Handbook of Size Exclusion Chromatography and Related ...

Table 3 Best Columns for the Separation of Double-Stranded DNA
Fragments
of the samples to be separated. Tables 2and 3summarize the best columns in
relation to molecular weight range.
7 ELUANT
Molecular weight range Best column
,40,000 (,60) a
G2000SW or G3000SW
40,000–80,000 (60–120) G3000SW
80,000–250,000 (120–400) G4000SW
250,000–800,000 (400–1,200) G5000PW
800,000–5,000,000 (1,200–7,000) G6000PW or DNA-PW
a
Values in parentheses are ranges in base pairs.
Source: Ref. 8.
Eluant ionic strength affects the elution volume and resolution in the SEC of
nucleic acids, and therefore it must be properly adjusted to obtain good results.
Figure 9shows the effect of eluant ionic strength on the elutionvolumes obtained
on TSKgel G3000SW,G4000SW,and G5000PW columns. Elution of both RNA
and DNA fragments is delayed by increasing the eluant ionic strength. Elution
volumesvarygreatlyinthelowionicstrengthregion,butathighionicstrengththe
elution volumes seem to become constant. Furthermore, the elution volumes of
small molecules are more markedly affected than those of large molecules. The
peak widths broaden with increasing eluant ionic strength, although slightly.
Accordingly,in general, an eluant ionic strength of 0.3–0.5 may be optimum.
When an eluant of low ionic strength is used, the exclusion limits of the columns
are considerably lowered. The main source of variation in elution volume with
eluant ionic strength is probably the repulsive ionic interaction between samples
and column packing materials, because both nucleic acids and TSKgel SW and
PW are negatively charged. TSKgel SW is based on silica and contains some
residualsilanolgroupsonitssurface,whereasTSKgelPWisbasedonhydrophilic
synthetic resin and contains some carboxyl groups. Most other commercially
available columns for aqueous SEC are also negatively charged, and the
phenomenon of increasing elution volume with increasing eluant ionic strength
has been observed on them, too. Other sources may also be responsible in some
cases. For example, elutionvolumes increase regularly with eluant ionic strength,
eveninthehighionicstrengthregion,whereionicinteractionsshoulddiminish,in
the case of 16S and 23S rRNAs (see 16S rRNA in Fig. 9b). The retardation of
© 2004 by Marcel Dekker, Inc.