Dye Quality Control Reference Material Kit 4
[ CARE AND USE MANUAL ]
Example: If the dye is seen in the waste line 15 seconds before
the valve clicks to the collection position, the delay time is
calculated as follows:
Actual splitter collector delay = 30 – 15 = 15 seconds.
5. In the Timing tab of the FractionLynx method, change the Splitter/
Collector delay to the value just calculated, and rerun the
experiment to ensure that the delay time is correct.
6. Repeat the procedure for all flow rates, such as 10, 15, 20,
or 25 mL/min.
7. For the MS-based system, complete the procedure for APCI and
ESI if you are using both probes.
8. Perform a chromatography test of the entire system, as
described below.
c. Performing a Chromatography Test
To perform the chromatography test, an inlet method using the
parameters provided in this procedure needs to be created. The
XTerra 19 mm x 50 mm column is used to perform the tests in the
manual. Scale the method appropriately for different flow rates and/
or differently-sized columns. Use the flow rate that will be used for
purification.
To create an inlet method for the system test:
1. Set up the Inlet Editor to run a 10 minute method labeled “Dye.”
2. Set up the Waters 2545 binary gradient module to use
these parameters:
• Mobile Phase A: Water with 0.1% TFA or 0.2% formic acid
• Mobile Phase B: Acetonitrile with 0.1% TFA or 0.2%
formic acid
• Flow Rate: specify the same rate that you used in the delay
timing test.
• Run Time: 10 minutes
• Gradient conditions as specified in Table 2
Table 2. Gradient Conditions for the System Test
Time A% B% Curve Number
T = 0 minutes 95 5 6
T = 1 minutes 95 5 6
T = 7 minutes 30 70 6
T = 7.5 minutes 5 95 6
T = 8.5 minutes 5 95 6
T = 9 minutes 95 5 6
T = 10 minutes 95 5 6
3. In the Inlet Editor, set the wavelength range you want to
acquire, based on your detector type:
• For a Waters 2998 PDA Detector, in the Inlet Editor, set the
wavelength range to 210 nm to 650 nm.
• For a Waters 2489 dual wavelength absorbance detector,
in the Inlet Editor, set the single channel wavelength to
418. Set the dual channel wavelength to 590.
4. For an MS-directed system, create a mass spectrometer method
with these parameters, and then set it to run for 10 minutes:
• Set up a Scan Function: 150 to 500 amu
• ES+
• Centroid Data
• 0.5 second scan time with a 0.1 second Inter Scan Delay
• Cone Voltage = 35 V
5. In the MassLynx sample list, perform these tasks:
• Specify an injection volume of 100 μL.
• For an MS-directed system, enter 227.1 in the Mass A col-
umn, 282.1 in the Mass B column, and 371.2 in the Mass C
column. In the Fraction Trigger 1 column, enter Mass A. In
the Fraction Trigger 2 column, enter Mass B. In the Fraction
Trigger 3 column, enter Mass C.
(5 pages)
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