Nano-HPLC

Advances in Capillary and Nano HPLC Technology for Drug Discovery and Development... 

FIGURE 14.12 Fully automated nano HPLC-MS with autosampler injection of large sample volume using two parallel online sample trapping, concentrating, desalting, and filtering columns. Pumps 1 and 2 transfer sample from loop of the autosampler into trap column 1 while pumps 3 and 4 elute sample in trap column 2 into the analytical column then to MS. 

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

Splitting the solvent flow, delivered by the pumps, down to the required flow rate for nano-HPLC is achieved by inserting a variety of devices between the pumps and the injector. While Chervet... 

Martin, S. E., Shabanowitz, J., Hunt, D. R, and Marto, J. A., Subfemtomole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI Eourier transform ion cyclotron resonance mass spectrometry, Analytical Chemistry 72(18), 4266 274, 2000. 

Cappiello, A., Eamiglini, G., Eiorucci, C., Mangani, R, Palma, P, and Siviero, A., Variable-gradient generator for micro- and nano-HPLC, Analytical Chemistry 75(5), 1173-1179, 2003. 

Mitulovic, G., Smolnch, M., Chervet, J. R, Steinmacher, I., Kungl, A., and Mechtler, K., An improved method for tracking and redncing the void volnme in nano HPLC-MS with micro trapping columns. Analytical and Bioanalytical Chemistry 376(7), 946-951, 2003. 

Schmid, D., Behnke, B., Metzger, J., and Kuhn, R., Nano-HPLC-mass spectrometry and MEKC for the analysis of oligosaccharides from human milk, Biomedical Chromatography 16(2), 151-156, 2002. 

Le Bihan, T., Robinson, M. D., Stewart, 1. L, and Figeys, D., Definition and characterization of a trypsinosome from specific peptide characteristics by nano-HPLC-MS/MS and in silico analysis of complex protein mixtures. Journal of Proteome Research 3(6), 1138-1148, 2004. 

Pitchayawasin, S., Isobe, M., Kuse, M., Franz, T., Agata, N., and Ohta, M., Molecular diversity of cereu-lide detected by means of nano-HPLC-ESl-Q-TOF-MS, International Journal of Mass Spectrometry 235(2), 123-129, 2004. 

Wagner, Y, Sickmann, A., Meyer, H. E., and Daum, G., Multidimensional nano-HPLC for analysis of protein complexes. Journal of the American Society for Mass Spectrometry 14(9), 1003-1011, 2003. 

Miniaturization of HPLC-ICP-MS is an important issue in bioanalytical chemistry when small amounts of sample (e.g., single cells) need to be investigated.33 ICP-MS (with an octopole collision cell) in combination with nano-HPLC (75 pun column) was optimized for the detection of selenopeptides in a selenium-yeast protein digest after 100-fold preconcentration on a C18 capillary precolumn (300 (im column for salt removal and cleanup).34 Under identical separation and preconcentration conditions, electrospray MS/MS (using Nanospray qQqToF-MS - QSTAR from Applied... 

With respect to sample preparation, it is necessary to develop effective and fast procedures involving only a few steps in order to avoid contamination, reduce analysis time and to improve the quality of analytical work. Microsampling and the use of smaller sample sizes is required and also the further development of analytical techniques. In particular, there is a need for the development of online and/or hyphenated techniques in ICP-MS. Microsampling combined with the separation of small amounts of analytes will be relevant for several chromatographic techniques (such as the development of micro- and nano-HPLC). There is a demand for further development of the combination of LA-ICP-MS as an element analytical technique with a biomolecular mass spectrometric technique such as MALDI- or ESI-MS for molecular identification and quantification of protein phosphorylation as well as of metal concentrations, this also enables the study of post-translational modifications of proteins, e.g. phosphorylation. 

Alternative approaches to improve chromatography and thereby enhance signal-to-noise ratio include the use of ultrapressure liquid chromatography (UPLC) (Dear et al., 2006), where chromatographic resolution can be dramatically increased (Castro-Perez et al., 2004, 2005 Plumb et al., 2004), or nano-HPLC (Schmidt et al., 2003), which may serve to reduce ion suppression effects. For offline use, the 96-well plate approach (typically the Scintiplate) will normally be used in combination with direct mass spectrometric infusion at conventional flow rates (5-10 xL/min). This approach enables multiple experiments to be performed in order to... 

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. 

Figure 3.1 A complete chip-based nano-HPLC machine.

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]. 

Nanoanalyses relay on the most sensitive, efficient, and reproducible detectors. The art of hyphenation of the detectors with nano-HPLC is the most important and crucial aspect in separation science at the trace level. In spite of the use of many detectors in nano-LC, the sensitivity of detection at the nanolevel is still a challenging job. However, this task may be achieved by using very... 

The recorder also plays an important role in analysis by printing the chromatograms. Nowadays, all nano-HPLC instruments are computer based and data... 

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