Waters Styragel Columns - Manual (Page 6)

[ Care and Use ManUal ]
Styragel Columns 6
Table 2. Recommended Sample Concentrations
Ultrahigh-molecular-weight polymers
Shearing is also a factor to be considered with most ultrahigh-molecular-weight
polymers. The effect of shearing may not be as easily observed as it is with
narrow molecular-weight standards. Ultrahigh-molecular-weight polyolefin frac-
tions are subject to possible shearing and incipient precipitation. Use especially
dilute solutions of these fractions (for example, 0.02 percent) to minimize
precipitation.
Iv. usIng the column
This chapter describes:
• Chromatography guidelines
• Calibrating the column
a. Chromatography Guidelines
Gel permeation chromatography columns have a finite lifetime directly related
to their care and use. Column life is reduced by contamination from samples
and eluents, frequent solvent changeover, improper handling and storage, and
temperature cycling.
Guidelines for column use
When using Waters Styragel columns, observe the following guidelines:
• For best resolution and maximum column life, do not exceed a flow
rate of 1.0 mL/min or the backpressure exceed 3.5 MPa (500 psi,
35 atm) per column (corrected for system backpressure).
• For solvent efficient columns, best resolution and maximum
column life, do not let the flow rate exceed 0.3 mL/min or the
backpressure exceed 3.5 MPa (500 psi, 35 atm) per column
(corrected for system backpressure). Normal flow rate for these
columns is 0.3 mL/min.
• Protect the column from vibration and mechanical shock.
• Minimize temperature cycling.
• Protect the column from rapid changes in pressure that can result
from rapidly changing the composition of the solvent.
• When changing to a solvent with a different viscosity, it may be
necessary to adjust the flow rate to stay below the operating
backpressure specification of 3.5 MPa (500 psi, 35 atm) per
column.
• Avoid precipitation by dissolving samples in the mobile phase.
• Always use high-quality HPLC solvents.
• Dedicate columns to specific applications. Frequent switching of
samples and solvents accelerates column deterioration and loss
of resolution.
• Using highly aqueous mobile phases may damage the resin and is
not recommended.
b. Calibrating the Column
Whenever you replace a single column or a complete column bank, generate a
new calibration curve to ensure the reproducibility of your application. Figure
2, Figure 3, and Figure 4 show typical calibration curves by column family. The
calibration curves were obtained with polystyrene standards.
Molecular-weight Range Sample Concentration
0 to 25,000 <0.25%
25,000 to 200,000 <0.1 %
200,000 to 2,000,000 <0.05%
Above 2,000,000 <0.02%
Styragel ® HR
Molecular W
eight
Styragel ® HR 0.5
Styragel ® HR 1
Styragel ® HR 2
Styragel ® HR 3
Styragel ® HR 4
Styragel ® HR 5
Styragel ® HR 6
Styragel ® HR 4E
Styragel ® HR 5E
Elution Volume (mL)
Styragel ® HR
Molecular W
eight
Styragel ® HR 0.5
Styragel ® HR 1
Styragel ® HR 2
Styragel ® HR 3
Styragel ® HR 4
Styragel ® HR 5
Styragel ® HR 6
Styragel ® HR 4E
Styragel ® HR 5E
Elution Volume (mL)
Figure 2. Calibration Curves for Waters Styragel HR Columns