[ CARE AND USE MANUAL ]
Preparative Chromatography Mix Standard
CONTENTS
I. INTRODUCTION
a. The Purpose of a QCRM
b. Determining your QCRM Criteria
c. What Affects your QCRM Result
II. STORAGE & STABILITY
III. USING THE PREPARATIVE CHROMATOGRAPHY MIX
IV. QCRM TESTING
V. TROUBLESHOOTING
VI. ORDERING INFORMATION
I. INTRODUCTION
The Quality Control Reference Material (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 preparative chromatography mix is 5 mg/mL each of Diclofenac
sodium salt, Diphenhydramine hydrochloride, and Flavone in DMSO.
This standard mix should be used to confirm the benchmark
performance of your Preparative/purification system. This particular
QCRM is a precisely formulated mix that includes a void marker,
neutral, acidic and basic compounds. These compounds were
vigorously tested and evaluated and chosen because they provide
the following advantages:
– Well-separated
– Easily visually identified
– Acceptable for use on a variety of column
Preparative Chromatography Mix
a. The Purpose of a QCRM:
Waters recommends to benchmark your chromatographic system with
a QCRM prior to system usage when there is confidenence your system
is in good working order. It is recommended to run and save the
initial results and continue to compare your QCRM results to the
previous benchmark any critical assay is run, and after any hardware,
column or mobile phase changes.
The QCRM 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 the benchmark
results over time will provide an understanding of system typical
variability. Trending of the same QCRM result on multiple systems
will provide the typical variability of those systems. Trending of the
same QCRM result on systems in laboratories in different locations
will provide the typical variability from across locations. Setting
specification for QCRM results of a system, multiple systems or
1
[ CARE AND USE MANUAL ]
between laboratories 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.
Determining your QCRM Criteria:
QCRM criteria should be determined based on specific requirements.
As mentioned above, specifications should not be set until the
variability of the system population is understood. The criteria and
specifications should allow it to be determined if the QCRM results
indicate that the system is functioning as expected or outside of
expectation. Typical criteria might include any of the following:
retention time reproducibility, peak area reproducibility, peak tailing
plate count, peak resolution, mass accuracy range, sensitivity
or response.
b. What affects your QCRM result:
The goal of the QCRM specifications and criteria will be to
indicate that the system is functioning as expected or outside
of expectation.
The system is comprised of many interdependent components
working together to produce results to an expected specification.
An issue with any one component can produce erroneous final
results. All components performing correctly will produce results
within an expected variability. Any changes or technical issues
within any one of the system components (hardware, software, or
chemical) may add variability to the QCRM result. Potential causes
of variability in QCRM results may include the following: mobile
phase preparation, column performance, tubing size, system
component performance (pump, injector, detector), temperature
control, data collection rate, integration.
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.
II. STORAGE AND STABILITY:
The compounds are stable through the expiration date listed
as provided in 1 mL amber ampule before opening. This product
is for one time usage. The integrity of the standard can not be
guaranteed if stored after first use.
III. USING THE PREPARATIVE CHROMATOGRAPHY MIX
For preparative chromatographic analysis on a 19 x 50 mm column the
Preparative Chromatography Standard mix was injected at 10 µL. The
injected quantity should be scaled for other column diameters.
Sample chromatography for the Preparative Chromatography
Standard is shown in Figure 1. Note that the use of different column
stationary phases and/or column dimensions will have a effect on
the separation. On different column chemistries or dimensions, the
method may need to be modified or re-developed to obtain sufficient
resolution. To properly transfer the separation across column
dimensions, use the Prep Calculator. www.waters.com/prepcalculator.
The table below indicated the approximate retention times obtained
for the compounds when using the specified chromatographic method
in Figure 1, as well as the m/z criteria for each compound.
Approximate RT (min)
Compound Type
Diclofenac
sodium salt
Diphenhydramine
hydrochloride
Flavone Neutral 223.07 4.3
Table 1: Preparative Mix UV and MS
Acid 296.02 4.6
Base 256.17 2.8
MS
(M+ H)
(XSelect™ CSH™ C18, 5 µm,
19 x 50 mm)
220 nm
Preparative Chromatography Mix 2
[ CARE AND USE MANUAL ]
91
91
Figure 2 shows an example of the
chromatography obtained for the
preparative mix via UV and MS
when the method in Figure 1 is
using an XSelect™ CSH C18, 5 µm,
19 x 50 mm.
8.0e-1
6.0e-1
AU
4.0e-1
2.0e-1
0.0
12345678
99
%
-1
1
2345678
IV. QCRM TESTING
The use of reference standards for QCRM testing should allow the
analyst to track important instrument analytical parameters such as
peak width, peak area, retention time, and peak resolution. Each of
these important parameters can be tracked and evaluated using control
charts. The use of a high quality reference standard allows the analyst
to reliably measure and track these parameters.
QCRM testing should be performed on a regular basis for each
instrument/analyst combination or instrument per test method. The
data should be collected and entered into a control chart allowing
the analyst to evaluate the system performance over time. The use of
performance control charts has been a staple of analytical chemistry
quality control. The most common form of the control charting is to
track the analytical results and statistically analyze the data to a 99%
(3 standard deviations) or 95% confidence interval (2 standard
deviations) confidence interval around the mean of the data to
esta blish upper control limits (UCL) and lower control limits (LCL).
The initial criteria to establish a mean, standard deviation and
control limits involves analyzing a reference material a minimum
of 7 times to establish an initial estimate of precision and bias. This
provides the analyst with sufficient data to be statistically valid. The
analyses should be carried over the course of several days to provide
a more realistic view of the system variability. The frequency
of analyzing system performance will be dependent on the stability
of the analysis and the analytes. QCRM should always be evaluated
after maintenance has been performed, or when changes to the
system or analytical procedure have been made.
2
3
1
1. Diphenhydramine
2. Flavone
3. Diclofenac
220 nm
+
TIC; ES
The example in Table 1 uses retention time monitoring to establish
a set of control limits for the purpose of monitoring on-going
system performance.
Table 1: Reference Standard Retention Time Data Example
Analysis Peak Retention Time (mins)
1 7.10
2 7.11
3 7.12
4 7.09
5 7.08
6 7.10
7 7.11
8 7.13
9 7.10
10 7.11
Mean 7.11
Standard Deviation 0.0136
LCL 7.08
UCL 7.13
The standard reference material was analyzed 10 times yielding
the above retention times. The mean retention time and standard
deviation were calculated and from this the UCL and LCL limits
were determined. The control limits represent a 95% confidence
interval (2 standard deviations) for the data. The control chart
in Figure 2 was then produced to establish that the instrument
retention times are in control.
0 min
0 min
Preparative Chromatography Mix 3
[ CARE AND USE MANUAL ]
Figure 2: Retention Time Control Chart
1 Initial 25.00 95.0 5.0 Initial
2 0.10 25.00 95.0 5.0 6
3 6.10 25.00 5.0 95.0 6
4 7.10 25.00 5.0 95.0 6
5 7.20 25.00 95.0 5.0 6
6 10.10 25.00 95.0 5.0 6
Time
(min)
Flow
(mL/min)
A = 0.1% TFA in water
B = 0.1% TFA in acentonitrile
%A %B Curve
The establishment of control limits provides data as to the current
capabilities of the system. Control charting allows the quality
professional to compare instrument performance to the required
method specifications.
The process of continuous quality improvement can also be tracked
using control charts. When improvements are made to a method,
control charts allow you to see that the changes you have made are
effective and having the desired impact. The control chart will also
allow you to track trends over time. By observing the data trending
higher or lower over time, you can take preventative action prior to
having an out of specification result.
V. TROUBLESHOOTING:
The Preparative Chromatography Standard contains an acidic, basic
and neutral compound. Due to the vastly different properties of
stationary phases, not all compounds will behave similarly on each
column. For instance, basic compounds often have poor peak
shape on reversed-phase columns at neutral pH due to increased
interactions between the charged bases and silanols on the surface
of the stationary phase. For this reason, it is very important to
benchmark the performance of the Preparative Chromatography
Standard on a new column and functioning system. This will help
to identify whether poor peak shape issues are due to compound
interactions on the column, or failing column/system performance.
Failure to meet QCRM criteria will result in the need to troubleshoot
the system. Some chromatographic issues may be easily resolved,
for instance, a missing peak may simply be due to co-elution of
two peaks. However, most issues such as poor peaks shape, tailing
peaks, retention time shifts and poor peak response, to name a few,
may be due to a variety of causes that can be difficult to pinpoint.
For a detailed and comprehensive guide to troubleshooting, please
refer to the HPLC Troubleshooting Guide (WA20769) on the
Waters website.
Control charting can be employed for each QCRM criteria; peak
retention time, peak area, peak width, and peak resolution. Control
charts allow quality control professionals to establish statistically
significant criteria to monitor and control their HPLC analyses
thereby avoiding criteria that are too stringent or set arbitrarily.
Summary
The use of high quality reference standards specifically designed for
the system analysis, provide a controlled, consistent, and reliable
measure of system performance. Regular use of reference standards
and control charting the data provides improved monitoring of system
performance and system robustness, while at the same time
providing assurance that any results produced are high quality,
reliable, and reproducible.
References
1) Taylor, J.K., “Quality A ssurance of C hemical Measurements”, Lewis Publishers, 1987
2) Smith, G.M., “Statistic al Process C ontrol and Quality Improvement”, 3rd edition,
Prentice Hall, 1998
3) Ahuja, S. and Dong, M.W., “Handbook of Pharmaceutical Analysis by HP LC”, Elsevier
Inc., 2005
Preparative Chromatography Mix 4
VI. ORDERING INFORMATION
Description Part Number
Preparative/Purification Chromatography
Mix Standard
Thank you for choosing a QCRM from Waters. The standards are
manufactured in our ISO 9001 ISO 17025 facility. Each standard
is manufactured to ensure optimal reproducibility from lot to lot. A
Waters QCRM can be depended on for its’ accuracy. This removes
one variable from your system variability and provides you the most
dependable starting point for your testing.
If the QCRM box shows significant damage, notify the carrier and
your supplier at once and retain evidence of shipping damage so that
a claim can be made.
186006703
[ CARE AND USE MANUAL ]
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Preparative Chromatography Mix 6
[ CARE AND USE MANUAL ]
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