Dionex 062998, Product Manual, Page: 16 / 25

DNAPac PA200 Document No. 065036 Page 16 of 25
5.7 Effect of Terminal Base on Selectivity
5.7.1 Selectivity in Sodium Chloride (NaCl) Gradients
The influence of the 5’ and 3’ terminal base on retention is shown below for NaCl eluents. These examples
consist of mixed-base oligonucleotide (ODN) 25 mers with identical sequence except for the 3’ and 5’ terminal
bases. The samples are chromatographed at pH 9, 10 & 11.
In each panel, the top 4 traces show elution of ODNs with identical 5’ termini, and altered only at the 3’ base.
The bottom 4 traces show elution of ODNs with identical 3’ termini, and altered only at the 5’ base. The
middle trace is common to both sets.
Using NaCl as the salt, all of the ODNs differing only at the 3’ base are resolved at pH 9 or 10. The ODNs
differing only at the 5’ base are also at least partially resolved at pH 9 or 10. In each case, C contributes the
least to retention at either the 5’ or 3’ end, and G contributes the most.
The relative contributions of A and T at either end are pH dependent. At pH 9, A contributes more than T but
at the higher pH values it contributes less.
Addition of solvent (e.g., CH
3
CN) tends to reduce retention, and minimize hydrophobic interactions. This may
in some cases improve selectivity and resolution, (chart not shown).
CHART 8 Effect of pH on Retention (by Terminal Base):
15 mM/mL NaCl gradient, 0% CH
3
CN, 5’X-G
6
C
3
A
5
T
9
-Y3’, pH 9-11
5.5 6.5 7.5 8.5
-1
140
mA
Time (min)
7
6
5
4
3
2
1
Flow: 1.20 ml/min
X=C, Y=A
X=A, Y=A
X=T, Y=A
X=G, Y=A
X=C, Y=T
X=C, Y=C
X=C, Y=G
pH 9
pH 11
21.5 22.5 23.5 24.5
-1
140
mA
Time (min)
7
6
5
4
3
2
1
Flow: 1.20 ml/min
X=C, Y=A
X=A, Y=A
X=T, Y=A
X=G, Y=A
X=C, Y=T
X=C, Y=C
X=C, Y=G
pH 10
13.5 14.5 15.5 16.5
-1
140
mA
Time (min)
7
6
5
4
3
2
1
X=C, Y=A
X=A, Y=A
X=T, Y=A
X=G, Y=A
X=C, Y=T
X=C, Y=C
X=C, Y=G
Flow: 1.20 ml/min
5.5 6.5 7.5 8.5
-1
140
mA
Time (min)
7
6
5
4
3
2
1
Flow: 1.20 ml/min
X=C, Y=A
X=A, Y=A
X=T, Y=A
X=G, Y=A
X=C, Y=T
X=C, Y=C
X=C, Y=G
pH 9
pH 11
21.5 22.5 23.5 24.5
-1
140
mA
Time (min)
7
6
5
4
3
2
1
Flow: 1.20 ml/min
X=C, Y=A
X=A, Y=A
X=T, Y=A
X=G, Y=A
X=C, Y=T
X=C, Y=C
X=C, Y=G
pH 10
13.5 14.5 15.5 16.5
-1
140
mA
Time (min)
7
6
5
4
3
2
1
X=C, Y=A
X=A, Y=A
X=T, Y=A
X=G, Y=A
X=C, Y=T
X=C, Y=C
X=C, Y=G
Flow: 1.20 ml/min