Chip HPLC Systems

Agilent Technologies currently offers commercial nanospray HPLC-ChipR and HPLC-Chip CubeR MS and MS/MS systems. Chips come with standard Cis packing or can be custom packed with standard HPLC column materials. They are aimed at proteomic labeling studies, and small molecule separations. 

The advantage of generic LC/MS run conditions is that it allows the preparation of an LC/MS separation database that can be referenced for compound mixtures from anywhere in the development and manufacturing process cycle. It trades off resolution for consistency, speed, and a decrease in methods development times. It permits creation of a computer-searchable database of information for all of the compounds being investigated in the company. The mass spectrometer provides sensitivity and resolution gain as well as information on retention times and molecular weights. 

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Micro-injections in micro-flow and nano-flow systems are done with injectors in which the external sample loop is replaced with the internal fixed volume within the injector body. HPLC-on-a-chip systems also build the column into the injector body. The internal path within the injector body is abladed with a laser, packed with micro-packing material, and this serves as the separating media. The injector inlet is connected to the pumping system and the outlet to the detector. Sample is loaded into an internal loop in the load position, then injected onto the chip HPLC by turning the injector. Obviously, in a system like this sample size is very limited and the detector is usually a highly sensitive mass spectrometer. 

Chip HPLC—Nano-flow, micro-sample HPLC system in which the packed column resides within the body of the injector. Originally touted as the HPLC of the future for nano-level LC/MS, but its potential has been slow to materialize. 

Several modules had to be developed before a functional microfluidic HPLC system could be assembled. In a series of papers, the Sandia National Laboratory group demonstrated high-pressure connectors for introduction of liquids, fabrication of integrated polymer checking microvalve elements, on-chip high-pressure picoliter injector, and eventually a complete HPLC chip. ... 

A typical high-performance liquid chromatography (HPLC) system consists of two pumps, a mixer, an injector, a guard colunm, a separation column, a detector, an electrospray nozzle, and a mass spectrometer. The two pumps and the mixer allow establishment of desired solvent gradients. The injector is used to inject a small amount of sample into the column. In all separation techniques, definition of a small volume of injected sample is cmcial in preventing adverse broadening of peaks and a consequent loss in compmient resolution. The injected compounds are separated in the separation colunm and then detected via a simple detector or a mass spectrometer. All of the parts of the HPLC system have been miniaturized, and several research groups have been able to demonstrate on-chip HPLC separations. 

Another technique that shows potential for use in the EDM assay arena is the nanoelectrospray technology. Nanoelectrospray has been available for many years but has recently become available in a convenient chip-based system that can be used for both quantitative and qualitative applications (Dethy et al., 2003 Ackermann and Dethy, 2005 Wickremsinhe et al., 2005, 2006). The main advantage of this technique is that it is fairly easy to implement (it can be interfaced directly to many triple quadrupole MS systems) and it provides a fast assay time (about 20—30 s/sample). Van Pelt et al. (2003) described the use of the nanoelectrospray system for the analysis of Caco-2 samples. More reeently, Balimane et al. (2005) demonstrated that a chip-based nanoelectrospray system could be used to assay PAMPA samples in a high-throughput maimer. In this report, the samples for 10 test compounds were assayed by HPLC UV or nanoelectrospray and the permeability results from the two assays were found to be similar for this test set. 

The chip micro reactor ([R 6]) was only one part of a complex serial-screening apparatus [20]. This automated system consists of an autosampler (CTC-HTS Pal system) which introduces the reactant solutions in the chip via capillaries. A pumping system (p-HPLC-CEC System) serves for fluid motion by hydro dynamic-driven flow. A dilution system [Jasco PU-15(5)] is used for slug dilution on-chip. The detection system was a Jasco UV-1575 and analysis was carried out by LC/MS (Agilent 1100 series capLC-Waters Micromass ZQ). All components were on-line and self-configured. 

The application of polymer monoliths in 2D separations, however, is very attractive in that polymer-based packing materials can provide a high performance, chemically stable stationary phase, and better recovery of biological molecules, namely proteins and peptides, even in comparison with C18 phases on silica particles with wide mesopores (Tanaka et al., 1990). Microchip fabrication for 2D HPLC has been disclosed in a recent patent, based on polymer monoliths (Corso et al., 2003). This separation system consists of stacked separation blocks, namely, the first block for ion exchange (strong cation exchange) and the second block for reversed-phase separation. This layered separation chip device also contains an electrospray interface microfabricated on chip (a polymer monolith/... 

For reference methods, HPLC with various detectors has become the standard reference technique for analysis of food additives, but new developments in this area are mainly linked to detector technology. Diode array detectors have not totally met the expectations of food analysts in terms of their specificity and LC-MS is likely to fill the gap. Specific detection with biosensor chips may also have a future for certain analyses. The use of combined LC-MS/DAD systems is... 

Elevated temperatures up to 100°C have also been used with integrated HPLC chips that include both a column and an electrochemical detector [83]. This system used a resistive heater and a mobile phase preheater and was used to separate o-phthaldialdehyde amino acid derivatives. 

K. Killeen, H. Yin, D. Sobek, R. Brennen, T.V.D. Goor, Chip-LC/MS HPLC-MS using polymer microfluidics. Presented at Seventh International Conference on Micro Total Analysis Systems (microTAS 2003). Squaw Valley, California, October 5-9, 481 (2003). 

All the ions studied were successfully separated and the chromatogram was obtained with the on-chip conductivity detector (Fig. 6.8). The peaks were sharp due to the small column dimensions and minimized dead volume of the integrated system and separation occurred within 90 seconds. Other important applications of nano-HPLC are summarized in Table 6.1. 

Fig. 15. Schematic layout of the HPLC chip a layout of the channel system, b cross section along the channel axis, c cross section along the detector cell axis. IS sample and mobile phase inlet, S split injector, OS outlet for rejected sample and mobile phase, C separation channel, F frit, D optical detector cell, OD outlet to waste, P positioning grooves for optical fibers (reprinted with permission from [84]. Copyright 1995 John Wiley)...

Reichmuth, D.S., Shepodd, T.J., Kirby, B.J., RP-HPLC microchip separations with subnanoliter on-chip pressure injections. Micro Total Analysis Systems 2003, Proceedings 7th ftTAS Symposium, Squaw Valley, CA, Oct. 5-9, 2003, 1021-1024. 

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