Waters Spherisorb Columns User Manual
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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
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.
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
Waters Spherisorb Columns 1
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Table 1. Spherisorb Column Physical Characteristics
Chemistry
Silica Spherical 3, 5 and 10 80 220 0.50 n/a n/a
ODS2 (C
ODS1 (C
ODSB (C
C
C
C
Nitrile (CN) Spherical 3, 5 and 10 80 220 0.50 3.1 no
Amino (NH
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
) - Fully End Capped Spherical 3, 5 and 10 80 220 0.50 11.5 yes
18
) - Partially End Capped Spherical 3, 5 and 10 80 220 0.50 6.2 no
18
) - Base De-activated Spherical 5 80 220 0.50 11.5 yes*
18
8
6
1
) Spherical 3, 5 and 10 80 220 0.50 1.9 no
2
Particle
Shape
Spherical 3, 5 and 10 80 220 0.50 5.8 yes
Spherical 3, 5 and 10 80 220 0.50 4.7 yes
Spherical 3, 5 and 10 80 220 0.50 2.2 no
Particle Size
(µm)
Pore Size
(Å)
Surface Area
2
(m
/g)
Pore Volume
(cc/g)
% Carbon
Load
Endcapped
* polar endcapping
i. GettinG started
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
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.
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
Waters Spherisorb Columns 2
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.
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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
Length
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
Column Internal Diameter (mm)
1.0 2.1 3.0 4.6 10 20
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 3
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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
®
columns or Sep-Pak
up the sample before analysis. Link to www.waters.com/sampleprep
cartridges of the appropriate chemistry to clean
solid-phase extraction cartridges/
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 combina-
tion with elevated temperatures will lead to shorter column lifetimes.
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 chro-
matography, less salt in the case of ion exchange) than the mobile
phase for the best peak shape and sensitivity.
Waters Spherisorb Columns 4
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