Waters ACQUITY UPLC CSH Columns User Manual
[ Care and Use ManUal ]
ACQUITY UPLC CSH COLUMNS
Contents
I. GettInG started
a. Column Connectors
b. Column Installation
c. Column Equilibration
d. eCord Installation
e. Initial Column Efficiency Determination
f. VanGuard Pre-Columns
II. CoLUMn Use
a. Sample Preparation
b. pH Range
c. Solvents
d. Pressure
e. Temperature
III. CoLUMn CLeanInG, reGeneratInG and storaGe
a. Cleaning and Regeneration
b. Storage
IV. IntrodUCInG eCord InteLLIGent CHIP teCHnoLoGY
a. Introduction
b. Installation
c. Manufacturing Information
d. Column Use Information
Thank you for choosing a Waters ACQUITY UPLC® CSH™ column.
ACQUITY UPLC CSH columns feature Waters Charged Surface
Hybrid (CSH) Technology which provides excellent peak shape,
high efficiency and loading capacity for basic compounds when
using acidic, low ionic strength mobile phases. T his same particle
™
technology is used in the XSelect
thus enabling seamless transferability between HPLC and UPLC
system platforms. The ACQUIT Y UPLC CSH packing materials were
designed specifically for use with the ACQUITY UPLC systems and
are manufactured in a cGMP, ISO 9001 certified manufacturing
facility using ultra pure reagents. Each batch of ACQUITY UPLC CSH
material is tested chromatographically with acidic, basic and neutral
analytes and the results are held to narrow specification ranges to
assure excellent, reproducible performance. Every column is individu-
ally tested and a Performance Chromatogram and Certificate of Batch
Analysis are provided on the eCord
ACQUITY UPLC CSH columns were designed and tested specifically
for use on ACQUIT Y UPLC systems. ACQUITY UPLC CSH columns will
exhibit maximum chromatographic performance and benefits ONLY
when used on holistically-designed ACQUITY UPLC systems since these
systems and columns were created and designed to operate together.
For these reasons, Waters cannot support the use of ACQUITY UPLC
columns on any system other than an ACQUITY UPLC system.
CSH family of HPLC columns,
™
intelligent chip.
®
V. addItIonaL InForMatIon
a. Tips for Maximizing ACQUITY UPLC CSH Column Lifetimes
b. Recommended Flow Rates and Backpressures for
ACQUITY UPLC CSH Columns
ACQUITY UPLC CSH Columns 1
[ Care and Use ManUal ]
I. GettInG started
Each ACQUITY UPLC CSH column comes with a Certificate of
Analysis and Performance Test Chromatogram embedded within
the eCord intelligent chip. The Certificate of Analysis is specific to
each batch of packing material contained in the ACQUIT Y UPLC CSH
columns and includes the gel batch number, analysis of unbonded
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. T hese data should
be stored for future reference.
a. Column Connectors
ACQUITY UPLC systems utilize tubing and connectors which have
been designed to meet stringent tolerance levels and to minimize
extra column volumes.For information on system tubing and
connectors, please refer to the ACQUIT Y UPLC System Operator’s
Guide (Part Number 71500082502).
b. Column Installation
Note: The flow rates given in the procedure below are for typical 2.1 mm i.d. by 50 mm
length 1.7 µm columns. Scale the flow rate up or down accordingly based upon the flow
rate and pressure guide provided in Section V (Additional Information).
1. Purge the pumping system of any buffer-containing mobile phases and
connect the inlet end of the column to the injector outlet.
c. Column Equilibration
ACQUITY UPLC CSH 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 (refer to Table 1
for a list of column volumes). The column may be considered thermally
equilibrated once a constant backpressure is achieved.
Table 1. Empty Column Volumes in mL (multiply by 10 for flush solvent volumes)
Cloumn Length
(mm)
30 - 0.1 0.2
50 0.04 0.2 0.4
100 0.08 0.4 0.8
150 0.12 0.5 1.0
1.0 mm 2.1 mm 3.0 mm
Internal Diameter
To avoid precipitating mobile-phase buffers on your column or in
your system, flush the column with five column volumes of a water/
organic solvent mixture, using the same or lower solvent content as
in the desired buffered mobile phase. (For example, flush the column
and system with 60% methanol in water prior to introducing 60%
methanol/40% buffer mobile-phase.)
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.
2. Flush column with 100% organic mobile phase (methanol or
acetonitrile) 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
equilibration.
4. Gradually increase the flow rate as described in step 2.
5. Once a steady backpressure and baseline have been achieved, proceed
to the next section.
Note: If mobile-phase additives are present in low concentrations (e.g., ion-pairing
reagents), 100 to 200 column volumes may be required for complete equilibration. In
addition, mobile phases that contain formate (e.g., ammonium formate, formic acid,
etc.) may also require longer initial column equilibration times.
ACQUITY UPLC CSH Columns 2
e. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it. T his test may
consist of:
a. an analyte test mixture that is commonly used in your laboratory,
and /or
b. an analyte mixture as found on the “Performance Test
Chromatogram” which accompanied your column.
Note: If b. is performed, the isocratic efficiencies measured in your laboratory may be
less than those given on the Waters “Performance Test Chromatogram”. This is normal.
The Waters isocratic column testing systems have been modified in order to achieve
extremely low system volumes. This presents a more challenging test of how well the
column was packed. This guarantees the highest quality packed column. These special
testing systems have been modified to such an extent that they are not commercially
viable and have limited method flexibility other than isocratic column testing.
[ Care and Use ManUal ]
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 perfor-
mance over time.
f. VanGuard Pre-Columns
VanGuard™ Pre-columns are 2.1 mm i.d. x 5 mm length guard column
devices designed specifically for use in the ACQUIT Y UPLC systems.
VanGuard Pre-columns are packed with the same chemistries and frits
as our 2.1 mm i.d. ACQUITY UPLC CSH columns. VanGuard Pre-
columns are designed to be attached directly to the inlet side of an
ACQUITY UPLC CSH column.
Note: In order to ensure void-free and leak-free connections, the VanGuard Pre-column is
shipped with the collet and ferrule NOT permanently attached. Care must be taken when
removing the O-ring that holds these two pieces on the pre-column tubing.
™
ACQUITY UPLC® Column
Place wrench here
Ferrule
VanGuard
Collet
Flow
Pre-Column
Place wrench here
Installation Instructions
1. Remove VanGuard Pre-column from box and shipping tube and remove
plastic plug.
2. Orient pre-column so that male end is facing up and carefully remove
rubber O-ring that holds collet and ferrule in place during shipping
(collet and ferrule are not yet permanently attached).
3. Orient ACQUITY UPLC CSH column perpendicular to work surface so
that column inlet is on the bottom (column outlet on top).
4. From below, insert VanGuard Pre-column into ACQUITY UPLC
CSH column inlet and hand-tighten (collet and ferrule are not yet
permanently attached).
5. While pushing the VanGuard Pre-column into the column inlet,
turn assembled column and pre-column 180˚ so that pre-column
is now on top.
6. Tighten with two 5/16” wrenches placed onto ACQUIT Y UPLC CSH
column flats and VanGuard Pre-column hex nut (male end) as shown
above.
7. Tighten 1/4 turn to set collet and ferrule.
8. Check that ferrule is set by loosening connection and inspecting
the ferrule depth. A properly set ferrule depth will resemble other
connections in the ACQUITY UPLC system.
9. Reattach pre-column, apply mobile-phase flow and inspect for leaks.
II. CoLUMn Use
To ensure the continued high performance of ACQUITY UPLC CSH
columns, follow these guidelines:
a. Sample Preparation
1. Sample impurities often contribute to column contamination. One
®
option to avoid this is to use Oasis
®
columns or Sep-Pak
cartridges of the appropriate chemistry to clean
solid-phase extraction cartridges/
up the sample before analysis. For more information,
visit www.waters.com/sampleprep
2. It is preferable to prepare the sample in the operating mobile phase
or a mobile phase that is weaker than the mobile phase for the best
peak shape and sensitivity.
3. If the sample is not dissolved in the mobile phase, ensure that the
sample, solvent and mobile phases are miscible in order to avoid
sample and/or buffer precipitation.
4. Filter sample with 0.2 µm membranes to remove particulates. If the
sample is dissolved in a solvent that contains an organic modifier (e.g.,
acetonitrile, methanol, etc.) ensure that the membrane material does
not dissolve in the solvent. Contact the membrane manufacturer with
solvent compatibility questions. Alternatively, centrifugation for 20
minutes at 8000 rpm, followed by the transfer of the supernatant
liquid to an appropriate vial, could be considered.
b. pH Range
Please see Table 2 below for information on pH range and suggested
operating temperatures.
ACQUITY UPLC CSH Columns 3
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