Waters UPLC C18 User Manual
[ Care and Use ManUal ]
ACQUItY UPLC® ost C
18
*ost: oLIgonUCLeotIde sePArAtIon teChnoLogY
Thank you for choosing an ACQUITY UPLC® OST C18 column designed
®
specifically for use on Waters ACQUITY UPLC
tion of detritylated oligonucleotides on Waters second generation of
™
hybrid-silica BEH Technology
particles are based on the well estab-
lished method on ion-pair, reversed-phase chromatography. ACQUITY
®
OST C18 columns are available in several configurations to
UPLC
address different application needs. Your ACQUITY UPLC
packing material is manufactured in a cGMP, ISO 9002 certified plant
using ultra pure reagents. Each batch of ACQUITY UPLC
material is chromatographically tested with acidic, basic and neutral
analytes and the results are held to narrow specification ranges to
assure excellent, reproducible performance. In addition, every column
is individually tested and the associated Performance Test Chromatogram and Certificate of Acceptance information is available through
™
the attached eCord
intelligent chip technology.
Note: Optimum performance of ACQUITY UPLC
best assured using an appropriately configured Waters ACQUITY UPLC
System (e.g., See Section II, g). Consequently, use of ACQUITY UPLC
columns on conventional HPLC systems is not recommended.
OST C
18
System. The separa-
®
OST C
®
OST C18
®
OST C18 columns is
18
Contents
I. GETTING STARTED
a. Column Connectors
b. Column Installation
c. Column Equilibration
d. eCord™ Installation
e. Initial Column Efficiency Determination
II. COLUMN USE
a. Sample Preparation
b. Recommended Mobile Phases
c. Recommended Injector Wash Solvents
d. pH Range
e. Pressure
f. Temperature
g. Mixer Options
®
h. Flow Rate
®
III. COLUMN CLEANING, REGENERATING AND STORAGE
a. Cleaning and Regeneration
b. Storage
IV. ECORD™ INTELLIGENT CHIP TECHNOLOGY
a. Introduction
b. Installation
V. ADDITIONAL INFORMATION
a. Band Spreading Minimization
[ Care and Use ManUal ]
I. GETTING STARTED
Each ACQUITY UPLC® OST C18 column comes with Certificate of
Analysis and a Performance Test Chromatogram embedded within
™
the eCord
the batch of packing material contained in the ACQUITY UPLC
C
18
particles, analysis of bonded particles, and chromatographic results
and conditions. The Performance Test Chromatogram is specific to
each individual column and contains such information as gel batch
number, column serial number, USP plate count, USP tailing factor,
capacity factor, and chromatographic conditions. These data should
be stored for future reference.
a. Column Connectors
The ACQUITY UPLC
compression screws that have been designed to meet stringent
tolerance levels and minimize extra column volumes. Optimized
column inlet tubing (part # 430001084) is supplied with the
ACQUITY UPLC
clearly marked with a blue shrink tube marker. Insert the opposite
end of the tubing into the ACQUITY UPLC
compression fitting using two 5/16-inch wrenches. For information
on the correct column outlet tubing, please refer to the relevant
detector section in the ACQUITY UPLC
(part # 71500082502).
b. Column Installation
1. Purge the pumping system of any buffer-containing mobile
phases and connect the inlet end of the column to the
injector outlet.
2. Flush column with 100% organic mobile phase
(acetonitrile with TEAA or methanol for TEA-HFIP ion-pairing
method) by setting the pump flow rate to 0.1 mL/min and
increase the flow rate to 0.5 mL/min over 5 minutes.
3. When the mobile phase is flowing freely from the column
outlet, stop the flow and attach the column outlet to the
detector. This prevents entry of air into the detection system
and gives more rapid baseline equilibration.
intelligent chip. The Certificate of Analysis is specific to
®
OST
column and includes the gel batch number, analysis of unbonded
®
system utilizes tubing and gold plated
®
system. The inject valve end of the tubing is
®
column and tighten the
®
System Operator’s Guide
c. Column Equilibration
®
ACQUITY UPLC
OST C18 columns are shipped in 100% acetonitrile.
It is important to ensure mobile phase compatibility before changing
to a different mobile phase system. Equilibrate the column with a
minimum of 10 column volumes of the mobile phase to be used for
the oligonucleotide separation.
Note: When mobile phase additives are present in low concentrations
(e.g., TEAA or TEA-HFIP ion-pairing reagents), 100 to 200 column
®
volumes may be required for complete ACQUITY UPLC
OST C18
column equilibration.
Table 1. Empty Column Volumes in mL (multiply by 10 for flush solvent volumes)
Column Length (mm) Internal Diameter 2.1 mm
50 0.2
100 0.4
150 0.5
™
d. eCord
Installation
The eCord™ button should be attached to the side of the column heater
™
module. The eCord
button is magnetized and does not require specific
orientation.
e. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it.
Waters recommends using a suitable solute mixture, as found in
the “Performance Test Chromatogram”, to analyze the column
upon receipt.
2. Determine the number of theoretical plates (N) and use this
value for periodic comparisons.
3. Repeat the test at predetermined intervals to track column
performance over time. Slight variations may be obtained
®
on two different UPLC
systems due to the quality of the
connections, operating environment, system electronics,
reagent quality, column condition, and operator technique.
4. Gradually increase the flow rate as described in step 2.
5. Once a steady backpressure and baseline at 260 nm have been
achieved, proceed to the next section.
2
[ Care and Use ManUal ]
0 minutes 28
ACQUITY UPLC OST C18 column performance can be tested with
the MassPREP OST Standard (P/N 186004135), a quality controlled
synthetic oligonucleotide sample consisting of 15, 20, 25, 30 and
35 mer deoxythymidine. Approximately 0.1 nmol of each oligonucleotide was injected onto the ACQUITY UPLC OST C18 column.
Refer to P/N 715001677 for more information on sample prep for
the MassPREP OST Standard. Smaller peaks eluting prior to the main
peaks are failure, by-product sequences from the synthesis.
30
20
15
25
35
II. COLUMN USE
To ensure the continued high performance of your ACQUITY UPLC®
columns, follow these guidelines:
OST C
18
a. Sample Preparation
1. Dissolve the detritylated synthetic oligonucleotide sample in
Mobile Phase A (e.g., 0.1 M TEAA). For example, a 0.05 - 0.2 µmole
scale synthesis can be prepared in 0.1 mL of 0.1 M TEAA.
Proportionately larger or smaller volumes of 0.1M TEAA is required
when dissolving samples from different scale syntheses. Due to
the nature of gradient separations, relatively large volumes of sample
(in low organic strength eluent) can be injected and concentrated onto
the head of the column before beginning the gradient elution program.
2. Samples must be completely in solution and free of
particulates. Remove all particles from the sample
(Controlled Pore Glass Synthesis Support, etc.), which may
block the inlet column frit, increase the operating pressure,
and shorten the column life time. Sample contamination with
high concentration of salts and/or detergents may also interfere
with analysis.
Figure 1: Separation using the MassPREP OST Standard on ACQUITY
UPLC OST C18
UPLC® System: Waters ACQUITY U PLC® System with installed 425 µL mixer
(see Section II, g), PDA Detector with standard UV cell
Sample Injected: Approximately 0.1 nmol of Mass PREP OS T Standard (P/N 186004135)
diluted in 0.1 M TE AA
Column: Waters ACQUIT Y UPLC® OST C18 column, 1.7µm (2.1 x 50 mm)
Mobile P hases: A: 0.1 M TEAA,
B: Acetonitrile / 0.1M TEAA, 20/80, v/v
Flow rate: 0.2 mL/min
Column Temp.: 60 ˚C
Gradient delay: 0.45 mL
Gradient: 40 to 59.5% B in 26 minutes (8-11.9% acetonitrile,
0.15% acetonitrile per minute)
Detection: 260 nm, 5 scans per second
3. To remove particulates the sample may be filtered with a
0.2 μm membrane. Be sure that the selected membrane is
compatible and does not dissolve with the selected mobile
phase diluent. Contact the membrane manufacturer with
solvent compatibility questions. An alternative method of
particulate removal involves centrifugation for 20 minutes
at 8,000 rpm, followed by the transfer of the supernatant liquid
to an appropriate vial.
b. Recommended Mobile Phases
The most common ion-pair mobile phase for synthetic oligonucleotide separations is based on Triethylammonium Acetate (TEAA).
This mobile phase can be prepared by titrating Glacial Acetic Acid
aqueous solution with Triethylamine (TEA).
Note: To maximize column life, it is ESSENTIAL that all prepared OST
mobile phases be filtered through a solvent compatible, 0.2 µm mem-
brane and contained in bottles that are clean and particulate free.
TEAA
1 L of 0.1 M TEAA may be prepared as follows:
1) Perform work in a hood.
3
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