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The analysis of complex mixtures of closely related species is quickly becoming a bottleneck in the development of new drug substances, reflecting the ever-increasing complexity of both fundamental biology and the therapeutics used to treat disease. Two-dimensional liquid chromatography (2D-LC) is emerging as a powerful tool to achieve substantial improvements in peak capacity and selectivity. However, 2D-LC suffers from several limitations, including the lack of automated multicolumn setups capable of combining multiple columns in both dimensions. Herein, we report an investigation into the development and implementation of a customized online comprehensive multicolumn 2D-LC-DAD-MS setup for screening and method development purposes, as well as analysis of multicomponent biopharmaceutical mixtures. In this study, excellent chromatographic performance in terms of selectivity, peak shape, and reproducibility were achieved by combining reversed-phase (RP), strong cation exchange (SCX), strong anion exchange (SAX), and size exclusion chromatography (SEC) using sub-2-μm columns in the first dimension in conjunction with several 3.0 mm × 50 mm RP columns packed with sub-3-μm fully porous particles in the second dimension. Multiple combinations of separation modes coupled to UV and MS detection are applied to the LC × LC analysis of a protein standard mixture, intended to be representative of protein drug substances. The results reported in this study demonstrate that our automated online multicolumn 2D-LC-DAD-MS workflow can be a powerful tool for comprehensive chromatographic column screening that enables the semi-automated development of 2D-LC methods, offering the ability to streamline full visualization of sample composition for an unknown complex mixture while maximizing chromatographic orthogonality. [ABSTRACT FROM AUTHOR]

The ability of human serum albumin to capture unbound copper under different clinical conditions is an important variable potentially affecting homeostasis of this element. Here, we propose a simple procedure based on size-exclusion chromatography with on-line UV and nitrogen microwave-plasma atomic-emission spectrometry (MP-AES) for quantitative evaluation of Cu(II) binding to HSA upon its glycation in vitro. The Cu-to-protein molar ratio for non-glycated albumin was 0.98 ± 0.09; for HSA modified with glyoxal (GO), methylglyoxal (MGO), oxoacetic acid (GA), and glucose (Glc), the ratios were 1.30 ± 0.22, 0.72 ± 0.14, 0.50 ± 0.06, and 0.95 ± 0.12, respectively. The results were confirmed by using ICP-MS as an alternative detection system. A reduced ability of glycated protein to coordinate Cu(II) was associated with alteration of the N-terminal metal-binding site during incubation with MGO and GA. In contrast, glycation with GO seemed to generate new binding sites as a result of tertiary structural changes in HSA. Capillary reversed-phase liquid chromatography with electrospray-ionization quadrupole-time-of-flight tandem mass spectrometry enabled detection and identification of Cu(II) coordinated to the N-terminal metal-binding site (Cu(II)-DAHK) in all tryptic digests analyzed. This is the first report confirming Cu(II)-DAHK species in HSA by means of high-resolution tandem mass spectrometry, and the first report on the use of MP-AES in combination with chromatographic separation. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Online comprehensive two-dimensional liquid chromatography (online LCxLC) presents high peak capacity compared with one-dimensional liquid chromatography, and reasonable operation time compared with an offline or stop-flow mode. Among various combinations, coupling of two reversed-phase (RPxRP) columns generates a high peak capacity product rate. Its wide applicability made RPxRP a promising technique in separation of complex samples. This review discusses the practical considerations in development and application of an RPxRP system, including systematic investigation of column stationary phase chemistry, column combinations, mobile phase system, interface, gradient types, and achieving fast analysis in LCxLC. In addition, many efforts are given to methods that increase the fraction coverage because this is the main obstacle caused by the correlated separation mechanism in RPxRP. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

rapid and easy method has been proposed, optimized and evaluated for quantitative determination at trace level of a representative group of non-polar pesticides in fat samples. The method includes n-hexane-saturated acetonitrile extraction, fat precipitation by cooling pre clean-up followed by dispersive solid-phase extraction (d-SPE) based on QuEChERS procedure clean-up. Determination was performed by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode. Efficiency of the d-SPE clean-up step was evaluated by comparison with fat oxidation treatment and gel permeation chromatography. Different combinations of d-SPE extraction reagents and sample amounts were tested in order to minimize matrix co-extractives and interferences. Best recoveries were obtained with 1200 mg of MgSO, 400 mg of end-capped C, 400 mg of PSA and 1 g of sample amount. SIM method, matrix effect, precision, and accuracy were evaluated with spiked pork fat samples for 38 representative pesticides. Results of this study showed that this technique is applicable in routine analysis for its application into monitoring programs. It simplifies time-consuming clean-up steps and allows a satisfactory long-term chromatographic performance. [ABSTRACT FROM AUTHOR]

Size-exclusion chromatography (gel filtration chromatography or gel permeation chromatography) in conjunction with online synchrotron radiation solution small-angle X-ray scattering optics, absorbance, and/or refractive index detectors was further assessed by application of biological macromolecules, such as the hollow sphere protein complex, apoferritin, and a linear polysaccharide, pullulan. The net X-ray scattering patterns of the eluted 24-mer molecule of apoferritin showed the specific character for the hollow spherical shape. The chromatographic (time-resolved) X-ray scattering data of the linear polysaccharide pullulan revealed the flexible chain structure during the chromatographic separation in an aqueous solution. These further applications demonstrated that the present measurement technique will be useful for not only the determination of the radius of gyration value of less than about 10 nm and molecular weight below several hundred thousand but also for the structural characterization of the various macromolecules during the chromatography. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

We synthesized poly(lactide- co-succinimide) (PLS) copolymers with the ratio of lactide to succinimide units of 3:1 and 6.5:1 and studied their elution behavior by size exclusion chromatography with an on-line light-scattering detection. Since the copolymers contain a certain amount of carboxyl groups, they behave as ionomers in N, N-dimethylacetamide (DMAc) and show a typical polyelectrolyte (PE) effect. The PE effect was eliminated by the addition of simple electrolyte like LiBr, H3PO4, or both in DMAc. The efficiency of the additive decreases in the order: LiBr > LiBr + H3PO4 > H3PO4. The ionic strength of the 0.1 M LiBr/DMAc was high enough for the onset of hydrophobic interactions of PLS lactic acid segments intermolecularly as well as with the column packing material. The drawback of the LiBr + H3PO4/DMAc solvent system is a rather high intensity of the system peaks, which are imposed on the right side of the copolymer signal. System peaks strongly influence the determination of number and to a lesser extent the weight average molar masses of PLS copolymers. An addition of only H3PO4 in high enough concentration to DMAc (0.05 and 0.1 M) successfully eliminated the PE effect of the 6.5:1 PLS copolymer. On the contrary, the PE effect of the 3:1 PLS copolymer having higher charge density compared to 6.5:1 PLS copolymer cannot be entirely canceled out in any of the H3PO4/DMAc solutions examined. [ABSTRACT FROM AUTHOR]

Silica colloids were separated by size-exclusion chromatography and monitored by fluorimetric and UV detection. In the former means of detection, silica colloids were visualized by light-scattering. The signal intensity based on the light scattering increased with increasing size of the silica colloids. The maximum intensity was observed at excitation wavelengths around 270–290nm. In UV detection, silica colloids were visualized based on turbidimetry, and the signal intensity also increased with increasing size of the silica colloids and with decreasing detection wavelength. The signal intensities for both light-scattering and turbidimetric detection were a linear function of the concentration of the silica colloids. The detection limit at S/ N = 3 for 78-nm colloids was 0.06 ppm for light-scattering detection whereas the LOD was 2.3 ppm for UV detection. Effects of mobile phase conditions and flow rate on resolution and peak shape were examined. Use of phosphate buffer allowed the separation of silica colloids of different sizes in size-exclusion chromatography. [ABSTRACT FROM AUTHOR]

Size exclusion chromatography (SEC) was coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS) for speciation study of trace metals in cancerous thyroid tissues in comparison to healthy thyroids aimed to estimation of changes in metalloprotein speciation in pathological tissue. The study showed a presence of species binding Cu, Zn, Cd and Pb in healthy thyroid tissue with a good reproducibility of chromatographic results, whereas the same species could not be detected in cancerous tissues. Thus, remarkable differences with respect to metal-binding species were revealed between healthy and pathological thyroid samples, pointing out a completely different distribution of trace metals in cancerous tissues. The metal-binding species could not be identified in the frame of this work because of a lack of appropriate standards. Nevertheless, the results obtained confirm the suitability of SEC-ICP-MS for monitoring of changes in trace metal distribution in cancerous tissue and will help to better understand the role of metal-containing species in thyroid pathology. [ABSTRACT FROM AUTHOR]

A new one-step cleanup procedure, based on size-exclusion chromatography (SEC), usable for the extracts from accelerated solvent extraction (ASE), Soxhlet extraction, or ultrasonic extraction (USE), is described. The method is suitable for the determination of polycyclic aromatic hydrocarbons (PAHs), especially from very complicated plant matrices (e.g. pine needles, deciduous leaves, mosses). The main improvement compared with previous conventional procedures is that analyte peaks barely overlap with matrix peaks in the chromatograms and that it is a very rapid and simple one-step procedure with clearly improved analytical performance. Essential advantages of this SEC procedure are the sharper GC–MS chromatograms for the PAH fraction at retention times between 9.2 and 12.0 min, distinctly separated substance peaks resulting in better analysis, shorter running times, and lower solvent consumption. [ABSTRACT FROM AUTHOR]

The article focuses on size-exclusion chromatography (SEC), also known as gel-permeation chromatography (GPC) or gel-filtration chromatography (GFC) which is considered the premier method for determining the molar mass averages and distribution of polydisperse macromolecules. It then discusses in detail oligomeric SEC, high-speed SEC, multi-detector SEC and SEC as part of multi-dimensional separations.