High-Performance SEC

Commercially available packings for high performance SEC of proteins. 

Molecular weight range reported by manufacturer for globular proteins. 

The fractionation range for proteins In the random coll conformation Is approximately one-third of the limiting values for globular proteins. 

The minimum molecular weight ratio determined by Ffannkoch et al. (6) In a direct comparison of high performance SEC columns. 

SynChrom, Inc., Linden, Indiana EM Laboratories, Elmsford, New York 

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Polymer Laboratories employs their own individual design of column hardware, which was developed to give low dispersion, high-performance SEC columns. The components used in the design are specified to very low tolerances dimen-... 

High-performance SEC packings have more modest phase ratios of 0.5 to... 

This limitation is offset by the high efficiencies and rapid analysis times of high-performance SEC. It should be evident that as support pore diameter and pore volume increase, the amount of solid material in the particle will be reduced, compromising the mechanical strength of the support matrix. 

The low molecular diffusion coefficients of proteins and other biopolymers reduces the efficiency of mass transfer and compromises efficiency as flow rate is increased. Therefore, high-performance SEC columns are usually operated at modest flow rates, e.g., 1 ml/min or less. However, operation at very low flow rates is undesirable due to excessive analysis times, loss of efficiency due to axial analyte diffusion, and the risk of poor recovery due to analyte adsorption. 

BARTH Nonsize Exclusion Effects in High-Performance SEC... 

Both the time needed for a separation and the amount of dilution of the sample constituents have been drastically reduced with the introduction of high performance SEC (HPSEC). However, non-size exclusion effects tend to be more severe when chemically bonded phases are used. Fortunately, as the problems are identified and understood new column materials have been developed to replace the older ones. In a few articles (Cassidy, 1981 Drull, 1984) the usefulness of HPSEC for the fractionation of metal che-... 

High-performance SEC columns for nucleic acid separation... 

Unlike silica, polymer-based particles are readily available in smaller pore sizes. Small pore size silicas, such as Merck 40 or Davisil 20, are not commercially available in the 5-10 am particle size range suitable for high-performance SEC. Syloid 63, a food additive produced by W. R. Grace, is an irregular 9 rm particle size silica with 22 A pores and 0.4 ml/g pore volume. Its broad particle size distribution does not make it readily suitable for high-performance SEC of small molecules. 

Although size separation was pioneered by the use of Sephadex (Pharmacia Fine Chemicals) columns in aqueous systems (gel filtration), the development of high-performance SEC for water-soluble low-molecular-mass compounds (see also Chapter 7) has been disappointing. Most columns for aqueous use have been designed for separation of large biochemical molecules such as proteins and peptides. Complex interactions, such as adsorption, or ionic effects which lead to a non-SEC mechanism, may occur. The essential simplicity of SEC is therefore lost. 

Since the elution volumes of polymers on High Performance SEC columns span only a few millilitres, great precision in the flow rate determination Is called for so as to be able to discriminate with a high degree of accuracy between the different peaks eluted. As a way of overcoming this problem, elution data has been given in time units rather than in volume units. ... 

Figure 1. High performance SEC of protein standards In nondenaturing phosphate buffer (A), guanidine hydrochloride (B), and SDS (C).

Figure 2. Calibration curves derived for high performance SEC of proteins using a TSK 3000SW column in phosphate buffer (B guandlne hydrochloride (t - - C), and SDS (A - A - A). K values were determined by Individual analysis of the followfng protein standards as described for Figure 1 phosphorylase a (93,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), trypsin inhibitor (20,100) and a-lactalbumln (14,400), the gtianldlne hydrochlrodle and SDS profiles also display the K values determined for EIAV p26, pl5, and p9 (20). All chromatogfaphy conditions are as described in Figure 1.

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