[ 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
V.ADDITIONAL INFORMATION
a.Tips for Maximizing ACQUITY UPLC CSH Column Lifetimes
b.Recommended Flow Rates and Backpressures for ACQUITY UPLC CSH Columns
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. This same particle technology is used in the XSelect™ CSH family of HPLC columns, thus enabling seamless transferability between HPLC and UPLC® system platforms. The ACQUITY 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 individually tested and a Performance Chromatogram and Certificate of Batch Analysis are provided on the eCord™ intelligent chip.
ACQUITY UPLC CSH columns were designed and tested specifically for use on ACQUITY 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.
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[ CARE AND USE MANUAL ]
I. GET TING START ED |
c. Column Equilibration |
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 ACQUITY 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. These 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 ACQUITY 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.
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.
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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 |
|
Internal Diameter |
|
(mm) |
1.0 mm |
2.1 mm |
3.0 mm |
|
|||
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30 |
- |
0.1 |
0.2 |
|
|
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50 |
0.04 |
0.2 |
0.4 |
|
|
|
|
100 |
0.08 |
0.4 |
0.8 |
|
|
|
|
150 |
0.12 |
0.5 |
1.0 |
|
|
|
|
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.
e. Initial Column Efficiency Determination
1.Perform an efficiency test on the column before using it. This 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 performance 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 ACQUITY 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 Precolumns 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 |
VanGuard™ Pre-Column |
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Place wrench here |
Collet |
Place wrench here |
Ferrule |
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Flow
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 ACQUITY 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® solid-phase extraction cartridges/ columns or Sep-Pak® cartridges of the appropriate chemistry to clean 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.
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