Search

• Explain the separation mechanism of ultrasound-assisted chromatography (UAC). • Comparatively analyzed ultrasound effects on C18 PEEK and SS columns. • Distinguished the differences between acoustic and temperature effects in UAC. • Ultrasound can be utilized as an external factor in chromatography. Based on the previous ultrasound-assisted chromatography (UAC) studies on plastic and stainless steel (SS) columns, this study explores the UAC mechanism by comparatively analyzing ultrasound effects on plastic and SS columns with C18 stationary phase when separating a mixture of polycyclic aromatic hydrocarbons (PAHs) under various ultrasound intensities. The results showed a substantial difference in H values between the PEEK and SS columns under the influence of ultrasound agitation. Specifically, for the pyrene peak, as the ultrasonic intensity increased from 0% to 100% of 900 W, the H values of the SS column slightly rose from 8.82 μm to 9.86 μm. Conversely, the corresponding values for the PEEK column exhibited a significant 12-fold increase from 11.5 μm to 134 μm. The findings demonstrated poor penetration of ultrasound energy through the SS column, and the temperature rise of the medium induced by the ultrasound was the primary contributing factor to PAH separation. However, ultrasound easily penetrated through the plastic column, resulting in acoustic cavitation within the C18 polyether ether ketone (PEEK) column. Cavitation induced heat generation and contributed to a decrease in retention time and the magnitude of peak broadening or distortion, depending on the specific ultrasonic energy. Based on the estimated change in inlet temperature of the PEEK column due to an acoustic effect, the comparison with temperature effects under non-sonic conditions consistently demonstrated a stronger acoustic effect in reducing the retention time, by 2–9%, depending on specific peaks and pairs. We revisited the previously described separation mechanism of ultrasound-assisted ion chromatography and conjoined with our findings to infer and establish a thorough explanation for the previously unexplained separation mechanism of chiral separation and size exclusion chromatography by UAC using SS columns. [ABSTRACT FROM AUTHOR]

Plant polysaccharides have numerous medicinal functions. Due to the differences in their origins, regions of production, and cultivation conditions, the quality and the functions of polysaccharides can vary significantly. They are macromolecules with large molecular weight (MW) and complex structure, and pose great challenge for the analytical technology used. Taking Dendrobium officinale (DO) from various origins and locations as model samples. In this investigation, mechanochemical extraction method was used to successfully extract polysaccharides from DO using water as solvent, the process is simple, fast (40 s) and with high yield. The MWs of the intact saccharides from calibration curve and light scattering measurement were determined and compared after separation with size exclusion chromatography (SEC). The large polysaccharide was acid hydrolyzed to oligosaccharides and the products were efficiently separated and identified using liquid chromatography coupled to a high resolution tandem mass spectrometry (LC–MS 2 ). Obvious differences were observed among LC–MS 2 chromatograms of digested products, and the chemical structures for the products were proposed based on accurate mass values. More importantly, isomeric digested carbohydrate compounds were explored and characterized. All the chromatographic and mass spectrometric results in this study provided a multi-dimensional characterization, fingerprint analysis, and molecular structure level assessment of plant polysaccharides. [ABSTRACT FROM AUTHOR]

• Capillary-channeled polymer fibers provide high throughput as 2D in 2DLC. • Fiber columns in 2D eliminates the need for UPLC hardware in the second dimension. • SEC x RP with fiber columns provides for potential implementation for ESI sampling. • PET and PP fibers exhibit different levels of selectivity, under RP conditions. • RP x RP with fiber columns provides orthogonality in chemical information. Polymeric materials are readily available, durable materials that have piqued the interest of many diverse fields, ranging from biomedical engineering to construction. The physiochemical properties of a polymer dictate the behavior and function, where large polydispersity among polymer properties can lead to problems; however, current polymer analysis methods often only report results for one particular property. Two-dimensional liquid chromatography (2DLC) applications have become increasingly popular due to the ability to implement two chromatographic modalities in one platform, meaning the ability to simultaneously address multiple physiochemical aspects of a polymer sample, such as functional group content and molar mass. The work presented employs size exclusion chromatography (SEC) and reversed-phase (RP) chromatography, through two coupling strategies: SEC x RP and RP x RP separations of the water-soluble polymers poly(methacrylic acid) (PMA) and polystyrene sulfonic acid (PSSA). Capillary-channeled polymer (C-CP) fiber (polyester and polypropylene) stationary phases were used for the RP separations. Particularly attractive is the fact that they are easily implemented as the second dimension in 2DLC workflows due to their low backpressure (<1000 psi at ∼70 mm sec−1) and fast separation times. In-line multi-angle light scattering (MALS) was also implemented for molecular weight determinations of the polymer samples, with the molecular weight of PMA ranging from 5 × 104 to 2 × 105 g mol−1, while PSSA ranges from 105 to 108 g mol−1. While the orthogonal pairing of SEC x RP addresses polymer sizing and chemistry, this approach is limited by long separation times (80 min), the need for high solute concentrations (PMA = 1.79 mg mL−1 and PSSA = 0.175 mg mL−1 to yield comparable absorbance responses) due to on-column dilution and subsequently limited resolution in the RP separation space. With RP x RP couplings, separation times were significantly reduced (40 min), with lower sample concentrations (0.595 mg mL−1 of PMA and 0.05 mg mL−1 of PSSA) required. The combined RP strategy provided better overall distinction in the chemical distribution of the polymers, yielding 7 distict species versus 3 for the SEC x RP coupling. [ABSTRACT FROM AUTHOR]

A comparison is made using size-exclusion chromatography (SEC) of synthetic polymers between fully porous particles (FPPs) and superficially porous particles (SPPs) with similar particle diameters, pore sizes and equal flow rates. Polystyrene molecular weight standards with a mobile phase of tetrahydrofuran are utilized for all measurements conducted with standard HPLC equipment. Although it is traditionally thought that larger pore volume is thermodynamically advantageous in SEC for better separations, SPPs have kinetic advantages and these will be shown to compensate for the loss in pore volume compared to FPPs. The comparison metrics include the elution range (smaller with SPPs), the plate count (larger for SPPs), the rate production of theoretical plates (larger for SPPs) and the specific resolution (larger with FPPs). Advantages to using SPPs for SEC are discussed such that similar separations can be conducted faster using SPPs. SEC using SPPs offers similar peak capacities to that using FPPs but with faster operation. This also suggests that SEC conducted in the second dimension of a two-dimensional liquid chromatograph may benefit with reduced run time and with equivalently reduced peak width making SPPs advantageous for sampling the first dimension by the second dimension separator. Additional advantages are discussed for biomolecules along with a discussion of optimization criteria for size-based separations. [ABSTRACT FROM AUTHOR]

• Simulated moving bed chromatography can be performed at a microliter scale. • Custom-made 3D printed valve reduces overall dead volume to 358 µL. • Pressure-driven closed-loop flow control is a suitable flow source. • Desalting level of up to 99% for continuous protein desalting • Feed flow rate as low as 15 µL/min. In the 1960s, chromatography processes were revolutionized by the invention of simulated moving bed chromatography. This method not only enhances the separation performance and resin utilization in comparison to batch-chromatography, it has also a much lower buffer consumption. While simulated moving bed chromatography nowadays is applied for a wide range of industrial applications, it was never transferred to the micro-scale (in regards to column and system volume). In our opinion a micro simulated moving bed chromatography system (µSMB) would be a useful tool for many applications, ranging from early process development and long term studies to downstream processing of speciality products. We implemented such a µSMB with a 3D printed central rotary valve and a microfluidic flow controller as flow source. We tested the system with a four zone open loop setup for the separation of bovine serum albumin and ammonium sulfate with size exclusion chromatography. We used four process points and could achieve desalting levels of BSA ranging from 94% to 99%, with yields ranging form 65% to 88%. Thus, we were able to achieve comparable results to common lab scale processes. With a total dead volume of 358 µL, including all sensors, connections and the valve, this is, to the best of our knowledge, the smallest SMB system that was ever built and we were able to perform experiments with feed flow rates reaching as low as 15 µL/min. [ABSTRACT FROM AUTHOR]

Two graft copolymer samples of identical average composition were synthesized by grafting living polystyrene anions onto a broadly distributed PMMA backbone. Size exclusion chromatography (SEC) with only RI-detection, SEC with viscometry and light scattering detection, SEC with UV and RI dual detection, gradient chromatography and 2-dimensional chromatography were applied to compare the information that can be obtained by the different techniques. While only limited information was retrieved by conventional SEC or SEC with molar mass sensitive detection, SEC with UV and RI revealed different chemical heterogeneity of the samples. Using gradient chromatography and 2-dimensional chromatography it was possible to identify non-grafted side chains and unreacted parent PMMA besides the actual graft copolymer molecules. While in one sample a heavily grafted product was formed besides non-grafted PMMA, the second sample did not contain ungrafted PMMA but a graft product of lower grafting density. The different product distributions were explained by the different synthetic procedures. [ABSTRACT FROM AUTHOR]

Surface plasmon resonance (SPR) is an optical technique that measures biomolecular interactions. Stand-alone SPR cannot distinguish different binding components present in one sample. Moreover, sample matrix components may show non-specific binding to the sensor surface, leading to detection interferences. This study describes the development of coupled size-exclusion chromatography (SEC) SPR sensing for the separation of sample components prior to their on-line bio-interaction analysis. A heterogeneous polyclonal human serum albumin antibody (anti-HSA) sample, which was characterized by proteomics analysis, was used as test sample. The proposed SEC-SPR coupling was optimized by studying system parameters, such as injection volume, flow rate and sample concentration, using immobilized HSA on the sensor chip. Automated switch valves were used for on-line regeneration of the SPR sensor chip in between injections and for potential chromatographic heart cutting experiments, allowing SPR detection of individual components. The performance of the SEC-SPR system was evaluated by the analysis of papain-digested anti-HSA sampled at different incubation time points. The new on-line SEC-SPR methodology allows specific label-free analysis of real-time interactions of eluting antibody sample constituents towards their antigenic target. [ABSTRACT FROM AUTHOR]

Band broadening in size-exclusion chromatography (SEC) is always present to some extent. Broadening effects on averages such as the weight- and number average molecular weights ( M W ¯ and M n ¯ respectively) are minimal with modern SEC systems. However, broadening distorts the shape of the true molecular weight distribution (MWD), which causes problems if one wants to compare the detailed form of the MWD to a model. An addition to current methods for overcoming this problem is presented. One starts with a sufficiently wide range of samples whose exact values of M n ¯ and M W ¯ have been measured by non-SEC methods (e.g. by fluorimetry and light scattering, respectively, of the sample without size separation). A true (unbroadened) molecular weight distribution for a sample can be obtained by deconvolution (here using a maximum-entropy algorithm) by fitting SEC data for these samples to these exact M n ¯ and M W ¯ values to find the values of the parameters in a sufficiently flexible assumed broadening function. This was modelled using simulated band broadening and subsequent deconvolution, with the broadening parameters least-squares fitted to the “exact” sets of values of M n ¯ and M W ¯ . The results show that if these M n ¯ and M W ¯ values are for a series of broad (not narrow) standards covering a sufficient range of molecular weight, then after deconvolution, a good representation of the original molecular weight distribution used in the simulation is obtained. The method should prove useful for water-soluble polymers, for which it is often difficult to obtain narrow standards of a wide range of molecular weight, as required in a number of well-established methods for correcting for band broadening. [ABSTRACT FROM AUTHOR]

• SEC packing materials with three different bondings were prepared. • The three materials were characterized and compared. • Non-specific interactions were systematically studied. • The new HO-PEO phase was clearly the most favourable one. • A fast platform method (3 min analysis time) has been developed for protein SEC. Size exclusion chromatography has become an essential tool for the protein therapeutics industry. Conceptually, it is a simple form of chromatography that is driven by entropy and sieving effects. An ideal size exclusion column would exhibit no adsorptive interactions between its internal surfaces and the solutes being analysed, but that is not easily achieved. To this end, we have studied the utility of three unique packing materials in pursuit of additional column chemistries that might be less prone to interacting with proteins. These packing materials were each prepared from bridged ethylene hybrid organic/inorganic particles but uniquely derivatized into either hydroxy terminated PEO bonded, methoxy terminated PEO bonded, or diol bonded packing materials. All three materials were packed into column hardware modified with hydrophilic hybrid surface technology (h-HST) so that packing material effects could be more clearly observed without any influence from the secondary interactions that can originate from metal hardware. Non-specific interactions were compared for various challenging protein samples in the presence of ammonium acetate (volatile) and phosphate buffered saline (non-volatile) buffers. It was reconfirmed that the h-HST column hardware mitigates a majority of non-desired secondary interactions. However, during studies on hydrophobic interactions, the new hydroxy terminated PEO packing material showed clear benefit to obtaining higher apparent recoveries to better ensure accurate aggregate quantitation. Further experiments were explored to show that a hydroxy terminated PEO column could be effectively paired with a mobile phase comprised of standard strength phosphate buffered saline to make a fast platform method capable of baseline resolving monoclonal antibody monomer and aggregate peaks within a 3 min analysis time. [ABSTRACT FROM AUTHOR]

Average molecular weights and polydispersity indexes are some of the most important parameters considered in the polymer characterization. Usually, gel permeation chromatography (GPC) and multi angle light scattering (MALS) are used for this determination, but GPC values are overestimated due to the dispersion introduced by the column separation. Several procedures were proposed to correct such effect usually involving more complex calibration processes. In this work, a new method of calculation has been considered including diffusion effects. An equation for the concentration profile due to diffusion effects along the GPC column was considered to be a Fickian function and polystyrene narrow standards were used to determine effective diffusion coefficients. The molecular weight distribution function of mono and poly disperse polymers was interpreted as a sum of several Fickian functions representing a sample formed by only few kind of polymer chains with specific molecular weight and diffusion coefficient. Proposed model accurately fit the concentration profile along the whole elution time range as checked by the computed standard deviation. Molecular weights obtained by this new method are similar to those obtained by MALS or traditional GPC while polydispersity index values are intermediate between those obtained by the traditional GPC combined to Universal Calibration method and the MALS method. Values for Pearson and Lin coefficients shows improvement in the correlation of polydispersity index values determined by GPC and MALS methods when diffusion coefficients and new methods are used. [ABSTRACT FROM AUTHOR]

• 2D SEC-CEX-MS allows multiplex analysis of mAb aggregates and charge variants in a single method. • 2D SEC-CEX-MS streamlines product development in industry. • 2D SEC-CEX-MS is applied to understand mAb forced degradation pathways. Monoclonal antibodies (mAbs) are extremely complex due to the presence of structural modifications resulting from enzymatic and chemical reactions such as glycosylation, glycation, deamidation, isomerisation, oxidation, aggregation and fragmentation. Size and charge variants analysis are carried out from the early stages of drug development throughout product lifetime to investigate product degradation pathways and optimise process conditions. However, conventional analytical workstreams for size and charge variant characterization are both time and sample demanding, requiring the application of multiple analytical methods. This study presents the development of a novel 2D-LC/MS approach combining both aggregate and charge variant profiling of a mAb candidate in a single method. Aggregate quantification was performed in the first dimension (1D) by size exclusion chromatography SEC, followed by online fraction transfer of the monomer peak to the second dimension (2D) by a heart-cutting for charge variant analysis by cation exchange chromatography (CEX). Aiming to maximise the information obtained from minimal sample and time required for analysis, a salt-based separation with UV detection was developed for supporting the processing of a large number of samples to facilitate high-throughput process development (HTPD). In addition, a mass spectrometry (MS) compatible SEC-CEX separation was developed enabling online charge variant peak identification. This study presented the ability to multiplex mAb size and charge variants analysis by coupling SEC with CEX in a 2D-LC set-up. To date, this is the first 2D SEC-CEX-UV(-MS) application for intact mAb analysis. [ABSTRACT FROM AUTHOR]

This study investigated the experimental conditions needed to obtain molecular weight distribution (MWD) of ultrahigh MW poly(ethylene oxide) (PEO) using size exclusion chromatography (SEC) hyphenated with a multiangle light scattering photometer (MALS) and a differential refractive index detector (RI). Ultrahigh MW components yielded non-linear angular dependency in scattered light intensities. The first-order linear fitting using Zimm formalism resulted in significant differences in MW depending on if the signals from detector 4 to 16 were used in the fitting or only five low-angles from 4 to 8 were used. In contrast, no significant differences in MW were obtained for lower MW PEO samples (equal or less than 1000 KDa) between the two fitting approaches. It was thus proposed to use only the five low-angles to derive MW for a sample with broad polydispersity including both ultrahigh and low MW components. The SEC separation was done using one column designed for ultrahigh MW polymer separation connected with another mixed-bed column. The ultrahigh MW column allowed separation and characterization of polymeric components in the MW range between 10 and 50 million Dalton (MDa) and the size range between 300 and 600 nm in radius of gyration (R g). Online calibration curves were obtained from the linear fittings of MW as a function of elution volume. MW polydispersity was derived from the online calibration curve showing that the ultrahigh MW PEO had higher polydispersity than the lower MW samples. The double logarithmic plot of radius of gyration versus MW indicated that both ultrahigh MW and low MW PEO would adopt expanded coil conformations in the aqueous solution. [ABSTRACT FROM AUTHOR]

• The method suggests an efficient purification technique for production NSP free FMDV vaccine by 2D-SEC-AEC accompanied with mathematical modeling. • Q-Sepharose XL virus licensed showed a desirable performance for capturing step by providing higher volume loadability than conventional SEC media. • In proposed methodology FMDV could be obtained with purity higher than 85% and 77.9% virus yield. • The present study describes application of MALDI-TOF MS as a complementary tool for evaluation purity and characterization of purified FMDV after a chromatographic procedure. The production of high-quality purified virus particles in high quantities for vaccine preparation requires a scalable purification procedure in the downstream step. A purification scheme based on combined strong anion-exchange and size exclusion chromatography (2D-AEC-SEC) was developed for the production of non-structural protein-free foot and mouth disease vaccine, and the whole procedure was accomplished with 77.9% virus yield. Additionally, a mathematical modeling and a simulation approach based on a plate model of chromatography were developed and matched with the experimental chromatography data to improve prediction of retention behavior and save time in the development of the downstream scale-up method. The purified pooled virus fraction obtained from the final polishing step had a purity higher than 85% based on analytical size exclusion analysis. Moreover, more than 90.1% of residual DNA (rDNA) was removed from the purified vaccine. The analysis of purified virus particles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), dynamic light scattering (DLS), high performance size exclusion chromatography (HP-SEC), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and transmission electron microscopy (TEM) provided clear evidence of purity and demonstrated that the final product is structurally spherical, intact particles qualified for formulation as a vaccine product. [ABSTRACT FROM AUTHOR]

• Isolation of mcDNA by size-exclusion chromatography is presented. • mcDNA is isolated with high purity and recovery. • The applicability of the method to the isolation of other mcDNA is demonstrated. Minicircle DNA (mcDNA) is the new cutting-edge technology which researchers have been exploring for gene therapy and DNA vaccination. Although it presents enormous advantages in comparison to conventional plasmid DNA regarding bioactivity and safety, its challenging isolation from parental plasmid and miniplasmid has been setting back its launching in biomedical sciences. In this work, it is demonstrated the use of a simple size exclusion chromatographic method for the isolation of supercoiled mcDNA. Sephacryl S-1000 SF matrix was explored under different conditions (flow, peak fractionation volume and sample loading) to achieve the best performance and retrieve a mcDNA sample devoid of other bacterial contaminants or plasmid species resultant from the recombination process. This isolation methodology resulted in 66.7% of mcDNA recovery with 98.1% of purity. In addition, to show the robustness of the method, the potential of using this matrix for the isolation of a larger mcDNA was also evaluated. Upon adjusting the flow or the column volume, the larger mcDNA molecule was also successfully isolated. Overall, a simple and effective strategy has been established for the isolation of supercoiled mcDNA, underlining the potential of size exclusion chromatography in mcDNA separation. [ABSTRACT FROM AUTHOR]