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Handbook of Size Exclusion Chromatography and Related ...

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composition/architecture/molar mass in the column C#1 effluent. Thus Sin the<br />

RSRabbreviationdenotes“switching”<strong>and</strong>“storage”becausecolumnC#1effluent<br />

canwaitintheRSRsystemforreinjectionintocolumnC#2.Averyimportantrole<br />

<strong>of</strong> the RSR system is the defined reintroduction (second R) <strong>of</strong> (reconcentrated)<br />

fractions from column C#1 into column C#2 so that retention volumes <strong>of</strong><br />

macromolecules leaving the second dimension separation column can be exactly<br />

identified.<br />

The pecularities <strong>of</strong> sample purification, as well as their reconcentration,<br />

storage, <strong>and</strong> reinjection, <strong>and</strong> also <strong>of</strong> eluent exchange by means <strong>of</strong> RSR systems<br />

will be discussed in Secs 7, 8, <strong>and</strong> 9.<br />

Various experimental arrangements <strong>of</strong> 2D-HPLC <strong>of</strong> polymers are possible<br />

<strong>and</strong> the complexity<strong>of</strong> the particular instrument applied depends on theseparation<br />

problem to be solved. The most simple 2D-HPLC apparatus utilizes two different<br />

columns with just one pump P#1 <strong>and</strong> one (set <strong>of</strong>)detector(s) D#2 while the RSR<br />

system is simplified or fully ab<strong>and</strong>oned. The complicated 2D-HPLC systems<br />

comprise two (systems <strong>of</strong>)columns C#1 <strong>and</strong> C#2, an isocratic pump plus a<br />

complete gradient making device P#1 <strong>and</strong> P#2, further amulticolumn/multivalve<br />

RSR system, <strong>and</strong> two series <strong>of</strong> detectors D#1 <strong>and</strong> D#2 (Sec. 11).<br />

3 RETENTION MECHANISMS IN LIQUID<br />

CHROMATOGRAPHY OF MACROMOLECULES<br />

In any HPLC separation, the retention volume VR <strong>of</strong> an analyte is determined by<br />

the distribution constant K <strong>of</strong> sample molecules between a certain part <strong>of</strong> the<br />

eluent <strong>and</strong> column filling. K is expressed as a ratio <strong>of</strong> sample concentration in the<br />

(quasi) stationary phase CS <strong>and</strong> the free mobile phase CM. The free mobile phase is<br />

situated in the interstitial volume <strong>of</strong> the column packed with particulate material or<br />

in the flow-through channels <strong>of</strong> the monolithic column. The volume <strong>of</strong> the SEC<br />

stationary phase corresponds to the mobile phase within the separation pores, as<br />

well as to that situated near the outer surface <strong>of</strong> the column packing particles or<br />

near the surface <strong>of</strong> the transport channels <strong>of</strong> a monolith. In other words, we deal<br />

with the mobile phase volume from which macromolecules are partially or fully<br />

excluded. The stationary phase in the interactive (enthalpic) HPLC mainly<br />

includes the outer <strong>and</strong> inner (situated within the separation pores) column filling<br />

surface on which or near which adsorption, or ionic effects <strong>of</strong> analyte molecules<br />

occur. Enthalpic partition <strong>of</strong> analyte molecules takes place between the (quasi)<br />

stationary liquid phase <strong>and</strong> the mobile phase provided these two phases have<br />

different natures or compositions. Phase separation <strong>of</strong> macromolecules usually<br />

takes place in the mobile phase. The stationary phase can be either chemically<br />

bonded to an appropriate particulate or monolithic carrier, or formed by the<br />

stagnant molecules <strong>of</strong> eluent adsorbed on the column filling surface.<br />

© 2004 by Marcel Dekker, Inc.

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