Capillary HPLC separation

FIGURE 4.14 Capillary HPLC separation of anhydrolutein I isomers on a C30 phase. (From Hentschel, P. et al., J. Chromatogr. A, 285, 2006. With permission.)... 

The capillary HPLC separation from a selected protein spot provides a base-peak profile shown in Figure 6.2A. The base-peak profile is similar to a total ion current (TIC) profile, but it contains only the most abundant mass spectral peak in each scan. The chromatogram is simplified and the contributions from background ion abundances are eliminated, resulting in an enhanced signal-to-ion ratio for an improved visualization of data. The molecular mass for each component is labeled along with corresponding amino acid residues. This format provides a comprehensive approach for peak selection and peptide identification. 

Figure 6.2 Identification of proteins separated by 2-DGE. (A) Base-peak profile from the capillary HPLC separation from a selected protein spot. (.B) Mass spectrum of the second major chromatographic peak (residue 60-70). (C) LC/MS/MS product ion spectrum of m/z 451.3. (Reprinted with permission from Amott et al., 1995. Copyright 1995 American Chemical Society.)...

Capillary Electrochromatography Another approach to separating neutral species is capillary electrochromatography (CEC). In this technique the capillary tubing is packed with 1.5-3-pm silica particles coated with a bonded, nonpolar stationary phase. Neutral species separate based on their ability to partition between the stationary phase and the buffer solution (which, due to electroosmotic flow, is the mobile phase). Separations are similar to the analogous HPLC separation, but without the need for high-pressure pumps, furthermore, efficiency in CEC is better than in HPLC, with shorter analysis times. 

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. 

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). 

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. 

Capillary electrophoresis separates by differences in charge while reversed-phase HPLC separates on the basis of hydrophobicity. It turns out that these proteins have wide differences in hydrophobicity. 

HPLC separation was carried out by using a 2.1 x 100 mm capillary column with a stationary phase of ODS-AQ Cig. A flow rate of 200. ilmin 1 was employed with a linear gradient from 0 to 35% solvent B over 40 min and then to 100% B over 10 min. (Solvent A was 5% aqueous acetonitrile containing 0.05% trifluoroacetic acid (TFA), while solvent B was 80% aqueous acetonitrile containing 0.05% TFA.)... 

Huber s group recently prepared poly(styrene-co-divinylbenzene) monolithic columns in the capillary format using tetrahydrofuran/decanol mixtures as poro-gen. These columns were tested for the HPLC separation of protein digests followed by ESI MS detection enabling protein identification [129]. This technique represents an important contribution to the currently emerging techniques for studying of proteomes as it is more convenient and accurate to use than the classical 2-D gel electrophoresis. 

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... 

With capillary electrophoresis (CE), another modern primarily analytically oriented separation methodology has recently found its way into routine and research laboratories of the pharmaceutical industries. As the most beneficial characteristics over HPLC separations the extremely high efficiency leading to enhanced peak capacities and often better detectability of minor impurities, complementary selectivity profiles to HPLC due to a different separation mechanism as well as the capability to perform separations faster than by HPLC are frequently encountered as the most prominent advantages. On the negative side, there have to be mentioned detection sensitivity limitations due to the short path length of on-capillary UV detection, less robust methods, and occasionally problems with run-to-run repeatability. Nevertheless, CE assays have now been adopted by industrial labs as well and this holds in particular for enantiomer separations of chiral pharmaceuticals. While native cyclodextrins and their derivatives, respectively, are commonly employed as chiral additives to the BGEs to create mobility differences for the distinct enantiomers in the electric field, it could be demonstrated that cinchona alkaloids [128-130] and in particular their derivatives are applicable selectors for CE enantiomer separation of chiral acids [19,66,119,131-136]. 

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]. 

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. 

The temperature stability of monolithic stationary phases based on alkyl methacrylate monomers in capillary HPLC has also been reported [103]. These columns allowed the separation time to be reduced by over 10-fold at temperatures up to 80°C. The upper-temperature limit for these columns was not reported. 

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