Waters Oligonucleotide Separation Technology ACQUITY UPLC C18 Column - Manual (Page 4)

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[ method guidelines ]
Mobile Phase B change per min) is required. In these situations, it is
recommended that the organic solvent concentration in Mobile Phase
Line B be reduced by “premixing” with a measured amount of Mobile
Phase A (e.g., Mobile Phase A= 0.1 M TEAA and Mobile Phase B=
Acetonitrile /0.1M TEAA, 20/80, v/v).
Use of a 425 µL mixer (Part # 205000403) specifically designed
for shallow UPLC
® gradient separations is recommended when the
solvent premixing technique (detailed above) is not used and when
Mobile Phase B contains either 100% acetonitrile (for TEAA ion-pair-
ing method) or 100% methanol (for TEA-HFIP ion-pairing method).
In addition, the Solvent Delivery System Outlet Tube Assembly (Part
Number 430001486) is required for 425 µL mixer installation onto a
standard ACQUITY UPLC
® System.
Note: 1) The 425 µL mixer introduces an additional delay volume to gra-
dient separations. For ultra-fast OST analyses, the smaller 50 µL mixer
should be used with the described premixed mobile phase technique.
2) The recommended flow rate for oligonucleotide separations performed
on a 2.1 x 50 mm ACQUITY UPLC
® OST C
18
column is 0.2 mL/minute.
When faster flow rates are desired for separations, use of the 425 µL
mixer with installed Outlet Tubing Assembly is recommended.
VI. generAL ConsIderAtIon In deV eLoPIng
sePArAtIons
Separation of detritylated synthetic oligonucleotides by ion-pair,
reversed-phase chromatography uses very shallow gradients. With
both TEAA and TEA-HFIP ion-pairing systems, a rate of strong eluent
change between 0.1-0.25 % Acetonitrile (or Methanol) per minute is
recommended. However, the formation of shallow gradients can place
performance demands on LC pumps and mixers that can compromise
the quality of the separation. Consequently, it is strongly advised that
Mobile Phase B formulation contain a premix blend of aqueous and
organic solvents (e.g., Mobile Phase A= 0.1 M TEAA and Mobile Phase
B= Acetonitrile / 0.1 M TEAA, 20/80, v/v) to minimize potentially inad-
equate solvent mixing that can compromise component resolution.
As illustrated in Figures 3-4, these analyses were performed with the
following mobile phases:
Mobile Phase A: 0.1 M TEAA
Mobile Phase B: Acetonitrile containing 0.1 M TEAA, 20:80 (v:v)
The 0.1% acetonitrile concentration change per min (from an initial 5
to 10% acetonitrile concentration) over 50 minutes was programmed
as specified below:
Table 1: Acetonitrile Concentration Change
Time % A % B
Actual Acetonitrile (ACN)
Concentration
0 min 75 25 5%
50 min 50 50 10%
Examples:
For the initial 5% Acetonitrile concentration:
B. Initial %B = desired ACN %/Volume Fraction of ACN in Mobile
Phase So, initial %B = 5%/0.2 = 25%
For the final 10% Acetonitrile concentration:
B. Final %B = desired ACN %/Volume Fraction of ACN in Mobile
Phase 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 2.1 x 50 mm column at a 0.2 mL/min
flow) are recommended for labeled oligonucleotides or for short,
5-15mer sequences. Separations of longer sequences are typically
performed using more shallow gradient slopes (e.g. 0.15% ACN change
per minute on a 2.1 x 50 mm column at a 0.2 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 sample 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