Waters Oligonucleotide Separation Technology XBridge OST C18 Columns - Manual (Page 5)

5
[ method guidelines ]
As illustrated in Figures 5 through 7, these analyses were performed
with the following mobile phases:
Mobile Phase A: 0.1 M TEAA
Mobile Phase B:
Acetonitrile (ACN) containing 0.1 M TEAA, 20:80 (v:v)
The 0.1% ACN / min gradient change from an initial 5 to 10% Acetonitrile
concentration over 50 minutes was programmed as specified in Table 1:
Table 1
Time % A % B Actual Acetonitrile (ACN) Concentration
0 min 75 25 5%
50 min 50 50 10%
Example:
For the initial 5% Acetonitrile concentration:
Initial %B = desired ACN % / Volume Fraction of ACN in Mobile Phase
B. So, initial %B = 5% / 0.2 = 25%
For the final 10% Acetonitrile concentration:
Final %B = desired ACN % / Volume Fraction of ACN in Mobile Phase
B. So, final %B = 10% / 0.2 = 50%
With TEAA mobile phases, the unmodified oligonucleotides elute
within a 7-10 % ACN gradient window. However, C and G rich oligo-
nucleotide sequences are generally less retained (i.e., elute within a
5-8% ACN gradient window) than A and T rich sequences (i.e., elute
within a 8-11% ACN gradient span). When using a shallow gradient,
the total length of analysis for an unknown sample sequence may be
excessive. Use of a fast scouting gradient with a 1% ACN per minute
change is recommended in such cases. Information gathered from this
scouting analysis can then be used to create a more appropriate and
time efficient set of gradient conditions for the particular sample.
Gradient slope has a direct impact on the achievable oligonucleotide
component resolution (along with the type of ion-pairing agent,
sequence, and oligonucleotide modification). Steeper gradients (e.g.,
1% ACN change per minute on a 4.6 x 50mm column at a 1.0 mL/min
flow) are recommended for labeled oligonucleotides or for short, 5-15
mer sequences. Separation of longer sequences are typically performed
using more shallow gradient slopes (e.g. 0.15% ACN change per minute
on a 4.6 x 50mm column at a 1.0 mL/min flow).
The organic solvent concentration at initial sample loading conditions
has to be well chosen. If the initial organic solvent strength is too
high, then some desired oligonucleotide sequences may be unretained.
In the other extreme, when the gradient starts with too low an organic
concentration, the analysis is excessively long without the benefit
of enhanced component resolution. A suitable gradient separation
method can be approximated from the oligonucleotide base (C, G, A,
and T) composition. The initial gradient is typically adjusted while
keeping the gradient slope constant.
Table 2: Suggested Gradient Conditions for Non-Standard Detritylated
Synthetic Oligonucleotide Sequences
Gradient 1
[Standard
oligonucleotides (1)]
Gradient 2
[High GC content or short
oligonucleotides (2)]
Gradient 3
[High AT content or long
oligonucleotides (3)]
Initial % ACN 7.00% 5.25% 7.50%
Final % ACN 10.75% 9.00% 12.50%
Gradient Length(4) 15 min 15min 20min
1: Standard oligonucleotides: 10 – 30mers
2: Short oligonucleotides: Less than 10mer
3: Long oligonucleotides: 30 – 60mers
4: Assuming use of a 2.1 x 50mm XBridge ™ OST C
18
column at a flow
of 0.2 mL/min and a separation temperature of 60 ˚C.
The retention of single and dual dye-labeled oligonucleotides is dictated
by the nature of label. For example, the retention of 25 mer oligonu-
cleotide increases according to the type of label attached as follows:
no label<6FAM<
Vi. analysis oF ModiFied oligonuCleotides
XBridge ™ OST C
18
columns are suitable for analysis of unmodified as
well as modified detritylated oligodeoxyribonucleotides and oligoribo-
nucleotides. Phosphorothioate and 2’-O -alkyl modified oligonucleotides
can also be analyzed with IP-RP-HPLC method. However, these full
length oligonucleotide products are usually more difficult to resolve
from their shorter length failure sequences. The recommended ion-pair
system for phosphorothioate oligonucleotide analysis is TEA-HFIP (see
Recommended mobile phases). An example of a 25mer phosphorothioate
oligonucleotide analysis is shown in Figure 5.