that may be present, even in medium-resolution SEC, where incomplete resolution occurs. The on-line coupling of modern RP-HPLC for biopolymers with LALLS detection represents a major step forward in the ability of bioanalytical chemists to determine the nature (monomer versus higher-order aggregate) of such materials. Other classes of biopolymers should prove suitable for studies with the same RP-HPLC—LALLS—UV approaches.

Non-aqueous size-exclusion chromatography coupled on-line to reversed-phase high-performance liquid chromatography: Interface development and applications to the analysis of low-molecular-weight contaminants and additives in foods

An interface has been developed which permits the on-line coupling of size-exclusion chromatography in tetrahydrofuran with aqueous reversed-phase high-performance liquid chromatography. The interface isolates the required size exclusion chromatography fraction and dilutes it with water to ensure reconcentration of analytes on the reversed-phase column prior to gradient elution. Operational parameters and the influence of analyte polarity have been examined in detail. A predictive system is presented for determining the applicability of the system to any analyte, based on solute retention times on an ODS phase eluted with a methanol—water gradient. The method is illustrated with examples of direct analyses of crude lipid extracts from a snack product for 2,6-di-tert.-4-methylphenol and from chocolate for dibutyl phthalate. Detection limits of ca. 0.5 mg/kg have been achieved.