Waters Oligonucleotide Separation Technology XBridge OST C18 Columns - Manual (Page 7)

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[ method guidelines ]
Once the separation has been optimized, one begins preparing for the
preparative separation. The steps to successfully scale a separation
from an analytical to a preparative column, containing the same packing
material composition, are detailed below
Step A: Calculate the flow rate for use on the Preparative column.
Preparative Column Flow Rate =
Analytical Column Flow Rate x (Diameter of Prep Column) 2
(Diameter of Anal Column) 2
Step B: To get similar chromatography, the gradient elution profile
should be created on both columns using the same number of column
volumes. When the analytical and preparative columns are of the
same length, as is recommended for this application, then the gradient
duration should be the same.
Note: This assumes use of the same flow rate linear velocity for both
runs as calculated above. For preparative runs, it is also important to
note that an initial gradient delay is required to allow the entire sample
to load onto the head of the column prior to beginning chromatography.
Step C: The last calculation involves determining how much sample
can be loaded on the preparative column. This calculation compares
the relative volumes of the two columns assuming that both columns
are the same length as recommended for this application.
Preparative Column Sample Load =
Previously Determined Analytical Column Sample Load x (Diameter of Prep Column) 2
(Diameter of Anal Column) 2
Table 3: XBridge ™ OST C
18
Column Selection Guide for Detritylated
Oligonucleotide Purification
Column (mm) Approx Mass Load (µmoles)** Flow Rate (mL/min)
2.1 x 50 0.04 0.2
4.6 x 50 0.20 1.0
10.0 x 50 1.00 4.5
19.0 x 50* 4.00 16.0
30.0 x 50* 9.00 40.0
50.0 x 50* 25.00 110.0
* XBridge ™ OST C
18
Custom Column
** Values are only approximate and vary depending on detritylated
oligonucleotide length, base composition, and “heart-cutting” fraction
collection method used
Figure 7 shows the separation of 90 nmoles of a detritylated 30 mer
deoxythymidine crude reaction mixture on a 4.6 x 50mm XBridge
™
OST C
18
column. The collection interval is suggested by the lines. Due
to partial column overloading, the N -1, N -2... impurities are partially
displaced and elute earlier than expected. With the proper hearth-cutting
technique, 95-98% purity is typically achieved for 15-35 mer oligo-
nucleotides at this purification scale.
Figure 7: Purification of a Detritylated 30mer Deoxythymidine Sample
HPLC system: Waters BioAlliance ™ 2796, PDA Detector with micro UV cell
Sample: Crude detritylated 30mer oligothymidine, 200 nmole dissolved in
100 µl of mobile phase A, 45 µl was injected on column
Column: XBridge ™ OST C
18
, 2.5 µm (4.6 x 50mm)
Mobile phase: A: 0.1M TEAA with 400 mM HFIP
B: Acetonitrile/0.1M TEAA, 20/80 (v/v)
Flow rate: 1.0 mL/min
Column Temp.: 60 ˚C
Gradient delay: 0 mL (compensated)
Gradient: 35 to 65% B in 24 minutes (7-13% ACN, 0.25% ACN per minute)
Detection: 260 nm, 2 scans per second
Table 4: Ordering Information
Description Particle Size Pore Size Dimension Part No.
XBridge ™ OST C
18
2.5 μm 135Å 2.1 x 50 mm 186003952
XBridge ™ OST C
18
2.5 μm 135Å 4.6 x 50 mm 186003953
XBridge ™ OST C
18
2.5 μm 135Å 10.0 x 50 mm 186003954
Custom
XBridge ™ OST C
18
-- -- -- 186003955