General methods for flash chromatography using disposable columns.
Ontology highlight
ABSTRACT:
SUBMITTER: Stevens WC
PROVIDER: S-EPMC2780649 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Advances in phosphopeptide enrichment methods enable the identification of thousands of phosphopeptides from complex samples. Current offline enrichment approaches using TiO(2), Ti, and Fe immobilized metal ion affinity chromatography (IMAC) material in batch or microtip format are widely used, but they suffer from irreproducibility and compromised selectivity. To address these shortcomings, we revisited the merits of performing phosphopeptide enrichment in an HPLC column format. We found that Fe-IMAC columns enabled the selective, comprehensive, and reproducible enrichment of phosphopeptides out of complex lysates. Column enrichment did not suffer from bead-to-sample ratio issues and scaled linearly from 100 ?g to 5 mg of digest. Direct measurements on an Orbitrap Velos mass spectrometer identified >7500 unique phosphopeptides with 90% selectivity and good quantitative reproducibility (median cv of 15%). The number of unique phosphopeptides could be increased to more than 14,000 when the IMAC eluate was subjected to a subsequent hydrophilic strong anion exchange separation. Fe-IMAC columns outperformed Ti-IMAC and TiO(2) in batch or tip mode in terms of phosphopeptide identification and intensity. Permutation enrichments of flow-throughs showed that all materials largely bound the same phosphopeptide species, independent of physicochemical characteristics. However, binding capacity and elution efficiency did profoundly differ among the enrichment materials and formats. As a result, the often quoted orthogonality of the materials has to be called into question. Our results strongly suggest that insufficient capacity, inefficient elution, and the stochastic nature of data-dependent acquisition in mass spectrometry are the causes of the experimentally observed complementarity. The Fe-IMAC enrichment workflow using an HPLC format developed here enables rapid and comprehensive phosphoproteome analysis that can be applied to a wide range of biological systems.
| S-EPMC4288255 | biostudies-literature
Project description:We report on a potential method to separate sugars by using the specific interaction between fullerenes and saccharides in liquid chromatography (LC). Aromatic rings with high electron density are believed to interact strongly with saccharides due to CH-π and/or OH-π interactions. In this study, the fullerene-bonded columns were used to separate saccharides by LC under aqueous conditions. As a result, 2-aminobenzamide-labeled glucose homopolymer (Glcs) was effectively separated by both C60 and C70 columns in the range of Glc-1 to Glc-20 and high blood glucose level being retained in greater quantity. Furthermore, similar separations were identified by LC-mass spectrometry with non-labeled glucose homopolymers. Theoretical study based on molecular dynamics and DFT calculation demonstrated that a supramolecular complex of saccharide-fullerene was formed through CH-π and/or OH-π interactions, and that the interactions between saccharide and fullerene increase with the increase units of the saccharide. Additionally, the C60 column retained disaccharides containing maltose, trehalose, and sucrose. In this case, it was assumed that the retention rates were determined by the difference of the dipole moment in each saccharide. These results suggest that the dipole-induced dipole interaction was dominant, and that maltose-with the higher dipole moment-was more strongly retained compared to other disaccharides having lower dipole moment.
| S-EPMC7429847 | biostudies-literature
Project description:With the emergence of wearable or disposable electronics, there grows a demand for a flash memory realizable on various flexible substrates. Nevertheless, it has been challenging to develop a flash memory that simultaneously exhibits a significant level of flexibility and performance. This is mainly due to the scarcity of flexible dielectric materials with insulating properties sufficient for a flash memory, which involves dual dielectric layers, respectively, responsible for tunneling and blocking of charges. Here we report ultra-flexible organic flash memories based on polymer dielectrics prepared by initiated chemical vapor deposition. Using their near-ideal dielectric characteristics, we demonstrate flash memories bendable down to a radius of 300 μm that exhibits a relatively long-projected retention with a programming voltage on par with the present industrial standards. The proposed memory technology is then applied to non-conventional substrates, such as papers, to demonstrate its feasibility in a wide range of applications.Flexible flash memory is crucial to modern electronics, but its fabrication is challenging in the absence of suitable dielectric materials. Here, Lee et al. realize organic memory with retention over 10 years using tunneling and blocking dielectric layers prepared by initiated chemical vapor deposition.
| S-EPMC5620045 | biostudies-literature
Project description:Trypsin is the most widely used enzyme in proteomic research due to its high specificity. Although the in-solution digestion is predominantly used, it has several drawbacks, such as long digestion times, autolysis, and intolerance to high temperatures or organic solvents. To overcome these shortcomings trypsin was covalently immobilized on solid support and tested for its proteolytic activity. Trypsin was immobilized on bridge-ethyl hybrid silica sorbent with 300Å pores, packed in 2.1×30mm column and compared with Perfinity and Poroszyme trypsin columns. Catalytic efficiency of enzymatic reactors was tested using N?-Benzoyl-l-arginine 4-nitroanilide hydrochloride as a substrate. The impact of buffer pH, mobile phase flow rate, and temperature on enzymatic activity was investigated. Digestion speed generally increased with the temperature from 20 to 37°C. Digestion speed also increased with pH from 7.0 to 9.0; the activity of prototype enzyme reactor was highest at pH 9.0, when it activity exceeded both commercial reactors. Preliminary data for fast protein digestion are presented.
| S-EPMC5497570 | biostudies-literature
Project description:Proteomic studies of circulating vesicles are hampered by difficulties in purifying vesicles from plasma and serum. Isolations are contaminated with high-abundance blood proteins that may mask genuine vesicular-associated proteins and/or simply provide misleading data. In this brief report, we explored the potential utility of a commercially available size exclusion chromatography column for rapid vesicle purification. We evaluated the performance of the column, with cancer cell line conditioned medium or healthy donor plasma, in terms of removing non-vesicular protein and enriching for vesicles exhibiting exosome characteristics. Serial fractions revealed a peak for typical exosomal proteins (CD9, CD81 etc.) that preceded the peak for highly abundant proteins, including albumin, for either sample type, and harvesting only this peak would represent elimination of >95% of protein from the sample. The columns showed good reproducibility, and streamlining the workflow would allow the exosome-relevant material to be collected in less than 10 minutes. Surprisingly, however, subsequent post-column vesicle concentration steps whilst resulting in some protein loss also lead to low vesicle recoveries, with a net effect of reducing sample purity (assessed by the particle-to-protein ratio). The columns provide a convenient, reproducible and highly effective means of eliminating >95% of non-vesicular protein from biological fluid samples such as plasma.
| S-EPMC4376847 | biostudies-literature
Project description:In many areas of application, key objectives of chemical separation and analysis are to minimize the sample quantity while maximizing the chemical information obtained. Increasing measurement sensitivity is especially critical for proteomics research, especially when processing trace samples and where multiple measurements are desired. A rich collection of technologies has been developed, but the resulting sensitivity remains insufficient for achieving in-depth coverage of proteomic samples as small as single cells. Here, we combine picoliter-scale liquid chromatography (picoLC) with mass spectrometry (MS) to address this issue. The picoLC employs a 2-?m-i.d. open tubular column to reduce the sample input needed to greatly increase the sensitivity achieved using electrospray ionization (ESI) with MS. With this picoLC-MS system, we show that we can identify ?1000 proteins reliably using only 75 pg of tryptic peptides, representing a 10-100-fold sensitivity improvement compared with the state-of-the-art liquid chromatography (LC) or capillary electrophoresis (CE)-MS methods. PicoLC-MS extends the limit of separation science and is expected to be a powerful tool for single cell proteomics.
| S-EPMC7279521 | biostudies-literature
Project description:A silica-bound C-butylpyrogallol[4]arene chromatographic stationary phase was prepared and characterised by thermogravimetric analysis, scanning electron microscopy, NMR and mass spectrometry. The chromatographic performance was investigated by using C60 and C70 fullerenes in reverse phase mode via flash column and high-pressure liquid chromatography (HPLC). The resulting new stationary phase was observed to demonstrate size-selective molecular recognition as postulated from our in-silico studies. The silica-bound C-butylpyrogallol[4]arene flash and HPLC stationary phases were able to separate a C60- and C70-fullerene mixture more effectively than an RP-C18 stationary phase. The presence of toluene in the mobile phase plays a significant role in achieving symmetrical peaks in flash column chromatography.
| S-EPMC8198677 | biostudies-literature
Project description:Therapeutic drug monitoring, which is used to determine appropriate drug doses, is critical in pharmacological therapy. In this study, we developed thermoresponsive chromatography columns with various cationic properties for effective therapeutic drug monitoring. Thermoresponsive cationic copolymer poly(N-isopropylacrylamide-co-n-butyl methacrylate-co-N,N-dimethylaminopropyl acrylamide) (P(NIPAAm-co-BMA-co-DMAPAAm))-modified silica beads, which were used as the chromatographic stationary phase, were prepared by modifying the radical initiator of the silica beads, followed by radical polymerization. Characterization of the prepared silica beads demonstrated that thermoresponsive polymers with various cationic properties successfully modified the beads. The elution behavior of several steroids in the prepared bead-packed columns at various temperatures indicated that the optimal column operating temperature was 30 °C. Appropriate measurement conditions for 13 drugs were investigated by varying the cationic properties of the columns and the pH of the mobile phase. Drug concentrations in serum samples were determined using the developed columns and mobile phases with a suitable pH. Voriconazole concentrations in human serum samples were determined using the developed columns with all-aqueous mobile phases. We anticipate that the developed chromatography columns can be used for therapeutic drug monitoring because drug concentrations can be measured using all-aqueous mobile phases that are suitable in clinical settings.
| S-EPMC9329465 | biostudies-literature
Project description:Despite an outstanding agent for control of Lepidoptera, the diamide insecticide cyclaniliprole (CYCP) is a suspected carcinogen. In the present study, an analytical method was developed for the determination of CYCP in six fruits and vegetables (apple, grape, peach, bell pepper, lettuce, and tomato) using ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry. Sample preparation was carried out by the acetonitrile-salting-out extraction followed by simple and fast cleanup of disposable pipette extraction tip containing styrene divinyl benzene and/or graphitized carbon black. Satisfactory linearity (r > 0.99) was obtained in the calibration range of 0.001-1 µg mL-1. Matrix effects decreased from -9.9--17.9% to -1.0--7.6% after the cleanup. The recoveries of CYCP at three spike levels (0.01, 0.1, and 1 mg kg-1) from different matrices were between 75.7% and 111.5%, with the intra-day (n = 5) and inter-day (n = 15) relative standard deviations lower than 12.1%. The limit of quantification was 0.01 mg kg-1. The developed method provides a good reference for routine monitoring of CYCP in these fruits and vegetables.
| S-EPMC9570770 | biostudies-literature
Project description:Cryotherapy is a commonly used office procedure that causes destruction of tissue by cryonecrosis due to rapid freezing and thawing of cells. The limitation in treating plantar warts and deeper dermal lesions is that the freeze time should be longer to penetrate deeper, which results in collateral damage to normal skin surrounding the lesion. This results in unwanted side effects of prolonged pain, blistering and haemorrhage and increased healing time. The cone spray technique was used to reduce collateral damage, but deeper penetration is difficult to achieve. An innovative technique using disposable biopsy punches is described that ensures deeper freezing as compared to the plastic cone. The metal cutting edge of the punch enters deeper into the lesions as the liquid nitrogen is passed, sparing damage to surrounding skin.
| S-EPMC4134645 | biostudies-literature