Capillary HPLC

Although the OTHdC has several unique applications in polymer analysis, this technique has several limitations. First, it requires the instrumentation of capillary HPLC, especially the injector and detector, which is not as popular as packed column chromatography at this time. Second, as discussed previously, the separation range of a uniform capillary column is rather narrow. Third, it is difficult to couple capillary columns with different sizes together as SEC columns. 

Pusecker K, J Schewitz, P Gfrorer, L-H Tseng, K Albert, E Bayer (1998) On-line coupling of capillary electrochromatography, capillary electrophoresis, and capillary HPLC with nuclear magnetic resonance spectroscopy. Anal Chem 70 3280-3285. 

Microbore and packed capillary HPLC column technology has not yet met the requirements for breakthrough of new technologies [554]. On commercial instruments in general efficiency and detectability with microbore columns are lower than with normal-bore columns. Microbore and capillary HPLC suffer from... 

The ionspray (ISP, or pneumatically assisted electrospray) LC-MS interface offers all the benefits of electrospray ionisation with the additional advantages of accommodating a wide liquid flow range (up to 1 rnl.rnin ) and improved ion current stability [536]. In most LC-MS applications, one aims at introducing the highest possible flow-rate to the interface. While early ESI interfaces show best performance at 5-l() iLrnin, ion-spray interfaces are optimised for flow-rates between 50 and 200 xLmin 1. A gradient capillary HPLC system (320 xm i.d., 3-5 xLmin 1) is ideally suited for direct coupling to an electrospray mass spectrometer [537]. In sample-limited cases, nano-ISP interfaces are applied which can efficiently be operated at sub-p,Lmin 1 flow-rates [538,539]. These flow-rates are directly compatible with micro- and capillary HPLC systems, and with other separation techniques (CE, CEC). 

Compatibility with capillary HPLC, RPLC and NPLC... 

Capillary HPLC (usually SEC or LCCC), coupled semi off-line with MALDI-ToFMS for oligomer and end-group characterisation, may be operated using collection modules precoated with matrix material. 

Capillary HPLC-MS has been reported as a confirmatory tool for the analysis of synthetic dyes [585], but has not been considered as a general means for structural information (degradant identification, structural elucidation or unequivocal confirmation) positive identification of minor components (trace component MW, degradation products and by-products, structural information, thermolabile components) or identification of degradation components (MW even at 0.01 % level, simultaneous mass and retention time data, more specific and much higher resolution than PDA). Successful application of LC-MS for additive verification purposes relies heavily and depends greatly on the quality of a MS library. Meanwhile, MB, DLI, CF-FAB, and TSP interfaces belong to history [440]. 

Hieda et al. determined theophylline, theobromine, and caffeine in human plasma and urine by gradient capillary HPLC with frit fast atom bombardment (FAB) mass spectrometry with 7-ethyl theophylline as the internal standard.64... 

The instrumental setup for capillary HPLC-NMR coupling is shown in Figure 4.6. The capillary pump is connected via 50 pm capillaries between the capillary HPLC pump, the UV detector, and the NMR flow probe. 

FIGURE 4.6 Instrumental setup for capillary HPLC-NMR coupling. (From Hentschel, P. et al., J. Chromatogr. A, 285, 2006. With permission.)... 

Krucker, M., Lienau, A., Putzbach, K., Grynbaum, M. D., Schuler, P., and Albert, K. 2004. Hyphenation of capillary HPLC to microcoil 11 NMR spectroscopy for the determination of tocopherol homologues. Anal. Chem. 76 2623-2628. 

Svec, F. (2004b). Organic polymer monoliths as separation phases for capillary HPLC. J. Sep. Sci. 27, 1419-1430. 

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. 

Syringe pumps driven by screw mechanisms were popular in the 1960s because of their inherent precision and pulseless flow characteristics. Their disadvantages are higher manufacturing costs and the problems associated with syringe refill cycles. Syringe pumps are currently used in specialized systems for microbore and capillary HPLC. 

The application of the improved MS techniques presented above with highly resolving separation methods, such as 2-D electrophoresis, capillary HPLC, and CE, resulted in the creation of a new science, proteomics63 While genomics, described by DNA databases, represents the ground stage of the cell, the study of the differential status of the cell, due to various stimuli or disease states, reflects the functional expression of protein products or proteomics. Proteomics studies are aimed at identifying the proteome, the network of proteins that define the... 

Utilization of capillary columns in conjunction with micro ESI devices is becoming a new trend in the field of LC/MS. Capillary HPLC has become a particularly important technique in situations where the supply of analyte is limited, such as in proteomic analysis. According to studies conducted by Smith et al., only one in a hundred thousand of analyte molecules present in solution eventually reach mass detection in a conventional ESI interface. Smith et al. attributed this poor electrospray... 

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).

The peptides generated by proteolysis are separated using reverse-phase HPLC to minimize mass overlap and ionization suppression caused by ion competition in the electrospray source [40]. The optimized LC gradient parameters efficiently separate peptides while minimizing loss of deuterium through back exchange with solvent. Increased sensitivity can be achieved by using capillary HPLC columns and nanoelectrospray methods [47]. 

Encinar, J. R., Schaumloffel, D., Ogra, Y., and Lobinski, R., Determination of selenomethionine and selenocysteine in human serum using speciated isotope dilution-capillary HPLC-inductively coupled plasma collision cell mass spectrometry, Analytical Chemistry 76(22), 6635-6642, 2004. 

Ogra, Y. and Suzuki, K. T., Speciation of selenocompounds by capillary HPLC coupled with ICP-MS using multi-mode gel filtration columns. Journal of Analytical Atomic Spectrometry 20(1), 35-39, 2005. 

Polatajko, A., Encinar, J. R., Schaumloffel, D., and Szpunar, J., Quantification of a selenium-containing protein in yeast extract via an accurate determination of a tryptic peptide by species-specific isotope dilution capillary HPLC-ICP MS, Chemia Analityczna 50(1), 265-278, 2005. 

Ballihaut, G., Tastet, L., Pecheyran, C., Bouyssiere, B., Donard, O., Grimaud, R., and Lobinski, R., Biosynthesis, purification and analysis of selenomethionyl calmodulin by gel electrophoresis-laser ablation-ICP-MS and capillary HPLC-ICP-MS peptide mapping following in-gel tryptic digestion. Journal of Analytical Atomic Spectrometry 20(6), 493 99, 2005. 

Dugo, R, Favoino, O., Presti, M. L., Luppino, R., Dugo, G., and Mondello, L., Determination of antho-cyanins and related components in red wines by micro- and capillary HPLC, Journal of Separation Science 27(17-18), 1458-1466, 2004. 

Plematl, A., Demelbauer, U. M., Josic, D., and Rizzi, A., Determination of the site-specific and isoform-specific glycosylation in human plasma-derived antithrombin by lEF and capillary HPLC-ESl-MS/MS, Proteomics 5(15), 4025-4033, 2005. 

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