DNAPac PA200
Document No. 065036
Page 15 of 25
5.6 Effect of Temperature on Oligonucleotide Retention
Elevated temperature is often used to limit or eliminate Watson-Crick, and poly-G hydrogen bonding within, and
between oligonucleotides that have self-complementary sequences.
NOTE: Dionex does NOT recommend combining the use of elevated temperatures with high pH elution
systems. Such conditions will accelerate degradation of the DNAPac PA200 stationary phase.
At relatively low pH, 9 or below, increased temperature may have mixed effects on nucleic acid retention.
Nucleoside monophosphates and very short, 2-3 base, oligonucleotides may exhibit decreased retention times at
elevated temperatures. Nucleoside triphosphates and oligonucleotides greater than a few bases long usually exhibit
increased retention as the temperature increases. The chart below illustrates the influence of increased temperature
at constant pH (8). As the chromatographic temperature increases, retention of the oligonucleotides also increases,
in this case by an average of ~ 2.7 min per 10°C.
CHART 7 Effect of Temperature on Retention of Oligonucleotides
d(AC)
10-11
: pH 8, 5 mM NaClO
4
/ mL
0.0
4.0
8.0
12.0
16.0
20.0
0
100
d(AC)x-xi 25 °C
d(AC)x-xi 35 °C
d(AC)x-xi 45 °C
d(AC)x-xi 55 °C
mAU
min
4
3
2
1
WVL:260 nm
Flow: 1.20 ml/min
0.33M NaClO4:
21.2 %
51.5
75.0
22.1