High nano-HPLC

Introduces microparallel liquid chromatography (LC), ADME/PK high-throughput assay, MS-based proteomics, and the advances of capillary and nano-HPLC technology... 

Micro- and nano-HPLC systems (Fig. 15.11) rely on small-diameter and capillary columns packed with high-efficiency packing materials and very slow flow rates to produce concentrated solutions and sharp chromatography peaks to feed electrospray interfaces for mass spectrometers. 

Basically, no special devices have been developed to handle mobile phases in nano-HPLC. However, our experience dictates that reservoirs small in volume and of high quality glass are preferred for this purpose. Solvent containers should be air tight and free from any contamination. The use of helium gas, through a sparging device, may be beneficial for degasification of solvents in nano-HPLC, which can improve check valve reliabilities, especially at nano flow, and diminish baseline noise in UV detection. Besides, each reservoir should be equipped with a shutoff valve for efficient helium consumption [9]. 

MHC-Associated Peptide Proteomics (MAPPs). MAPPs is a comparably novel technology that allows direct sequence analysis of potential T cell epitopes and integrates three steps (Kropshofer and Spindeldreher, 2005) (i) coculture of human DCs and therapeutic protein in vitro, mimicking uptake and processing of biotherapeutics in vivo (ii) extraction of HLA-peptide complexes and peptide elution and (iii) high-throughput sequence analysis of DC-associated peptide epitopes by a combination of nano HPLC and mass spectrometry. 

Solving this problem will be an important step forward as speciation moves into new fields of application where highly precise isotope ratios are required, be it for single-shot laser ablation, CE, GC or nano-HPLC methods. Future research will be focused on these techniques. 

The speed of analysis in HPLC is a potential bottleneck for complex sample analysis. Various approaches such as utilizing short columns at high flow rates and the recent focus on 1.5 to 2 /an particles have been reported to increase the speed of analysis. Multidimensional chromatographic approaches have also been demonstrated to increase the throughput of HPLC. The five major parameters that may affect the speed of capillary and nano LC are discussed below. 

The development of microbore chromatography and nano-flow HPLC has greatly increased interest in detectors suitable for sub-microliter peak volumes. These include LIE, EC, and MS spectrometry detectors. Zare first described the application of lasers for detection in analytical chemistry in 1984, outlining the potential for the use of lasers in analytical science, especially their application to HPLC [20]. This was followed in 1988 by another review that focused more on HPLC applications and provided examples of the high sensitivity possible with LIE detection [21], Another good review was written by Rahavendran and Karnes outlining the usefulness of LIE in pharmaceutical analysis [22],... 

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]. 

Planar chromatography, also known as Thin Layer Chromatography (TLC), is a technique related to HPLC but with its own specificity. Although these two techniques are different experimentally, the principle of separation and the nature of the phases are the same. Due to the reproducibility of the films and concentration measurements. TLC is now a quantitative method of analysis that can be conducted on actual instruments. The development of automatic applicators and densitometers has lead to nano-TLC, a simple to use technique with a high capacity. 

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. 

In addition to HPLC, microchips have also been used in other modalities of liquid chromatography, including capillary electrochromatography and mi cel -lar electrokinetic chromatography. Many workers have attempted to achieve nano separations at high speed of different molecules with high efficiency, reproducibility, and low detection limits. The state of the art of separation in these modalities is discussed in this chapter, with special emphasis on their applications, optimization, and mechanisms of separation. 

Because of their well-recognized physicochemical properties (relatively high hydrophobicity, cationic character, and short length) reversed-phase, size-exclusion, and cation exchange chromatographies are particularly appropriate to purify bioactive peptides from the immune system of invertebrates. The sensitivity of HPLC, MS, Edman degradation, and liquid growth inhibition assays allow one to use from narrow (e.g., 2.1-mm internal diameter) down to micro-or nano-columns. 

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