Automated Trapping Column Exchanger for High-Throughput Nanoflow Liquid Chromatography (original) (raw)
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Column switching (CS) emerged as a strategy for direct injection of raw biological samples and currently is the more versatile technique for the fully automated integration of the sample preparation and the chromatography analysis. At the miniaturized scale, CS addresses the matrix complexity and allows the injection of sample volumes larger than those supported by the capillary/nano analytical columns. Detectability can be significantly improved while preserving the analytical capillary/nano column and the proper functioning of the instrument. In the last two decades, fully miniaturized CS systems have been under continuous development. Innovative polymeric, inorganic, and nanomaterials-based extraction phases have been introduced and exploited in diverse column formats, including particle-packed, monolithic, fiber-packed, and open tubular, extraction media. This paper reviews the more recent advances in microextraction column technology for fully miniaturized column-switching systems. Modern sorptive phases and extraction will be described, discussing their potentialities, advantages, and limitations. The overview will emphasize the significance of those developments in bioanalytical applications.
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b S Supporting Information T he complexity and broad dynamic range of the proteome presents great analytical challenges in global proteomic profiling. Because of its high-sensitivity, high resolving power, and the robustness of the instrumentation for polypeptide analysis, the nano-HPLCÀMS system has developed into the major analytical platform for proteomics research. 1À4 Chromatographic performance is considered to be the critical factor for the success of qualification and quantitation in proteomics using nano-HPLCÀMS. However, the reproducibility and lifetime of the nano-HPLC columns is rather limited in comparison with the conventional HPLC columns. The nano-HPLC columns can be easily damaged by the small particles (such as polyacrylamide gel residues) or hydrophobic molecules (such as detergents or undigested proteins). 5,6 In the fabrication of new nano-HPLC capillary columns to replace damaged ones, the method used to retain the packing particles is still a key technology. Currently, most nano-HPLC columns are made by packing particles into a tapered tip or frit capillary. To prepare a tapered tip capillary, one end of the column is heat-drawn to create a small aperture (10À20 μm), thus creating a "keystone" effect to retain the packing material; the tapered tip of the column also acts as an emitter of electrospray ionization (ESI). 7À11 The fabrication of the tapered tip capillary is simple and has minimized postcolumn dead volume, which reduces band broadening due to longitudinal diffusion. However, if the tip is cracked or contaminated, the whole column has to be discarded and replaced with a new one to maintain the ionization efficiency. Because of the reduced outer diameter at the tapered end, the tapered tip capillary cannot be used for the fabrication of trap columns.
Evolution in miniaturized column liquid chromatography instrumentation and applications: An overview
The purpose of this article is to underline the miniaturized LC instrumental system and describe the evolution of commercially available systems by discussing their advantages and drawbacks. Nowadays, there are already many miniaturized LC systems available with a great variety of pump design, interface and detectors as well as efficient columns technologies and reduced connections devices. The solvent delivery systems are able to drive the mobile phase without flow splitters and promote gradient elution using either dual piston reciprocating or syringe-type pumps. The mass spectrometry as detection system is the most widely used detection system; among many alternative ionization sources direct-EI LC-MS is a promising alternative to APCI. In addition, capillary columns are now available showing many possibilities of stationary phases, inner diameters and hardware materials. This review provides a discussion about miniaturized LC demonstrating fundamentals and instrumentals' aspects of the commercially available miniaturized LC instrumental system mainly nano and micro LC formats. This review also covers the recent developments and trends in instrumentation, capillary and nano columns, and several applications of this very important and promising field.
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Journal of Biochemical and Biophysical Methods, 2004
Small-volume chromatographic columns are only able to generate narrow peaks when flow rates, injection volume and instrument components, such as detector, connecting tubing and fittings, are matched to the peak dispersion from the column. Criteria for the proper design of chromatographic instrumentation are therefore derived from a general model on total dispersion. The performance of such a system is then experimentally evaluated from applications run on narrow-bore, small-volume columns. In order to achieve flow rates that match the dimensions of such columns, a new concept for electronic flow control (EFC) is introduced. A theoretical optimization of column efficiency and throughput is discussed and the results verified with practical examples on short, narrow-bore columns packed with small, porous and superficially porous particles. For complex sample mixtures, the concept of peak capacity is introduced and applied to orthogonal separation principles in multiple chromatographic dimensions through column switching techniques.
ELECTROPHORESIS, 2008
An automated nano-LC-MS/MS platform without trap column was established, which only used a 20 cm lauryl methacrylate-ethylene dimethacrylate (LMA-EDMA) monolithic capillary column to allow preconcentration and separation of peptides. The monolithic column had the advantages of good permeability and low backpressure resulting in higher flow rates for capillary columns. Tryptic digests of bovine albumin and yeast protein extract were tested using the monolithic column system. High proteomic coverage using this approach were demonstrated in this study. Furthermore, peptide samples extracted from mouse liver were separated by using the monolithic column system combined with size-exclusion chromatography prefractionation. This monolithic column system might be a promising alternative for the automated system previously using a trap column for routine proteome and peptide profiling analysis.
Continuous sampling and analysis by on-chip liquid/solid chromatography
Sensors and Actuators B: Chemical, 2007
This paper describes a lab-on-a-chip device for continuous liquid/solid chromatography measurements. Chromatographic separations of phenolic test solutions as well as of vitamins are illustrating the ability and versatility of the system. The dependence of the peak height and width with respect to the injected plug and the saturation limits of the microchip column have been investigated and good correlation to the theoretical predictions have been observed.
Instrumentation for hand-portable liquid chromatography
Journal of Chromatography A, 2014
Liquid chromatography (LC) has lagged behind gas chromatography (GC) in developments related to hand-portable instrumentation. In this work, a new battery-operated (24V DC) nano-flow pumping system with a stop-flow injector was developed and integrated with an on-column UV-absorption detector (254nm) that was reduced in size to an acceptable weight and power usage for field operation. The pumping system, which includes nano-flow pump, stepper motor and high-pressure valve weighs only 1.372kg (3lbs) and can generate up to 110.32MPa (16,000psi) pressure. A major advantage of this pump is that it does not employ a splitter, since it was specifically designed for capillary column use. The volume capacity of the pump is 24μL, and a sample volume as low as 10nL can be injected. Flow rate calibration (300nL to 6.12μL per min) was performed, and an accuracy >99.94% was obtained. The percent injection carry-over was found to be low (RSD 0.31%), which makes it practical for quantitative analysis. The detector linear range and limit of detection (LOD) were determined using sodium anthraquinone-2-sulfonate. A linear regression coefficient (R) of 0.9996 was obtained for a plot of log peak area versus log concentration over the range of 3.2μM to 6.5mM, and the LOD (S/N=3) was found to be 7.8fmol (0.13μM). The short term noise of the detector is comparable to commercially available detectors (∼10(-5)AU). In this work, the system was tested in the laboratory using regular line power (120V AC) with an AC to DC adapter. Reversed-phase isocratic separations were performed using a 15.5cm×75μm i.d. fused silica capillary column containing a monolithic stationary phase synthesized from 1,6-hexanediol dimethacrylate. Good retention time repeatability (RSD 0.09-0.74%) was obtained for a mixture containing an unretained marker (i.e., uracil) and a homologous series of alkyl benzenes.
Molecular & Cellular Proteomics, 2013
We report the development and characterization of a novel, vendor-neutral ultra-high pressure-compatible (ϳ10,000 p.s.i.) LC-MS source. This device is the first to make automated connections with user-packed capillary traps, columns, and capillary emitters. The source uses plastic rectangular inserts (referred to here as cartridges) where individual components (i.e. trap, column, or emitter) can be exchanged independent of one another in a plug and play manner. Automated robotic connections are made between the three cartridges using linear translation powered by stepper motors to axially compress each cartridge by applying a well controlled constant compression force to each commercial LC fitting. The user has the versatility to tailor the separation (e.g. the length of the column, type of stationary phase, and mode of separation) to the experimental design of interest in a cost-effective manner. The source is described in detail, and several experiments are performed to evaluate the robustness of both the system and the exchange of the individual trap and emitter cartridges. The standard deviation in the retention time of four targeted peptides from a standard digest interlaced with a soluble Caenorhabditis elegans lysate ranged between 3.1 and 5.3 s over 3 days of analyses. Exchange of the emitter cartridge was found to have an insignificant effect on the abundance of various peptides. In addition, the trap cartridge can be replaced with minimal effects on retention time (<20 s).