Eluant systems

In choosing eluants for SEC of proteins two considerations must be taken into account. First, the eluant must eliminate the adsorption of proteins to the column packing though ionic and/or hydrophobic interactions. As manipulation of the eluant composition to decrease one of these potential interactions may actually enhance the other interaction, a careful balance of ionic strength and polarity are required for optimum SEC of proteins. 

Identical samples of a standard protein mixture containing equal amounts of phosphorylase b (P, 93,000). bovine serum albumin (A, 67,000), ovalbumin (0, 43,000), carbonic anhydrase (C, 30,000), trypsin Inhibitor (T, 20,100) and a-lactalbumln (L, 14,400) were dissolved and chromatographed In the appropriate buffer as described In Montelaro et al. (20). The chromatograms represent fifty pi samples containing 50 pg of the protein mixture analyzed on a Waters TSK 3000SW column In the Indicated eluant system at a flow rate of 0.4 ml/mln, and the absorbance monitored at 280 nm. The void volume (V ) of the column was determined to be at 12 min and the total column volume (V ) at 27 min. 2-ME, mercaptoethanol. 

Ionic strength can be used to affect protein separations by nonideal size exclusion chromatography (8). 

Although the phosphate buffer-NaCl system Is versatile and widely employed for nondenaturing protein SEC, the nonvolatile nature of the salts complicates subsequent concentration or buffer exchange of separated proteins. For these purposes we have employed a O.IM ammonium acetate buffer, pH 5.0, in place of the phosphate-NaCl buffer and observed equal resolution of the protein standards and virtually Identical values for individual proteins (Table 3). However, the ammonium acetate can be removed by lyophlllzatlon, thereby facilitating concentration of minute quantities of protein and/or 

Data adapted from Montelaro et. al. (20), except for the results obtained for ammonium acetate which are unpublished observations. Protein analyses were performed on a TSK 3000SW column equilibrated and eluted with different buffers under otherwise Indentlcal conditions. 

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The eluant systems used in adsorption chromatography are based on nonpolar solvents, commonly hexane, containing a small amount of a polar additive, such as 2-propanol. When the sample is applied, solute molecules with polar functionality will bond to the active sites on the packing they will subsequently be displaced by the polar modifier molecules of the eluant as the chromatogram is developed, and will pass down the column to be re-adsorbed on fresh sites. The ease of displacement of solute molecules will depend on their relative polarities. More polar molecules will be adsorbed more strongly, and hence will elute more slowly from the column. A system as described in Figure 4.1 may be used. It was the type of apparatus first used for chromatographic separations of the kind familiar today. 

In iterative approaches only the three apex isoelutropic solvents are run before data evaluation. On the basis of the CRF values an eluant system is predicted which offers potentially the optimum separation. This approach reduces the number of eluant compositions studied to about four or five. 

Unknown(s) powdered drug preparation, for instance a proprietary analgesic formulation, containing two or more of identified analgesics. Eluants system 1, comprising dichloroethane and acetic acid in the ratio of 120 1 system 2, comprising cyclohexane, dichloroethane and acetic acid in the ratio of 40 60 0.8. 

Table 5 Typical Eluant Systems for Synthetic Water-Soluble Polymers...

Accuracy of protein molecular weight determinations In different eluant systems. 

However, suitable eluant systems are still required to analogously calculate the phase volume ratio in ion chromatography. 

Infra-red Umited sensitivity, and restricted to non-aqueous eluant systems. 

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