Natural Resources Canada
CFS - GLFC
STANDARD OPERATING PROCEDURE
Title: Agilent 1100 HPLC
SOP Number: M52506/029/002
Effective Date:
TITLE: Agilent 1100 HPLC
APPROVING OFFICIALS:
DD / MM / YY
Approved By: _________________
Concurred By: _________________
Prepared By: _________________
Management
QAU Manager
___________
___/___/___
___/___/___
___/___/___
SIGNIFICANT CHANGES FROM PREVIOUS VERSION:
-this SOP has been revised to include a section for significant changes from
the previous version.
- quality control check samples are re-analyzed at the end of the sequence
to allow assessment of accuracy and precision for samples in matrix.
- a new definition has been added to differentiate between instrumental
verification and analytical method validation.
- section 2.5 has been re-subtitled to specify that it pertains to instrument
verification.
- section 2.7 has been revised to require copies of verification records to be
maintained in the study manual as well as in the instrument log book.
1.0 INTRODUCTION
1.1 Purpose
To assure calibration, operation and maintenance of the Agilent 1100 HPLC
(“Tigger”) and associated Chemstation (“Thelma”) used for instrument
control, data acquisition and data evaluation.
1.2 Scope
This study-specific SOP is a GLFC management directive that shall be
followed by all study personnel using the Agilent 1100 HPLC (“Tigger”) and
the associated Chemstation (“Thelma”) in the conduct of a GLP compliant
study.
1.3 Definitions
Analyte(s) – the specific compound or compounds of interest to the analyst
conducting qualitative or quantitative analysis.
Archivist - The person specified by management who is responsible for the
management of the archive(s).
Calibration – in the context of this document, calibration refers to the
process of determining response factors used to calculate absolute
component concentrations by injection of specially prepared calibration
standards.
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STANDARD OPERATING PROCEDURE
Title: Agilent 1100 HPLC
SOP Number: M52506/029/002
Document Control Official (DCO) - The person specified by management
who is responsible for controlling designated documents, including SOPs.
Effective Date - The date from which the procedures given in a SOP are to
be implemented.
Good Laboratory Practice (GLP) – A quality management system set forth
by the Organization for Economic Cooperation and Development (OECD) to
assure the quality and integrity of the processes and conditions under which
non-clinical health and environmental safety studies are planned,
performed, monitored, recorded, archived and reported.
GLPsave Register – a specific protected checksum, encoded binary file,
saved automatically at the end of each analysis when selected in the run
time checklist which contains information on the signal, logbook, integration
results table, quantification results table, instrument performance and data
analysis method which can be used to prove originality, quality, and
authenticity of the data and quality performance of the instrument.
HPLC system - in the context of this document is defined as being
comprised of the Agilent 1100 HPLC (“Tigger”), its modular components
including autosampler, thermostated columns compartment, column
switching valve, binary pump, vacuum degasser, diode array detector (DAD)
and the associated Chemstation (“Thelma”).
Logbook – A detailed chronicle of all maintenance, repair and/or calibration
conducted on a specific instrument used in generation, analysis or sample
measurement to produce raw data.
Maintenance – Routine actions taken on an instrument or piece of
equipment to assure continuous performance according to accepted quality
standards and specifications.
Method – in the context of this document, method refers to a detailed
description of all parameters required for specific and reproducible
acquisition and analysis of data, including pre-run and post-run tasks such
as calibration and integration.
MSDS – Material Safety Data Sheet, a summary description of a chemical,
reagent or substance prepared by the manufacturer or supplier and
required by WHMIS legislation to inform workers about procedures required
to safely work with the material.
NA - Not Applicable.
Quality Assurance Unit (QAU) - Persons designated by management to
ascertain that the study is in compliance with current OECD Principles of
Good Laboratory Practice, and who are completely independent from the
conduct of the study for which they are performing quality assurance
functions.
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STANDARD OPERATING PROCEDURE
Title: Agilent 1100 HPLC
SOP Number: M52506/029/002
Repair – Periodic replacement, change or renovation of parts of an
instrument or piece of equipment to compensate for accidental breakage or
wear and to assure good operating condition of the instrument or
equipment.
Study Manual - 3-ring binder(s) or similar device(s) prepared in
accordance to this SOP and which is used to compile and organize all
information pertinent to a specific study.
Standard Operating Procedures (SOPs) - Management directives
describing administrative or technical routine procedures conducted in a
laboratory or field operation that are not specified in detail in the study plan
or test guidelines.
Study Director – The individual responsible for the overall conduct of the
non-clinical health and environmental safety study.
Study Specific Technical SOP – A SOP describing a routine technical
procedure relating to a unique piece of equipment (e.g., using a custombuilt piece of equipment) or to a specific work unit and not normally not
required/followed by other departments at GLFC. The SD (or PI, if
applicable) of the work unit that developed the SOP is responsible for its
validity and for ensuring that personnel engaged in this activity function in
compliance with described procedures.
Test Facility Management – The individual who has the authority and
formal responsibility for the organization and functioning of the test facility
according to current OECD Principles of Good Laboratory Practice.
Validation – the process of evaluating analytical methods to ensure
compliance with requirements in terms of accuracy, precision and
reproducibility of results
Verification – the process of checking and confirming correct functioning of
an analytical instrument or other piece of equipment and associated
software typically achieved by running a standard verification test and
comparing results against expected values to determine pass or fail status.
1.4 Safety
1.4.1
1.4.2
1.4.3
All persons must be familiar with emergency procedures as
described in the CFS-GLFC Emergency and Fire Safety Manual,
prior to working in a laboratory using hazardous materials. WHMIS
training is recommended.
Persons using hazardous materials must use minimum personal
protective equipment as stipulated in the current version of SOP
No. M52506/014.
Persons using hazardous materials must do so in accordance with
the pertinent MSDS sheets and WHMIS labels. These items are
normally stored on a shelf in the laboratory in accordance with the
current version of SOP No. M52506/015.
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1.4.4
1.4.5
1.5
STANDARD OPERATING PROCEDURE
Title: Agilent 1100 HPLC
SOP Number: M52506/029/002
Decanting, transfer or other handling of large (>500 mL) containers
of solvents must be conducted in an approved, operational fume
hood, as set forth in the current version of SOP No. RS9701/001.
Preparation of calibration or analytical samples must be conducted
in an approved operational fume hood, as set forth in the current
version of SOP No. RS9701/001.
Materials
Materials required for calibration and routine operation of the HPLC
system include:
(a)
Agilent 1100 HPLC “Tigger” (degasser, photo diode array
detector, fluorescence detector, autosampler, pump, and
column compartment) column switching valve and
chemstation “Thelma”.
(b)
Laser Jet printer, ink cartridge and paper.
(c)
Solvent or solvent mixture of choice.
(d)
Solvent reservoir bottles
(e)
HPLC vials/inserts and caps suitable for use with Agilent
1100 series autosampler.
(f)
4 L Winchester bottle for collection of waste mobile phase.
(g)
Analytical column of choice.
(h)
Pre-columns or cartridges suitable for use with selected
analytical column
(i)
0.22 um nylon membrane filters (solvents, aqueous)
(j)
Filter stones to attach to pump tubing in solvent reservoir
bottles
(k)
Instrument logbooks for “Tigger” and “Thelma”
(l)
Replacement parts required for routine maintenance and
repair (located in drawers under the instrument in F166C)
(m)
Reference manuals (refer to section 8.0 (f))
(n)
Analytical standards
(o)
Disposable pipettes and bulbs
2.0 PROCEDURES
2.1 Initial Installation, Setup and Verification
2.1.1
Initial installation, setup and verification procedures must be
conducted by or under the guidance of a qualified technical
representative of the manufacturer.
2.2 Routine Maintenance and Repair
2.2.1
2.2.2
Routine maintenance and repair actions shall be conducted in
accordance with reference manuals for individual components as
listed in section 8.0 (f). All maintenance and repair actions shall be
recorded in the instrument logbooks.
Should any maintenance or repair action require opening of
capillary tubes or fittings, there may be exposure to solvents,
therefore these procedures should be performed in accordance
with the current version of SOP No. M52506/014.
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2.2.3
STANDARD OPERATING PROCEDURE
Title: Agilent 1100 HPLC
SOP Number: M52506/029/002
A common problem in HPLC analyses is microbial or other
contamination of solvents used in the mobile phase and
subsequent plugging of filters stones, frits or other small orifice
components in the system. The problem is particularly common
with aqueous buffers. To minimize this potential, all aqueous
buffers must contain 4% methanol. To further limit microbial
growth, amber reservoir bottles should be used or clear bottles
should be covered with tinfoil. Filter all solvents through minimum
0.22 μm filters.
2.3 Routine Start-Up, Calibration and Operation
2.3.1
2.3.2
2.3.3
2.3.4
2.3.5
2.3.6
2.3.7
2.3.8
2.3.9
Power to all HPLC system components is routinely left on; check to
ensure all component modules have power by observing front
panels for yellow (standby) LED lights.
Open the faceplate of the thermostated column compartment and
check to ensure that the appropriate guard and analytical columns
required for your application as stated in the study plan are in place
and that the column switching valve is set to deliver mobile phase
to that column.
Check to ensure that appropriate solvents, (as specified in the
study plan or analytical method) are in place and that reservoirs
contain sufficient volume for the run. Refill solvent reservoirs if
necessary using high purity (HPLC grade or equivalent) water or
solvents. All solvents used for HPLC analysis will be obtained
directly from Winchester bottles located in the solvent storage
cabinet in F166D. All water and solvents used in HPLC analysis
will be filtered prior to use through a new 0.22 μm filter.
Check the mobile phase waste bottle (4 L winchester bottle)
located in the labeled cupboard below the instrument to ensure
sufficient capacity.
Replace with empty winchester bottle if
necessary and dispose of full waste bottle in accordance with the
current version of SOP No. M52506/006.
Turn on the computer and monitor of the chemstation “Thelma”.
Login to the chemstation using the designated password.
Click the icon labeled as “Tigger online” to load chemstation
software.
Once the software loads, ensure that the system is in method and
run control view mode or change to this view by clicking the view
menu in the upper toolbar and choosing method and run control
from the drop down box.
Ensure that the appropriate method (as specified in the study plan
or chosen by operator where a study plan does not pertain) is
shown in the method file (*.M) box in the top portion of the method
run and control window. If the appropriate method is not shown,
reset by clicking “Method” and select “Load Method”. From this list
click on the appropriate method to be used.
Click on “Instrument” in the upper tool bar and select “System on”
to turn on the binary pumps. Allow the system to pump for a
minimum of 20 minutes prior to initiating an actual run sequence to
ensure that the system is fully purged and mobile phase solvents
are degassed. Observe tubes running from solvent reservoirs to
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CFS - GLFC
2.3.10
2.3.11
2.3.12
2.3.13
2.3.14
2.3.15
2.3.16
2.3.17
STANDARD OPERATING PROCEDURE
Title: Agilent 1100 HPLC
SOP Number: M52506/029/002
the binary pump module for bubbles. If bubbles are observed in
the tubes, purge and prime the system as described in the Agilent
1100 series binary pump reference manual.
While the pump is running, check that all components of the
system diagram on the method run and control page are colored
green. Any component colored in yellow or red is either not ready
or in error, requiring specific trouble shooting actions as specified in
the reference manual for that component or in the on-line help
available by clicking “Help” in the upper toolbar.
When all system components are functioning properly (colored
green) click on “Sequence” in the upper tool bar and choose
“Sequence Table”. Enter the specific parameters of the sequence
table as required for the application.
For quantitative analyses, the first 5 sample lines in the sequence
table will be utilized for system calibration by either external or
internal standardization techniques as specified in the study plan or
analytical method. Thus, the first five entries of the sequence table
will typically identify a calibration standard concentration series and
will specify “calibration” in the “Sample Type” column. For the
typical application, check to ensure that the first sequence table line
for the calibration standard shows “replace” in the response factor
(RF) and retention time (RT) update columns. Subsequent lines 25 for calibration standards should indicate “average” in these
column cells.
Complete the sequence table as required for specific applications.
Typically, this involves listing unique sample identities for each
sample to be analyzed. At minimum, for each set of 10 samples to
be analyzed a selected calibration standard (typically from the midpoint of the concentration series or a concentration that is similar to
that of the samples) is re-run at the mid-point and end of the
sequence and analyzed as if it were a sample to allow for a postrun check of accuracy and precision. In addition, quality control
check samples are re-analyzed at the end of the sequence to allow
assessment of accuracy and precision for samples in matrix.
For all analytical runs, the last sample run in the sequence will be a
blank mobile phase sample with an appropriate shutdown method
designated in the method column.
With the sequence table still open, load all samples into
appropriate positions in the autosampler tray and double check to
verify that vial position and sample identities in the tray match with
those in the sequence table.
Once the sequence table has been completed, click on
“Sequence” and choose “Save Sequence”. This should be done
every time a change is made to the sequence table.
Click on “Sequence” and select “Sequence parameters” from the
drop down list. Edit the sequence parameter table as necessary to
ensure that the operator name, data file prefix and counter values
are accurate. Also ensure that the files will be saved in the correct
location by looking at the Path. If the instrument is to be shut down
at the end of the run as stated in section 2.3.14, make sure that the
Post Sequence Command is checked and the macro is set to
“macroShutdown.mac”. In cases where the same data file prefix
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CFS - GLFC
STANDARD OPERATING PROCEDURE
Title: Agilent 1100 HPLC
SOP Number: M52506/029/002
is being used, ensure that the counter value is increased by one
unit from the last data file number so that previous files are not
overwritten. The last data file number with this prefix can be
determined by using the windows file explorer tool to view data files
in the appropriate subdirectory.
2.3.18 When sequence parameter editing is complete, view the baseline
in the on-line plot window to ensure a stable base line, and that all
system components are colored green. In cases where these
conditions are not met, troubleshooting in accordance with the
reference manuals or on-line help (available by clicking “Help” in
the upper toolbar) may be required.
2.3.19 If the baseline is stable and all systems are colored green choose
“RunControl” from the upper toolbar in the method and run control
view and choose “Run Sequence” to initiate the analytical run.
2.3.20 During the analysis, where practical, check the instrument status
periodically to ensure that no errors are indicated.
2.4 Shutdown Checks and Procedures
2.4.1
2.4.2
2.4.3
As a routine, shutdown of the instrument is performed automatically
by including a “shutdown” sample and invoking a specific
“shutdown method” in the sequence table and in the sequence
parameters as described in section 2.3.14 and 2.3.17 above.
Specific shutdown methods are created by a trained operator and
may vary with the application involved. However, each shutdown
method will at minimum turn off the binary pump, turn off the diodearray detector, and ensure that the guard and analytical columns
have been purged with a mobile phase appropriate for short or
longterm column storage.
In the case of a power outage during the course of a run the
system will shut down. In this event, samples should be removed
from the carousel and placed in appropriate storage as defined in
the study plan. Power buttons on the front of each system module
should be turned off. When power is fully restored, turn power
buttons on each instrument module back on and follow procedures
for routine startup, calibration and operation as outlined in section
2.3.
2.5 HPLC System Verification
2.5.1 The Certificate of Validation shipped with each software package is
to be retained in the instrument logbook.
2.5.2 Prior to use of the HPLC system for GLP compliant studies, system
verification checks must be run to demonstrate that the instrument
and associated software, or relevant components thereof, are
performing correctly at the time of analysis.
2.5.3 System verification with the Agilent Chemstation can be performed
by running a standard verification test following instructions from the
“Help Topics” section of the online “Help” system. Select “Contents”
and then “Verification View “.
2.5.4 Results of system verification tests must be printed and retained in
the instrument logbook.
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STANDARD OPERATING PROCEDURE
Title: Agilent 1100 HPLC
SOP Number: M52506/029/002
2.5.5 When running HPLC analysis for GLP compliant studies, the
GLPsave register shall be invoked by selecting this option in the Run
Time checklist. The GLP review function can be used to show that
chromatographic data are original, prove the quality of the analysis
from the instrument performance data and to demonstrate the
authenticity of the data interpretation.
2.5.6 Prior to validation of analytical methods and use of the HPLC system
in GLP compliant studies, a diode array detector (DAD) test will be
performed to assess the performance of the detector in terms of
intensity and wavelength calibration. To do this click “Help” on the
top toolbar, select “Help Topics”. View the “Contents” page, select
“Diagnosis View”, then “Agilent 1100 Diagnosis Test”, and then
select “Diode Array Detector Test Procedures”. Test results must
be saved by pressing the save function to retain results electronically
in the DADTest database as a file protected by a checksum, and
binary encoded to ensure that it is not changed.
2.5.7 Similarly, prior to validation of analytical methods requiring
fluorescence detection for GLP compliant studies, the fluorescence
detector (FLD) performance must be checked against specifications
published by the manufacturer and results documented in logbook
for “Tigger”. Performance tests should be conducted in accordance
with manufacturer instructions for “Verifying the Performance of Your
Detector” as outlined in the Fluoresence Detector – Info and Trouble
shooting manual.
2.5.8 Using automated functions of the Chemstation software, system
suitability is to be determined prior to and at the end of routine
analyses for a given GLP study. System suitability tests are
comprised of instrument and methods tests, which determine
precision for parameters of peak retention time, peak area, amount,
peak height, peakwidth at half height, peak symmetry, peak tailing,
capacity factor (k’), plate numbers, resolution, selectivity relative to
preceding peak, skew and excess. The mean, standard deviation,
coefficient of variation and confidence interval are calculated for
each parameter according to standard formulae as detailed in the
Agilent Chemstation Manual.
2.6 Calculations
NA
2.7 Documentation and Reporting
2.7.1 Any documentation pertaining to initial installation, system verification
or other diagnostic checking of the instrument hardware or software
is to be retained in the instrument logbook.
2.7.2 Any maintenance or repair actions taken on an instrument must be
recorded as set forth in the current version of SOP No. M52506/001.
Each maintenance and repair action must be recorded on the day
that the action was taken and initialed by the person performing the
maintenance or repair action.
2.7.3 Routine daily calibrations will be retained together with analytical
results in study manuals for the specific study in question.
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CFS - GLFC
STANDARD OPERATING PROCEDURE
Title: Agilent 1100 HPLC
SOP Number: M52506/029/002
2.7.4 Information and data pertaining to system verification conducted in
conjunction with a GLP compliant study will be retained with the
study specific records and copies maintained with the instrument
logbook.
3.0 DISTRIBUTION AND ARCHIVING
3.1 Distribution
3.1.1
3.1.2
3.1.3
Official copies of this SOP shall be made by the DCO and
distributed as follows:
(a) The original SOP and an exact copy shall be retained in the –
GLFC-GLP archive (i.e., copies 001 and 002).
(b) One copy shall be kept by the DCO in the DCO SOP manual
(i.e., copy 003).
(c) One copy shall be kept by the QAU manager in the QAU
manager SOP manual (i.e., copy 004).
The DCO may distribute additional copies of this SOP to other
individuals requiring training and or use on this instrument.
The DCO shall announce the availability of new versions of this
SOP within one week of the effective date, and shall distribute new
versions by requiring that old versions be exchanged for the new
ones.
3.2 Archiving
3.2.1
3.2.2
The Archivist shall maintain current and historical versions of this
SOP.
The study director, or PI where applicable, shall ensure that the
contents of the equipment logbook pertaining to GLP studies, are
archived annually. Verified copies of the material may be retained
by the study director or PI for historical reference.
3.3 Destruction of Outdated SOPs
With the exception of archived historical versions, the DCO shall ensure the
destruction of retired versions of this SOP by shredding.
4.0 ASSURING SOP VALIDATION AND COMPLIANCE
4.1 SOP Validation
The Study Director is responsible for assuring that this SOP is valid.
4.2 SOP Compliance
The Study Director is responsible for assuring that this SOP is followed.
5.0 REVISION OF THE SOP
5.1 Responsible Individual
The Study Director is responsible for initiating the revision process.
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CFS - GLFC
STANDARD OPERATING PROCEDURE
Title: Agilent 1100 HPLC
SOP Number: M52506/029/002
5.2 Revision Schedule
5.2.1
5.2.2
This SOP shall be revised when its provisions no longer agree with
current practices or applicable regulations.
If this SOP requires revision, it shall be revised according to current
GLFC procedures and approved within 45 calendar days of
initiation of the revision process.
6.0 CONTINGENCIES
When personnel find circumstances that do not permit compliance with this
SOP, they shall consult both the QAU manager and the Study Director.
7.0 CONFIDENTIALITY
All SOPs are considered confidential and shall be handled accordingly.
They are not to be copied or made available to outside parties without
proper safeguards and the approval of management. Distribution to
outside parties shall be conducted by the DCO.
8.0 REFERENCES
(a)
(b)
(c)
(d)
(e)
(f)
CFS-GLFC Emergency and Fire Safety Manual.
SOP No. M52506/001
SOP No. M52506/014
SOP No. M52506/015
SOP No. RS9701/001
Reference manuals for the HPLC system
(i) Agilent 1100 series binary pump
(ii) Agilent 1100 series autosampler
(iii) Agilent 1100 series thermostated column compartment
(iv) Column switching valve
(v) Agilent 1100 series vacuum degasser
(vi) Agilent 1100 series diode array detector
(vii) Agilent Chemstation
(viii) HP1046A Programmable Fluorescence Detector – Info and trouble
shooting manual
9.0 APPENDICES
NA
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