Resolution capacity, proteins

For best results, use the flow rate, injection volume, and column sample capacity guidelines in Table 3.11. Conditions outside these guidelines may be used, but poor resolution between proteins may result from extensive deviations from these guidelines. 

Carrier ampholyte-based IEF methods are commonly used in situations where very high resolution of proteins according to their pi values is not required. Several problems exist with the use of carrier ampholytes that limit their resolving power. These include the low and uneven ionic strength that results in smearing of the most abundant proteins in the sample, the uneven buffering capacity and conductivity, the unknown chemical environment, a low sample loading capacity, and a... 

The complex structures of proteins, which have different types of groups, loops and bridges, are responsible for the chiral resolution of racemic compounds. Therefore, a small change in the protein molecule results in a drastic change in enantioselectivity. A few reports are available that indicate that different protein structures are responsible for different chiral resolution capacities. One of the advantages of protein-based CSPs is that chiral chromatography is carried out under the reversed phase mode - that is, aqueous mobile phases are frequently used - and, therefore, there is considerable scope for optimizing chiral resolution. 

The effect of temperature on chiral resolution on protein phases is very cmcial, but few reports are available that deal with this effect. The loading capacities of protein CSPs may be understood from a = 2/ 1 and k = ks + k, where kg and correspond to selective and nonselective contributions. When the amount of the analyte on the CSP increases, the relative contribution of to the observed k values (ki and ka) also increases. The critical selective sites become saturated, resulting in a decrease in the separation factors, because k = Ogja, where and am are... 

Although the viscosity of the sample solution may affect the resolution, for practical reasons highly concentrated protein samples will give the best separations in the case of SEC with respect to the process economy. Although the actual loading capacity depends on the separation problem and on the... 

The resolution of these columns for protein mixtures, however, was comparably poor. The peak capacity for human serum albumin was near 3 during 20 min gradient elution. Improvement has been reached by covalent binding of PEI (M = 400-600) onto a 330 A silica of 5 pm particle size [38], The peak capacities of ovalbumin and 2a -arid glycoprotein were 30-40 (tgradienl = 20 min). Enhanced peak capacity and resolution probably were due to the more diffuse structure of PEI coupled to silane moieties than that of strictly adsorbed on silica and cross-linked (see Sect, 2.2). Other applications of covalently adsorbed PEI are discussed in Sect. 4.1. 

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