Waters µStyragel Column - Manual (Page 7)

[ Care and Use ManUal ]
µStyragel Column 7
INJEC
T
N = 25
2
( )
Test Procedure
1. Testing Instrument Band Spreading
The band spreading of a properly operating system should be less
than 150 µL. To determine the band spreading of your system:
1. Remove the column(s) from the system.
2. Connect the inlet and outlet tubing with a low dead-volume
union.
3. Inject the same sample as for a plate count determination. Use a
very fast chart speed to get a peak of easily measurable widths.
You may need to adjust the detector sensitivity to keep the peak
on scale.
4. Use the 5 sigma method, shown in Figure 3, to obtain a value
in µL.
5. Record the value. The band spreading of a properly operating
system should be less than 150 µL.
N = Column efficiency (plates) VR- Volume to peak apex (ml)
W = Volume at 4.4% of peak height (ml)
5 Sigma Method
Figure 6 illustrates the 5 sigma method, measuring the width at
4.4% peak height of diagram a Gaussian peak profile.
Figure 6: Method for Calculating Band Spreading
2. Column Efficiency Test
The column efficiency test described below may be used to calculate
a theoretical plate count, which is a measurement of column effi-
ciency within your system.
Solvents
It is not necessary to use the solvent that your column is shipped
in to determine the efficiency of your column. The solvent used in
normal operation is suitable. Initial plate counts determined using
solvents other than the one currently being used will have different
values.
Test columns individually using your normal operating solvent. To
perform the column efficiency test:
1. Slowly increase the flow rate to 1.0 mL/min.
2. Adjust the detector to an attenuation which achieves a peak of
70% full scale (noise level ≤0.5% full scale).
3. Set the recorder chart speed to 50 mm/min.
4. Refer to Table 6, Recommended Values for Column Efficiency
Test, for the sample to use when performing the efficiency test,
and for typical column efficiency results. For Ultrastyragel™
columns shipped in THF, use 1 µL of a 100% acetone solution
as a marker. For Ultrastyragel 100 and Linear columns shipped
in toluene, use 10 µL of a 5.0% dicyclohexylphthalate (DCHP)
solution as a marker made up in mobile phase.
5. Record the retention time, instrument settings, and column
configuration so you can reproduce them exactly for comparison
in the future.
6. Compute the plate count using the tangent method. Use these
results for comparison throughout the life of your column.
7. Replace any column that exhibits a plate count below the
acceptable range.