Size-exclusion chromatography selectivity

Examples of the application of size-exclusion chromatography to the analysis of proteins. The separation in (a) uses a single column that in (b) uses three columns, providing a wider range of size selectivity. (Chromatograms courtesy of Alltech Associates, Inc. Deerfield, IL). 

Conductivity detectors, commonly employed in ion chromatography, can be used to determine ionic materials at levels of parts per million (ppm) or parts per bUHon (ppb) in aqueous mobile phases. The infrared (ir) detector is one that may be used in either nonselective or selective detection. Its most common use has been as a detector in size-exclusion chromatography, although it is not limited to sec. The detector is limited to use in systems in which the mobile phase is transparent to the ir wavelength being monitored. It is possible to obtain complete spectra, much as in some gc-ir experiments, if the flow is not very high or can be stopped momentarily. 

In size exclusion chromatography, the mobile phase must be selected to totally solubilize the sample and eliminate all interactions of the solutes with the... 

To select a column for a particular analytical problem, the first step is to make a choice about the pore size(s) to be used for the separation. In general, one cannot expect that a single pore size will fulfill the needs of a separation. In size exclusion chromatography, it is more common that columns of different types are combined with each other to deliver the separation range needed for a particular analysis. Therefore, column banks with different pore sizes are frequently combined with each other to maximize the separation power for... 

Most size exclusion chromatography (SEC) practitioners select their columns primarily to cover the molar mass area of interest and to ensure compatibility with the mobile phase(s) applied. A further parameter to judge is the column efficiency expressed, e.g., by the theoretical plate count or related values, which are measured by appropriate low molar mass probes. It follows the apparent linearity of the calibration dependence and the attainable selectivity of separation the latter parameter is in turn connected with the width of the molar mass range covered by the column and depends on both the pore size distribution and the pore volume of the packing material. Other important column parameters are the column production repeatability, availability, and price. Unfortunately, the interactive properties of SEC columns are often overlooked. 

This chapter makes no distinction between gel-permeation chromatography (GPC) and size exclusion chromatography (SEC). We make mention of specific analysis conditions wherever possible. We have attempted to include a variety of conditions but by no means should this chapter be considered a comprehensive review of conditions for analyzing polyacrylates. We have drawn extensively from our own experience in selecting examples. 

The instrumentation of HdC, including a pump, an injector, a column (set), a detector, and a recorder or computer, is very similar to size exclusion chromatography SEC). The essence of this technique is the column. There are two types of HdC columns open microcapillary tubes and a nonporous gel-packed column. This chapter emphasizes column technology and selection and the applications of this technique on the molecular weight analysis of macromolecules. 

A more complicated, but flexible, system has been reported by Blomberg et al. (46). Here, size exclusion chromatography (SEC), normal phase EC (NPLC) and GC were coupled for the characterization of restricted (according to size) and selected (according to polarity) fractions of long residues. The seemingly incompatible separation modes, i.e. SEC and NPLC, are coupled by using an on-line solvent-evaporation step. 

The two techniques differ in that HDC employs a nonporous stationary phase. Separation is affected as a result of particles of different size sampling different velocities in the interstitial spaces. Size exclusion chromatography is accomplished by superimposing a steric selection mechanism which results from the use of a porous bed. The pore sizes may vary over a wide range and the separation occurs as a result of essentially the same processes present in the gel permeation chromatography of macromolecules. 

Figure 4.27 Flow chart for coluwi selection based on sample type (m - molecular weight). PLC precipitation-liquid chromatography SEC = size-exclusion chromatography lEC - ion-exchange chromatography HIC hydrophobic interaction chromatography LSC liquid-solid chromatography RPC - reversed-phase liquid chromatography BPC (polar) bonded-phase chromatography and IPC - ion-pair chromatography.

Bedani, F., Kok, W.Th., Janssen, H.-G. (2006). A theoretical basis for parameter selection and instrument design in comprehensive size-exclusion chromatography x liquid chromatography. J. Chromatogr. A 1133, 126-134. 

It should be noted that discrepancies amidst available data may result from different methods of determination. For example, selected hydrodynamic radii of PAMAM dendrimers of Table 14.1 were obtained by two different experimental methods for generations 1 through 4 by dilute solution viscometry, while for generations 5 through 10 by size exclusion chromatography. 

Several affinity screening methodologies that include MS-based readout and work under protein-excess conditions have been developed in the past decade [1]. Some examples include affinity selection/mass spectrometry (ASMS Abbott Labs [10]), size exclusion chromatography with LC-ESI-MS (see Chapter 2 and 3 [11-19]), the use of coupled or non-coupled pulsed ultra-filtration/mass spectrometry (summarized in this chapter [11, 20-23]), restricted access phase chromatography (see Chapter 5 [24, 25]), capillary electrophoresis [26, 27], target shift mass spectrometry [28], and multitarget affinity/specificity screening (MASS, see Chapter 10 [29, 30]). 

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