Chromatographic performance of microfluidic liquid chromatography devices: Experimental evaluation of straight versus serpentine packed channels.

We prepared a series of planar titanium microfluidic (µLC) columns, each 100 mm long, with 0.15, 0.3 and 0.5 mm i.d.'s. The microfluidic columns were packed with 1.8 µm C18 sorbent and tested under isocratic and gradient conditions. The efficiency and peak capacity of these devices were monitored using a micro LC instrument with minimal extra column dispersion. Columns with serpentine channels were shown to perform worse than those with straight channels. The loss of efficiency and peak capacity was more prominent for wider i.d. columns, presumably due to on-column band broadening imparted by the so-called "race-track" effect. The loss of chromatographic performance was partially mitigated by tapering the turns (reduction in i.d. through the curved region). While good performance was obtained for 0.15 mm i.d. devices even without turn tapering, the performance of 0.3 mm i.d. columns could be brought on par with capillary LC devices by tapering down to 2/3 of the nominal channel width in the turn regions. The loss of performance was not fully compensated for in 0.5 mm devices even when tapering was employed; 30% loss in efficiency and 10% loss in peak capacity was observed. The experimental data for various devices were compared using the expected theoretical relationship between peak capacity Pc and efficiency N; (Pc-1) = N0.5 × const. While straight µLC columns showed the expected behavior, the devices with serpentine channels did not adhere to the plot. The results suggest that the loss of efficiency due to the turns is more pronounced than the corresponding loss of peak capacity.

Subzero Celsius separations in three-zone temperature controlled hydrogen deuterium exchange mass spectrometry.

Hydrogen deuterium exchange mass spectrometry (HDX MS) reports on the conformational landscape of proteins by monitoring the exchange between backbone amide hydrogen atoms and deuterium in the solvent. To maintain the label for analysis, quench conditions of low temperature and pH are required during the chromatography step performed after protease digestion but before mass spectrometry. Separation at 0°C is often chosen as this is the temperature where the most deuterium can be recovered without freezing of the typical water and acetonitrile mobile phases. Several recent reports of separations at subzero Celsius emphasize the promise for retaining more deuterium and using a much longer chromatographic gradient or direct infusion time. Here we present the construction and validation of a modified Waters nanoACQUITY HDX manager with a third temperature-controlled zone for peptide separations at subzero temperatures. A new Peltier-cooled door replaces the door of a traditional main cooling chamber and the separations and trapping column are routed through the door housing. To prevent freezing, 35% methanol is introduced post online digestion. No new pumps are required and online digestion is performed as in the past. Subzero separations, using conventional HPLC column geometry of 3µ m particles in a 1×50mm column, did not result in major changes to chromatographic efficiency when lowering the temperature from 0 to -20°C. There were significant increases in deuterium recovery for both model peptides and biologically relevant protein systems. Given the higher levels of deuterium recovery, expanded gradient programs can be used to allow for higher chromatographic peak capacity and therefore the analysis of larger and more complex proteins and systems.

Gas-phase hydrogen/deuterium exchange in a traveling wave ion guide for the examination of protein conformations.

Accumulating evidence suggests that solution-phase conformations of small globular proteins and large molecular protein assemblies can be preserved for milliseconds after electrospray ionization. Thus, the study of proteins in the gas phase on this time scale is highly desirable. Here we demonstrate that a traveling wave ion guide (TWIG) of a Synapt mass spectrometer offers a highly suitable environment for rapid and efficient gas-phase hydrogen/deuterium exchange (HDX). Gaseous ND(3) was introduced into either the source TWIG or the TWIG located just after the ion mobility cell, such that ions underwent HDX as they passed through the ND(3) on the way to the time-of-flight analyzer. The extent of deuterium labeling could be controlled by varying the quantity of ND(3) or the speed of the traveling wave. The gas-phase HDX of model peptides corresponded to labeling of primarily fast exchanging sites due to the short labeling times (ranging from 0.1 to 10 ms). In addition to peptides, gas-phase HDX of ubiquitin, cytochrome c, lysozyme, and apomyoglobin were examined. We conclude that HDX of protein ions in a TWIG is highly sensitive to protein conformation, enables the detection of conformers present on submilliseconds time scales, and can readily be combined with ion mobility spectrometry.

High-speed and high-resolution UPLC separation at zero degrees Celsius.

The conformational properties of proteins can be probed with hydrogen/deuterium exchange mass spectrometry (HXMS). In order to maintain the deuterium label during LC/MS analyses, chromatographic separation must be done rapidly (usually in under 8-10 min) and at 0 degrees C. Traditional RP-HPLC with approximately 3-mum particles has shown generally poor chromatographic performance under these conditions and thereby has been prohibitive for HXMS analyses of larger proteins and many protein complexes. Ultraperformance liquid chromatography (UPLC) employs particles smaller than 2 mum in diameter to achieve superior resolution, speed, and sensitivity as compared to HPLC. UPLC has previously been shown to be compatible with the fast separation and low temperature requirements of HXMS. Here we present construction and validation of a custom UPLC system for HXMS. The system is based on the Waters nanoACQUITY platform and contains a Peltier-cooled module that houses the injection and switching valves, online pepsin digestion column, and C-18 analytical separation column. Single proteins in excess of 95 kDa and a four-protein mixture in excess of 250 kDa have been used to validate the performance of this new system. Near-baseline resolution was achieved in 6-min separations at 0 degrees C and displayed a median chromatographic peak width of approximately 2.7 s at half-height. Deuterium recovery was similar to that obtained using a conventional HPLC and ice bath. This new system represents a significant advancement in HXMS technology that is expected to make the technique more accessible and mainstream in the near future.

Development of a conductivity-based photothermal absorbance detector for capillary separations.

A contactless conductivity-based absorbance detector has been developed for use with capillary separations. Detection is based on a photothermal process. As analytes pass through the detector they absorb light, producing a thermal perturbation. This thermal event results in a change in the solution conductivity. The measured change in conductivity is directly related to the absorption of light. The major advantage to this type of detector is that the measured absorbance is, to a first approximation, independent of optical path length, allowing small-diameter capillaries to be used. This approach combines the optical simplicity of traditional transmission-based instruments with the path length independence of similar refraction-based photothermal detectors. In addition to the initial development and characterization of the photothermal absorbance detector, multiphysical modeling of the heat transfer within the conductivity cell was performed.

Expanded electrical model of a contactless conductivity detector: development and verification.

A theoretical model of the contactless conductivity detector (CCD) has been developed consisting of a network of resistors and capacitors. The output of the model is compared to experimental results and to the output of a simpler model. Experimentally, a lock-in amplifier is added to the detection scheme of the contactless conductivity detector to provide a more sensitive method of signal isolation. The detector is assembled on a printed circuit board with the electrodes in a co-axial configuration. The electrodes are chosen to allow for use with fused silica capillaries in capillary electrophoresis. The use of a lock-in amplifier in place of a previous rectification/filtering circuit allows for an approximate 10-fold improvement in S/N. The detector shows a linear response to changes in excitation voltage and to changes in analyte concentration. Mass limits of detection of 60, 63, and 50 fg are determined for the inorganic cations potassium, sodium, and lithium, respectively (for a signal three times the level of the rms noise).