Waters AccQ-Tag Amino Acid Analysis Column - Manual (Page 3)

[ CARE AND USE MANUAL ] [ CARE AND USE MANUAL ]
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• Protect the column from rapid changes in pressure, which can
result from rapid changes in eluent composition or flow rate.
• Use water that has been purified with a Millipore ® Water System
capable of delivering 18 megohm water. De-ionized water may
contain organic compounds that alter column selectivity.
b. Efficiency Testing
Perform an efficiency test before attempting the first analysis. Run
the test sample using the conditions detailed in the following pages.
Monitor column performance by measuring efficiency regularly over
the life of the column.
There are four parts to performing the efficiency test:
1) Conditioning the column
2) Preparing the acenaphthene standard
3) Running the acenaphthene standard
4) Calculating column efficiency
If you experience problems during operation, repeat the efficiency test
and compare the results. Differences may indicate a column problem.
1) Conditioning the Column
Condition the column with acetonitrile/water (60%/40%) when you
install it. To condition the column:
1. Set the column heater temperature to 37 ˚C.
2. Set the pump flow rate to 0.0 mL/min.
3. Increase the flow rate gradually in 0.1 ml/min increments to
1.0 ml/min.
4. Equilibrate at 37 ˚C for 20 minutes.
2) Preparing the Acenaphthene Standard
The test standard is 0.05% acenaphthene in acetonitrile. Prepare
an acenaphthene sample by dissolving 0.05 gram acenaphthene in
100 ml acetonitrile.
3) Running the Acenaphthene Standard
1. Set the gain on the detector to obtain a peak between 50 and 70%
of full scale.
2. Inject 10 pl test standard and run the analysis for 10 minutes.
3. Record results, instrument settings, and instrument configuration
for future comparison.
Table 1. AccQ•Tag ™ Efficiency Test Conditions
4) Calculating Column Efficiency
Record the retention time of the acenaphthene peak. Measure the
width of the peak at 4.4% of peak height and perform the calculation
shown in Figure 2. The column efficiency (N) should yield >9,000
theoretical plates.
Figure 2. 5-Sigma Method Test Calculations
c. Equilibrating the Column for the AccQ•Tag ™ Method
1. Equilibrate the column in 100% AccQ•Tag ™ Eluent A for
10 minutes at 1 ml/min.
Test Sample Acenaphthene (0.05% in acetonitrile)
Mobile Phase Acetonitrile/water (60%/40%)
Flow Rate 1.0 ml/min
Injection Volume 10 μl
Detection
(Waters 470 Scanning
Fluorescence Detector)
Excitation wavelength - 250 nm
Emission wavelength - 395 nm
Gain - 10
Filter - 0.5
Temperature 37 ˚C
Run Time 10 minutes