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Handbook of Size Exclusion Chromatography and Related ...

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(exclusion) <strong>and</strong> enthalpic (interaction) retention mechanisms. The nonselective<br />

SEC retention <strong>of</strong> complex polymers concerning their three different molecular<br />

characteristics was described in the preceding section. Therefore, the second<br />

column,evenifselectiveenoughtoseparatepolymerspeciesmainlyorexclusively<br />

according to their composition, could not provide aset <strong>of</strong> information needed for<br />

fullcopolymercharacterizationbecauseeachfractionfromthesecondcolumnstill<br />

contained macromolecules with different molar masses.<br />

Concerning separation selectivity,we can define an important condition for<br />

asuccessful 2D-HPLC <strong>of</strong> complex polymers; namely different selectivities <strong>of</strong><br />

separation systems, <strong>and</strong> especially <strong>of</strong> the first dimension separation system C#1,<br />

towardone<strong>of</strong>themolecularcharacteristicstobedetermined.Thiscanbeachieved<br />

by:<br />

1. Full or at least substantial suppression <strong>of</strong> sample separation according<br />

toonemolecularcharacteristicwhileselectivity<strong>of</strong>separationaccording<br />

to the second characteristic remains essentially unchanged.<br />

2. Considerable enhancement <strong>of</strong> separation selectivity according to one<br />

molecularcharacteristicsothatitfairlyexceedsselectivity<strong>of</strong>separation<br />

according to the second characteristic.<br />

3. Suppression <strong>of</strong> separation according to one characteristic <strong>and</strong><br />

enhancement <strong>of</strong> separation according to another characteristic.<br />

Evidently, the latter, ideal case is difficult to reach. Most liquid<br />

chromatographic approaches directed to these goals are based on the controlled<br />

combinations, coupling, <strong>of</strong> various HPLC retention mechanisms within the same<br />

column (Sec. 5).<br />

Further features <strong>of</strong> combinations <strong>of</strong> various chromatographic methods<br />

include sample dilution <strong>and</strong> detectability.The latter aspects <strong>of</strong> two-dimensional<br />

separations together with efficiencies <strong>of</strong> both 2D partner procedures were<br />

theoretically analysed by Schure (8). In this broad discussion, he included gas<br />

chromatography, field flow fractionation, eluent gradient HPLC, SEC, <strong>and</strong><br />

capillary electrophoresis. The binary combinations <strong>of</strong> the latter three methods<br />

should give the best results.<br />

Sample dilution represents an important practical problem in 2D-HPLC.<br />

Fractions leaving the first dimension separation system C#1 (Fig. 3) may be too<br />

diluted to allow their quantitative detection. In this case, the reconcentration step<br />

must be introduced into the 2D-HPLC chromatograph. The corresponding system<br />

is denoted RSR in Fig. 3, where the first R st<strong>and</strong>s for “reconcentration.” If<br />

necessary, the RSR system should allow also for storage <strong>of</strong> fractions from system<br />

C#1 <strong>and</strong>/or sample solvent <strong>and</strong> mobile phase exchange in the second dimension<br />

system C#2. RSR enables direct forwarding <strong>of</strong> effluent from the column C#1 into<br />

the (set <strong>of</strong>) detector(s) for independent monitoring <strong>of</strong> sample concentration/<br />

© 2004 by Marcel Dekker, Inc.

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