Waters Dye QCRM Kit User Manual
[ CARE AND USE MANUAL ]
DYE QUALITY CONTROL REFERENCE MATERIAL KIT
CONTENTS
I. INTRODUCTION
a. MS-Directed Purification
b. The Purpose of a QCRM
c. What Affects Your QCRM Results
II. STORAGE AND STABILITY
III. RECOMMENDED USAGE OF THE DYE QCRM
a. Testing System Performance
b. Performing the Delay Timing Test Manually
c. Performing a Chromatography Test
IV. TROUBLESHOOTING
V. ORDERING INFORMATION
I. INTRODUCTION
The Quality Control Reference Materials (QCRM) portfolio is a unique
collection of standards and mixtures. These products allow users
to evaluate and benchmark the chromatography system before
analysis of critical material. The products in the portfolio are all
precisely formulated based on the expertise of Waters scientist.
The AutoPurification™ Dye QCRM contains 3 compounds including
Thionin, Thioflavin T, and Crystal Violet.
This standard mix is used to confirm the benchmark performance
of a Preparative/Purification System through fraction collection.
This particular QCRM is precisely formulated to allow the user to
verify correct fraction collection. This test is highly valuable to
perform upon first use/installation, and any time the system
has not been used or has had any changes. T hese compounds were
vigorously tested and evaluated and chosen because they provide
the following advantages:
• Well-separated
• Easily visually identified
515 makeup pump
(not s hown)
PC with MassLynx and
FractionLynx software
2767 sample
manager
Bottle
tray
2545 binary
gradient module
System fluidics
organizer
2998 PDA
detector
SQD2
detector
Dye Quality Control Reference Material Kit 1
a. MS-Directed Purification
The Waters MS-directed autopurification system uses the Waters
SQD2 Mass Detector as its primary detector.
UV-Directed SystemMS-Directed System
PC with MassLynx and
FractionLynx software
2767 sample
manager
Bottle
tray
2545 binary
gradient module
System fluidics
organizer
2489 UV
detector
[ CARE AND USE MANUAL ]
b. The Purpose of a Quality Control Reference Material
Waters recommends to benchmark a purification chromatographic
system with a QCRM prior to system usage when there is confidence
that the system is in good working order. It is recommended to run
and save the initial results and to continue to compare new QCRM
results to the previous benchmark before any critical assay is run,
and after any hardware, column, or mobile phase changes.
The QCRM standard benchmark result will be specific to the
performance of the system it is run on. All chromatographic systems
have some minor level of variability from run-to-run. Trending of
results over time is useful for defining variability on a single system,
multiple systems or on systems in different locations.
Setting specification for QCRM results should not be done without
sufficient data trending. Once variability is understood, QCRM
results will help determine the capability of the system to provide
reliable results.
c. What Affects Your Quality Control Reference
Material Results
The goal of the QCRM specifications and criteria will be to indicate
that the system is functioning as expected. The system is comprised
of many interdependent components working together to produce
results within an expected specification. An issue in any one of
the components can affect your QCRM result. Variability can
be attributed to hardware, software or chemical changes. All
components performing correctly will produce results within an
expected variability.
Sources of the variability that may affect system results include:
• Mobilephasepreparation • Temperaturecontrol
• Columnperformance • Datacollectionrate
• Tubingsize • Integration
• System component performance
(pump, injector, detector)
II. STORAGE AND STABILITY
The AutoPurification™ Dye QCRM contains 3 compounds including
Thionin, Thioflavin T, and Crystal Violet. The compounds are stable
in their original packaging, through the expiration date listed as
provided in 10 mL amber ampule before opening. This is intended
to be a one time use product. T he integrity of the standard can
not be guaranteed if stored after first use.
III. RECOMMENDED USAGE OF THE DYE QCRM
a. Testing System Performance
After installing FractionLynx™, it is recommended to test the system
to confirm that the instruments are functioning correctly, and that the
system produces the expected chromatographic results.
Testing involves using the Dye Kit (part number 716000765) to inject
dyes and subsequently collect them. A passing result for this system-
level test means that the system instruments function properly. If the
test results differ from those in the examples provided, ensure that
collection parameters are set appropriately.
The conditions defined in each of the following tests are designed
to help rapidly determine whether all the components function as a
system. Actual system performance (column load per run) depends
greatly on conditions such as the HPLC solvents or modifiers, and the
dimension of the columns.
For the MS-directed system test and the UV-directed system test,
many FractionLynx method parameters are identical, including the
gradient and UV detector parameters. The chromatogram examples
represent results that can be expected, although they can differ
somewhat from system to system.
b. Performing the Delay Timing Test Manually
Determine the delay time manually by injecting the dye standard
from the dye kit. The following test is conducted using a Waters
19 mm x 50 mm XTerra® column.
Differences in any of the components mentioned can result in system
to system variability of results even when each system’s components
are functioning correctly.
Dye Quality Control Reference Material Kit 2
[ CARE AND USE MANUAL ]
Note: When using a different size and type of column than the one
described here, the flow and injection volumes must be adjusted
accordingly. Pressure balancing and flow to the mass spectrometer
need to be performed at the purification flow rate. When using the
column isocratically, the retention time is approximately 90 to
120 seconds, based on the column size and flow rate.
Required materials:
• Stopwatch
• Mobile phases:
– A Water/TFA, 0.1% (v:v)
– B Acetonitrile/TFA, 0.1% (v:v)
• Sample dye kit, part number 716000765
Table 1. Test Dye Compounds Information
Item Thionin Acetate Thioflavin T Crystal Violet
m/z 227.1 282.1 371.2
UV
max
Formula C12H9N3SC2H4O
590 nm 418 nm 590 nm
C17H19CIN2S C25H30CIN
2
3
3. For MS-directed systems, create an MS method for a 3 minute scan
acquisition with electrospray + ve, and specify these parameters:
– Centroid = 150 to 550 amu scan for 0.5 seconds
– Inter-scan delay = 0.1 seconds
– Cone Voltage = 35 V.
To run the delay timing test
1. In the MassLynx sample list, specify these values:
• Specify the Injection Volume as 100 μL.
• Enter these values depending upon the detectors configured
in the system:
– For an MS-directed system, enter 227.1 in the Mass A column,
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.
– For a UV-directed system, enter 418 in the Wavelength
A column and 590 in the Wavelength B Column. In the
Fraction Trigger 1 column, enter Wavelength A for a PDA
detector or UV1 for a UV detector, such as a Waters 2489.
In the Fraction Trigger 2 column, enter Wavelength B for a
PDA detector or UV2 for a UV detector.
To set up the delay timing test:
1. In the FractionLynx method, set the split collector delay to
30 seconds.
2. In MassLynx™, create an inlet method with the following settings:
• Run at the required flow rate for 3 minutes with 10% A
and 90% B.
For UV-directed systems, set the UV detector to the following
parameters, and ensure that the connections on the UV Fraction
Manager correspond to the flow rate used for this test:
– Waters 2998 PDA Detector to scan from 200 to 600 nm
– Waters 2489 UV Detector to monitor 418 nm and 590 nm.
Dye Quality Control Reference Material Kit 3
Tip: UV1 and UV2 correspond to Channel A and Channel B wave-
lengths in the Inlet method. The setting in the Wavelength column is
for reporting purposes only; it does not control the 2489 detector. The
software uses the value set in the Fraction Trigger column, UV 1, UV 2,
against the value set in the Inlet Editor.
2. When the injection occurs, monitor the waste line from the
fraction collector valve. As soon as the dye appears in the
waste line of the fraction collector valve, start the stopwatch.
When you hear the fraction collector valve click to the dispense
position, stop the stopwatch. The dye should have eluted before
the collection valve opened.
4. Calculate the actual Splitter/Collector delay as follows:
The actual delay is equal to original Splitter/Collector delay
specified in the FractionLynx method (e.g., 30 seconds) minus
the time recorded on the stopwatch.
Loading...