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Implementation of high slurry concentration and sonication to pack high-efficiency, meter-long capillary ultrahigh pressure liquid chromatography columns.

ABSTRACT: Slurry packing capillary columns for ultrahigh pressure liquid chromatography is complicated by many interdependent experimental variables. Previous results have suggested that combination of high slurry concentration and sonication during packing would create homogeneous bed microstructures and yield highly efficient capillary columns. Herein, the effect of sonication while packing very high slurry concentrations is presented. A series of six, 1m×75?m internal diameter columns were packed with 200mg/mL slurries of 2.02?m bridged-ethyl hybrid silica particles. Three of the columns underwent sonication during packing and yielded highly efficient separations with reduced plate heights as low as 1.05.

SUBMITTER: Godinho JM

PROVIDER: S-EPMC5501974 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Implementation of high slurry concentration and sonication to pack high-efficiency, meter-long capillary ultrahigh pressure liquid chromatography columns.

Godinho Justin M JM Reising Arved E AE Tallarek Ulrich U Jorgenson James W JW

Journal of chromatography. A 20160801

Slurry packing capillary columns for ultrahigh pressure liquid chromatography is complicated by many interdependent experimental variables. Previous results have suggested that combination of high slurry concentration and sonication during packing would create homogeneous bed microstructures and yield highly efficient capillary columns. Herein, the effect of sonication while packing very high slurry concentrations is presented. A series of six, 1m×75μm internal diameter columns were packed with ...[more]

PMID: 27499108

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Project description:Column wall effects and the formation of larger voids in the bed during column packing are factors limiting the achievement of highly efficient columns. Systematic variation of packing conditions, combined with three-dimensional bed reconstruction and detailed morphological analysis of column beds, provide valuable insights into the packing process. Here, we study a set of sixteen 75?m i.d. fused-silica capillary columns packed with 1.9?m, C18-modified, bridged-ethyl hybrid silica particles slurried in acetone to concentrations ranging from 5 to 200mg/mL. Bed reconstructions for three of these columns (representing low, optimal, and high slurry concentrations), based on confocal laser scanning microscopy, reveal morphological features associated with the implemented slurry concentration, that lead to differences in column efficiency. At a low slurry concentration, the bed microstructure includes systematic radial heterogeneities such as particle size-segregation and local deviations from bulk packing density near the wall. These effects are suppressed (or at least reduced) with higher slurry concentrations. Concomitantly, larger voids (relative to the mean particle diameter) begin to form in the packing and increase in size and number with the slurry concentration. The most efficient columns are packed at slurry concentrations that balance these counteracting effects. Videos are taken at low and high slurry concentration to elucidate the bed formation process. At low slurry concentrations, particles arrive and settle individually, allowing for rearrangements. At high slurry concentrations, they arrive and pack as large patches (reflecting particle aggregation in the slurry). These processes are discussed with respect to column packing, chromatographic performance, and bed microstructure to help reinforce general trends previously described. Conclusions based on this comprehensive analysis guide us towards further improvement of the packing process.

Project description:Improvements in sample preparation, separation, and mass spectrometry continue to expand the coverage in LC-MS based lipidomics. While longer columns packed with smaller particles in theory give higher separation performance compared to shorter columns, the implementation of this technology above commercial limits has been sparse due to difficulties in packing long columns and successfully operating instruments at ultrahigh pressures. In this work, a liquid chromatograph that operates up to 35 kpsi was investigated for the separation and identification of lipid species from human plasma. Capillary columns between 15-50 cm long were packed with 1.7 µm BEH C18 particles and evaluated for their ability to separate lipid isomers and complex lipid extracts from human plasma. Putative lipid class identifications were assigned using accurate mass and relative retention time data of the eluting peaks. Our findings indicate that longer columns packed and operated at 35 kpsi outperform shorter columns packed and run at lower pressures in terms of peak capacity and numbers of features identified. Packing columns with relatively high concentration slurries (200 mg/mL) while sonicating the column resulted in 6-34% increase in peak capacity for 50 cm columns compared to lower slurry concentrations and no sonication. For a given analysis time, 50 cm long columns operated at 35 kpsi provided a 20-95% increase in chromatographic peak capacity compared with 15 cm columns operated at 15 kpsi. Analysis times up to 4 h were evaluated, generating peak capacities up to 410 ± 5 (n = 3, measured at 4σ) and identifying 480 ± 85 lipids (n = 2). Importantly, the results also show a correlation between the peak capacity and the number of lipids identified from a human plasma extract. This correlation indicates that ionization suppression is a limiting factor in obtaining sufficient signal for identification by mass spectrometry. The result also shows that the higher resolution obtained by shallow gradients overcomes possible signal reduction due to broader, more dilute peaks in long gradients for improving detection of lipids in LC-MS. Lastly, longer columns operated at shallow gradients allowed for the best separation of both regional and geometrical isomers. These results demonstrate a system that enables the advantages of using longer columns packed and run at ultrahigh pressure for improving lipid separations and lipidome coverage.

Project description:In recent years protein therapeutics have seen increasing use in the therapeutic arena. As with traditional small molecule drug substances, one is obligated to ensure purity and stability of the various dosage forms. With these higher molecular weight therapeutics a common approach for analytical characterization is enzymatic digestion followed by gradient elution liquid chromatography with mass spectrometry detection to create a peptide map (bottom-up protein analysis). Due to the difficult to separate mixtures frequently encountered, there is the need for advanced chromatographic systems featuring increased resolution and/or peak capacity that can be operated in the gradient elution format. Presently we describe an extreme ultra-pressure liquid chromatography (XUPLC) system that has been implemented as an in-house add-on to a commercial ultra-pressure chromatography system. This add-on allows operation at the 38 Kspi range, accommodates the use of capillary columns in excess of one meter packed with sub-2 ?m particles and can be operated in the gradient elution format. To evaluate the utility of this system, rat growth hormone was used as a model protein and was exposed to light (? 254 nm) to create a stress environment. When enzymatic digests of control and stressed protein were analyzed with the XUPLC system using MS detection, greater than 92% peptide coverage was achieved, including the identification some peptides where pre-oxidation of Met residues had occurred, as well as chemistry specifically related to the photolysis of protein disulfide linkages. When the same samples were analyzed by commercial UPLC and compared to the XUPLC results, the utility of the increased peak capacity available with the XUPLC was apparent as previously co-eluting peaks were now well resolved. In particular one specific degradation route was identified where a pair of isobaric cis/trans diastereomerically related peptides were well resolved by XUPLC while they were unresolved by UPLC. Clearly the use of this system operating at the higher pressure regime with long capillary columns is and will be useful in continued investigations of protein stability, especially in cases where only subtle differences in the amino acid residues have occurred during degradation.

Dataset Information

Implementation of high slurry concentration and sonication to pack high-efficiency, meter-long capillary ultrahigh pressure liquid chromatography columns.

Publications

Implementation of high slurry concentration and sonication to pack high-efficiency, meter-long capillary ultrahigh pressure liquid chromatography columns.

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