Fused-core Particles Research Articles

Optimized superficially porous particles for protein separations

Continuing interest in larger therapeutic molecules by pharmaceutical and biotech companies provides the need for improved tools for examining these molecules both during the discovery phase and later during quality control. To meet this need, larger pore superficially porous particles with appropriate surface properties (Fused-Core® particles) have been developed with a pore size of 400Å, allowing large molecules (<500kDa) unrestricted access to the bonded phase. In addition, a particle size (3.4μm) is employed that allows high-efficiency, low-pressure separations suitable for potentially pressure-sensitive proteins. A study of the shell thickness of the new fused-core particles suggests a compromise between a short diffusion path and high efficiency versus adequate retention and mass load tolerance. In addition, superior performance for the reversed-phase separation of proteins requires that specific design properties for the bonded-phase should be incorporated. As a result, columns of the new particles with unique bonded phases show excellent stability and high compatibility with mass spectrometry-suitable mobile phases. This report includes fast separations of intact protein mixtures, as well as examples of very high-resolution separations of larger monoclonal antibody materials and associated variants. Investigations of protein recovery, sample loading and dynamic range for analysis are shown. The advantages of these new 400Å fused-core particles, specifically designed for protein analysis, over traditional particles for protein separations are demonstrated.

A Fast Determination of Chlorophylls in Barley Grass Juice Powder Using HPLC Fused-Core Column Technology and HPTLC

This work presents a novel approach to qualitative and quantitative evaluation of chlorophyll content in food supplement using simple and fast separation techniques (high performance liquid chromatography (HPLC) and high performance thin layer chromatography (HPTLC)). For the first time, an Ascentis® Express C-18 HPLC column (100 × 4.6 mm; 2.7 μm), based on fused-core particles, was utilised for the analysis of chlorophyll a and chlorophyll b in green young barley grass juice powder extracts. The mobile phase was composed of methanol, water and ethyl acetate of 40:10:50 (v/v/v) at a flow rate of 1.5 mL/min. Chlorophylls were separated in time less than 6 min. The extraction procedure was optimised by using of 90 % of acetone in water, and the method was validated. The method precision was found in the range of 1.15–2.85 %, and the method recovery was established at three concentration levels. Calibration graphs were linear in the range of 0.75–25.5 mg/L, with correlation coefficients r 2 = 0.99958 for chlorophyll a and r 2 = 0.99961 for chlorophyll b. The comparative HPTLC fingerprint analysis of three samples from different continents showed only slight qualitative differences between the particular samples. The results demonstrated that the proposed methods can be used as fast and powerful techniques for the characterisation of barley juice extracts in quality control.

Fused-core particle technology in high-performance liquid chromatography: An overview

The advent of superficially porous particles (SPPs) for packed HPLC columns has changed the way that many practitioners have approached the problem of developing needed separations. The very high efficiency of such columns, combined with convenient operating conditions, modest back pressures and the ability to use conventional HPLC instruments has resulted in intense basic studies of SPP technology, and widespread applications in many sciences. This report contains an overview of the SPP technology first developed in 2006 by Advanced Materials Technology, Inc., for sub-3-μm particles, then expanded into a family of SPP products with different particle sizes, pore sizes and other physical parameters. This approach was designed so that each particle of the family could be optimized for separating a particular group of compounds, usually based on solute size.

Evaluation of a liquid chromatography method for quality control of methylated cyclodextrins

Halo C18 column (fused core particles) and Chromolith RP18 column (monolith) were evaluated in liquid chromatography in order to analyze methylated-β-cyclodextrins (Me-β-CD) with various degrees of substitution, DS such as the number of methyl groups per cyclodextrin ring. Chromolith RP18 enables a performing analysis of Me-β-CD with low DS but is not suitable for dimethyl-β-cyclodextrins (DM-β-CD). On the other hand, Halo C18 column allows an improved fingerprint of CDs having a DS from 4.9 up to a value major than 14 and avoiding the use of various chromatographic systems. Thus, liquid chromatography performed with this column and an evaporative light scattering detector can be used as a generic system for methylated CD analysis. Moreover, fused core particles of Halo C18 column enables a rapid analysis and liquid chromatography coupled with electrospray-mass spectrometry appears as a powerful tool to determine co-elution and to characterize various isomers of complex methylated-β-cyclodextrin mixtures.

Quality by Design Approach for the Separation of Naproxcinod and its Related Substances by Fused Core Particle Technology Column

This paper describes the development of a rapid, novel, stability-indicating gradient reversed-phase high-performance liquid chromatographic method and associated system suitability parameters for the analysis of naproxcinod in the presence of its related substances and degradents using a quality-by-design approach. All of the factors that affect the separation of naproxcinod and its impurities and their mutual interactions were investigated and robustness of the method was ensured. The method was developed using an Ascentis Express C8 150 × 4.6 mm, 2.7 µm column with a mobile phase containing a gradient mixture of two solvents. The eluted compounds were monitored at 230 nm, the run time was 20 min within which naproxcinod and its eight impurities were satisfactorily separated. Naproxcinod was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. Naproxcinod was found to degrade significantly in acidic and basic conditions and to be stable in thermal, photolytic, oxidative and aqueous degradation conditions. The degradation products were satisfactorily resolved from the primary peak and its impurities, proving the stability-indicating power of the method. The developed method was validated as per International Conference on Harmonization guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness.

Superficially porous silica particles with wide pores for biomacromolecular separations

Since 2006, columns of superficially porous particles (SPPs), often called Fused-core®, porous–shell or core–shell particles, have had serious impact on HPLC separations. These particles have pore diameters of about 100Å designed for separating small molecules. More recently, SPPs with 160–200Å pore diameter have been made available for separating peptides and small proteins. This report describes the effects of fused-core particle size, pore size, shell thickness and ligand type for the rapid, efficient separation of larger molecules such as intact proteins and other biomacromolecules up to at least 400kDa. Optimization of these parameters resulted in particles that show no restricted diffusion that would compromise separating efficiency for large biomolecules. The thin porous shell provides excellent mass transfer (kinetics) for these large molecules, resulting in superior separations compared to conventional totally porous particles. Sample loading capacity can be adjusted to allow good detection sensitivity for minor components in a complex mixture. Strong particle strength ensures the loading of stable, high-efficiency columns. Stationary phases with different alkyl ligands were tested to provide data on retention, column efficiency and peak shapes for proteins. The development of these new wide-pore fused-core particles now allows the HPLC separation of a wide range of molecules of different sizes with advantages of the SPP configuration.

Fast ultrahigh performance liquid chromatographic method for the simultaneous determination of 25 emerging contaminants in surface water and wastewater samples using superficially porous sub-3 μm particles as an alternative to fully porous sub-2 μm particles

The development of fused-core silica stationary phases was considered a breakthrough in column manufacturing. In this study, a fast LC–UV method was developed and validated for the simultaneous determination of 25 emerging contaminants in surface water and wastewater. The selected analytes belonged to various classes such as veterinary antibiotics, central nervous system stimulants, non-steroidal anti-inflammatory drugs, steroids and preservatives. The chromatographic separation was optimized in order to achieve suitable retention times and good resolution for all analytes in a single run using solvent gradient and flow rate gradient. All analytes eluted within 10min on a Kinetex® C18 column packed with fused core particles. Sample preparation was executed with solid-phase extraction on Oasis HLB cartridges. The method was validated by assessing linearity, selectivity, accuracy, precision, limits of detection and limits of quantification. Good recoveries were obtained for all analytes ranging from 67.5% to 97.0% with standard deviations not higher than 5.7%, except for acetaminophen, sulphanilamide and acetyl salicylic acid, for which lower recoveries were obtained. The detection limits ranged from 1.5 to 15μgL−1, while limits of quantification were in the range from 5 to 50μgL−1. The short analysis time achieved by this method allowed analysis of a large number of samples in a short time, minimizing organic solvent consumption and lowering LC solvents cost. This fast method offers benefits both environmentally and economically.

Performance characteristics of new superficially porous particles

Superficially porous particles (also called Fused-Core, core shell or porous shell particles) show distinct advantages over comparable totally porous particles for separating small molecules. Columns of Fused-Core particles exhibit very high efficiency because of superior eddy dispersion properties (smaller van Deemter A term). The efficiency for columns of 2.7μm Fused-Core particles actually rivals that for sub-2μm totally porous particles with only about one-half the back pressure. These Fused-Core particles show special advantages with larger molecules for fast separations at high mobile phase velocities because of superior mass transfer (kinetic) properties (smaller van Deemter C term). This report describes the effect of different particle size and porous shell thicknesses on chromatographic performance for Fused-Core particles. Particle characteristics can significantly affect factors of separation importance. For example, the reduced plate height of packed columns is affected by particle diameter. Interestingly, larger Fused-Core particles show smaller reduced plate heights than smaller Fused-Core particles. Also, porous shell thickness has a strong effect on solute retention as well as separation efficiency, and particle surface area has a direct influence on sample loading characteristics. Fused-Core particles with a wide range of physical characteristics have been developed that allows the preparation of stable, efficient packed columns.

Ultra high pressure in the second dimension of a comprehensive two-dimensional liquid chromatographic system for carotenoid separation in red chili peppers

A comprehensive normal-phase×reversed-phase (NP-LC×RP-LC) liquid chromatography system was developed, and applied for analysis of the intact carotenoid composition of red chili peppers, with photodiode array and mass spectrometry detection. A micro-bore cyano column (250mm×1.0mm, 5μm d.p.) was chosen for the first dimension (1D) separation, interfaced to a second dimension (2D) C18 column (30mm×4.6mm, 2.7μm d.p.) packed with fused-core particles. Subsequently, two columns of the same stationary phase were coupled serially for second dimension separation, and operated under ultra high pressure LC conditions (UHPLC), within a cycle time of 1.50 or 1.00min, and equal modulation times. Performances of the three different set-ups were evaluated, in terms of peak capacity values (nc), and afterwards corrected by taking into account both the under-sampling, and the orthogonality effects. After these adjustments, the peak capacity values were estimated as follows: nc 526, for the NP-LC×RP-LC system, nc 373, for the NP-LC×RP-UHPLC system with a 1.50min modulation time, nc 639, for the NP-LC×RP-UHPLC system, with a 1.00min modulation time. A total of 33 compounds were separated into 10 different chemical classes in the two-dimensional space, and identified by accurate IT-TOF (ion trap-time of flight) MS detection.

Determination of the thrombin inhibitor AZD0837 and its metabolites in human bile using mixed mode solid phase extraction and LC–MS/MS

A method for the determination of AZD0837 and its two metabolites AR-H069927 and AR-H067637 in human bile was developed and validated. All three analytes and their stable isotope-labeled internal standards were isolated from bile using solid phase extraction on a mixed mode reversed phase/anion exchange column. Elution was done at high ionic strength with 0.125M ammoniumacetate in 50% methanol. The extraction recoveries were >75%. Due to the high concentration of AR-H067637 a portion of the extract was diluted before injection on to the LC column, while undiluted extract was directly injected for the analysis of AZD0837 and AR-H069927. Chromatographic separation of all three analytes was achieved in a single system utilizing a C18 column based on fused core particle technology at high flow rate. The two metabolites were eluted when a gradient from 30 to 57% methanol was applied while the more hydrophobic pro-drug, AZD0837, eluted during a steeper second gradient from 57 to 80% methanol with the ammonium acetate concentration and acetic acid concentration kept constant at 3.8mmol/L and 0.1%, respectively. The total cycle time was 3.2min. Detection was performed using positive electrospray ionization tandem mass spectrometry. The linearity range was 0.02–20μmol/L for AZD0837 and AR-H069927, and 1–1000μmol/L for AR-H067637. The repeatability and the overall precision were less than 15% (RSD) and the accuracy was within the interval 93–100%.

Fast Chromatography in The Regulated Bioanalytical Environment: Sub-2-µM Versus Fused-Core Particles

Increased throughput and rapid data turnaround have necessitated the need for faster LC separations in the regulated bioanalytical environment; sub-2-μm and fused-core chromatography are two viable approaches to accomplish this. Sub-2-μm columns offer high efficiency and resolution separations at the cost of high system backpressure and are best suited for preclinical sample analysis, whereas fused-core columns offer slightly lower efficiency and resolution, but are more tolerant to dirty extracts (such as those seen in clinical samples) that may clog the inlet filter. For clinical sample analyses, sub-2-μm columns should be restricted to sample extracts obtained via LLE or SLE, whereas fused-core columns can be used with sample preparations techniques ranging from protein precipitation to SLE.

Determination of Betaine in Fructus Lycii Using Hydrophilic Interaction Liquid Chromatography with Evaporative Light Scattering Detection

E-mail: hansb@cau.ac.kr (S. B. Han), youmjr@cau.ac.kr (J.-R. Youm)Received November 17, 2011, Accepted December 12, 2011A simple new method was developed for the determination of betaine in Fructus Lycii using hydrophilicinteraction liquid chromatography with evaporative light scattering detection (HILIC-ELSD). Good chromato-graphic separation and reasonable betaine retention was achieved on a Kinetex HILIC column (2.1 × 100 mm,2.6 µm) packed with fused-core particle. The mobile phase consisted of (A) acetonitrile and (B) 10 mMammonium formate (pH 3.0)/acetonitrile (90/10, v/v). It was used with gradient elution at a flow rate of 0.7 mL/min. The column temperature was set at 27.5 °C and the injection volume was 10 µL. The ELSD drift tubetemperature was 50 °C and the nebulizing gas (nitrogen) pressure was 3.0 bar. Stachydrine, a zwitterioniccompound, was used as an internal standard. Calibration curve over 10-250 µg/mL showed good linearity (R

Fused core particles as an alternative to fully porous sub-2 μm particles in pharmaceutical analysis using coupled columns at elevated temperature

The chromatographic performance of columns packed with fused-core particles was compared to that of fully porous particles (sub-2 μm) at elevated temperature and extended column lengths. The study involved a comparison of chromatographic parameters such as retention, selectivity, resolution, efficiency and pressure drop. The analytes showed lower retention on fused core particles compared to sub-2 μm particles at low and high temperatures, with substantially lower backpressure. Van Deemter plots were constructed for both kinds of packing materials using single and coupled columns of different lengths at different temperatures. Lower backpressure across the fused core particles allowed two columns to be coupled at 30 °C to generate ∼66 000 theoretical plates, and three columns could be coupled at 60 °C generating ∼129 000 plates. On the other hand, only two sub-2 μm columns could be coupled at 60 °C generating ∼49 000 plates at the optimum flow rate. Fused-core particles showed similar efficiency to sub-2 μm particles at and below the optimum linear velocity, but with lower backpressure. As an application to pharmaceutical analysis, two mixtures of pharmaceuticals of different classes were separated to illustrate the possibility of using fused-core particles as an alternative to sub-2 μm particles in terms of speed and efficiency. The low back-pressure of the fused-core particles allowed fast separations to be performed without significant loss in efficiency or resolution and allowed column coupling to increase efficiency.

Application of Fused-Core Particle Column in Two Dimensional Reversed Phase - Reversed Phase LC/MS Analysis of Biological Samples. Impact of Extra-Column Volume

We demonstrate successful applicability of fused–core column in analysis of biological samples (diluted urine and SPE–pre-purified plasma or serum) using 2 dimensional chromatography. Implementation of fused-core material in the second dimension of 2D chromatographic platform allowed improving S/N above 4 fold in comparison to traditional porous C18 column. Sample load onto the first dimension (5 μm) eliminates limitations of sample volume to be injected. The main limitation factor of the fused-core material–low loading capacity was overcome by using 2D chromatography. We emphasize importance of reduction of post-column (extra column) volume in LC/MS applications utilizing fused-core columns.

Application of novel fused-core particle column in 2D RP/RP MS analysis of biological samples. Impact of extra-column volume

Application of novel fused-core particle column in 2D RP/RP MS analysis of biological samples. Impact of extra-column volume

Efficiency in supercritical fluid chromatography with different superficially porous and fully porous particles ODS bonded phases

The chromatographic efficiency, in terms of plate number per second, was dramatically improved by the introduction of sub-two microns particles with ultra-high pressure liquid chromatography (UHPLC). On the other hand, the recent development of superficially porous particles, called core–shell or fused-core particles, appears to allow the achievement of the same efficiency performances at higher speed without high pressure drops. CO2-based mobile phases exhibiting much lower viscosities than aqueous based mobile phases allow better theoretical efficiencies, even with 3–5μm particles, but with relative low pressure drops. They also allow much higher flow rates or much longer columns while using conventional instruments capable to operate below 400bar. Moreover, the use of superficially porous particles in SFC could enhance the chromatographic performances even more. The kinetic behavior of ODS phases bonded on these particles was studied, with varied flow rates, outlet (and obviously inlet) pressures, temperatures, by using a homologous series (alkylbenzenes) with 10% modifier (methanol or acetonitrile) in the carbon dioxide mobile phase. Results were also compared with classical fully porous particles, having different sizes, from 2.5 to 5μm. Superior efficiency (N) and reduced h were obtained with these new ODS-bonded particles in regards to classical ones, showing their great interest for use in SFC. However, surprising behavior were noticed, i.e. the increase of the theoretical plate number vs. the increase of the chain length of the compounds. This behavior, opposite to the one classically reported vs. the retention factor, was not depending on the outlet pressure, but on the flow rate and the temperature changes. The lower radial trans-column diffusion on this particle types could explain these results. This diffusion reduction with these ODS-bonded superficially porous particles seems to decrease with the increase of the residence time of compounds.

Fast high performance liquid chromatography separations for proteomic applications using Fused-Core® silica particles

The separation range of superficially porous particles (Fused-Core®) has been extended by design of particles with 160Å pores. These particles show superior kinetics (lower resistance to mass transfer), allowing fast separations of peptides and small proteins (molecular weights of <15,000). The high efficiency and relatively low back pressure of these 2.7μm Fused-Core particles has been maintained so that separations can be performed with conventional HPLC instruments. Longer columns can be used for higher resolution of complex mixtures of peptides, such as proteolytic digests. Highly reproducible separations of peptides at elevated temperatures with low pH mobile phases are maintained as a result of a stable bonded stationary phase. The utility of such highly stable materials is exemplified by separations of problematic amyloid peptides at low pH (TFA mobile phase) at an operational temperature of 100°C. To address the issue of poor peptide peak shape in formic acid-containing mobile phases we show that the addition of 10–20mM ammonium formate improves peak shape, retention and load tolerance of peptides. Use of the Fused-Core particle materials for separations of synthetic peptides and tryptic digests yields peak capacities that are comparable to those obtained using columns packed with sub-2-μm particles, but with less than one-half of the operating back pressure. A peak capacity of 530 was obtained in 150min on coupled columns of HALO Peptide ES-C18 with a combined length of 250mm.

Enhanced capabilities of separation in Sequential Injection Chromatography – Fused-core particle column and its comparison with narrow-bore monolithic column

In the Sequential Injection Chromatography (SIC) only monolithic columns for chromatographic separations have been used so far. This article presents the first use of fused-core particle packed column in an attempt to extend of the chromatographic capabilities of the SIC system. A new fused-core particle column (2.7 μm) Ascentis ® Express C18 (Supelco™ Analytical) 30 mm × 4.6 mm brings high separation efficiency within flow rates and pressures comparable to monolithic column Chromolith ® Performance RP-18e 100-3 (Merck ®) 100 mm × 3 mm. Both columns matches the conditions of the commercially produced SIC system – SIChrom™ (8-port high-pressure selection valve and medium-pressure Sapphire™ syringe pump with 4 mL reservoir – maximal work pressure 1000 PSI) (FIAlab ®, USA). The system was tested by the separation of four estrogens with similar structure and an internal standard – ethylparaben. The mobile phase composed of acetonitrile/water (40/60 (v/v)) was pumped isocratic at flow rate 0.48 mL min −1. Spectrophotometric detection was performed at wavelength of 225 nm and injected volume of sample solutions was 10 μL. The chromatographic characteristics of both columns were compared. Obtained results and conclusions have shown that both fused-core particle column and longer narrow shaped monolithic column bring benefits into the SIC method.

Evaluation of fused-core and monolithic versus porous silica-based C18 columns and porous graphitic carbon for ion-pairing liquid chromatography analysis of catecholamines and related compounds

This paper evaluates the performances of reversed-phase (RPLC) and ion-pairing chromatography (IPLC) coupled with UV detection for the analysis of a set of 12 catecholamines and related compounds. Different chromatographic columns (porous C18-silica, perfluorinated C18-silica, porous graphitic carbon, monolithic and fused-core silica-based C18 columns) were tested using semi-long perfluorinated carboxylic acids as volatile ion-pairing reagents. Much more promising results were obtained by IPLC than by RPLC and important improvements in analytes peak symmetry and separation resolution were observed when using the “fast chromatography” columns (monolithic and fused-core C18) under IPLC conditions. For UV detection, a satisfactory separation of the 12 selected analytes was achieved in less than 20 min by using a fused-core particles column (Halo C18) and a mobile phase composed of a 1.25 mM nonafluoropentanoic acid aqueous solution and methanol under gradient elution mode. The chromatographic method developed can be directly coupled with electrospray ionization tandem mass spectrometry (ESI–MS/MS) in positive ionization mode and 10 solutes among those selected can be observed. The presence of the acidic ion-pairing reagent in the mobile phase makes this system incompatible with negative ionization mode and thus unable to detect the two acidic compounds that only responded in negative mode. In terms of MS detection, Monolithic C18 column proved to be the best one to reach the lowest detection limits (LODs) (from 0.5 ng mL −1 to 10 ng mL −1 depending on the neurotransmitter). The applicability of the optimized LC–MS/MS method to a “real world” sample was finally evaluated. The presence of the matrix leads to signal suppression for several solutes and thus to higher LODs.

Comparison of different types of stationary phases for the analysis of soy isoflavones by HPLC

Nowadays, there are new technologies in high-performance liquid chromatography columns available enabling faster and more efficient separations. In this work, we compared three different types of columns for the analysis of main soy isoflavones. The evaluated columns were a conventional reverse phase particle column, a fused-core particle column, and a monolithic column. The comparison was in terms of chromatographic parameters such as resolution, asymmetry, number of theoretical plates, variability of retention time, and peak width. The lower column pressure was provided by the monolithic column, although lower chromatographic performance was achieved. Conventional and fused-core particle columns presented similar pressure. Results also indicate that direct transfer between particle and monolithic columns is not possible requiring adjustment of conditions and a different method optimization strategy. The best chromatographic performance and separation speed were observed for the fused-core particle column. Also, the effect of sample solvent on the separation and peak shape was evaluated and indicated that monolithic column is the most affected especially when using higher concentrations of acetonitrile or ethanol. Sample solvent that showed the lowest effect on the chromatographic performance of the columns was methanol. Overall evaluation of methanol and acetonitrile as mobile phase for the separation of isoflavones indicated higher chromatographic performance of acetonitrile, although methanol may be an attractive alternative. Using acetonitrile as mobile phase resulted in faster, higher resolution, narrower, and more symmetric peaks than methanol with all columns. It also generated the lower column pressure and flatter pressure profile due to mobile phase changes, and therefore, it presents a higher potential to be explored for the development of faster separation methods.