Chip HPLC

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. 

An H-filter was constructed to remove blood cells (see Figure 5.19). Subsequent off-chip HPLC analysis was conducted to determine the amount of an antibiotic, cephalosporin cephradine (0.2-100 pg/mL), in blood [596]. 

High-temperature HPLC (methanol at 120 °C, retention times in the range of seconds) and low temperature HPLC (5 to 10 °C), which can be used to analyze solute configurations, are techniques with practical advantages. However, they will for the time being remain in the research laboratories, like even bolder approaches such as the chip HPLC. 

FIGURE 47.4 Photograph of a microsystem fabricated via the multilayered structure route (top) and on-chip HPLC separation of cytochrome c digest using a lauryl methacrylate-based monolithic colnmn (bottom). (Reprinted with permission from Le Gac, S., et al., J. Chromatogr. B, 808, 3, 2(K)4. Copyright 2004 Elsevier B.V.) Conditions sample concentration 800 fmol, mobile phase A, 5% acetonitrile in 0.1% aqueons formic acid, mobile phase B, 95% acetonitrile in 0.1 % aqueous formic acid, gradient 5-50% B in A in 30 min, 50-95% B in A in 1 min, flow-rate 200 nL/min. 

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. 

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/... 

HPLC with microchip electrophoresis. Capillary RPLC was used as the first dimension, and chip CE as the second dimension to perform fast sample transfers and separations. A valve-free gating interface was devised simply by inserting the outlet end of LC column into the cross-channel on a specially designed chip. Laser-induced fluorescence was used for detecting the FITC-labeled peptides of a BSA digest. The capillary HPLC effluents were continuously delivered every 20 s to the chip for CE separation. 

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... 

Kapila et al. [501] used an on-line SFE-LC to determine chlorinated phenols in wood chips over the concentration range 1-500 mg/kg. Following the extraction, the sample was loaded into a sample loop of the HPLC and chromatographed using a conventional packed LC column and UV detector. 

Fig. 5.9 Design of the chip-based enzyme ESI-MS assay. MS instrument Ion-trap mass spectrometer (LCQ Deca, Thermo Electron). I Sample components/inhibitors injected by flow injection or eluting from capillary HPLC column. E Infusion pump delivering the enzyme cathepsin B. S infusion pump delivering the substrate Z-FR-AMC. Micro-chip design Vrije Universiteit Amsterdam. Micro-chip production Micronit Microfluidics BV (Enschede, The Netherlands).

Lab-on-a-chip/high-performance liquid chromatography 2005 LOC/HPLC 215 Collins, 13.4... 

A chip-based nanospray interface between an HPLC and the MS has been introduced by Advion Biosystems (Ithaca, NY). This instrument aligns a specialized pipette tip with a microfabricated nozzle, set in an arrayed pattern on a silicon wafer. The advantage of this interface is that each sample is sprayed through a new nozzle, thus virtually eliminating cross contamination. 

Although this section provides a brief description of most commonly nsed detectors for HPLC, most of the focus is on a few detection modes. Optical absorbance detectors remain the most widely nsed for HPLC, and are discnssed in some detail. We also focns on flnorescence, condnctivity, and electrochemical detection, as these methods were not widely nsed for HPLC in the past, bnt are especially well suited to micro- and nano-flow instrnments becanse of their high sensitivity in small sample volumes. Mass spectrometry has also come into wide and rontine nse in the last decade, but as it is the subject of another chapter, it will not be fnrther discnssed here. Miniaturization has been particularly important for capillary and chip-based electrophoresis, which often employs sub-nanoliter detection volnmes [36,37]. 

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. 

---