Elution profile size exclusion chromatography

Analytical size exclusion chromatography was performed on an Akta Purifier (Pharmacia) using a 24-ml Superose 6 HR 10/30 column (Pharmacia). 250-pl samples in AP-buffer (20mM Tris buffer, pH 8.0, 100 mM NaCl), with or without 0.1 % P-DM, were injected onto the column equilibrated with the same buffer and eluted at a flow rate of 0.3 ml min 1. Elution profiles were recorded at 220, 280 and 674 nm. 

Size exclusion chromatography (SEC) polymer elution profiles yield information regarding the molecular size distributions of polydisperse macromolecules. Polymer molecular weight distribution (MWD) represents an intrinsic property which provides direct correlation with many end-use physical properties and a universal criterion for polymer characterization (1). In order to convert elution profiles or chromatograms into MWD information proper calibration methods are required. SEC molecular weight calibration techniques represent experimental approaches for transformation of polymer elution profiles into MWD information and are dependent upon instrumentation, columns, and the polymer/solvent system under study. 

A more sensitive viscometer than the drop-time glass capillary method is also needed in size exclusion chromatography (SEC) such as the gel permeation chromatographic (GPC) analysis of polymer molecular weight distribution (MWD). In an SEC system, a concentration detector is commonly used for providing the weight concentration profile of the polymer elution curve. 

The data in the upper panel of Figure 9 show the elution profile obtained from the separation of acid extracts of uncooked, cooked, and cooked/stored meat by size exclusion chromatography. Uncooked meat has the greatest... 

Recovery, Purity and Amino Acid Composition of CMP. The elution profiles of the CMP powders obtained on size exclusion chromatography 23) are shown in Figure 6. Both CMP isolated from WPC and whey did not contain major... 

It has been shown that the major part of Ca and Mg are found in the soluble fraction (whey) of human milk, whereas only small amounts are present in insoluble caseins or in fat [11-13]. Calcium and Mg speciation studies in milk whey using size exclusion chromatography (SEC) combined with inductively coupled plasma atomic emission spectrometry (ICP-AES) [14] indicated that they were preferably associated with the milk nonprotein fraction. A further work [15] showed similar chromatographic profiles by SEC hyphenated with inductively coupled plasma mass spectrometry (ICP-MS), with Ca and Mg eluting in the lowmolecular-weight region (LMW) (<1.4 kDa). Thus, there seem to be weak associations, if any, of Ca2+ or Mg2+ with higher molecular mass biocompounds of milk. 

Figure 9 - Elution Profiles from Size Exclusion Chromatography-Studies of Pectins...

MW of 33-34 kDa. This concurs with an apparent MW of 35 kDa from the elution profile during size exclusion chromatography (not shown). The purest fractions containing protease activity are contaminated by a minor component of 40 kDa, but this does not co-elute with protease activity. A gel showing purification of the protease from pea leaves is shown in Fig. 2. In this case there are several bands in the purest preparation, none of which can be readily identified as the protease. The apparent MW of the pea enzyme from size exclusion chromatography was also 30-35 kDa. The spinach enzyme has a MW of 34 kDa as judged from size exclusion chromatography (4). 

Mlynar, J. Sarkenen, S. Renaissance in ultracentrifugal sedimentation equihbrium cahbrations of size exclusion chromatographic elution profiles. In Strategies in Size Exclusion Chromatography, Potschka, M. Dubin, P.L., Eds. ACS Symposium Series 635 ACS Washington, DC, 1996 379-400. 

Partially hydrolyzed poly(vinyl alcohol) polymer samples were separated using size exclusion chromatography and then characterized with respect to their degree of hydrolysis on a PLRPS column (ELSD, drift tube T = 100°C, N2 nebulizer gas 12.5L/min) using an 8-min 98/2 30/70 water/THF gradient [814]. Elution times and profiles were clearly different for each collected ftaction and plots of percent hydrolysis versus retention time were presented. 

The primary information obtained from SEC is not the molar mass, but the apparent concentration at an elution volume. Only with the matching SEC calibration and the concentration profile from the concentration detector can the molar mass average and the MMD be obtained. Size exclusion chromatography is therefore a relative method if no absolute detection is employed [1]. The SEC calibration is based on assigning a molar mass to an elution volume (calibration of x-axis). This is in contrast to HPLC, where the detector response (signal intensity and peak area) is calibrated and assigned to a concentration (calibration of y-axis). 

Mlynar, J., and Sarkanen, S., 1996, Renaissance in Ultracentrifugal Sedimentation Equilibrium Calibrations of Size Exclusion Chromatographic Elution Profiles. In Strategies in Size Exclusion Chromatography (M. Potschka and P. L. Dubin, eds.), ACS Symposium Series No. 635, American Chemical Society, Washington, D.C., pp. 379-400. 

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