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Handbook of Size Exclusion Chromatography and Related ...

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Figure 18 Schematic representation <strong>of</strong> the 2D-HPLC sample transfer system with two<br />

six-porttwo-wayvalves.Column#1worksincontinuousmode.Loops#1<strong>and</strong>#2arefilled<br />

alternatively. Both valves are operated simultaneously. Other symbols as in Fig. 7. For<br />

further explanation see the text.<br />

introduced into C#2 may also affect sample retention within column #2, stability<br />

<strong>of</strong> column #2, as well as detection <strong>of</strong> effluent from column #2. Therefore eluent<br />

exchange between C#1 <strong>and</strong> C#2 may bring several advantages.<br />

An important query <strong>of</strong> any 2D-HPLC procedure relates to sample dilution<br />

(98), which complicates detection <strong>of</strong> polymer in the column #2 effluent.<br />

Reinjection<strong>of</strong>alargenumber<strong>of</strong>dilutedfractionsfromcolumn#1intocolumn#2<br />

also prolongs totaltime <strong>of</strong> analysis. The reinjection <strong>of</strong> only the most concentrated<br />

parts <strong>of</strong> the column #1 fractions (heart cut approach) may bring about<br />

unintentional disregard <strong>of</strong> important information about the sample.<br />

It seems that several <strong>of</strong> the above problems can be solved by means <strong>of</strong> the<br />

FRE procedures (Fig. 20). The full retention–elution method allows storage <strong>of</strong><br />

fractionsfromcolumn#1<strong>and</strong>thuspracticallyindependentoperation<strong>of</strong>column#2.<br />

If only fraction storage is needed, “FRE columns” can be packed with nonactive<br />

nonporous particles to reduce diffusion-induced mixing within each fraction (99).<br />

Alternatively,FRE columns can be substituted by aset <strong>of</strong> capillary loops. FRE<br />

columnscanalsoservefor thereconcentration/focusing<strong>of</strong>fractionsfromthefirst<br />

dimensioncolumn<strong>and</strong>thusforatleastpartialexchange<strong>of</strong>samplesolventinjected<br />

into column #2. Separation in column #1 can be repeated <strong>and</strong> corresponding<br />

Figure 19 Eight-port two-way valve system for the 2D-HPLC sample transfer. Two<br />

loops L#1 <strong>and</strong> L#2 are filled alternatively. Other symbols as in Fig. 17. For further<br />

explanation see the text.<br />

© 2004 by Marcel Dekker, Inc.

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