DNAPac PA200
Document No. 065036
Page 14 of 25
5.5.4 Effect of pH on Selectivity
The figure below illustrates the influence of pH on oligonucleotide selectivity. Here the elution patterns of two
23-base oligonucleotides differing only in their 5’ and 3’ terminal bases are compared between pH 9 and pH
11.
The top trace for each pair of chromatograms has an additional T at the 5’ end of the molecule, and lacks the 3’
A. The oligonucleotide chromatographed in the bottom trace lacks the 5’ T, but has the 3’A. At pH 9 - 9.5
(bottom 2 pairs of traces) these oligos are unresolved.
At pH 10, the 5’ TG-3’G oligonucleotide is eluted earlier than the 5’ G-3’ GA oligonucleotide, and the two are
only partially resolved. However, at pH 10.5 and 11 this elution order is reversed, due to the relative
contributions of T and A to retention at these pH values.
The base composition of these 23-base oligos is 5’ X-G
4
C
4
A
3
T
7
-Y 3’, and optimal resolution is observed at pH
10.5.
CHART 6 Effect of pH on Selectivity
75
4
12
0
mAU
Time (Min)
10
9
8
7
6
5
4
3
2
WVL:260 nm
1
Flow: 1.20 ml/min
X=GA, Y=TGA, pH 9
X=TGA, Y=TG, pH 9
X=GA, Y=TGA, pH 9.5
X=TGA, Y=TG, pH 9.5
X=GA, Y=TGA, pH 10
X=TGA, Y=TG, pH 10
X=GA, Y=TGA pH 10.5
X=TGA, Y=TG, pH 10.5
X=GA, Y=TGA, pH 11
X=TGA, Y=TG, pH 11
5
6
7
9
10
11
8
75
4
12
0
mAU
Time (Min)
10
10
9
9
8
8
7
7
6
6
5
5
4
4
3
3
2
2
WVL:260 nm
1
1
Flow: 1.20 ml/min
X=GA, Y=TGA, pH 9
X=TGA, Y=TG, pH 9
X=GA, Y=TGA, pH 9.5
X=TGA, Y=TG, pH 9.5
X=GA, Y=TGA, pH 10
X=TGA, Y=TG, pH 10
X=GA, Y=TGA pH 10.5
X=TGA, Y=TG, pH 10.5
X=GA, Y=TGA, pH 11
X=TGA, Y=TG, pH 11
5
6
7
9
10
11
8