Column Frits Research Articles

Evaluation of hybrid surface technology for the analysis of the B-group vitamins by LC-ESI-MS/MS

Recently, a novel hybrid surface technology (HST) has been developed to mitigate metal analyte adsorption in liquid chromatography. The HST provides a hybrid organic–inorganic surface on the metal fluidic path, from injection to detector and including the column frits and wall, to mitigate the interaction between analytes and metals. Here the impact of the HST on the analysis of B group vitamins using liquid chromatography coupled with electrospray tandem mass spectrometry (LC-ESI-MS/MS) has been evaluated. Significant improvements in analyte intensity, limit of quantification (LOQ), carry-over, and peak shape were observed using an LC-ESI-MS/MS system and column that incorporated the HST. The key observed improvements include a 3–10 times increase in sensitivity (providing a lower LOQ) for riboflavin, thiamine, nicotinamide, FMN, PLP, and 5MTHF, no carry-over, and a more symmetrical peak for thiamine. When applied to the analysis of B group vitamins in energy drinks and B vitamin dietary supplement samples, the HST system demonstrated excellent accuracy and repeatability.

The critical role of mobile phase pH in the performance of oligonucleotide ion-pair liquid chromatography-mass spectrometry methods.

The critical role of mobile phase pH in the performance of oligonucleotide ion-pair liquid chromatography-mass spectrometry methods.

Impact of Nonspecific Adsorption to Metal Surfaces in Ion Pair-RP LC-MS Impurity Analysis of Oligonucleotides

Nonspecific adsorption has been a consistent challenge in the analysis of oligonucleotides. Nonspecific adsorption is a result of interactions between charged acidic analytes and adsorption sites present in metallic surfaces located in the fluidic path of chromatography systems. Due to their high surface area, adsorption to column frits is especially concerning. Poor peak shape, low recovery and compromised LOQ have been associated with this phenomenon. Alternative methods including substitution of stainless steel for different hardware materials and mobile phase additives have been explored in an attempt to minimize this issue. Chemical modification of metal surfaces using hybrid surface technology (HST) by-passes the limitation of stainless steel construction material by forming a hybrid organic/inorganic layer that acts as a barrier and limits nonspecific interactions. In this study we explore the implications of this new technology in sensitive analysis and determination of relative impurity levels of oligonucleotides. Higher relative impurity levels and better reproducibility were obtained with columns using HST.

Improving LC/MS/MS-based bioanalytical method performance and sensitivity via a hybrid surface barrier to mitigate analyte – Metal surface interactions

The accurate determination of the pharmacokinetics (PK) of a candidate drug molecule is critical in both drug discovery and development. Over the last 30 years, the sensitivity and selectivity of LC/MS has resulted in it being established as the technology of choice for these studies. However, unwanted chemical interactions between analyte(s) and the metal components in a chromatography system can result in poor peak shape and reduction in signal response, which can adversely affect the analysis of low concentrations of drugs and their metabolites in biological samples. This study evaluated the benefits of employing an inert hybrid surface technology (HST) applied to the metallic components in the LC flow path, column frits and column wall to mitigate these interactions. The results obtained were compared with that of an identical conventional LC for the bioanalysis of two steroid phosphate drugs (dexamethasone phosphate and hydrocortisone phosphate) and an epidermal growth factor receptor (EGFR) inhibitor (gefitinib) in human plasma. The results showed that for the two steroid phosphates, the peak width was reduced by 20%, peak tailing factors reduced by up to 30% and the assay sensitivity improved by factors of 7.5 and 10. This resulted in a significant improvement in the limit of detection. The new LC system also improved the reproducibility of peak integration for gefitinib, thereby reducing assay coefficients of variation (%CV) from greater than 10% to less than 5% at the lower limit of quantification.

Mitigation of analyte loss on metal surfaces in liquid chromatography

The adsorptive loss of acidic analytes in liquid chromatography was investigated using metal frits. Repetitive injections of acidic small molecules or an oligonucleotide were made on individual 2.1 or 4.6 mm i.d. column frits. Losses were observed for adenosine 5′-(α,β-methylene) diphosphate, 2-pyridinol 1-oxide and the 25-mer phosphorothioate oligonucleotide Trecovirsen (GEM91) on stainless steel and titanium frits. Analyte adsorption was greatest at acidic pH due to the positive charge on the metal oxide surface. Analyte recovery increased when a series of injections was performed; this effect is known as sample conditioning. Nearly complete recovery was achieved when the metal adsorptive sites were saturated with the analyte. A similar effect was achieved by conditioning the frits with phosphoric, citric or etidronic acids, or their buffered solutions. These procedures can be utilized to mitigate analyte loss. However, the effect is temporary, as the conditioning agent is gradually removed by the running mobile phase. Metal frits modified with hybrid organic/inorganic surface technology were shown to mitigate analyte-to-metal surface interactions and improve recovery of acidic analytes. Quantitative recovery of a 15–35 mer oligodeoxythymidine mixture was achieved using column hardware modified with hybrid surface technology, without a need for column conditioning prior to analysis.

High Performance Size Exclusion Chromatography and High-Throughput Dynamic Light Scattering as Orthogonal Methods to Screen for Aggregation and Stability of Monoclonal Antibody Drug Products

The presence of aggregates in monoclonal antibody (mAb) drug product (DP) formulations can present product quality challenges. Here we show that use of High Performance Size Exclusion Chromatography (HP-SEC), in conjunction with high-throughput dynamic light scattering (HT-DLS) analyses of mAb DPs can be a useful strategy to determine monomer content and the presence of aggregates under simulated stress conditions. This analytical approach was used to evaluate four commercially available mAb DPs under different conditions i.e.; original formulations, diluted, and thermo-mechanical stressed condition. Due to particle size limitations of HP-SEC columns, resulting in particles accumulating in the column frits prior to reaching the detector for analysis, there is a possibility that large mAb aggregates may not be detected. Both HP-SEC and HT-DLS were able to detect and resolve the mAb monomer (~10–12 nm) of the DPs in their recommended storage conditions. However, the ability to detect large aggregates (>40 nm) by both analytical methods differed, and HT-DLS was able to detect aggregates between 60 nm and 1400 nm under stress conditions. Our data indicates that HP-SEC, in conjunction with HT-DLS, may be beneficial to detect both mAb DP monomer content and multiple aggregate species (1–1000 nm) in the submicron size range.

Impact of frit dispersion on gradient performance in high-throughput liquid chromatography

The impact of porous frits (2.1 mm i.d. × 1 mm thick, 0.2 μm porosity, 20% void, 0.69 μL) in short 0.5–5 cm long × 2.1 mm i.d. columns packed with sub-2 μm superficially porous particles on gradient performance was investigated using a low dispersive i-class ACQUITY UPLC system (25 cm × 75 μm outlet tube + 250 nL optical cell). In order to maximize data accuracy, the sample dispersion through a single frit was measured from four independent methods: (1) plate height subtraction, (2) peak capacity versus efficiency plot, (3) flow reversal, and (4) direct measurement. Frit dispersion increases non-linearly with increasing flow rate. The corresponding volume variances of small molecules were measured at 0.16 ± 0.04, 0.24 ± 0.05, and 0.31 ± 0.06 μL2 at flow rates of 0.1, 0.2, and 0.3 mL/min, respectively. These observed variances are lower than and consistent with the maximum volume variance of 0 . 692 ∼ 0.5 μL2 expected for a mixer-like behavior. The peak capacity of short columns were then calculated for mixtures of peptides using a general model of gradient elution by considering (or not) the actual analyte dispersion taking place in the outlet frit, in the post-column connecting tube, and in the detection cell. The results show that the sole presence of the outlet frit is responsible for about 50% loss in peak capacity (relative to the expected gradient performance in absence of frit and post-column tube) for a 1 cm long column operated under standard gradient steepness. The actual structure and volume of column frits needs adjustment if one wants to take full advantage of the true performance of short high-throughput columns packed with sub-2 μm particles.

Determination of 5-nitroimidazole residues in milk by capillary electrochromatography with packed C18 silica beds

This work presents a novel methodology for analysing 5-nitroimidazole residues in milk samples by capillary electrochromatography using lab-made packed columns, produced by carrying out a high pressure packing procedure using acetone as driving solvent and C18 silica uncapped particles (5µm particle size) as packing material. Column frits resulted from sintering the proper stationary phase by heating the packed material for 20s with a nichrome ribbon (80% Ni–20% Cr, 28cm×2mm×0.2mm, electric resistance 1.3Ω) connected to a 7V AC power supply. Lab-made C18 silica packed capillaries (40cmx50µm i.d.) were employed for the determination of 5-nitroimidazole drugs. Milk samples were treated by a salting-out assisted liquid–liquid extraction followed by a solid phase extraction with Oasis®HLB cartridges prior to their injection. Samples were hydrodynamically injected into the column for 120s at 11.5bar. Afterwards eight 5-nitroimidazole compounds were separated in isocratic mode under an applied voltage of 27kV and a temperature of 30°C. The selected mobile phase consisted of a mixture 60:40 acetonitrile:ammonium acetate (2.5mM, pH=5). Separation was monitored at 320nm and it was performed in less than 15min. The method was characterized in terms of linearity (R2≥0.993) and precision (repeatability, RSD≤12.2% and reproducibility, RSD≤14.5%), obtaining detection limits lower than 29µg/L for all compounds under study.

Characterization of Group 5 dubnium homologs on diglycolamide extraction chromatography resins from nitric and hydrofluoric acid matrices

A diglycolamide-based resin was characterized for Group 5 (Ta, Nb and pseudo-homolog Pa) separation from Group 4 (Hf and Zr) and tri-valent actinides (Am). The batch uptake of the radionuclides of interest were determined for HNO3/HF matrices and the results were used to develop a column separation method that could be used for element 105 (Db) purification chemistry. Tantalum is significantly retained by the TODGA resin, while Pa/Nb show little to no retention. The affinity for Ta decreases as a function of increased [HNO3], while Zr/Hf (and Am) affinities increase under the same conditions. Adsorption of Ta to polypropylene vials and column frits became a significant focus of this work following several observations made during the course of the column separation method development. All of the Group 5 elements were separated from Group 4 and Am while Ta may be completely isolated from Nb/Pa with the TODGA resin with improvements in column parameters: column height and bed volume.

Do column frits contribute to the on-column, flow-induced degradation of macromolecules?

Flow-induced, on-column degradation is a major hindrance to the accurate characterization of ultra-high molar mass macromolecules and colloids. This degradation is a direct result of the large shear rates which are generated within the column, which cause chain scission to occur both in the interstitial medium and, it has been postulated, at the packing particle pore boundary. An additional putative source of degradation has been the column frits, though little experimental evidence exists to either support or refute this claim. To this effect, the present experiments examine the role of the frits in the degradation of high molar mass macromolecules. Two narrow dispersity polystyrene standards, the molar mass of which differs by a factor of two, were analyzed on three different size-exclusion chromatography (SEC) columns, each with frits of different pore size, at various flow rates. In the smallest pore size column, which also contained the smallest frits and which was packed with the smallest diameter particles, the larger standard was forced to degrade by increasing the flow rate of the mobile phase. During the course of the latter portion of the study, the inlet and the outlet frits were removed from the column, in stepwise fashion. It was concluded that neither frit played any appreciable role in the degradation. Results of our studies were applied to explain previously observed degradation in ultra-high pressure liquid chromatography of polymers. The general conclusion arrived at herein is that the column frits are likely to have a secondary role (as compared to interstitial and pore boundary stresses), or no role at all, in polymer degradation for cases where the frit radius is larger than or equal to the hydraulic radius rcof the column.

Analysis of complex polymers by multidetector field-flow fractionation

Field-flow fractionation (FFF) is a powerful alternative to column-based polymer fractionation methods such as size-exclusion chromatography (SEC) or interaction chromatography (IC). The most common polymer fractionation method, SEC, has its limitations when polymers with very high molar masses or complex structures must be analysed. Another limitation of all column-based methods is that the samples must be filtered before analysis and shear degradation of large macromolecules may be caused by the stationary phase and/or the column frits. Finally, the separation of very polar polymers may be a challenge because such polymers interact very strongly with the stationary phase, causing irreversible adsorption or other negative effects. This article reviews the latest developments in field-flow fractionation of complex polymers. It is demonstrated that some of the limitations of column-based chromatography can be overcome by FFF. When appropriate, results from column-based fractionations are compared with those from FFF fractionations to highlight the specific merits and challenges of each method. In addition to the fractionations themselves, various detector setups are discussed to show that different polymer distributions require different experimental procedures. Examples are given of the analysis of molar mass distribution, chemical composition, and microstructure. Advanced detector combinations are discussed, most prominently the very recently developed coupling to (1)H NMR. Finally, analysis of polymer nanocomposites by asymmetric flow field-flow fractionation (AF4)-FTIR is presented.

Kinetic performance of narrow-bore columns on a micro-system for high performance liquid chromatography

The kinetic performance of 0.5mm×50mm columns packed with 2.7μm Halo-C18 core–shell particles and 3μm EP-120-C18 fully porous particles fitted on an Eksigent LC-Express Ultra μHPLC system were measured. The instrument contribution to band broadening was obtained by directly connecting the injection valve and the detector cell with a short, narrow PEEKSIL tube. The connections between the column and the connecting tubes, the column endfittings and its frits contribute to band spreading and are responsible for a significant rear peak tailing, even for retained compounds, resulting in a significant loss of efficiency. Our results show that the μHPLC system could outperform the current VHPLC systems using 2.1mm I.D. columns packed with 1.7μm particles if it were using 0.5mm I.D. columns packed with 1μm particles, if it could operate at a few kbar pressure drop, and if the sum of the contributions of the instrument, column endfittings and the column frits to band dispersion were three times smaller than it is at present.

Deformation and degradation of polymers in ultra-high-pressure liquid chromatography

Ultra-high-pressure liquid chromatography (UHPLC) using columns packed with sub-2 μm particles has great potential for separations of many types of complex samples, including polymers. However, the application of UHPLC for the analysis of polymers meets some fundamental obstacles. Small particles and narrow bore tubing in combination with high pressures generate significant shear and extensional forces in UHPLC systems, which may affect polymer chains. At high stress conditions flexible macromolecules may become extended and eventually the chemical bonds in the molecules can break. Deformation and degradation of macromolecules will affect the peak retention and the peak shape in the chromatogram, which may cause errors in the obtained results ( e.g. the calculated molecular-weight distributions). In the present work we explored the limitations of UHPLC for the analysis of polymers. Degradation and deformation of macromolecules were studied by collecting and re-injecting polymer peaks and by off-line two-dimensional liquid chromatography. Polystyrene standards with molecular weight of 4 MDa and larger were found to degrade at UHPLC conditions. However, for most polymers degradation could be avoided by using low linear velocities. No degradation of 3-MDa PS (and smaller) was observed at linear velocities up to 7 mm/s. The column frits were implicated as the main sources of polymer degradation. The extent of degradation was found to depend on the type of the column and on the column history. At high flow rates degradation was observed without a column being installed. We demonstrated that polymer deformation preceded degradation. Stretched polymers eluted from the column in slalom chromatography mode (elution order opposite to that in SEC or HDC). Under certain conditions we observed co-elution of large and small PS molecules though a convolution of slalom chromatography and hydrodynamic chromatography.

Abstracts: Joint Meeting, II Latinamerican Symposium on Fetal-Maternal Interaction and Placenta and XIX Anual Meeting of The Chilean Society of Physiological Sciences (SCHCF)

Flow-induced, on-column degradation is a major hindrance to the accurate characterization of ultra-high molar mass macromolecules and colloids. This degradation is a direct result of the large shear rates which are generated within the column, which cause chain scission to occur both in the interstitial medium and, it has been postulated, at the packing particle pore boundary. An additional putative source of degradation has been the column frits, though little experimental evidence exists to either support or refute this claim. To this effect, the present experiments examine the role of the frits in the degradation of high molar mass macromolecules. Two narrow dispersity polystyrene standards, the molar mass of which differs by a factor of two, were analyzed on three different size-exclusion chromatography (SEC) columns, each with frits of different pore size, at various flow rates. In the smallest pore size column, which also contained the smallest frits and which was packed with the smallest diameter particles, the larger standard was forced to degrade by increasing the flow rate of the mobile phase. During the course of the latter portion of the study, the inlet and the outlet frits were removed from the column, in stepwise fashion. It was concluded that neither frit played any appreciable role in the degradation. Results of our studies were applied to explain previously observed degradation in ultra-high pressure liquid chromatography of polymers. The general conclusion arrived at herein is that the column frits are likely to have a secondary role (as compared to interstitial and pore boundary stresses), or no role at all, in polymer degradation for cases where the frit radius is larger than or equal to the hydraulic radius rc of the column.

Protein proteolysis and the multi-dimensional electrochromatographic separation of histidine-containing peptide fragments on a chip.

This paper reports a system for three-dimensional electrochromatography in a chip format. The steps involved included trypsin digestion, copper(II)-immobilized metal affinity chromatography [Cu(II)-IMAC] selection of histidine-containing peptides, and reversed-phase capillary electrochromatography of the selected peptides. Trypsin digestion and affinity chromatography were achieved in particle-based columns with a microfabricated frit whereas reversed-phase separations were executed on a column of collocated monolithic support structures. Column frits were designed to maintain constant cross sectional area and path length in all channels and to retain particles down to a size of 3 microm. Cu(II)-IMAC selection of histidine-containing peptides from standard peptide mixtures and protein digests followed by reversed-phase chromatography of the selected peptides was demonstrated in the electrochromatography mode. The possibility to run a comprehensive proteomic analysis by combining trypsin digestion, affinity selection, and a reversed-phase separation on chips was shown using fluorescein isothiocyanate-labeled bovine serum albumin as an example.

A radiochemical study of irreversible protein loss on high-performance liquid chromatography column frits

Much success has been achieved in the separation and purification of a wide range of proteins using various high-pressure liquid chromatography techniques. Quantitative analyses of proteins which require 100% mass recovery of the protein are still beset with problems, especially when the total injected amount of protein decreases to below 10 μg. Stainless-steel frits have been cited for their deleterious effects on chromatography in general. In addition, the frits have specifically been found to be a significant contributor to irreversible protein loss—particularly when protein sample sizes are on the order of 1 μg or less. The findings presented below should therefore be of concern to those using HPLC for protein work.