Waters Styragel Columns User Manual
[ Care and Use ManUal ]
styragel column
I. IntroductIon
This manual covers the care and use of the Waters Styragel® HR, HT, and HMW
families of Gel Permeation Chromatography (GPC) columns. Please take a few
moments to read this manual carefully. Follow the recommendations in this
manual to prolong column life and enhance chromatographic reproducibility.
This introduction describes the three families of Waters Styragel columns:
• Styragel HR
• Styragel HT
• Styragel HMW
Waters Styragel columns are packed with high-performance, fully porous, highly
cross-linked styrene-divinylbenzene copolymer particles. Their different charac-
teristics allow you to choose the column optimally suited to your application.
Styragel columns are shipped in three solvents: toluene, tetrahydrofuran (THF),
and dimethylformamide (DMF).
contents
I. IntroductIon
a. Styragel HR
b. Styragel HT
c. Styragel HMW
II. InstallIng the column Bank
a. Preparing the GPC/HPLC System
b. Installing the Columns
c. Repairing Damaged Compression Screw Assemblies
d. Equilibrating the Column Bank
III. PreParIng solvent and samPles
a. Preparing the Solvent
b. Changing Solvents
c. Preparing the Sample
Iv. usIng the column
a. Chromatography Guidelines
b. Calibrating the Column
Styragel Columns 1
v. care and maIntenance
a. Troubleshooting
b. Storing the Column
c. Efficiency Testing
1. Testing Instrument Band Spreading
2. Column Efficiency Test
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a. Waters Styragel HR
Use Waters Styragel HR columns for the high-resolution analysis of low-
molecular weight polymers, oligomers and additives. Packed with 5 µm
particles, Waters Styragel HR columns provide the high plate counts necessary
for this type of analysis.
b. Waters Styragel HT
Use Waters Styragel HT columns for the analysis of polymers with mid-range
molecular-weight distributions. They are the most versatile columns for
molecular-weight analysis. Waters Styragel HT columns are packed with 10 µm
particles to provide dependable performance over a wide range of temperatures
and solvents.
c. Waters Styragel HMW
Waters Styragel HMW columns are designed for the molecular-weight analysis
of ultrahigh-molecular-weight polymers. Their 20 µm particle size together with
the nominally 10 µm HMW frit design prevents the breakdown of ultrahigh
molecularweight polymers due to shear, which can occur with smaller particles.
This manual covers both column sizes 7.8 x 300 mm columns, and the solvent
efficient 4.6 x 300 mm columns. In sections with recommend flow rates or
spare parts, the 4.6 mm column conditions, or spare part follow immediately
after the 7.8 mm recommendations.
3. Flush the system to remove any microparticulates and old solvents.
Flush the injector loop if applicable.
Band spreading
The connection tubing and fittings in any chromatographic system contribute
to extra-column band spreading. Before installing the column, me sure your
system instrument band spreading (see Section on Testing Instrument Band
Spreading). If this test is not possible with your system, refer to your system
operator’s manual.
Narrow-Bore Chromatography for 4.6 mm Solvent Efficient Columns
The peak volume in a narrow-bore system is so small, it is critical to minimize
band spreading. Use the shortest tubing possible for all connections. It is not
necessary to use a microbore flow cell in your detector or to change your
conventional HPLC system in any way. Use 0.009-inch (0.25 mm) i.d. tubing
throughout the system.
b. Installing the Columns
When connecting columns in series, use the 0.009 inch (0.25 mm) i.d.
U-shaped column-joining tube supplied with each column.
Sequence of columns in a column bank
II. InstallIng the column Bank
This chapter describes:
• Preparing the GPC/HPLC system
• Installing the columns
• Repairing damaged compression screw assemblies
• Equilibrating the column
a. Preparing the GPC/HPLC System
Before attaching the columns in the flow path on a GPC/HPLC system, you
must first prepare the system:
1. Directly connect the system injector to the detector by replacing
the old columns with a zero-dead-volume union.
2. Convert the system to the solvent in which the columns have been
stored. For a new column set, this is the shipping solvent.
Generally, the results of an analysis are independent of the sequence in which
a column bank is arranged. However, to improve resolution and column life,
arrange the columns in order of decreasing pore size, with the column with
the largest pore size closest to the injector. This is recommended because:
• The columns with the larger pore sizes are more rugged and are
better able to tolerate the accumulation of extraneous materials.
• The species with the highest molecular-weight in the sample
contributes the most to the viscosity of the sample. If the largest
species is separated first, the viscosity decreases more quickly,
placing less strain on the column bank. In the case of ultrahigh MW
polymers, there is less shear on the sample.
Styragel Columns 2
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Installing columns in a column bank
To install your columns:
1. Remove the end plugs from each column and save them.
2. Connect the first column to the injector outlet tubing. Ensure that
solvent flow is in the direction shown by the arrow on the column
label. Finger-tighten the fittings, then tighten the fittings with a
wrench by another 1/4 turn.
3. Connect the next column to the previous column using the U-tubes
supplied with the columns. Ensure that solvent flow is in the
direction shown by the arrow on the column label. Thread the
inlet and outlet fittings of the U-tube until finger tight, then
tighten the fittings by another turn with a wrench.
4. Repeat step 3, until all columns are connected.
5. Connect the last column to the detector inlet tubing using steps
1 through 4.
c. Repairing Damaged Compression Screw Assemblies
To remove a damaged compression screw or a worn ferrule assembly:
Figure 1. Ferrule and Compression Screw Assembly
Ferrule 0.009-inch I.D. Tubing (0.25 mm) 0.130-inch gap (3.3 mm)
The distance between the end of the ferrule and the end of the U-tube may
differ for different column types. If you have used columns from another
manufacturer, it may be necessary to reset the ferrules, or make up a new
fitting (see Figure 1). Use 0.009-inch (0.25 mm) i.d. tubing for all lines
between the injector and detector. For Waters columns, the critical distance is
0.130 inch (2.25 mm).
d. Equilibrating the Column Bank
Equilibrate the columns when you install them and when you use them after
they have been stored. To equilibrate your column bank:
1. Set the pump flow rate at 0.0 mL/min, then turn on the pump.
1. Scribe the circumference of the tubing at the desired break point
using a tube cutter or a file with a cutting edge.
2. Grasp the tubing on both sides of the scribe mark with cloth-
covered pliers (to prevent marring the tube surface). Gently
work the tube back and forth until it separates at the scribe mark.
Ensure that the tubing end is straight, open, and free of burrs.
3. Slide the compression screw, followed by the ferrule (large end of
the taper first), over the tube. Properly bottom the tubing in the
fitting seat. If the tubing is not completely seated, the resulting
dead volume can lead to poor chromatographic results.
2. Increase the flow rate by 0.1 mL/min at 15 second intervals until
you reach the final flow rate.
3. Purge the columns with the shipping solvent until you obtain a
stable baseline.
Styragel Columns 3
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Flow rate and backpressure for 4.6 mm
Solvent Efficient Columns
For best resolution and maximum column life, do not allow the flow rate to
exceed 0.3 mL/min or the backpressure to exceed 3.5 MPa (500 psi, 35 atm)
per column.
Please note: The flow rate recorded on the Certificate of Analysis, included
with the column, may be higher than the guidelines provided. For maximum
column life in your lab, please follow the flow and back pressure guidance
we provide.
When using the HR 0.5, HR 1, and HR 2 columns, increase the flow rate in 0.1
mL/min increments at 30-second intervals until you reach 0.3 mL/min.
Defective columns
One or more defective columns in a series may cause the entire set of columns
to appear defective. One defective column can cause peak spreading that
cannot be overcome by any number of good columns. See Table 4, Table 5,
or Table 6 for column efficiency data.
Initial efficiency tests
Test your system and columns before the first analysis. Run a test sample
using the recommended parameters for your system and columns, and record
the results. These results serve as a baseline to compare future performance.
See Section on Efficiency Testing, for the procedures to determine the
efficiency of your system and columns.
a. Preparing the Solvent
Use clean solvent for reproducible results and maintenance-free operation. Use
solvents of LC-grade or better, filtered to remove micro particulate matter larger
than 0.45 µm. Refer to Waters catalog, filtration section for filter choice and
solvent compatibility chart.
b. Changing Solvents
Solvent compatibility
Waters Styragel columns are shipped in the solvent of your choice: toluene,
THF or DMF. Some applications require a different solvent. Changing solvents
works best between compatible solvents. Refer to the table below for solvent
compatibilities.
Note: The use of highly aqueous mobile phases may damage the resin and is
not recommended.
Table 1. Solvent Conversion Table
To convert to:
o-Dichlorobenzene Toluene
Trichlorobenzene Toluene Hexafluoroisopropanol THF
Phenol/TCB Toluene N-Methyl pyrrolidone DMF
y-Butyrolactone THF m-Cresol DMF
Use columns
shipped in:
To convert to:
To use Waters Styragel columns for high-temperature chromatography in
solvents like trichlorobenzene (TCB) or orthodichlorobenzene (ODCB), you
must convert the column to the selected solvent at elevated temperature.
Use columns
shipped in:
Save the chromatograms from these tests. For the column-efficiency test,
record the retention times, system settings, and all experimental conditions
so they may be reproduced exactly for future comparison.
III. PreParIng solvent and samPles
This chapter describes:
• Preparing the solvent
• Changing solvents
• Converting columns to high temperature solvents
• Preparing the sample
Styragel Columns 4
High-temperature conversion procedure
To convert the column bank to high-temperature operation:
1. Convert the system to the column shipping solvent at room
temperature.
2. Purge solvent efficient columns at 0.1 mL/min while gradually
increasing the temperature to 90 ˚C over a minimum of 3 hours.
Solvent Efficient Columns
(Purge solvent efficient columns at 0.1 mL/min while gradually
increasing the temperature to 90 ˚C over a minimum of 3 hours.)
Set the system to 55 ˚C when converting columns from THF to
HFIP or Y _ Butyrolactone.
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