[ CARE AND USE MANUAL ]
ACQUITY UPLC BEH Columns
CONT ENT S
I. INT RODUCTION
II. GET TING START ED
a.Column Connectors
b.Column Installation
c.Column Equilibration
d.eCord Installation
e.Initial Column Efficiency Determination
f.VanGuard™ Pre-Columns
III. COLUMN USE
a.Sample Preparation
b.pH Range
c.Solvents
d.Pressure
e.Temperature
IV. COLUMN CLEANING, REGENERATING AND STORAGE
a.Cleaning and Regeneration
b.Storage
V.INT RODUCING ECORD INT ELLIGENT CHIP T ECHNOLOGY
a.Introduction
b.Installation
c.Manufacturing Information
d.Column Use Information
VI. ADDITIONAL INFORMATION
a.Tips for Maximizing ACQUITY UPLC BEH Column Lifetimes
b.Recommended Flow Rates and Backpressures for Reversed-Phase ACQUITY UPLC BEH Columns
c.Getting Started with ACQUITY UPLC BEH
HILIC Columns
d. Getting Started with ACQUITY UPLC BEH Amide Columns
I. INT RODUCTION
Thank you for choosing a Waters ACQUITY UPLC® BEH Column. The ACQUITY UPLC BEH packing materials were designed specifically for use with the ACQUITY UPLC System and are manufactured in a cGMP, ISO 9001 certified plant using ultra pure reagents. Each batch of ACQUITY UPLC BEH 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 Columns were designed and tested specifically for use on the ACQUITY UPLC System. ACQUITY UPLC Columns will exhibit maximum chromatographic performance and benefits ONLY when used on the holistically-designed ACQUITY UPLC System since the ACQUITY UPLC System and column 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.
[ CARE AND USE MANUAL ]
II. GET TING START ED
Each ACQUITY UPLC BEH 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 BEH 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
The ACQUITY UPLC System utilizes tubing and gold plated compression screws which have been designed to meet stringent tolerance levels and to minimize extra column volumes.
Optimized column inlet tubing (Part Number 430001084) is supplied with the ACQUITY UPLC System. The inject valve end of the tubing is clearly marked with a blue shrink tube marker. Insert the opposite end of the tubing into the ACQUITY UPLC Column and tighten the compression fitting.
For information on the correct column outlet tubing, please refer to the relevant detector section in the ACQUITY UPLC System Operator’s Guide (Part Number 71500082502).
b. Column Installation
Note: The flow rates given in the procedure below are for a typical 2.1 mm i.d. by 50 mm length 1.7 µm column. 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.
c. Column Equilibration
ACQUITY UPLC BEH 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)
Column Length |
|
Internal Diameter |
|
(mm) |
1.0 mm |
2.1 mm |
3.0 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 |
|
|
|
|
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.)
For ACQUITY UPLC BEH HILIC Columns, flush with 50 column volumes of 50:50 acetonitrile:water with 10 mM final buffer concentration. For ACQUITY UPLC BEH Amide Columns, flush with 50 column volumes of 60:40 acetonitrile:aqueous. Prior to the first injection, equilibrate with 20 column volumes of initial mobile phase conditions (refer to Table 1 for a list of column volumes). See “Getting Started with ACQUITY UPLC BEH HILIC Columns” or “Getting Started with ACQUITY UPLC Amide Columns” for additional information.
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[ CARE AND USE MANUAL ]
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.
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.
f.VanGuard Pre-Columns
VanGuard Pre-columns are 2.1 mm ID x 5 mm length guard column devices designed specifically for use in the ACQUITY UPLC System. VanGuard Pre-columns are packed with the same UPLC chemistries and frits as our 2.1 mm ID UPLC columns. VanGuard Pre-columns are designed to be attached directly to the inlet side of an ACQUITY UPLC 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 |
Place wrench here |
Collet |
|
|
Ferrule |
Place wrench here |
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 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 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 precolumn is now on top.
6.Tighten with two 5/16” wrenches placed onto ACQUITY UPLC 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.
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[ CARE AND USE MANUAL ]
II. COLUMN USE
To ensure the continued high performance of ACQUITY UPLC BEH 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. Acetone should
not be used as a sample solvent/diluent unless a Hexane Tetrahydrofuran Compatibility Kit (P/N: 205000464) has been installed.
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
5.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.
6.For Hydrophilic Interaction Chromatography (HILIC) separations, the samples must be prepared in a high percentage of organic solvents (e.g., 95% acetonitrile). See “Getting Started with ACQUITY UPLC BEH HILIC Columns” or “Getting Started with ACQUITY UPLC Amide Columns” for additional information.
b. pH Range
The recommended operating pH range for ACQUITY UPLC BEH Columns is 1 to 12 for the C18, C8 and Phenyl chemistries; 2 to 11 for the Shield RP18 and BEH Amide chemistries; and 1 to 9 for the BEH HILIC chemistry. A listing of commonly used buffers and additives is given in Table 2. Additionally, the column lifetime will vary depending upon the operating temperature, the type and concentration of buffer used. For example, the use of phosphate buffer at pH 8 in combination with elevated temperatures will lead to shorter column lifetimes.
c. Solvents
To maintain maximum column performance, use high quality chromatography grade solvents. Filter all aqueous buffers prior to use through a 0.2 µm filter. Pall Gelman Laboratory Acrodisc® filters are recommended. Solvents containing suspended particulate materials will generally clog the outside surface of the inlet distribution frit of the column. This will result in higher operating pressure and poorer performance. See Section V for more information.
d. Pressure
ACQUITY UPLC BEH Columns can tolerate operating pressures up to 18000 psi (1241 bar or 124 MPa). Note: Working at the extremes of pressure, pH and/or temperature will result in shorter column lifetimes.
e. Temperature
Temperatures between 20 ˚C – 90 ˚C are recommended for operating ACQUITY UPLC BEH Columns in order to enhance selectivity, lower solvent viscosity and increase mass transfer rates. When operating at high pH, lower operating temperatures are recommended for longer column lifetime. Working at high temperatures (e.g. > 70 °C) may also result in shorter column lifetimes. Under HILIC conditions, ACQUITY UPLC BEH Amide Columns can be used at high pH and at high temperatures without issue (see recommended conditions in Getting Started with BEH Amide section).
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