Electrophoresis matrix effects

We therefore sought to evaluate reproducibility of shotgun proteomics in studies of archival FFPE tissue. Because FFPE samples are more complex than non-cross-linked samples, we evaluated FFPE human liver for analytical reproducibility and confidence in protein assignments.20 This complexity strengthens the argument for using high-resolution separations to maximize analyte concentration and minimize matrix effects. In this case, we used transient capillary isotachophoresis/capillary zone electrophoresis (cITP/cZE) in place of IEF to help address this effect. cITP/cZE has a resolution superior even to cIEF (90% of identified peptides in 1 fraction, 95% in 2 fractions or less for cITP/cZE, vs. 75% and 80%, respectively, for cIEF). 

The quality of a calibration also depends on the quality of the reference samples used. Glucuronic acid is a particular example the enzymatic method for this analysis is not reliable in that it lacks robustness. This problem has not been identified in the literature and is probably linked to non-identified matrix effects. The first FTIR calibrations were carried out using the enzymatic method as a reference and, even though gluconic acid has highly qualitative absorption characteristics, the results obtained by FTIR were unreliable. Subsequent calibrations carried out using more reliable methods such as capillary electrophoresis as a reference technique have resulted in FTIR analyses of high quality in terms of precision and accuracy. 

Unlike HPLC, sample preparation in CE requires careful thinking and strategy to obtain a good analysis. There is a relationship between the sample matrix and the separation buffer. Based on how the sample is prepared and how the separation buffer is selected, sample matrix effects can be both favorable and detrimental to the analysis. Matrix effects are different in capillary zone electrophoresis (CZE) from those observed in micellar electrokinetic capillary chromatography (MEKC). Understanding sample matrix effects is the first step in sample preparation in order to obtain a good separation by CE [1]. 

Shihabi, Z.K. Sample matrix effects in capillary electrophoresis II. Acetonitrile deproteinization. J. Chromatogr. A, 652, 471, 1993. 

Capillary electrophoresis (CE) has been used to separate a number of arsenic species, but up to now problems with matrix effects and the high detection limits do not permit its application to biological and environmental samples. 

Despite these difficulties (discussed in Section 5.3.3a) API sources are currently used widely in combination with a wide range of liquid chromatography methods, i.e. normal and reverse phase, isocratic and gradient, normal bore (4.6 mm i.d.) and capillary columns, as well as ESI with capillary electrophoresis and APCI with GC. In the context of trace level quantitation, API techniques are most often used in combination with reverse phase HPLC, and it is this combination that will be the main focus of discussions of matrix effects (Sections 5.1.1 and 5.3.6a) and of the more practical aspects in Sections 9.6 and 10.10.4.1d. In this regard it is worth noting here that in reverse phase chromatography using e.g., a Cjg-derivatized silica... 

Garcia LL, Shihabi ZK (1993) Sample matrix effects in capillary electrophoresis I. Basic considerations. J Chromatrography A 652(2) 465-469... 

Other separation techniques, such as capillary electrophoresis (CB) and supercritical fluid chromatography (SBC), have been shown to perform well for the separation of phenols. Several papers describing the use of CB for the separation of phenolic compoimds in water samples have been published lately [97-100]. The majority of these employ UV detection, but BSP-MS in the negative-ion mode [101] and indirect fluorescence detection [102] have also been used. In one study, a comparison between HPLC and CB was performed to assess their suitability for the determination of the 11 priority pollutants in water [16]. The authors claim that CB gave a shorter analysis time and smaller matrix effects. However, it was not possible to achieve the desired detection limits without a preconcentration on solid-phase material. 

In order to illustrate the effects of media structure on diffusive transport, several simple cases will be given here. These cases are also of interest for comparison to the more complex theories developed more recently and will help in illustrating the effects of media on electrophoresis. Consider the media shown in Figure 18, where a two-phase system contains uniform pores imbedded in a matrix of nonporous material. Solution of the one-dimensional point species continuity equation for transport in the pore, i.e., a phase, for the case where the external boundaries are at fixed concentration, Ci and Cn, gives an expression for total average flux... 

Satow, T., Machida, A., Funakushi, K., and Palmieri, R., Effects of the sample matrix on the separation of peptides by high performance capillary electrophoresis, HRC CC, 14, 276, 1991. 

Despite some refinements in the methods, the basic principles and protocols of gel electrophoresis have not changed appreciably since their introduction. Proteins are introduced into a gel matrix and separated by the combined effects of an electrical field, buffer ions, and the gel itself, which acts as a protein sieve. At the completion of the electrophoresis run, separated proteins in the gel are stained to make them visible, then analyzed qualitatively or quantitatively. The topic has been covered in numerous texts, methods articles, and reviews.1-11 In addition, apparatus and reagents for analytical and preparative gel electrophoresis are available from several suppliers. 

In standard FAB, the surface of the matrix solution is depleted of analyte and suffers from radiational damage during elongated measurements. Refreshment of the surface proceeds by diffusion (limited by the viscosity of the matrix) or evaporation. Continuous-flow fast atom bombardment (CF-FAB) continuously refreshes the surface exposed to the atom beam. [107,108] The same effect is obtained in slightly different way by the frit-fast atom bombardment (frit-FAB) technique. [109,110] In addition, both CF-FAB and frit-FAB can be used for online-coupling of liquid chromatography (LC, Chap. 12) [111] or capillary electrophoresis (CE) to a FAB ion source. [112]... 

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