DNAPac PA200
Document No. 065036
Page 21 of 25
6.3.2 Sodium Chloride Eluent Systems
The following separation represents a good starting guideline for developing sodium chloride (NaCl) based
methods for longer oligonucleotides.
In this example, deoxythymidine oligomers, 19 – 24 bases long, were injected onto a DNAPac PA200 column
and eluted according to the conditions listed below. At pH 12, this gradient is effective for resolving the “full-
length” oligonucleotide phosphodiesters, up to 25 bases long, from the n-1 components.
The same gradient of 15 mM NaCl per mL of eluent can also be used to resolve full length from n-1
components, between pH 8 and pH 12, using other buffered eluent systems, e.g., Tris, AMPS, Na
3
PO4, etc.
CHART 12 N, N-1 Separation of Deoxythymidine Oligomers
Conditions:
31.5 minute gradient of 330 to 900 mM NaCl in 20 mM NaOH (pH 12.4).
Flow rate:
1.2 mL/minute.
Injection volume: 4 µL.
Sample:
1 A
260
/mL solution of deoxythymidine oligomers, Temperature: 25°C.
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
0.0
15.0
mA
26
0
Time (min)
dT19
dT20
dT21
dT22
dT23
dT24
Flow: 1.20 ml/min
1.25M NaCl:
26.4 %
72.0
80.0
26.4
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
0.0
15.0
mA
26
0
Time (min)
dT19
dT20
dT21
dT22
dT23
dT24
Flow: 1.20 ml/min
1.25M NaCl:
26.4 %
72.0
80.0
26.4
72.0
80.0
26.4