Waters XSelect CSH HPLC Columns User Manual
[ CARE AND USE MANUAL ]
XSELECT CHARGED SURFACE HYBRID (CSH) COLUMNS
CONTENTS
I. GETTING STARTED
a. Column Installation
b. Column Equilibration
c. Initial Column Efficiency Determination
II. COLUMN USE
a. Guard Columns
b. Sample Preparation
c. Operating pH Limits
d. Solvents
e. Pressure
f. Temperature
III. SCALING UP/DOWN ISOCRATIC METHODS
IV. TROUBLESHOOTING
V. COLUMN CLEANING, REGENERATION
AND STORAGE
a. Cleaning and Regeneration
b. Storage
Thank you for choosing a Waters XSelect® CSH column. This HPLC
column features Waters Charged Surface Hybrid (CSH) Technology
which provides excellent peak shape, high efficiency and loading
capacity for basic compounds when using low ionic strength, acidic
mobile phases. T his same particle technology is used in the ACQUITY
®
CSH column family, thus enabling seamless transferability
UPLC
between HPLC and UPLC® system platforms. All XSelect CSH packing
materials are manufactured in a cGMP, ISO 9001:2000 certified plant
using ultra pure reagents. Each batch of XSelect 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 Test Chromatogram is provided with
each column along with the Certificate of Acceptance.
VI. CONNECTING THE COLUMN TO THE HPLC
a. Column Connectors and System Tubing Considerations
b. Measuring System Band-spreading Volume and System Variance
c. Measuring Gradient Delay Volume (or Dwell Volume)
VII. ADDITIONAL INFORMATION
a. Use of Narrow-Bore (3.0 mm i.d.) Columns
b. Impact of Band-spreading Volume on 2.1 mm i.d.
Column Performance
c. Non-Optimized vs. Optimized LC/MS/MS System:
System Modification Recommendations
[ CARE AND USE MANUAL ]
I. GETTING STARTED
Each XSelect CSH column comes with a Certificate of Analysis and
a Performance Test Chromatogram. The Certificate of Analysis is
specific to each batch of packing material contained in the XSelect
CSH column and includes the 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 information such
as: batch number, column serial number, USP plate count, USP
tailing factor, retention factor, and chromatographic conditions.
This data should be stored for future reference.
a. Column Installation
Note: The flow rates given in the procedure below are for a typical 5 µm packing
in a 4.6 mm i.d. column. Scale the flow rate up or down accordingly based upon
the i.d., length, particle size and backpressure of the XSelect CSH column being
installed. See Scaling Up/Down Isocratic Separations section for calculating
flow rates when changing column i.d and/or length. See Connecting the Column
to the HPLC section for a more detailed discussion on HPLC connections.
1. Purge the pumping system of any buffer-containing mobile phases
and connect the inlet end of the column to the injector outlet. An
arrow on the column identification label indicates the correct
direction of solvent flow.
b. Column Equilibration
XSelect 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 listing of empty column volumes).
To avoid precipitating out 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 HPLC system with 60% methanol in water
prior to introducing 60% methanol/40% buffer mobile phase).
c. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it in the desired
application. 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 t his value for
periodic comparisons.
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 1 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.
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.
3. Repeat the test at predetermined intervals to track column
performance over time. Slight variations may be obtained on
two different HP LC systems due to the quality of the connections,
operating environment, system electronics, reagent quality, column
condition and operator technique.
Table 1: Empty Column Volumes in mL (multiply by 10 for flush
solvent volumes)
Column Internal Diameter (mm)
Column
Length
(mm)
20 - 0.07 0.14 0.33 - - - - 30 - 0.10 0.21 0.50 - 2.4 8.5 - 50 0.04 0.17 0.35 0.83 2.4 3.9 14 35 98
100 0.08 0.35 0.71 1.7 4.8 7.8 28 70 150 0.12 0.52 1.0 2.5 7.2 12 42 106 294
250 - 0.87 1.8 4.2 - 20 70 176 490
1.0 2.1 3.0 4.6 7.8 10 19 30 50
2
XSelect CSH HPLC Columns
[ CARE AND USE MANUAL ]
II. COLUMN USE
To ensure the continued high performance of XSelect CSH columns, follow these guidelines:
a. Guard Columns
Use a Waters guard column of matching chemistry and particle size between the injector and main column. It is important to use a highperformance matching guard column to protect the main column while not compromising or changing the analytical resolution.
Guard columns need to be replaced at regular intervals as determined by sample contamination. When system backpressure steadily
increases above a set pressure limit, it is usually an indication that the guard column should be replaced. A sudden appearance of split
peaks is also indicative of a need to replace the guard column.
b. Sample Preparation
1. Sample impurities often contribute to column contamination. One option to avoid this is to use Waters Oasis® solid-phase extraction cartridges/columns
or Sep-Pak® cartridges of the appropriate chemistry to clean up the sample before analysis.
2. It is preferable to prepare t he sample in the operating mobile p hase or a mobile phase that is weaker (less organic modifier) 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 filters to remove particulates. If the sample is dissolved in a solvent that contains an organic modifier (e.g.,
acetonitrile, methanol, etc.) ensure that the filter material does not dissolve in the solvent. Contact the filter manufacturer with solvent
compatibility questions. Alternatively, centrifugation for 20 minutes at 8,000 rpm, followed by the transfer of the supernatant liquid to an
appropriate vial, could be considered.
c. Operating pH Limits
The recommended operating pH limits for XSelect CSH columns are listed in Table 2. A listing of commonly used buffers and additives is given
in Table 3. Additionally, the column lifetime will vary depending upon the operating temperature, type and concentration of buffer used.
Table 2: Recommended pH and Temperature Limits for XSelect CSH columns
Columnn Name Particle Size (µm) Pore Diameter (Å) Surface Area (m2) pH Limits
XSelect CSH C
XSelect CSH Phenyl-Hexyl 2.5, 3.5, 5 135 185 1-11 80 45 2.3 14
XSelect CSH Fluoro-Phenyl 2.5, 3.5, 5 135 185 1-8 80 45 2.3 10
Note: Working at the extremes of pH, temperature and/or pressure will result in shorter column lifetimes.
18
2.5, 3.5, 5, 10 135 185 1-11 80 45 2.3 15
Temperature Limits (°C)
Low pH High pH
Ligand Density
(µmol/m2)
% Carbon
3
XSelect CSH HPLC Columns
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