[ CARE AND USE MANUAL ]
Waters Spherisorb columns
Contents
I.Getting started
a.Column Installation
1.Reversed-Phase Columns
2.Normal-Phase Columns
b.Column Equilibration
1.Reversed-Phase Columns
2.Normal-Phase Columns
c.Initial Column Efficiency Determination
II.COLUMN USE
a.Guard Columns
b.Sample Preparation
c.pH Range
d.Solvents
e.Pressure
f.Temperature
III. SCALING UP/DOWN ISOCRATIC METHODS
Iv. TROUBLESHOOTING
V.COLUMN CLEANING, REGENERATING AND STORAGE
a.Cleaning and Regenerating
1.Reversed-Phase Columns
2.Normal-Phase Columns
b.Storage
1.Reversed-Phase Columns
2.Normal-Phase Columns
VI. CONNECTING THE COLUMN TO THE HPLC
a.Column Connectors and System Tubing Considerations
b.Band Spreading Minimization
c.Measuring System Bandspreading Volume & System Variance
d.Measuring System Volume
VII. ADDITIONAL INFORMATION
a.Use of Narrow-Bore (≤3.0 mm i.d.) Columns
b.Impact of Bandspreading Volume on 2.1 mm i.d. Column Performance
c.Non-Optimized vs. Optimized LC/MS/MS System: System Modification Recommendations
d.Guard Cartridges and Columns Assembly
Thank you for choosing a Waters Spherisorb® column. Spherisorb analytical columns are durable, high-efficiency chromatographic columns, featured in thousands of references in chromatographic literature. The wide range of column lengths and diameters offers you exceptional flexibility in optimizing methods and reducing solvent consumption. Follow the guidelines in this manual to obtain the best performance, reproducibility and durability from your analytical columns and cartridges.
Waters Spherisorb Columns |
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[ CARE AND USE MANUAL ]
Table 1. Spherisorb Column Physical Characteristics
Chemistry |
Particle |
Particle Size |
Pore Size |
Surface Area |
Pore Volume |
% Carbon |
Endcapped |
|
Shape |
(µm) |
(Å) |
(m2/g) |
(cc/g) |
Load |
|||
|
|
|||||||
Silica |
Spherical |
3, 5 and 10 |
80 |
220 |
0.50 |
n/a |
n/a |
|
|
|
|
|
|
|
|
|
|
ODS2 (C18) - Fully End Capped |
Spherical |
3, 5 and 10 |
80 |
220 |
0.50 |
11.5 |
yes |
|
ODS1 (C18) - Partially End Capped |
Spherical |
3, 5 and 10 |
80 |
220 |
0.50 |
6.2 |
no |
|
ODSB (C18) - Base De-activated |
Spherical |
5 |
80 |
220 |
0.50 |
11.5 |
yes* |
|
C8 |
Spherical |
3, 5 and 10 |
80 |
220 |
0.50 |
5.8 |
yes |
|
C6 |
Spherical |
3, 5 and 10 |
80 |
220 |
0.50 |
4.7 |
yes |
|
C1 |
Spherical |
3, 5 and 10 |
80 |
220 |
0.50 |
2.2 |
no |
|
Nitrile (CN) |
Spherical |
3, 5 and 10 |
80 |
220 |
0.50 |
3.1 |
no |
|
|
|
|
|
|
|
|
|
|
Amino (NH2) |
Spherical |
3, 5 and 10 |
80 |
220 |
0.50 |
1.9 |
no |
|
Phenyl |
Spherical |
3, 5 and 10 |
80 |
220 |
0.50 |
2.5 |
no |
|
|
|
|
|
|
|
|
|
|
OD/CN (Mixed Mode) |
Spherical |
5 |
80 |
220 |
0.50 |
5.0 |
yes |
|
|
|
|
|
|
|
|
|
|
SAX |
Spherical |
3, 5 and 10 |
80 |
220 |
0.50 |
4.0 |
no |
|
|
|
|
|
|
|
|
|
|
SCX |
Spherical |
3, 5 and 10 |
80 |
220 |
0.50 |
4.0 |
no |
|
|
|
|
|
|
|
|
|
* polar endcapping
I. GET TING START ED
Each Spherisorb column comes with a Performance Test Chromatogram. This Performance Test Chromatogram is specific to each individual column and contains the following information: gel batch number, column serial number, USP plate count, USP tailing factor, capacity factor, and chromatographic conditions. The performance test chromatogram should be stored for future reference.
a. Column Installation
Note: The flow rates given in the procedure below are for a typical 4.6 mm i.d. column. Scale the flow rate up or down accordingly based upon the column i.d., length, particle size, and backpressure of the Spherisorb column being installed. See “Scaling Up/Down Isocratic Separations” for calculating flow rates when changing column i.d. and/or length. See “Connecting the Column to the HPLC” for a more detailed discussion on HPLC connections.
1. Reversed-Phase Columns
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 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.
2. Normal-Phase Columns
Note: It is assumed that your system has been used for reversed-phase chromatography. If this is not the case, you can start with step 3.
1.Purge the pumping system of any buffer containing mobile phases.
2.Flush the system thoroughly with acetonitrile.
3.Switch the system over to the mobile phase that you are planning to use in normal-phase chromatography.
4.Connect the column and equilibrate it with the mobile phase.
Note: Equilibration with the mobile phase may require a larger amount of solvent than in reversed-phase chromatography.
Waters Spherisorb Columns |
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[ CARE AND USE MANUAL ]
b. Column Equilibration
Spherisorb columns are shipped in test mobile phase. 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 2 for a listing of empty column volumes).
1. Reversed-Phase Columns
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.)
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.
2. Normal-Phase (Spherisorb Silica, Amino, Cyano) Column
Spherisorb normal-phase (NP) columns are delivered in 96% heptane / 4% isopropyl alcohol. Care should be taken not to pass any mobile phase through the column that might cause a precipitate. Spherisorb NP columns are compatible with water and all common organic solvents, provided that solvent miscibility is accounted for.
Equilibrate normal-phase silica columns in the mobile phase. Very small quantities of water in the mobile phase can dramatically affect the activity of normal-phase packings. For good reproducibility, ensure that the mobile phase always has the same water content.
It is difficult and usually unnecessary to completely eliminate the water from the mobile phase. Dry mobile phases can take a very long time to equilibrate the column. A water content of 50 percent of saturation is recommended for most applications.
To equilibrate your column:
1.Starting at 0.0 mL/min, increase the flow rate in 0.1 mL/min increments to 1.0 minutes.
2.Purge the column with the mobile phase until you obtain a stable baseline.
3.Verify that retention times and peak areas for a standard are stable by comparing 2-3 replicate consecutive injections
Before you perform the first analysis on your new column, perform an efficiency test to confirm the performance of the column.
Table 2. Empty Column Volumes in mL (multiply by 10 for flush solvent volumes)
Column |
|
Column Internal Diameter (mm) |
|
|||||
Length |
1.0 |
|
2.1 |
3.0 |
4.6 |
10 |
|
20 |
|
|
|
|
|
|
|
|
|
20 mm |
- |
|
0.07 |
0.14 |
0.33 |
- |
|
- |
|
|
|
|
|
|
|
|
|
30 mm |
- |
|
0.1 |
0.2 |
0.5 |
- |
|
- |
|
|
|
|
|
|
|
|
|
50 mm |
0.1 |
|
0.2 |
0.3 |
0.8 |
- |
|
- |
|
|
|
|
|
|
|
|
|
100 mm |
0.1 |
|
0.4 |
0.7 |
1.7 |
- |
|
- |
|
|
|
|
|
|
|
|
|
150 mm |
0.1 |
|
0.5 |
1.0 |
2.5 |
12 |
|
24 |
|
|
|
|
|
|
|
|
|
250 mm |
- |
|
0.9 |
1.8 |
4 |
20 |
|
40 |
|
|
|
|
|
|
|
|
|
c. Initial Column Efficiency Determination
1.Perform an efficiency test on the column before using it. Waters recommends using a suitable solute mixture, as found in the “Performance Test Chromatogram”, to analyze the column upon receipt. However, if the column is used only for a single routine assay, it may be more convenient to test the column under these assay conditions. Keep a record of the initial column performance.
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.
Waters Spherisorb Columns |
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[ CARE AND USE MANUAL ]
II. COLUMN USE
To ensure the continued high performance of Spherisorb columns, follow these guidelines:
a. Guard Columns
Use a Waters Spherisorb guard column of matching chemistry and particle size between the injector and main column. It is important to use a 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 or other changes in chromatographic performance 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 Oasis® solid-phase extraction cartridges/ columns or Sep-Pak® cartridges of the appropriate chemistry to clean up the sample before analysis. Link to www.waters.com/sampleprep
2.It is preferable to prepare the sample in the operating mobile phase or a mobile phase that is weaker (less organic modifier in the case of reversed-phase chromatography, less polar modifier in the case of normal-phase chromatography or hydrophilic-interaction chromatography, less salt in the case of ion exchange) 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. 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 8,000 rpm, followed by the transfer of the supernatant liquid to an appropriate vial, could be considered.
c.pH Range
The recommended operating pH range for Spherisorb columns is 2 to 8. A listing of commonly used buffers and additives is given in Table 3. Additionally, the column lifetime will vary depending upon the operating temperature, and 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.
Waters Spherisorb Columns |
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