DNAPac PA200
Document No. 065036
Page 20 of 25
6.3 Phosphodiester
Analysis
6.3.1 Sodium Perchlorate Eluent Systems
The following separation represents a good starting guideline for developing sodium perchlorate (NaClO
4
)
based methods for longer oligonucleotides.
In this example, phosphorylated deoxycytosine oligomers, 19 – 24 bases long, were injected onto a DNAPac
PA200 column and eluted according to the conditions listed below. At pH 8, 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 5 mM NaClO
4
per mL of eluent can also be used to resolve full length from n-1
components between pH 8 and pH 12, using other buffers, e.g., AMPS, Na
3
PO4, etc.
CHART 11 N, N-1 Separation of Phosphorylated Deoxycytosine Oligomers
Conditions:
22 minute gradient from 70 to 202 mM NaClO
4
in 20 mM Tris buffered eluent at pH 8.
Flow rate:
1.2 mL/minute.
Injection volume: 6 µL.
Sample:
1 A
260
/mL solution of phosphorylated deoxycytosine oligomers, Temperature: 25°C
0
4
80
12
16
20
24
0.
20.
P(dC)
19-24
mA
260
Time (min)