[ CARE AND USE MANUAL ]
XSELECT CHARGED SURFACE HYBRID (CSH) COLUMNS
CONT ENT S
I. GET TING START ED
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 ISOC RATIC MET HODS
IV. T ROUBLESHOOTING
V.COLUMN CLEANING, REGENERATION AND STORAGE
a.Cleaning and Regeneration
b.Storage
VI. CONNECTING T HE COLUMN TO T HE 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
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. This same particle technology is used in the ACQUITY UPLC® CSH column family, thus enabling seamless transferability 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.
[ CARE AND USE MANUAL ]
I. GET TING START ED
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.
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.
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 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 HPLC 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)
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Column Internal Diameter (mm) |
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Column |
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Length |
1.0 |
2.1 |
3.0 |
4.6 |
7.8 |
10 |
19 |
30 |
50 |
(mm) |
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20 |
- |
0.07 |
0.14 |
0.33 |
- |
- |
- |
- |
- |
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30 |
- |
0.10 |
0.21 |
0.50 |
- |
2.4 |
8.5 |
- |
- |
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50 |
0.04 |
0.17 |
0.35 |
0.83 |
2.4 |
3.9 |
14 |
35 |
98 |
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100 |
0.08 |
0.35 |
0.71 |
1.7 |
4.8 |
7.8 |
28 |
70 |
- |
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150 |
0.12 |
0.52 |
1.0 |
2.5 |
7.2 |
12 |
42 |
106 |
294 |
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250 |
- |
0.87 |
1.8 |
4.2 |
- |
20 |
70 |
176 |
490 |
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2 |
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XSelect CSH HPLC Columns |
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[ 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 the sample in the operating mobile phase 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 |
Temperature Limits (°C) |
Ligand Density |
% Carbon |
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Low pH |
High pH |
(µmol/m2) |
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XSelect CSH C18 |
2.5, 3.5, 5, 10 |
135 |
185 |
1-11 |
80 |
45 |
2.3 |
15 |
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XSelect CSH Phenyl-Hexyl |
2.5, 3.5, 5 |
135 |
185 |
1-11 |
80 |
45 |
2.3 |
14 |
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XSelect CSH Fluoro-Phenyl |
2.5, 3.5, 5 |
135 |
185 |
1-8 |
80 |
45 |
2.3 |
10 |
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Note: Working at the extremes of pH, temperature and/or pressure will result in shorter column lifetimes.
3 |
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XSelect CSH HPLC Columns |
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