Maximum efficiency protein separations

Resolution is a measure of the relative separation achieved between two chromatographi-cally distinct materials, and maximum resolution is the primary goal of any purification step. This discussion covers the main theoretical parameters that affect ability to resolve components of a protein sample into chromatograph i-cally distinct forms, thereby providing a basis on which to judge chromatographic results. The main parameters affecting resolution are selectivity, efficiency, and capacity. More detailed... 

A plot of the temperatures required for clouding versus surfactant concentration typically exhibits a minimum in the case of nonionic surfactants (or a maximum in the case of zwitterionics) in its coexistence curve, with the temperature and surfactant concentration at which the minimum (or maximum) occurs being referred to as the critical temperature and concentration, respectively. This type of behavior is also exhibited by other nonionic surfactants, that is, nonionic polymers, // - a I k y I s u I Any lalcoh o I s, hydroxymethyl or ethyl celluloses, dimethylalkylphosphine oxides, or, most commonly, alkyl (or aryl) polyoxyethylene ethers. Likewise, certain zwitterionic surfactant solutions can also exhibit critical behavior in which an upper rather than a lower consolute boundary is present. Previously, metal ions (in the form of metal chelate complexes) were extracted and enriched from aqueous media using such a cloud point extraction approach with nonionic surfactants. Extraction efficiencies in excess of 98% for such metal ion extraction techniques were achieved with enrichment factors in the range of 45-200. In addition to metal ion enrichments, this type of micellar cloud point extraction approach has been reported to be useful for the separation of hydrophobic from hydrophilic proteins, both originally present in an aqueous solution, and also for the preconcentration of the former type of proteins. 

With respect to resolving power (theoretical number of plates) and separation time, no doubt, capillary electrophoresis (CE) is the ultimate separation technique for complex peptide samples, and its combination with ESI MS online as well as MALDI MS off-line has been demonstrated many times [222-229]. The main reason why CE-MS, in contrast to nano-LC-MS, has not become a widespread method for protein and peptide analysis is the maximum total sample volume that can be separated by CE. In contrast to nano-LC, where many himdred microhters of dilute sample can be loaded without compromising separation power, the performance of CE directly depends on the sample volume and works best if only 50 nL or less is loaded. Recently, however, it has been reahzed that this requirement of CE is perfectly matched by nano-LC, which provides efficient sample concentration, and that the two techniques can be combined online upfront ESI or MALDI MS. For this purpose, a microfluidic chip was developed that enables, on demand, on-hne transfer (loading) of nano-LC fractions to an orthogonal CE separation channel, the effluent of which is either analyzed online by ESI MS or off-line by MALDI MS [230-232]. 

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