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Handbook of Size Exclusion Chromatography and Related ...

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elution step may be sufficient for sample separation. The one-step approaches are<br />

called also “on-<strong>and</strong>-<strong>of</strong>f procedures” or full retention–elution methods (FRE). The<br />

best known FRE method utilizes an adsorption retention mechanism <strong>and</strong> is also<br />

called the full adsorption–desorption (FAD) method. The present state <strong>of</strong><br />

development <strong>of</strong> the FAD method is described in the recent review (90) <strong>and</strong><br />

therefore only basic ideas will be repeated here <strong>and</strong> some new information about<br />

this powerful approach will be added.<br />

FRE procedures are similar to solid phase extraction, which is well known<br />

in HPLC <strong>of</strong> low molar mass substances. However, a unique ability <strong>of</strong><br />

macromolecules is utilized in FRE, namely to be quantitatively <strong>and</strong> irreversibly<br />

immobilized within a column filling under particular experimental conditions. The<br />

immobilization is so strong that polymer is not eluted by any volume <strong>of</strong> mobile<br />

phase (“infinitive retention volume”). On the other h<strong>and</strong>, a sudden change <strong>of</strong><br />

experimental conditions quantitatively releases either the whole polymer sample<br />

or its particular fraction from the FRE column. Sample immobilization <strong>and</strong> its<br />

controlled release seem to be easiest when the adsorption retention mechanism is<br />

applied. The high affinity adsorption isotherm (Fig. 15) is applicable to many<br />

polymers <strong>of</strong> medium <strong>and</strong> high polarity. FAD procedures work below saturation<br />

onset, that is, at the situation when virtually all macromolecules are attached to the<br />

adsorbent surface.<br />

It was shown that the adsorptive attachment <strong>of</strong> polymer species is a very fast<br />

process (20). The sample residence time within a FAD column is as short as a few<br />

seconds <strong>and</strong> is fully sufficient for trapping virtually all macromolecules within<br />

adsorbent under mobile phase flow (<strong>and</strong> therefore under intensive mixing)<br />

conditions. Similarly, detachment <strong>of</strong> macromolecules is fast <strong>and</strong> quantitative<br />

Figure 15 Typical course <strong>of</strong> a high affinity adsorption isotherm for macromolecules.<br />

© 2004 by Marcel Dekker, Inc.

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