Handbook of Size Exclusion Chromatography and Related ...

Figure 10 Dependence of HETP on flow rate for RNAs on a TSKgel G4000SW twocolumn
system and for DNA fragments on a TSKgel G5000PW two-column system (each
column 60 cm 7.5 mm ID). (From Ref. 8.)
elution in the high ionic strength region may be attributed to the adsorption of
samples on column packing materials by hydrophobic interaction.
8 FLOW RATE
Figure 10 shows the dependence of height equivalent to a theoretical plate (HETP)
on flow rate observed in the SEC of RNA and DNA fragment on 7.5 mm ID
columns. The HETP decreased with decreasing flow rate. Especially with highmolecular-weight
samples, such as 16S rRNA and a DNA fragment of 383 base
pairs, the HETP was significantly dependent on flow rate and reached a minimum
at flow rates lower than 0.1 mL/min. Flow rates of 0.3–0.5 mL/min seem to be a
good compromise when separation time and resolution are taken into
consideration.
9 CONCLUSIONS
A wide range of nucleic acids including RNAs, DNA fragments, plasmids, and
oligonucleotides can be separated effectively by SEC on the basis of molecular
size. Accordingly, it is possible to adopt SEC as an alternative to gel electrophoresis
for analytical purposes. Furthermore, because the separated components
in samples can be recovered easily and yet almost quantitatively by collection of
column effluent, SEC should be superior to gel electrophoresis for preparative
© 2004 by Marcel Dekker, Inc.