Zeml-2010b Eng LC Oqp Qa

　　　　　　　　　　　　　　　　　　　　ZEML-2010B
　　　　　　
Document No.:
SHIMADZU HPLC SYSTEM
Operational Qualification
Operational Qualification Protocol
We here by confirm that all contents (including changes) of the operational qualification procedures described in this
document have been approved in the presence of the performer, reviewer and the manager.
・ Performer Signature Date
Print / /
Title
Certificate No.
(Shimadzu-certified Performer only)
Company
・ Reviewer Signature Date
Print / /
Title
Company
・ Manager Signature Date
Print / /
Title
Company
Operational Qualification Table of Contents
Table of Contents
1. Definitions.........................................................................................................................3
1-1 Documentation..........................................................................................................................3
1-2 Scope.........................................................................................................................................4
1-3 Entries and Revisions................................................................................................................4
2. Preliminary Checks.....................................................................................7
2-1 Designation and Recording of Reviewer...................................................................................7
2-2 Performer Identification & Approval...........................................................................................7
2-3 Planning Items to be Evaluated in Operational Qualification....................................................7
2-4 Calibration Certification and Instruments Used.........................................................................8
2-5 Other Tools Used.......................................................................................................................9
2-6 Before Inspections...................................................................................................................10
3. Operational Qualification Outline...............................................................11
3-1 Outline of Inspection Procedures for Components..................................................................11
3-2 Outline of System Inspection Procedures...............................................................................17
4. Operational Qualification Protocol.............................................................18
4-1 Solvent Delivery Module..........................................................................................................18
4-1-1 LC-20AD/20AT....................................................................................................................18
4-1-2 LC-20AB..............................................................................................................................22
4-2 On-line Degasser.....................................................................................................................26
4-2-1 DGU-20A3/20A5...................................................................................................................26
4-3 Low Pressure Gradient Flow Control Valve.............................................................................27
4-3-1 Low Pressure Gradient Flow Control Valve........................................................................27
4-4 Gradient Mixer.........................................................................................................................28
4-5 UV-VIS Detector......................................................................................................................29
4-5-1 SPD-20A/20AV....................................................................................................................29
4-6 Spectrofluorometric Detector...................................................................................................34
4-6-1 RF-10AXL/RF-10AXL Super...............................................................................................34
4-7 Refractive Index Detector........................................................................................................37
4-7-1 RID-10A...............................................................................................................................37
4-8 Column Oven...........................................................................................................................40
4-8-1 CTO-20A/20AC/10Avp/10ACvp/10ASvp............................................................................40
4-9 System Controller....................................................................................................................44
4-9-1 CBM-20A/20Alite.................................................................................................................44
4-10 Autosampler............................................................................................................................46
4-10-1 SIL-20A/20AC.....................................................................................................................46
4-11 Data Processing Unit...............................................................................................................49
4-11-1 LCsolution...........................................................................................................................49
4-11-2 CLASS-VP..........................................................................................................................60
4-12 Photodiode Array Detector......................................................................................................64
4-12-1 SPD-M20A..........................................................................................................................64
4-12-2 SPD-M10Avp.......................................................................................................................67
4-13 System.....................................................................................................................................70
4-13-1 System with UV-VIS Detector.............................................................................................70
4-13-2 System with Photodiode Array Detector.............................................................................76
4-13-3 System with Spectrofluorometric Detector..........................................................................83
4-13-4 System with Refractive Index Detector...............................................................................87
5. Change List.....................................................................................................................91
Page 2 of 97 Rev. 3.12

Operational Qualification Definitions
1. Definitions
1.1 Documentation
1. This document stipulates the procedures and the documentation of the Operational Qualification (OQ)
of the Shimadzu High-performance Liquid Chromatograph. When all procedures are executed and all
items pass the inspection, it is verified that the system operates to satisfy the intended purpose.
The contents of this document must be checked and approved by the Performer, Reviewer and
Manager before implementing the Operational Qualification.
2. The following are established as the responsible parties in the execution of inspections.
・Performer
The Performer is the individual who actually performs the procedures stipulated in the Installation
Qualification Protocol and the Operational Qualification Protocol.
The Performer shall be an individual who, having been trained by the Quality Assurance Department
of the Analytical & Measuring Instruments Division of Shimadzu Corporation, has been certified as
an IQ/OQ inspector by the General Manager of the Quality Assurance Department, or an individual
recognized by the Manager to be equipped with the ability to conduct this inspection.
・Reviewer
The Reviewer is an individual designated by the Manager charged with responsibility for the
system.
The Reviewer verifies the contents of the Installation Qualification Protocol and Operational
Qualification Protocol. In addition, the Reviewer shall attend the inspection procedures together with
the Performer, and evaluate the procedures.
The Manager can serve concurrently as the Reviewer.
・Manager
The Manager is the individual responsible for administering the entire HPLC system being
inspected.
As well as verifying the contents of the Installation Qualification Protocol and Operational
Qualification Protocol, the Manager shall also grant final approval to the inspection procedures.
3. The Performer shall first check the content of this document. After checking all items, the Performer
shall sign and date the cover sheet of this document and submit this document as the Operational
Qualification Protocol report. (OQ must be performed by a trained and knowledgeable individual in
accordance with 21 CFR Part 211 Section 211.25 (cGMP, USA promulgated 29 September 1978)).
4. The Reviewer shall first check the content of this entire document. After checking all items, the
Reviewer shall sign and date the cover sheet of this document as approval of the Operational
Qualification Protocol.
5. The Manager shall first check the content of this entire document. After checking all items, the
Manager shall enter a unique document control number in the Document No. column at the top right
of the cover sheet, and then sign and date the cover sheet of this document as final approval of the
Operational Qualification Protocol.
6. This document is comprised of 1. Definitions, 2. Preliminary Checks, 3. Operational Qualification

Outline, 4. Operational Qualification Protocol, and 5. Change List. All of these sections must be
included.
7. This document contains important information and any form of duplication, photography, or
reproduction without the permission of the General Manager of the Quality Assurance Department,
Analytical & Measuring Instruments Division, Shimadzu Corporation, is prohibited.
Rev.3.12 Page 3 of 97
1.2 Scope
The Operational Qualification Protocol shall include the following.
・Operation check on each unit
Due to the modular nature of the system, the operation of each unit is checked separately.
・Operation check on overall system
Confirm that the system controller and workstation control each unit during analysis and that the
analysis results meet the prescribed criteria.
・Software and firmware check
The Operational Qualification also checks the software and firmware provided with each module.
The firmware checking is conducted based on version display and the Software Certificate of
Compliance. The software and firmware must be properly managed and change procedures must be
properly clarified
Analysis methods and data acquisition systems such as networks are not verified in the OQ of this system.
These must be separately verified by the customer.
1.3 Entries and Revisions

1. Making Entries
Use a black, permanent ballpoint pen, or equivalent, when recording results.
Names should be clearly printed.
Confirm that all items, except “Remarks” columns, are completed, and that there are no omissions.
Draw a diagonal line (from bottom left to top right) across all blank columns. (When making no entries, it
is not necessary to enter the “Change List No.__”, “Date” or “Signature”, or to enter the reason for the
change in the Change List of this document.)
Columns in which nothing has to be written in have been filled in with “N/A” in advance.
If choices are available within an item, place a checkmark in the box to select the appropriate decision.
2. Rounding of numeric values

If there are more significant figures in the measurement results than the specified criteria, round off the
result value at the number of significant figures required for the criteria. For example, if the criterion is
defined down to the first decimal place, round off the figure at the second decimal place of the result
value.
3. Deletion and correction of mistakenly filled items

To correct mistaken entries, delete the entire entry by drawing a diagonal line through it and enter the
correction in the margins. To correct numerical values or symbols (checkmark), draw 2 horizontal lines
across the mistake and re-write the entry. Moreover, write the date, and the signature, and the reason for the
change. (When deletion and correction of mistakenly filled items, it is not necessary to enter the “Change
List No.__”, “Date” or “Signature”, or to enter the reason for the change in the Change List of this
document.)
4. Changing or deleting steps, procedures or acceptance criteria

If there are sections of the steps or procedures which will not be applied, draw a diagonal line through the
entire item and write “Change List No. __” above the line and assign a number to it. Date and sign it.
Enter the necessary information in the Change List of this document.
When making a change, delete the existing description as explained above, enter the change point, and
then record the reason for the change in the Change List.
5. Revising entries
If the need to alter the contents of an entry arises, change the entry in accordance with “3. Deletion and
correction of mistakenly filled items”, and then write the reason for the change in the Change List of this
document.
When adding a new description, write it in the margins of the relevant page, and write “Change List No.
_”, the date and signature. Then, write the reason for the change in the Change List.
6. Handling Problems
Any problems that are identified in the Operational Qualification must be investigated and appropriate
actions must be taken. All such actions must be documented and be approved by the Manager.

7. In the event of a Fail

If troubles occur during an inspection and the item fails the inspection, place a checkmark in the “Fail”
box and describe the problem in the margins of the relevant page. Write “Change List No. _” and date and
sign the entry. Enter an outline of the problem in the Change List.
After the problem is solved, describe the solution and the result (“Pass”) in the margins of the relevant
page. Write “Change List No.__” and date and sign the entry. Enter the summary of the solution in the
Change List of this document.
8. Attaching data
Attached data are added by assigning page numbers as “A, B, C…” for each inspection item. If, for
example, there is a total of 3 sheets for attachment A, notate the pages as “73-A-1/3 - A-3/3”, ensuring that
no numbers are omitted. The Performer shall date and sign the attached data.
9. Other
Every item entered into the Change List shall be approved by the Reviewer and Manager.
Additional Information Significant Figures

The concept of significant figures is outlined below.
<What are Significant Figures?>

Significant figures are digits that are statistically significant in a measurement. The last digit of a significant
figure includes an error.
If we take, for example, a measured value 5.67, an error is included in the final digit, “7”. Despite the error in
the final digit “7”, the previous digit “6” is correct, and so the number of significant digits is three.
When making measurements, it is important to ascertain the number of significant digits for each measuring
instrument. For instruments with a digital display, the measurement sensitivity generally extends to the last
displayed digit, and so the last digit is considered to represent the lowest significant digit. However, the
sensitivity must be checked in the Instruction Manual.
For instruments with an analog display, the lowest significant digit corresponds to 1/10th of the smallest
increment on the scale. For example, measuring the length of an object with a ruler marked in 1-mm
increments permits measurement to the nearest 0.1 mm.
<Calculating Significant Figures>

Calculations based on measured values (such as calculating a mean value) result in more digits than the
number of significant digits used in the calculation. Such values must be rounded off to the number of
significant digits when the data is presented in reports. However, when a mean value, for example, is used in
further calculations, these calculations yield intermediate values with a larger number of digits, which are not
rounded during the calculations. No rounding for the purposes of bringing the figure in line with the
prescribed number of significant figures is conducted until the final result is obtained.
When quantities are being added or subtracted, the number of decimal places (not significant digits) in the
answer should be the same as the largest number of decimal places in the numbers being added or subtracted.
In a calculation involving multiplication or division, the number of significant digits in an answer should
equal the least number of significant digits in the numbers being multiplied, divided etc.
Uncertain value
(underlined) Uncertain value
(underlined)
Uncertain value
(underlined)
The value 1.25 was measured only to
the 1/100 digit position. Consequently,
the result becomes 5.62 (i.e., three
significant digits). As the digit "0" contains uncertainty,
the result becomes 13.0 (to three
significant digits).
<Format for Clearly Stating the Number of Significant Digits>

For example, the value 10,000 can be represented in the following ways to indicate different numbers of
significant digits.
Five significant digits 1.0000 x 104
Four significant digits 1.000 x 104
Three significant digits 1.00 x 104
Two significant digits 1.0 x 104
One significant digit 1 x 104
<References>
1. Writing a value as 0.00123000 indicates that it is accurate to six significant digits.
2. Subtracting 1.230 (four significant digits) from 1.231 (four significant digits) produces the result 0.001 ,
which is accurate to one significant digit (1 x 10-3).
3. Dividing 2.462 (four significant digits) by 1.231 (four significant digits) produces the result 2.000, which is
accurate to four significant digits (not 2).
4. If the number of significant digits of the measured result exceeds the number of significant digits of the pass
criteria, round the final result off to the number of significant digits of the pass criteria.

Operational Qualification Preliminary Checks
2. Preliminary Checks
2.1 Designation and Recording of Reviewer
Before conducting this inspection, the Manager shall designate the Reviewer. Record the Reviewer information
in the report. The Manager may also double as the Reviewer.
2.2 Performer Identification & Approval

The performer must be an individual who is trained and has adequate knowledge of this operational
qualification procedure. The Quality Assurance Department of the Analytical & Measuring Instruments
Division of Shimadzu Corporation performs such education, and recognizes individuals who are qualified to
perform the IQ/OQ procedures. An individual who is recognized as having been trained and who possesses
sufficient knowledge is certified by the General Manager of the Quality Assurance Department as a qualified
IQ/OQ Inspector, as proven by the certificate. A person who is recognized as having the ability to perform this
inspection may also perform this operational qualification procedure.
Before performing this operational qualification, the Performer’s qualification shall be confirmed and recorded
in the report.
2.3 Planning Items to be Evaluated in Operational Qualification

Prior to implementing this operational qualification, check the system configuration, determine the items to be
evaluated, record them in Operational Qualification Record and have them approved.
2.4 Calibration Certification and Instruments Used

The instruments and reagents used in the inspections are listed below.
When instruments controlled by the Shimadzu service company are to be used, copies of the Certificate of
Traceability and the Quality Assurance sheet will be submitted. However, if the assured performance
satisfies the required specifications, calibrated instruments controlled by the customer may be used in the
inspections.
(1) Items prepared by the Shimadzu Service Company
(1) Instruments for which the traceability certificate will be submitted
Certificates of traceability are attached to the following instruments controlled by the Shimadzu service
company.
* Thermometer and Recorder

The thermometer and recorder used for the Set Temperature Accuracy check on the Column Oven and
Autosampler Sample Cooler shall have a precision rating of 1.0C in the temperature range required for the
unit inspection (0C to 50C) as a guaranteed performance specification.
* Resistance temperature sensor

The resistance temperature sensor used for the Set Temperature Accuracy check on the Column Oven shall
conform to JIS Class A and have a precision rating of 0.5C in the temperature range required for the unit
inspection (0C to 50C).
* Thermocouple
The thermocouple used for the Set Temperature Accuracy check on the Column Oven and Sample Cooler shall
conform to JIS Class 1 and have a precision rating of 0.6C in the temperature range required for the unit
inspection (0C to 50C).
* DC voltage/current generator
The DC voltage/current generator used for the Chromatopac Hardware Validation check shall have a
precision rating of 0.15% at the test point as a guaranteed performance specification.
* Stopwatch
The stopwatch used for the pump Flow rate Accuracy check shall have a precision rating of 5'30" 0.3 s as a
guaranteed performance specification.
(2) Instrument and reagents for which Quality Assurance sheet is submitted upon request
A Quality Assurance sheet shall be submitted for the following instruments and reagents if requested by the
customer.
* Flasks
The 5 mL measuring flask used for the pump Flow rate Accuracy check.
(The amendment to the Measurement Law on November 1st 1993 excluded this item from the scope of the
Measurement Law.)
* Standard reagents for performance inspection

Acridine and anthracene (10 mg/L acetonitrile) 2 mL x 5 vials [228-32996-02]
For repeatability tests of system with spectrofluorometric detector
Caffeine aqueous solution, set of five concentrations 10 mL each ［228-45725-91］
For UV-VIS detector absorbance display, data output display, absorbance linearity and system
repeatability tests
Note:　［228-45725-91］ includes [228-45725-01](10mg/L)/ [228-45725-02](15mg/L)/
[228-45725-03](20mg/L)/ [228-45725-04](25mg/L)/ [228-45725-05](30mg/L).
Caffeine aqueous solution (250 mg/L) 50mL ［228-45725-06］
For gradient concentration accuracy test and system repeatability test of RID system
Glycerine (0.872 g/L water) 100 mL x 1 vial [228-32996-05]
For RID span tests
*In OQ report, place a checkmark in the box to select the appropriate decision for notes, and record the contents
for notes if necessary.

(2) Items to be prepared by the customer
* Electronic balance
A calibrated balance capable of measuring down to 0.001 g is required.
2.5 Other Tools Used

The tools, reagents etc. used in this inspection are indicated below.
These have been divided into items to be prepared by the customer, and items to be provided by the Shimadzu
service company.
(1) Items prepared by the customer

* Equipment
Glassware
* Solvents
Water For HPLC 2L
2-propanol Special Class reagent 1L
Methanol For HPLC 2L
Acetonitrile For HPLC 2 L (for system with RF detector only)
(2) Items prepared by the Shimadzu Service Company

* Equipment
Resistor tube, 4 m x 0.1 mm ID [228-32722-92] ; for pump, autosampler, system test
Low-pressure mercury lamp set [200-38423] ; for detector
Mercury lamp holder [228-24876] ; for SPD-10A(V) vp
Mercury lamp holder [228-34170-91] ; for SPD-M20A/10Avp
Mercury lamp holder [228-34478-91] ; for RF-10AXL
Teflon block ASSY [228-34319-91] ; for RF-10AXL
Syringe (10 mL) [046-00001] ; for detector
Syringe adaptor (standard accessory) [228-15672-91] ; for detector
Coupling1.6C (standard accessory) [228-16004-03] ; for detector
Male nut PEEK (standard accessory) [228-18565] ; for detector
Plug [228-16006] ; for RID-10A
Thermocouple fixing jig [228-35497-91] ; for CTO-10ASvp only
Aluminum tape (e.g., 3M Co., #425, width 18mm)[018-20072-01] ; for column oven
Thermocouple fixing jig [228-38106-91] ; for RF-10AXL Super only
Glass vial (1mL/1.5mL/4mL) ; for autosampler
Cap ; for autosampler
Septum ; for autosampler
Syringe (100L) ; for manual injector
Shim-pack VP-ODS 150 mm x 4.6 mm ID [228-34937-91] ; reverse-phase distribution column for
system test, or LUNA C18(2)
2.6 Before Inspections

Follow the procedure below to wash the flow channels before commencing the inspection.
(1) Remove the column and connect a resistor tube instead.
(2) Pump 2-propanol to wash the flow channels.
If the mobile phase previously used has no phase solubility with 2-propanol or if salt deposition is expected
from the buffer solution, wash the flow channels with an appropriate intermediate washing solution, and then
replace it with 2-propanol.
Consider the instrument history when determining the washing solution flow rate and the washing times.
Page 10 of 97 Rev. 3.12

Operational Qualification Operational Qualification Outline
3. Operational Qualification Outline

The inspection procedures for each of the regulated components and for the system are outlined below.
3.1 Outline of Inspection Procedures for Components

1. Solvent Delivery Unit LC-20AD/20AT/20AB
No. Item Outline
1 Display, LED test Checks the display and LED operation.
2 ROM, RAM self-diagnostic test Checks that ROM and RAM memory operates normally.
3 Firmware version check Checks the firmware version.
4 Time program test Checks the time program functions.
5 Solvent delivery stability test Checks that the pump pulsation meets the standards.
6 Pressure limiter test Checks the pressure limiter operation.
Checks the flow rate accuracy
7 Flow rate accuracy test (0.50mL/min,1.00mL/min,2.00mL/min) using a measuring
flask.
8 Leak sensor test Checks the leak sensor operation.
2. On-line Degasser DGU-20A3/20A5/DGU-3A/4A

No. Item Outline
1 Vacuum pump check Checks that the vacuum pump operates normally.
3. Low Pressure Gradient Flow Control Valve (For LC-20AD/20AT)/FCV-10AL

No. Item Outline
1 Solenoid valve test Checks that the valve switches correctly.
4. Gradient Mixer
No. Item Outline
This gradient concentration test is conducted as part of the
1 Gradient concentration test
gradient concentration test in the system inspections described.
5. UV-VIS Detector SPD-20A/20AV

No. Item Outline
Checks the operation of the display, LED, drive, and
Initialization test and ROM, RAM
1 wavelength accuracy test using the Hg emission line. Checks
self-diagnostic test
that ROM and RAM memory operates normally.
4 Lamp energy test Checks the brightness of the light sources used.
5 Lamp lighting time test Checks the accumulated time each lamp has lit.
Checks the wavelength accuracy in the UV range using a
6 Wavelength accuracy test
mercury lamp or caffeine standard solution.
Absorbance display/data output Checks that the displayed absorbance value equals the signal
7
test value output to the data-processing unit.
6. Spectrofluorometric Detector RF-10AXL/10AXL Super

No. Item Outline
1 Initialization test Checks the display, LED, and home position operation.
5 Lamp lighting time test Checks that the lamp lighting time is within the set time.
Uses the water Raman peak intensity and noise measurements
6 Sensitivity and S/N check
to check the sensitivity and S/N ratio.
Checks the firmware version number of the cell temperature
ROM, RAM self-diagnostic test
7 control unit, and the operation of ROM and RAM memory.
(Cell temperature control unit)
(RF-10AXL Super only)
Checks that temperature control precision and accuracy of the
Temperature control function
8 temperature-controlled cell unit is within the criteria range at a
check
temperature of 25C. (RF-10AXL Super only)
7. Refractive Index Detector RID-10A

No. Item Outline
1 Initialization test Checks that initialization completes without error.
4 Lamp energy test Checks that the lamp energy exceeds the acceptance criteria.
5 Lamp lighting time test Checks that the lamp lighting time is within the specified time.
6 Auto-zero test Checks that the auto-zero function works normally.
Temperature control function Checks that the temperature control function works normally.
7
check
8. Column Oven CTO-20A/20AC/10Avp/10ACvp/10ASvp

No. Item Outline
1 Display and LED test Checks the display and LED operation.
Checks the difference between the set temperature and actual
Set temperature accuracy (room
5 temperature at (room temperature - 5C). (CTO-
temp. - 5C)
20AC/10ACvp/10ASvp only)
Temperature adjustment accuracy Checks the temperature adjustment accuracy at (room
6
(room temp. - 5C) temperature - 5C). (CTO-20AC/10ACvp/10ASvp only)
Set temperature accuracy Checks the difference between the set temperature and actual
7
(40/50C) temperature at 40/50C.
8 Temperature adjustment accuracy Checks the temperature adjustment accuracy at 40/50C.
Page 12 of 97 Rev. 3.12

(40/50C)
9. System Controller CBM-20A/20Alite

No. Item Outline
2 Firmware version check Checks the contents and firmware version.
3 System control Checks the System Controller control operations.
Checks that the memory is backed up when the power is
4 Memory backup
turned off.
10. Autosampler SIL-20A/20AC/10ADvp

No. Item Outline
1 Display and LED test Checks the display and LED operation.
4 Basic operation check Checks that the basic operations are conducted correctly.
Checks the accuracy of the injected volume using 5μL, 10μL
and 50μL injections. However, evaluation of the injection
5 Injected volume accuracy test
volume accuracy for the 5μL and 10μL injections is taken
from the system repeatability test data.
Determines the difference between the set temperature and
Sample cooler set temperature
7 actual temperature of the sample cooler. (Only for SIL-20A
accuracy
and SIL-10ADvp with sample cooler.)
Page 14 of 97 Rev. 3.12

11. Data Processing Unit LCsolution

No. Item Outline
Personal Computer
1-1 Computer boot-up test Checks that the computer boots up and runs normally.
1-2 Time and date check Checks that the PC time and date are correct.
1-3 Hard disk drive test Checks that the hard disk operates correctly.
1-4 Print driver check Checks the print driver test page and the version number.
1-5 Network connection test Checks that the network connection is normal.
LCsolution
Checks that the software is not changed to an inappropriate
2-1 Software version check
version.
2-2 Program alteration check Conducts the program alteration check.
Checks the Shimadzu User Authentication Tool connection
Shimadzu User Authentication using database access and log registration. (This test is
2-3
Tool connection check conducted only if the Shimadzu User Authentication Tool is
used with LCsolution.)
Checks that the security functions operate, including
2-4 Security function tests
restrictions on the number of password characters.
Password log management Checks that a duplicated password cannot be used.
2-4-1
function check
Number of password characters Checks that the restriction on the minimum number of
2-4-2
check password operates.
2-4-3 Failed login detection Confirms that a failed login is detected.
Confirms that a user is locked out when the designated
2-4-4 User lockout function check
number of login failures is exceeded.
2-4-5 Failed login log Confirms that a failed login attempt is registered in the log.
Confirms that software use is restricted according to
2-5 User access rights check
differences in registered user access rights.
2-6 System policy function check Checks the LCsolution system policy function.
2-7 Audit trail check Checks the audit trail functions.
2-8 Screen lock function check Checks the screen lock function.
Test of function to prevent Confirms that a duplicate user name cannot be registered.
2-9
duplicate user registration
Checks the LCsolution peak integration, peak identification
from retention times, quantitative calculations using peak
2-10 Software function checks areas and peak heights, column performance parameter
calculations for each Pharmacopoeia, and the output of data
information, calibration curve information, and set parameters.
2-10-1 Peak integration Checks the peak integration functions.
Checks the quantitation calculation functions using the peak
2-10-2 Peak area percentage method
area percentage method.
Checks the quantitation calculation functions using the
2-10-3 Internal standard method
internal standard method.
Checks the quantitation calculation functions using the
2-10-4 External standard method
external standard method.
12. Data Processing Unit CLASS-VP

No. Item Outline
Personal Computer
1-1 Computer boot-up test Checks that the computer boots up and runs normally.
1-2 Time and date check Checks that the PC time and date are correct.
1-3 Hard disk drive test Checks that the hard disk operates correctly.
1-4 Print driver check Checks the print driver test page and the version number.
1-5 Network connection test Checks that the network connection is normal.
CLASS-VP
Checks that the software is not changed to an inappropriate
2-1 Software version check
version.
2-2 Program alteration check Conducts the program alteration check.
Page 16 of 97 Rev. 3.12

13. Photodiode Array Detector SPD-M20A/M10Avp

No. Item Outline
Checks the wavelength accuracy using the emission lines of a
2 Wavelength accuracy test
mercury lamp and deuterium lamp.
3 Lamp energy test Checks the brightness of the light sources used.
4 Lamp lighting time test Checks the accumulated time each lamp has lit.
3.2 Outline of System Inspection Procedures

1. System with UV-VIS Detector
No. Item Outline
Checks the concentration accuracy of the gradient system.
(Only for gradient system)
2 Noise/drift test Checks that the noise and drift values meet the criteria.
Checks the system repeatability of peak area and retention
3 UV-VIS repeatability test
time for different sample injection volumes.
2. System with Photodiode Array Detector

No. Item Outline
Checks the concentration accuracy of the gradient system.
2 Noise/drift test Checks that the noise and drift values meet the criteria.
3 PDA repeatability test
time for different sample injection volumes.
3. System with Spectrofluorometric Detector

No. Item Outline
1 RF repeatability test
time.
4. System with Refractive Index Detector

No. Item Outline
Measures the noise and drift while the flow cell is filled with
1 Noise/drift test
water.
2 RID repeatability test
time.
Page 18 of 97 Rev. 3.12

Operational Qualification LC-20AD/20AT Inspection Procedure
4. Operational Qualification Protocol

4.1 Solvent Delivery Module
4.1.1 LC-20AD/20AT
1. Introduction
This section presents the standard operating procedure (SOP) for Operational Qualification of the Solvent
Delivery Unit LC-20AD/20AT. Refer to the Instruction Manual for details about the operation procedure
and to clarify uncertain points.
Follow section 1-3 Entries and Revisions to change or delete the contents of the procedures.
2. Inspection Procedures
Test 1
(Item) Display, LED test
(Outline) Checks the display and LED operation.
(Procedure) 1) Turn on the power.

2) Check that all display dots and key panel LEDs light after the power is turned on.
(Criteria) All display dots and key panel LEDs light.
Test 2
(Item) ROM, RAM self-diagnostic test
(Outline) Checks that ROM and RAM memory operates normally.
(Procedure) 1) Turn on the power and press the [VP] key several times to display <VALIDATION>.
2) Press the [func] key several times to display <MEMORY CHECK>.
3) Press the [Enter] key.
(Criteria) Display shows “RAM OK/ROM OK”.
Test 3
(Item) Firmware version check
(Outline) Checks the firmware version.
(Procedure) 1) Press the [VP] key several times to display <PRODUCT INFO>.
2) Press the [func] key several times to display <S/W ID>.
3) The version number appears on the display.
(Criteria) The version number is displayed.

The version number matches the controlled version number.
Test 4
(Item) Time program test
(Outline) Checks the time program functions.
(Procedure) 1) Press the [edit] key to enter the time program edit mode.
2) Enter the following program step.
0.1 STOP
3) Press the [run] key and check that the prog run LED lights for six seconds and then
goes out.
(Criteria) Program executes normally.
Test 5
(Item) Solvent delivery stability test
(Outline) Checks that the pump pulsation meets the standards.
(Procedure) 1) Connect a 4 m x 0.1 mm ID resistor tube to the pump outlet.

2) Completely replace the mobile phase in the flow line with water.
3) Press the [func] key several times until the <PARAMETER> function is displayed,
and then press the [Enter] key.
4) Repeatedly press the [func] key until <COMP> is displayed, and record the currently
set value.
5) Enter 0.45 for <COMP>.
6) After pressing the [CE] key several times to return to the initial screen, press the [VP]
key several times to enter the <VALIDATION> group.
7) Press the [func] key several times and select <PULSE CHECK>. Enter “0.2:LC-
20AD”/“0.3:LC-20AT” and press the [Enter] key.
8) Press the [Enter] key to start the check, and at the point that the check is completed,
read the displayed value.
9) Check that the value falls within the criteria.
10) When the test is complete, revert <COMP> to the original value.
(Criteria) LC-20AD　　Pulsation ≦ 0.20 MPa (2.0kgf/cm2)

LC-20AT　　Pulsation ≦ 0.30 MPa (3.1kgf/cm2)
<Reference>１ kgf/cm2 = 0.098 MPa
Page 20 of 97 Rev. 3.12

Test 6
(Item) Pressure limiter test
(Outline) Checks the pressure limiter operation.

3) Set flow rate to 1 mL/min, P.MAX to 20MPa and P.MIN to 0MPa, and pump water
for one minute.
4) Monitor the pump pressure for one minute.
5) Add 1.0MPa to and subtract 1.0MPa from the measured pressure value monitored in
step 4), and use these values to set P.MAX and P.MIN, respectively.
6) Change the flow rate to 1.5 mL/min. Check that the pump stops and a P.MAX error is
displayed when the monitored pressure value exceeds P.MAX.
7) Press the [CE] key to reset the error.
8) Set the flow rate to 1.000 mL/min. again and pump water. Check that the pressure
stabilizes.
9) Change the flow rate to 0.5 mL/min. Check that the pump stops and a P.MIN error is
displayed several tens of seconds after the pressure decreases below the P.MIN value.
(Criteria) P.MIN and P.MAX errors are displayed.
Test 7
(Item) Flow rate accuracy test
(Outline) Checks the flow rate accuracy using a measuring flask.

3) Repeatedly press the [func] key to display < PARAMETER >. Press the [Enter] key.
4) Repeatedly press the [func] key until <COMP> is displayed, and after recording the
currently set value, enter 0.45 for <COMP>.
5) Set the flow rate to 0.5000 (for 20AT, 0.500) mL/min. and press the [pump] key to
start pumping water.
6) Wait five minutes until the pumping stabilizes.
7) Insert the resistor tube outlet into a 5 mL measuring flask.
8) Measure the time until the water reaches the line.
9) Set the flow rate to1.0000 (for 20AT, 1.000) mL/min. and press the [pump] key to
10) Repeat steps 6) through 8).
11) Set the flow rate to 2.0000 (for 20AT, 2.000) mL/min. and press the [pump] key to
13) After completing the test, display <COMP> as described in steps 3) and 4), and
return the set value to the recorded value.
(Criteria) Flow rate measured value (set flow rate 0.500 mL/min) = 0.50 ±0.01 mL/min
Flow rate measured value (set flow rate 1.000 mL/min) = 1.00 ±0.02 mL/min
Test 8
(Item) Leak sensor test
(Outline) Checks the leak sensor operation.
Press the [back] key to display <LEAK SENSOR TEST>.
2) Wet the temperature sensor under the leak sensor with cotton wool soaked in water.
3) Press the [Enter] key after 10 seconds.
(Criteria) “SENSOR GOOD” appears on the display.
Page 22 of 97 Rev. 3.12

Operational Qualification LC-20AB Inspection Procedure
4.1.2 LC-20AB
1. Introduction
This section presents the standard operating procedure (SOP) for Operational Qualification of the Solvent
Delivery Unit LC-20AB. Refer to the Instruction Manual for details about the operation procedure and to
clarify uncertain points.
Test 1

Test 2
Test 3
(Procedure) 1) Press the [VP] key several times to display <PRODUCT INFO>.
2) Press the [func] key several times to display <S/W ID>.

Test 4
0.1 STOP
goes out.
Test 5
(Item) Solvent delivery stability test
(Outline) Checks that the pump pulsation meets the standards.

3) Press the [func] key several times until the <PARAMETER> function is displayed,
4) Repeatedly press the [func] key until <ACOMP> is displayed, and record the
currently set value.
5) Enter 0.45 for <ACOMP>.
6) Record the current <BCOMP> value and change it to 0.45.
7) After pressing the [CE] key several times to return to the initial screen, press the [VP]
key several times to enter the <VALIDATION> group.
8) Press the [func] key several times to display <PULSE CHECK A>, enter 0.2, and
press the [Enter] key.
9) Press the [Enter] key to start the check, and at the point that the check is completed,
take the reading of the displayed value.
10) Check that the value falls within the criteria. Press the [Enter] key.
11) Press the [func] key to display <PULSE CHECK B>, and repeat the same procedure
as for <PULSE CHECK A>.
12) When the test is completed, return <ACOMP> and <BCOMP> settings to their
original values.
(Criteria) Pulsation ≦ 0.20 MPa (2.0kgf/cm2)
<Reference>１ kgf/cm2 = 0.098 MPa
Page 24 of 97 Rev. 3.12

Test 6
(Item) Pressure limiter test
(Outline) Checks the pressure limiter operation.
(Procedure) 1) Connect a 4 m x 0.1 mm ID resistor tube to the pump outlet.．

3) Set the flow rate to 1mL/min, B% to 0%, P.MAX to 20MPa and P.MIN to 0MPa, and
pump water for three minutes.
4) Monitor the pump pressure.
5) Set P.MAX 1.0 MPa higher than the pressure monitored at 4) above, and P.MIN
1.0MPa lower than the monitored pressure.
6) Change the flow rate to1.5 mL/min. Check that the pump stops and a P.MAX error is
displayed when the monitored pressure value exceeds P.MAX.
7) Press the [CE] key to reset the error.
8) Set the flow rate to 1 mL/min. again and pump water. Check that the pressure
stabilizes.
9) Change the flow rate to 0.5 mL/min. Check that the pump stops and a P.MIN error is
displayed several tens of seconds after the pressure decreases below the set P.MIN
value.
(Criteria) P-MIN and P-MAX errors are displayed.
Test 7
(Item) Flow rate accuracy test
(Outline) Checks the flow rate accuracy using a measuring flask.

3) Repeatedly press the [func] key to display < PARAMETER >. Press the [Enter] key.
4) Repeatedly press the [func] key until <ACOMP> is displayed, and after recording
the currently set value, enter 0.45 for <ACOMP>.
5) Press the [func] key to display <BCOMP>. Record the current value and change it to
0.45.
6) Set the flow rate to 0.5000 mL/min., B% to 0.0%, and press the [pump] key to start
pumping water.
7) Wait five minutes until the pumping stabilizes.
8) Insert the resistor tube outlet into a 5 mL measuring flask.
9) Measure the time until the water reaches the line and calculate the flow rate.
10) Set the flow rate to 1.0000 mL/min. and press the [pump] key to start pumping
water.
12) Set the flow rate to 2.0000 mL/min. and press the [pump] key to start pumping
water.
14) Set the flow rate to 0.5000mL/min, B% to 100, and press the [pump] key to start
pumping water.
15) Repeat steps 7) through 13) to check the accuracy of the pump B flow rate.
16) After completing the test, display <ACOMP> and <BCOMP> as described in steps
3) and 5), and return the set values to the original values.
(Criteria) Flow rate measured value (set flow rate 0.500 mL/min) = 0.50 ±0.01 mL/min
Test 8
(Procedure) 1) Turn on the power and press the [VP] key several times do display <VALIDATION>.
Press the [back] key to display <LEAK SENSOR TEST>.
Page 26 of 97 Rev. 3.12

Operational Qualification DGU-20A3/20A5 Inspection Procedure
4.2 On-line Degasser
4.2.1 DGU-20A3/20A5
1. Introduction
This section presents the standard operating procedure (SOP) for Operational Qualification of the On-line
Degasser DGU-20A3/20A5. Refer to the Instruction Manual for details about the operation procedure and to
2. Inspection Outline
Test 1
(Item) Vacuum pump check
(Outline) Checks that the vacuum pump operates normally.
(Procedure) 1) Turn on the pump that is connected to the online degasser.

2) Check that the red LED flashes, green LED flashes, and then the green LED
illuminates.
3) Check that the unit enters the standby status and that the red LED to detect leak does
not light.
(Criteria) Green LED lights; red LED does not light.
Operational Qualification Low Pressure Gradient Flow Control Valve Inspection Procedure
4.3 Low Pressure Gradient Flow Control Valve
4.3.1 Low Pressure Gradient Flow Control Valve
1. Introduction
This section presents the standard operating procedure (SOP) for Operational Qualification of the Low
Pressure Gradient Flow Control Valve incorporated into the LC-20AD/AT. Refer to the Instruction
Manual for details about the operation procedure and to clarify uncertain points.
Test 1
(Item) Solenoid valve test
(Outline) Checks that the valve switches correctly. The following procedures are implemented from
the solvent delivery unit LC-20AD or LC-20AT connected
(Procedure) 1) Press the [func] key of the connected pump repeatedly until <SYSTEM> is
displayed.
2) Press the [func] key several times to display <SYS>, and record the currently set
value.
3) Set the value to 1 to set the isocratic mode, and return to the initial screen.
4) Set the flow rate to 1mL/min.
5) Repeatedly press [func] until <PARAMETER> is displayed, press [Enter], and then
set <SV> to 1.
6) Lift all of the suction tubes out of the reservoir.
7) Start solvent delivery, and check that mobile phase A is flowing by the appearance of
bubbles in the mobile phase A suction tube.
8) Using the same procedure as in step 5), set <SV> to 2, 3, and 4 in sequence, and
check that mobile phases B, C and D are flowing by the appearance of bubbles in the
respective suction tubes. Confirm that valve switching is performed correctly.
9) Return the solvent delivery mode set in steps 1) and 2) to its original state.
(Criteria) Valve switches correctly.
Page 28 of 97 Rev. 3.12

Operational Qualification Gradient Mixer Inspection Procedure
4.4 Gradient Mixer
1. Introduction
This section presents the standard operating procedure (SOP) for Operational Qualification of the Gradient
Mixer. Refer to the Instruction Manual for details about the operation procedure and to clarify uncertain
points.
Test 1
(Item) Gradient concentration test
This gradient concentration test is conducted as part of the gradient concentration test in the system
inspections described.
Operational Qualification SPD-20A/20AV Inspection Procedure
4.5 UV-VIS Detector
4.5.1 SPD-20A/20AV
1. Introduction
This section presents the standard operating procedure (SOP) for Operational Qualification of the UV-VIS
Detector SPD-20A/20AV. Refer to the Instruction Manual for details about the operation procedure and to
Test 1
(Item) Initialization test and ROM, RAM self-diagnostic test
(Outline) Checks the operation of the display, LED, drive, and wavelength accuracy test using the
Hg and D2 emission lines. Checks that ROM and RAM memory operates normally.

3) Wait for the initialization test to complete and the results to be displayed.
4) Verify that <CHECK GOOD> is displayed on the screen.
(Criteria) “CHECK GOOD” appears on the display.
Test 2
(Procedure) 1) Press the [VP] key to display <PRODUCT INFO>, and press the [func] key several
times.

Page 30 of 97 Rev. 3.12

Test 3
0.1 STOP
goes out.
Test 4
(Item) Lamp energy test
(Outline) Checks the brightness of the light sources used.
(Procedure) ・ D2 lamp test

1) Press the [func] key to display <PARAMETER>. Press the [Enter] key.
2) Set the wavelength to 220nm and the Lamp to D2.
3) After pressing the [CE] key to display <PARAMETER>, press the [func] key
repeatedly to display <MONITOR>.
4) Press the [Enter] key, and record the displayed <REF EN> value.
・ W lamp test
5) For the SPD-20AV, continue by checking the W lamp brightness.
After pressing the [CE] key to return to the <MONITOR> display, press the [func]
key to display <PARAMETER>.
6) Press the [Enter] key, and set the wavelength to 540nm, and the lamp to W.
7) After pressing the [CE] key to display <PARAMETER>, press the [func] key
repeatedly to display <MONITOR>.
8) Press the [Enter] key, and record the displayed <REF EN> value.
9) Press the [func] key several times to display <CONTROL>. And press the
[Enter]key. Next, press the [func] key several times to display <W POWER>,and
record the displayed <W POWER> value.
(Criteria) D2 lamp energy at 220 nm ≧ 400 mV

W lamp energy at 540 nm ≧ 500 mV
Test 5
(Item) Lamp lighting time test
(Outline) Checks the accumulated time each lamp has lit.
(Procedure) 1) Press the [func] key several times until the < ＭＯＮＩＴＯＲ > function is displayed,
2) Press the [func] key to display <D2 TIME>. Record the D2 lamp lighting time
3) With the SPD-20AV, further press the [func] key to display <W TIME>. Record the
W (tungsten) lamp lighting time. (display units: hours).
(Criteria) D2 TIME≦ 2, 000h

W TIME≦ 2, 000h
Test 6
(Item) Wavelength accuracy test
(Outline) Checks the wavelength accuracy in the UV range using a mercury lamp or caffeine
standard solution.
(Method) Select either the method using the mercury lamp or the method using the caffeine
standard solution.
(Refer to Measurement Methods and Pass Criteria in the Additional Information below.)
(Procedure 1) 1) Turn on the power and fill the flow cell with water.
Mercury lamp 2) If the unit is in 2-wavelength simultaneous measurement mode, change it to 1-
method wavelength chromatogram mode.
3) Press the [func] key several times to display <CONTROL>, and then press the
[Enter] key.
4) <WAVE CHECK> will be displayed, so press the [Enter] key to execute the
wavelength accuracy check.
5) After the wavelength check is completed, confirm that <CHECK GOOD> is
displayed, and record the wavelength variance from 656nm and 254nm, respectively.
(Criteria) 254 nm and 656 nm emission peak   1 nm
(Procedure 2) 1) Turn on the power while holding down the [func] key.
Method using 2) Press the [func] key several times until the <FACTORY> function is displayed, and
caffeine then press the [Enter] key.
3) Press the [func] key several times until the <RESPONSE> function is displayed, and
then enter “1”.
4) Press the [func] key several times until the <LAMP> function is displayed, and then
enter “1(D2)”.
5) Press the [func] key several times until the <ZWAVE> function is displayed, and then
enter “1(F)”.
6) Fill the flow cell with water.
7) Change the wavelength setting to 350 nm, return to the initial screen, and press the
[zero] key.
8) Inject a 20 mg/L caffeine solution into the flow cell.
9) Set the wavelength to [266 nm] and record the displayed value.
10) Similarly, change the wavelength from [267 nm] to [277 nm] in 1 nm steps, and
record the corresponding displayed values.
11) Determine the wavelength where the maximum displayed value occurs.
(Criteria) Maximum absorption wavelength is 272  2 nm.
Page 32 of 97 Rev. 3.12

<Additional (Measurement Methods and Pass Criteria)

Information> ・Measurement method for the wavelength accuracy check
SPD-20A(V) uses zero-order light and the 486.0 nm and 656.1 nm emission lines of a
deuterium lamp (D2 lamp) for wavelength calibration. Strict calibration at these wavelengths
ensures wavelength accuracy across the UV range. Wavelength calibration using the emission
lines of a deuterium lamp (D2 lamp) is prescribed in the JIS standards, as follows:
JIS K0115 Rules for absorbance analysis

5.2.1 Wavelength Scale Calibration (2) Method using lamp emission lines
Measure the light intensity of a low-pressure mercury lamp or deuterium discharge tube
to determine the wavelengths exhibiting the maximum intensity, and compare with the
emission line wavelengths to calibrate the wavelength scale.
This method is used for the wavelength accuracy test in Test 3 Initialization Test.
The wavelength accuracy is ensured by the method above. However, this Wavelength
Accuracy Test (Test 6) confirms the wavelength accuracy using reference wavelengths in the
UV range that is often used for actual analysis.
・Mercury Lamp and Caffeine Pass Criteria

The low-pressure mercury lamp with an emission line at 253.65 nm and caffeine with
maximum absorbance at 272 nm can both be used as the reference wavelengths in the UV
range. Caffeine was also adopted as it is stable in storage and is easy to obtain and handle.
The wavelength accuracy in the UV range is determined by comparing these wavelengths
with the measured peak wavelengths.
The pass criteria for the low-pressure mercury lamp is 2541 nm and for caffeine is 2722
nm. In contrast with the monochromatic absorption line of the mercury lamp, the caffeine
maximum absorbance has some width, which makes accurate measurement of the peak
wavelength difficult. For this reason, the pass criterion is set lower for caffeine.
・Selecting the Mercury Lamp or Caffeine Method

The use of the mercury lamp is recommended to determine the wavelength accuracy more
precisely.
Use the caffeine method if a mercury lamp is unavailable or to make measurements in a
situation similar to actual detector operation.
(Additional Information)
Use the mercury lamp designated by Shimadzu for the wavelength accuracy test. The test
evaluation is based on a direct comparison of the emission line (natural physical constant)
unique to the lamp. The following document prescribes the use of natural physical constants
for traceability.
Recommended Practice
Medical Products And Pharmaceutical Calibration Control System
July 10, 1986, National Conference of Standards Laboratories
“5.2.2 Additional Traceability documentation (as required)
The use of calibration standards such as natural physical constants, derived standards,
standard reference materials, or internal standards which are not traceable to national
standards, will require adequate documentation to describe the validity of the
calibration standards used.”
<Additional (ZWAVE and SIG MODE Functions)

Information> <ZWAVE> and <SIG MODE> functions used for wavelength accuracy measurement are
described below.
・ ZWAVE
Sets whether zero correction of the output display (abs display) and output voltage is
conducted when the wavelength setting is changed.
Set <ZWAVE> to “1” to prevent zero compensation during wavelength accuracy
measurements, as the energy intensity at each wavelength is compared.
・ SIG MODE
Switches between the signal type (absorbance, sample cell light intensity, reference cell
light intensity, etc.) output to the output display (abs display) and output voltage terminals.
Set <SIG MODE> to “5” to display the light intensity passing through the sides of the
sample cell on the output display (abs display).
Test 7
(Item) Absorbance display/data output test
(Outline) Checks that the displayed absorbance value equals the signal value output to the data-
processing unit.
(Procedure) 1) Check that the D2 lamp is turned on.

2) Set <AUX RANGE> to 1AU/V on the data processor or the analyzer main unit.
3) Set the data-processing unit full-scale to about 1.00 V. For an absorbance display, set
full-scale to 1.00 AU.
4) Fill the flow cell with methanol.
5) Set the wavelength to 272 nm and press the [zero] key.
6) Inject the caffeine solution into the flow cell.
7) Read the data-processing unit signal value and record the value in AU units.
8) Read the absorbance value displayed on the screen, and calculate the ratio with
respect to the data-processing unit signal value.
Signal value ratio = displayed absorbance value/output signal value
(Criteria) Digital signal 1.00  0.01

Analog signal 1.00  0.02
Test 8
(Procedure) 1) Turn on the power and press the [VP] key several times. Press the [func] key several
times to display <LEAK SENSOR TEST>.
(Criteria) <SENSOR GOOD> appears on the display.
Page 34 of 97 Rev. 3.12

Operational Qualification RF-10AXL/RF-10AXL Super Inspection Procedure
4.6 Spectrofluorometric Detector
4.6.1 RF-10AXL/RF-10AXL Super
1. Introduction
This section presents the standard operating procedure (SOP) for Operational Qualification of the
Spectrofluorometric Detector RF-10XL/RF-10AXL Super.
Refer to the Instruction Manual for details about the operation procedure and to clarify uncertain points.
Test 1
(Item) Initialization test
(Outline) Checks the display, LED, and home position operation.
(Procedure) 1) Turn on the power and wait for the initialization test to complete.
(Criteria) The initialization test ends normally.
Test 2

2) Initialization and memory check are conducted.
(Criteria) “ROM FAILURE” or “RAM FAILURE” is not displayed.
Test 3
(Procedure) 1) After the power is turned on, the version number appears on the display.

Operational Qualification RF-10AXL/RF-10AXL Super Inspection
Procedure
Test 4
(Procedure) 1) Press the [shift] and [edit] keys to enter the time program edit mode.
0.1 STOP
goes out.
Test 5
(Outline) Checks that the lamp lighting time is within the set time.
(Procedure) 1) Press the [func] key several times to display <Xe TIME>.
2) Record the lamp lighting time. (Units: hours)
(Criteria) Lamp lighting time ≦ 500
Test 6
(Item) Sensitivity and S/N check
(Outline) Uses the water Raman peak intensity and noise measurements to check the sensitivity and
S/N ratio.
(Procedure) 1) Turn on the power and wait at least 30 minutes for the amplifier to stabilize.
2) Fill the RF-10AXL flow cell with water.
3) Press the [func] key to display the <S/N CHECK> screen.
4) Press the [Enter] key to automatically set the following parameters:
EX 350nm
EM 450nm
Gain 2 (x 4)
SCAN FILE 0
SPC TYPE 2 (EM)
EM SCAN BGN 350nm　
EM SCAN END 450nm
SCAN SPD 3 (MEDIUM)
RARIO 1 (ON)
Range 4 (x 4)
sens 2 (MED)
rsp 3 (1.5S)
5) Press the RF-10AXL [Enter] key.

6) When the scan is complete, press the [Enter] key to start the noise measurements.
7) The Raman peak intensity and S/N ratio are displayed sequentially when the noise
measurements are completed in approximately 15 minutes. Record these values.
Page 36 of 97 Rev. 3.12

(Criteria) 40  Raman peak height F ≦ 85
S/N ≧ 300
Operational Qualification RF-10AXL/RF-10AXL Super Inspection Procedure
Test 7
(Item) ROM, RAM self-diagnostic test (Cell temperature control unit)
(Outline) Checks the cell temperature control unit firmware version number, and checks that the
ROM and RAM memory operates normally.
(Procedure) 1) Turn on the instrument in which the cell temperature unit is installed.
2) The version number is displayed on the display.
3) Record the displayed version number.
4) Press the [func] key to display the <MEMORY CHECK> screen, and press the
[Enter] key to execute the ROM, RAM check.
5) Confirm that “ROM OK/ RAM OK” is displayed.

“ROM OK / RAM OK” is displayed.
Test 8
(Item) Temperature control function check
(Outline) Checks that temperature control precision and accuracy of the temperature-controlled cell
units are within the criteria ranges at a temperature of 25C.
(Procedure) 1) Remove the cover from the sample compartment, mount the thermocouple on the
raised part of the heat block using the thermocouple retaining jig, and place the
cover back on the sample compartment.
2) After turning on the RF-10AXL, turn on the instrument in which the cell
temperature control unit is installed while pressing the [func] key.
3) Press the [func] key to display the <SET TEMP> screen, set the temperature of the
temperature controller to 25C, and press the [CTRL] key to start temperature
control.
4) After the “Ready” lamp lights on the temperature controller display panel, wait at
least 10 more minutes.
5) Press the [func] key to display the <TEMPERATURE TEST> screen, and press the
[Enter] key to execute the test.
6) When the test is completed, confirm that “STABILITY GOOD” is displayed, and
record the DT value displayed.
7) Also record the thermometer value. (Record the measured temperature after
performing temperature correction based on the temperature measurement deviation
indicated in the temperature sensor test report.)
8) Continue by pressing the [Enter] key to display the <REF TEMP> screen, and enter
the above-mentioned actual measured temperature.
9) Confirm that “ACCURACY GOOD” is displayed.
(Criteria) “STABILITY GOOD” is displayed (temperature variation width dT ≦ 0.2 C)

“ACCURACY GOOD” is displayed (temperature accuracy 25 C  2 C)
Page 38 of 97 Rev. 3.12
Operational Qualification RID-10A Inspection Procedure
4.7 Refractive Index Detector
4.7.1 RID-10A
1. Introduction
This section presents the standard operating procedure (SOP) for Operational Qualification of the Refractive
Index Detector RID-10A.
Refer to the Instruction Manual for details about the operation procedure and to clarify uncertain points.
Note: Hold the cell temperature at 40C during the test.
Test 1
(Item) Initialization test
(Outline) Checks that initialization completes without error.
(Procedure) 1) Initialization is conducted automatically after the power is turned on. Check if an
error message appears.
(Criteria) System initialization completes without error.
Test 2


Test 3
(Procedure) 1) Press the [shift] + [edit] keys to enter the time program edit mode.
0.1 STOP
3) Press the [run] key.
4) Check that the prog run LED lights for six seconds and then goes out.
Test 4
(Outline) Checks that the lamp energy exceeds the acceptance criteria.
(Procedure) 1) Fill the flow cell with water.

2) Press the [func] key several times to display <TOTAL EN>.
3) Record the displayed value.
(Criteria) Lamp energy  6000
Test 5
(Outline) Checks that the lamp lighting time is within the specified time.
(Procedure) 1) Press the [func] key several times to display <LAMP TIME>.
2) Record the lamp lighting time. (Units: hours)
(Criteria) LAMP TIME ≦ 20000 h
Test 6
(Item) Auto-zero test
(Outline) Checks that the auto-zero function works normally.
(Procedure) 1) Press the [zero] key and read the displayed RI value.
2) Press the [zero] key again and read the displayed RI value.
(Criteria)  0.01 displayed after auto-zeroing.
Page 40 of 97 Rev. 3.12

Test 7
(Item) Temperature control function check
(Outline) Checks that the temperature control function works normally.
(Procedure) 1) The inspection is conducted at a controlled temperature of 40C. If the unit was used
at some other temperature, set the controlled temperature to 40C when the power is
turned on. If room temperature exceeds 28C, set the controlled temperature to (room
temperature + 12C).
2) Press the [func] key several times.
3) Enter “40.0” for <CELL TEMP>.
4) Press the [CE] key to return to the initial screen.
5) Press the [func] key several times to display <CELL TEMP>(temperature set value).
Press the [func] key again to display <ACT TEMP>(measured temperature).
6) Calculate and record the difference between <ACT TEMP> and < CELL TEMP>.
(Criteria) ACT TEMP value is (CELL TEMP  0.1 C)
Operational Qualification CTO-20A/20AC/10Avp/10ACvp/10ASvp Inspection Procedure
4.8 Column Oven
4.8.1 CTO-20A/20AC/10Avp/10ACvp/10ASvp
1. Introduction
This section presents the standard operating procedure (SOP) for Operational Qualification of the Column
Oven CTO-20A/20AC/10Avp/10ACvp/10ASvp. Refer to the Instruction Manual for details about the
operation procedure and to clarify uncertain points.
Test 1

Test 2
(Procedure) 1) Turn on the power. Press the [VP] key several times to display <Validation>.
Test 3
(Procedure) 1) Press the [VP] key several times to select <PRODUCT INFO>. Press the [func] key
several times.

Page 42 of 97 Rev. 3.12

Test 4
0.1 STOP
goes out.
Test 5
(Item) Set temperature accuracy (room temp. - 5C)
(Outline) Checks the temperature adjustment accuracy at (room temperature - 5C).

(CTO-20AC/CTO-10ACvp/CTO-10Asvp only)
(Procedure) 1) Turn on the unit. When unit initialization is complete, press the [func] key to display
<AMBIENT> and measure room temperature. Record the measured temperature.
2) Set the oven temperature to (room temperature -5C).
3) Insert a calibrated thermometer into the piping hole in the oven. For CTO-10ASvp,
insert a calibrated temperature sensor through the hole in the oven and attach it to the
heat block inside the unit using a thermocouple fixing jig.
4) Wait 20 minutes after the temperature stabilizes at (room temperature 5C). Record
the thermometer reading. However, record the temperature sensor reading after
correcting it using the temperature measurement deviation value shown in the
temperature sensor inspection report.
(Criteria) Set temperature accuracy: Set temperature  2.0  C
<Additional Information> How to calculate the correction value

(temperature measurement deviation)
[Correction value] If the required correction value is A3, then:
　　　　
A2 A3= A2-A1 　×(T3-T1) ＋A1
A3 T2-T1
T1 * This expression is used to calculate the correction

［℃］ value within each region of deviation, as the correction
T3 T2
A1 value can be considered linear within each region.
These corrections are the deviations (C) from the JIS
Table stated in the thermocouple or resistance
　　　　
Test 6
(Item) Temperature adjustment accuracy (room temperature - 5C)
(Outline) Checks the temperature adjustment accuracy at room temperature - 5C.

(CTO-20AC/CTO-10ACvp/CTO-10ASvp only)
(Procedure) 1) Turn on the unit. When unit initialization is complete, set <READY RANGE> to 1.0
and <WAIT TIME> to 5. Set the oven temperature to (room temperature - 5C) and
press the [oven] key.
2) Wait approximately 10 minutes after the temperature stabilizes.
3) Press the [VP] key several times to select <VALIDATION>. Press the [func] key
several times to display <TEMPERATURE TEST>.
4) Press the [Enter] key. Confirm “STABILITY GOOD” appears, and Temperature
deviation dT is within Criteria.
(Criteria) Temperature deviation dT ≦ 0.2  C at (room temperature - 5 C)
Test 7
(Item) Set temperature accuracy (40/50C)
(Outline) Checks the difference between the set temperature and actual temperature at 40/50C.
Select either 40C or 50C.
(Procedure) 1) Turn on the unit. When unit initialization is complete, set the oven temperature to
40C or 50C.
2) Insert a calibrated thermometer into the piping hole in the oven. For CTO-10ASvp,
insert a calibrated temperature sensor through the hole in the oven and attach it to the
heat block inside the unit using a thermocouple fixing jig.
3) Wait approximately 10 minutes after the temperature stabilizes at the set temperature.
Record the thermometer reading. However, record the temperature sensor reading
after correcting it using the temperature measurement deviation value shown in the
(Criteria) Set temperature accuracy: Set temperature  2.0  C
How to calculate the correction value

<Additional Information> (temperature measurement deviation)
［Correction value］ If the required correction value is A3, then:

A2-A1
A2 A3= 　　 ×(T3-T1) ＋A1
A3 T2-T1
T1 * This expression is used to calculate the correction

T3 value within each region of deviation, as the correction
T2 ［℃］
A1 value can be considered linear within each region.
These corrections are the deviations (C) from the JIS
　　　　
Page 44 of 97 Rev. 3.12

Test 8
(Item) Temperature adjustment accuracy (40/50C)
(Outline) Checks the temperature adjustment accuracy at 40/50C. Select either 40C or 50C.
(Procedure) 1) Turn on the unit. When unit initialization is complete, set <READY RANGE> to 1.0
and <WAIT TIME> to 5. Set the oven temperature to 40C or 50C and press the
[oven] key.
2) Wait approximately 10 minutes after the temperature stabilizes.
3) Press the [VP] key several times to display <VALIDATION>. Press the [func] key
several times to display <TEMPERATURE TEST>.
4) Press the [Enter] key. Confirm “STABILITY GOOD” appears, and Temperature
deviation dT is within Criteria.
(Criteria) Temperature deviation dT ≦ 0.2  C at 40 C/50 C
Test 9
(Outline) Checks the leak sensor operation. Select either 40C or 50C.
(Procedure) 1) Set the oven temperature to 40C or 50C and wait for the temperature to stabilize.
(No setting required for CTO-10ASvp.)
2) Press the [VP] key several times and press the [func] key several times to display
<LEAK SENSOR TEST>.
<CTO-20A/20AC/10Avp/10ACvp>
3) Inject 100 L methanol into the oven through the piping hole at the bottom-right of
the oven.
5) Check that “SENSOR GOOD” appears on the display within 20 seconds.
<CTO-10ASvp>
8) Check that “SENSOR GOOD” appears on the display.
Operational Qualification CBM-20A/20Alite Inspection Procedure
4.9 System Controller
4.9.1 CBM-20A/20Alite
1. Introduction
This section presents the standard operating procedure (SOP) for Operational Qualification of the System
Controller CBM-20A/20Alite. Refer to the Instruction Manual for details about the operation procedure and
to clarify uncertain points.
Test 1

2) At startup, the memory check is executed, and the unit should start up normally.
(Criteria) Unit starts up normally.
Test 2
(Outline) Checks the contents and firmware version.
(Procedure) 1) With the instrument running, start Internet Explorer on the connected PC.
2) Specify the system controller IP address for the URL.
3) When the connection is made, record the version number displayed under the
Shimadzu logo.
4) Log into this system controller.
5) Click the Instrument Configuration tab, and record the system controller version
number (firmware version number) displayed in the <Instrument Configuration>
screen.

Page 46 of 97 Rev. 3.12

Operational Qualification CBM-20A/20Alite Inspection Procedure
Test 3
(Item) System control
(Outline) Checks the system controller control operations.
(Procedure) 1) Connect to the system controller in the same way as described in Test 2.
2) Set the following analysis sequence:
Rack No.: 1 (Setting not required for autosamplers other than SIL-20A/AC)
SAMPLE No. From: 1, SAMPLE No. To: 1
Injection/Vial: 1, Injection Volume: 1
Method: - (No setting), Run Time: 1.00
3) [Isocratic system]
Click the [pump] button in the <Perform Analysis> screen, and set the flow rate to
0.5mL/min.
[Low-pressure gradient system]
1mL/min and B concentration to 50%.
[High-pressure gradient system]
1mL/min and B concentration to 50%.
4) In <Perform Analysis>, click the detector button, and perform the following settings.
[SPD] Set <WAVE> to 250.
[RF-10AXL] Set <EX. WAV> to 300, and <EM. WAV> to 400.
[RID-10A] Set <CELL.T> to 35.
5) Click [OK] to close the parameter setting screen, and click the [analysis start] button.
6) The system runs according to the programmed sequence.
(Criteria) The set sequence runs correctly.
Test 4
(Item) Memory backup
(Outline) Checks that the memory is backed up when the power is turned off.
(Procedure) 1) When the main unit is running, turn the power off and then on again.
2) Start Internet Explorer on the PC and connect to the system controller.
3) Check that the analysis program for the test 3 is saved.
4) When the inspection is complete, revert all conditions to their original state.
(Criteria) All input parameters are saved in memory.
Operational Qualification SIL-20A/20AC Inspection Procedure
4.10 Autosampler
4.10.1 SIL-20A/20AC
1. Introduction
Autosampler SIL-20A/20AC. Refer to the Instruction Manual for details about the operation procedure and
to clarify uncertain points.
Test 1

Test 2
3) Press the [Enter] key.．
Test 3
(Procedure) 1) Press the [VP] key to display <PRODUCT INFO>.

2) Press the [func] key several times.

Page 48 of 97 Rev. 3.12

Test 4
(Item) Basic operation check
(Outline) Checks that the basic operations are conducted correctly.
(Procedure) 1) Press the [VP] key several times to display <VALIDATION>.

2) Press the [func] key several times to display <POSITION SENS>.
3) Press the [Enter] key to run auto diagnosis.
4) Check that self diagnostics are executed and <SENS GOOD> is displayed.
Test 5
(Item) Injected volume accuracy test
(Outline) Checks the accuracy of the injected volume using 5μL, 10μL and 50μL injections.
However, evaluation of the injection volume accuracy for the 5μL and 10μL injections is
based on the system repeatability test data.
(Procedure) 1) Provide the following equipment and reagents to evaluate the 50L injection volume
accuracy.
a) An isocratic system or a gradient system
b) Mobile phase and washing liquid: Water (Degas the mobile phase and washing
liquid with the on-line degasser before use.)
c) Column: Resistor tube (4 m x 0.1 mm ID)
d) Sample: Water
e) Balance: Calibrated balance able to weigh down to 0.001g
2) Connect the system pipes as described in the unit instruction manual.
3) Connect the resistor tube at the column mounting position.
4) Wash the flow channels with a suitable solvent.
5) Replace the mobile phase and washing liquid with water and conduct purging.
6) Set the pump flow rate to 1.000 (1.0000 for LC-20AD/20AB) mL/min. and start the
pump.
7) Check that mobile phase flows from the detector outlet and that the flow channels do
not leak.
8) Set the following analysis conditions:
Injection volume: 50L
Number of injections: 10
Analysis time: 1min
9) Place 1 mL water into a vial and weigh it with a calibrated balance. Record the
weight.
10) Mount the vial in the sample rack and conduct analysis.
11) After analysis, weigh the vial with a calibrated balance and record the weight.
12) Determine the difference in vial weight before and after analysis to calculate the
injected volume accuracy.
Injected volume accuracy (%) = ((mass difference of the vial)/0.5-1)  100
0.5 indicates the theoretical mass difference of a vial.
13) After the system repeatability test is completed, use that data to calculate the actual
injection volume of 5μL and 10μL injections using the following equation.
Actual injection volume (μL) = (vial mass difference) / 10  (average peak area) /
(average peak area with 50 μL injection)  1000
14) Using the following equation, calculate the injection volume accuracy of 5μL and
10μL injections.
Injected volume accuracy (%) = ((actual injected volume) / (set injected volume)
-1)) x 100
(Criteria) Injected volume accuracy ±5.0%
Test 6
(Procedure) 1) Turn on the power and press the [VP] key several times. Press the [func] key several
times to display <LEAK SENSOR CHECK>.
Test 7
(Item) Sample cooler set temperature accuracy test
(Outline) Determines the difference between the sample cooler set temperature and actual
temperature.
This test is performed only for the SIL-20AC.
(Procedure) 1) Transfer 1mL of water to a 1.5mL glass sample vial, and set the vial in position 32 of
the standard accessory 1.5mL cooling rack.
2) Mount the rack in the main unit. Insert the sensor into the central vial mounted at
step 1) above. Pass the measurement sensor through the septum and put the sensor tip
in contact with the base of the vial. Ensure that the tip of the temperature
measurement sensor is not bent during these operations. In addition, take care with
the sensor cable routing to ensure that temperature measurement sensor is not pulled
out of the vial.
3) Set the sample cooler temperature to 4C and start temperature control. Leave the
autosampler door closed.
4) After about 1 hour, confirm that the sample cooler temperature has dropped to 4C.
5) Wait at least 30 minutes after the temperature drops to 4C and then read and record
the thermometer temperature. However, record the temperature sensor reading after
correcting it using the temperature measurement deviation value stated in the
(Criteria) At room temperature 30C max. and RH 60% max.

Set temperature accuracy: Set temperature  3.0 C
<Additional Information>
How to calculate the correction value
(temperature measurement deviation)
［Correction value］
If the required correction value is A3, then:
A2 A2-A1
A3 A3= 　　 ×(T3-T1) ＋A1
T2-T1
T1
* This expression is used to calculate the correction
T3 T2 ［℃］ value within each region of deviation, as the correction
A1
value can be considered linear within each region.
These corrections
A2-A1 are the deviations (C) from the JIS
Page 50 of 97 T2-T1
temperature sensor inspection report. Rev. 3.12
Operational Qualification LCsolution Inspection Procedure
4.11 Data Processing Unit
4.11.1 LCsolution
1. Introduction
Workstation LCsolution. Refer to the Instruction Manual for details about the operation procedure and to
・User Registration of Performer
Before conducting this Operational Qualification, the system administrator must complete user registration
for the Performer who conducts the work (OQ User). Account registration of the Performer is also necessary
to use Windows and Shimadzu application software. Disable the Performer's user registration when all
operations are complete.
・Windows Account Registration

Register two accounts in Windows. Create one account for the analyst who will belong to the group having
normal authority, and the other for the individual who will have administrator authority. The account
pertaining to the analyst authority group is used for checking Shimadzu Corporation application software,
including LCsolution and CLASS-Agent Manager software, while the account pertaining to the
administrator authority group is used for checking the computer etc.
・CLASS-Agent Manager Account Registration

A user with User Registration authority starts the CLASS-Agent Manager and logs in. Click [Tools] -> [User
Administration] to open the [User Administration] screen. If the OQ User is already created but disabled
(displayed gray), select the required user and click [Active/Invalid] to enable this user. Click [Edit] and
check that the authority shown below is set for the user. To register a new user, click [New] and register the
user as shown in the table below.
・LCsolution Account Registration

A user with System Administrator starts the LCsolution User Administration and logs in. Register the
LCsolution Users as shown in the table below. If the OQ user is already created, check that the
administration shown below is set for the user.
＜OQ User Registration＞
Login ID User name Password CLASS-Agent Manager LCsolution
(User ID) Administration Administration
OQ_USER_1 OQ_Operator ******* Operator, but with Add Group Name “OQ”
additional “Approve” to “Group Property” in
Permission for Agent Groups, without the right
Manager to edit method
(instrument parameters).
Add “OQ” to “User
Property” in “Users”,
with the right to use
connected instruments.
OQ_USER_2 OQ_Approver ******* Administrator System Administrator
* (User ID) Indicates name used with LCsolution
The administrator should issue the initial user password and inform the user of it.
　　　
Page 52 of 97 Rev. 3.12

Test 1-1
(Item) Computer boot-up test
(Outline) Checks that the computer boots up and runs normally. Self test at the time of computer startup
checks for memory errors.
(Procedure) 1) Turn on the PC.

2) Check that Windows starts up normally.
(Criteria) Windows starts up normally.
Test 1-2
(Item) Time and date check
(Outline) Checks that the PC time and date are correct.
(Procedure) 1) Open the Windows Control Panel.

2) Double-click [Date/Time] in the Control Panel to display [Date/Time Properties].
3) Check that the date and time are correct. Check the time against an accurate clock, such
as the telephone time service.
(Criteria) Date and time are correct.
Test 1-3
(Item) Hard disk drive test
(Outline) Checks that the hard disk drive operates correctly.
(Procedure) The user using this test must login to Windows with Administrator authorization. Moreover,
during procedure execution of 6) of the following, do not open and close a file.
1) All open windows are closed.

2) Open the Explorer.
3) Right-click of the hard disk drive used and select [Properties].
4) [Tools] tab is clicked.
5) It clicks [Check Now…] under “Error-checking”.
6) Check is not put into “Check disk options”, and [start] is clicked.
(Criteria) “Disk Check Complete.” is displayed.
Test 1-4
(Item) Print driver check
(Outline) Checks the print driver test page and the version number.
(Procedure) 1) Select [Printers] ([Printers and FAX] for Windows XP) from the Windows Control Panel
to display the printers used.
2) Right-click on the printer used and select [Properties].
3) Click the [Print Test Page] button in the [General] tab.
4) Check that the printed test page is correct and record the print driver version number
printed on it.
(Criteria) Test page printed correctly.

The driver version number matches the controlled version number.
Test 1-5
(Item) Network connection test
This inspection is required only for network connected PCs.
(Outline) Checks that the network connection is normal between the PC and the reference computer
such as the server computer.
(Procedure) 1) Run the Command Prompt and enter “C: ∖>PING***.***.***.***”

(substituting the IP address of the server computer or other reference computer for
“***.***.***.***”).
2) Data communication is conducted with the designated computer and the results are
displayed.
3) Check that no packets were lost.
4) Maximize the screen. Hold down the [Alt] key and press [PrtSc] to copy the screen.
5) Paste into the Windows Paint accessory and print.
(Criteria) No packets lost.
Test 2-1
(Item) Software version check
(Outline) Checks that the software is not changed to an inappropriate version.
(Procedure) 1)　Check the version number printed in the program alteration check results from Test 2-2.
2)　Check that the version number matches the controlled version number.
(Criteria) The version number matches the controlled version number.
Page 54 of 97 Rev. 3.12

Test 2-2
(Item) Program alteration check
(Outline) Conducts the program alteration check.
(Procedure) 1) Run Postrun from the LCsolution Launcher. Login as the “OQ_USER_2” test user.
2) Select [Check the Program Files] from the [Tools] menu. Click [Execute] in the [Check
the Program Files] screen.
3) File comparison is conducted.
Click on [Browse] to display the results. Click [Print] and then attach the hardcopy
printout to the OQ record.
(Criteria) Total “Pass” is displayed for every item.
Test 2-3
(Item) Shimadzu User Authentication Tool connection check
(Outline) Checks the Shimadzu User Authentication Tool connection using database access and log
registration.
* This test is conducted only if the Shimadzu User Authentication Tool is used with
LCsolution.
(Procedure) 1) Open the Windows Control Panel and double-click on the Shimadzu User Authentication
Tool icon to open the main menu.
2) Record the server name displayed at the bottom of the main menu and confirm that the
connected database is correct.
* Conduct steps 3) and 4) below after conducting Test 2-4-4.
3) Log in to [View event log] as “OQ_USER_2” from the main menu. Set the test date
(Period) and test computer name (PC name) in [Condition]. Search the log and print out
the results.
4) Confirm that the user lockout generated in Test 2-4-4 is registered and that the software
name is “LCPrun.exe”.
(Criteria) Server name for the connected database is correct.

Login possible from [View event log].
Lockout by LCPrun.exe is registered.
Test 2-4 Security function test

(Item) 2-4-1 Password log management function check
2-4-2 Number of password characters check
2-4-3 Failed login detection
2-4-4 User lockout function check
2-4-5 Failed login log
(Outline) Checks that the security functions operate, including restrictions on the number of password
characters.
(Preparation) Before conducting this test, set the following items in the LCsolution system policy.
Item Setting
(1) Minimum Password Length (Check only; set to non-zero value.)
(2) Lockout Settings [for User]
Login Attempt Limit (Check only; set to non-zero value.)
Lockout Duration 1 minute
(3) Lockout Settings [for PC]
Login Attempt Limit Set to the individual user lockout
settings value, or higher
(4) # of Levels for Duplicate Password Checking 10
(Procedure) 1) Select [User Administration] in the LCsolution Launcher. Login as the “OQ_USER_1”
test user.
2) Enter the current password, and enter the same password as the new password.
3) Confirm that the same password is not accepted.
4) Enter the current password and enter a new password that is shorter than the minimum
number of characters set for LCsolution system policy.
5) Confirm that the change is not accepted, as the password is too short.
6) Enter a password equal in length to the minimum number of characters and confirm that
the change is accepted.
7) Exit LCsolution user management.
8) Select [Postrun] from the LCsolution Launcher.
9) Enter “OQ_USER_1” as the user ID.
10) Enter a password that is not set and click OK.
11) Check that login is not permitted and a message is displayed warning that the password is
incorrect.
12) Repeat steps 8) to 11) for the number of times designated for “Login Attempt Limit” in
the individual user lockout settings in the LCsolution system policy.
13) After the designated numbers of login failures, confirm that OQ_USER_1 is locked out
and cannot log in for the designated lockout time.
14) After the designated lockout time elapses, select LCsolution Postrun again. Enter
“OQ_USER_1” as the login ID and correctly enter the set password.
15) Confirm that login is possible.
16) Exit LCsolution Postrun. Select [Log Browser] from the LCsolution Launcher and login
as “OQ_USER_2”.
17) Display the log. If the Shimadzu User Authentication Tool is used with LCsolution, select
“User Authentication Server Log” as the log type. Otherwise, select [Each Instrument
Log] -> (PC name) -> [None]. Confirm that the login lockout in the procedure above and
the corresponding time appear in the displayed log.
18) If the # of Levels for Duplicate Password Checking and Login Lockout Duration settings
were changed, return them to their original settings.
Page 56 of 97 Rev. 3.12

(Criteria)
2-4-1 Duplicated passwords cannot be used.
2-4-2 A password shorter than the minimum length is not accepted while a password
satisfying the minimum length is accepted.
2-4-3 Login is not possible with an incorrect password but possible with the correct password.
2-4-4 The user is locked out for the specified lockout time.
2-4-5 The login lockout is registered in the log.
Test 2-5
(Item) User access rights check
(Outline) Confirms that software use is restricted according to differences in registered user access
rights.
(Procedure) 1) Run [Offline Editor] for Analysis 1 (or equivalent, if name was changed) from LCsolution
Launcher. Login with the user ID “OQ_USER_2”.
2) Confirm that the analyzer parameters can be changed in the Instruments Parameters View
in the Data Acquisition screen, and then exit [Offline Editor].
3) Run [Offline Editor] again and login with the user ID “OQ_USER_1”. Confirm that the
Instrument Parameters cannot be changed in the Instruments Parameters View in the Data
Acquisition screen.
(Criteria) OQ_USER_2 can edit Instrument Parameters but OQ_USER_1 cannot edit them.
Test 2-6
(Item) System policy function check
(Outline) Checks the LCsolution system policy function.
The system policy function is verified by checking that the system policy settings for the audit
trail and save/overwriting of methods function correctly.
(Procedure) 1) Run [System Policy] from the LCsolution launcher. Login with the user ID
“OQ_USER_2”.
2) If no check mark appears by “Apply audit trail function when creating method file” or
“Prohibit overwriting of other method files”, enter a check mark and click [OK]. (Revert
to the original settings at the end of the test.)
3) Run [Offline Editor] for Analysis 1 from LCsolution Launcher. Login with the user ID
“OQ_USER_2”. Open the Data Acquisition screen.
4) Select [File] -> [New Method File] to create a new method file. Select [File] -> [Method
File Properties]. (The new file can be created using a template.)
5) Ensure that a check mark appears in the “Apply to the method file” and “Apply to the
data file acquired with this method file” checkboxes on the [Audit Trail] tab on the
displayed screen.
6) Select [File] -> [Save Method File As] and save the method file with the name
OQ_Method_xxx.lcm (where xxx is any number).
7) The following procedure is required for Test 2-7 Audit Trail Check.
Make arbitrary changes to the method parameters for the OQ_Method_xxx.lcm method
file and save it over the existing method file. A screen prompts for the input of the reasons
for the change. Enter “Audit trail test” and save.
8) Select [File] -> [New Method File] to create a new method file.
9) Select [File] -> [Save Method File As] and save the method file with the name of the file
saved previously.
Confirm that a message states that “You cannot overwrite the existing file” and that the
file is not overwritten.
10) Exit LCsolution [Offline Editor]. Run System Policy from the LCsolution launcher again
and revert all settings to their original values.
(Criteria) A check mark appears for the audit trail settings in a newly created method file.
Overwriting other method files is prohibited.
Test 2-7
(Item) Audit trail check
(Outline) Checks the audit trail functions.
(Procedure) 1) Run [Offline Editor] for Analysis 1 from LCsolution Launcher. Login with the user ID
“OQ_USER_2”. Open the Data Acquisition screen.
2) Open the method file OQ_Method_xxx.lcm saved during Test 2-6.
3) Select [File] -> [Audit Trail] from the menu.
4) Check that the audit trail details (Date&Time, Modification, Reason) are displayed
correctly. Click [Print] on the screen to print the audit trail.
(Criteria) Audit trail details (Date&Time, Modification, Reason) are displayed correctly.
Test 2-8
(Item) Screen lock function check
(Outline) Checks the screen lock function.
(Procedure) 1) While logged in to [Offline Editor] as “OQ_USER_2”, select [Window] -> [Lock] from
the menu to apply the screen lock.
2) Click on the locked [Offline Editor], and confirm that a User Authentication window to
unlock the screen appears.
3) Enter the user ID “OQ_USER_1” and the correct password in the user authentication
window and confirm that the screen cannot be unlocked.
4) Enter the user ID “OQ_USER_2” and the correct password in the user authentication
window and confirm that the screen is unlocked.
5) Exit [Offline Editor].
(Criteria) The Screen lock function operates.

“OQ_USER_1” cannot unlock the screen but “OQ_USER_2” can unlock the screen.
Page 58 of 97 Rev. 3.12

Test 2-9
(Item) Test of function to prevent duplicate user registration
(Outline) Confirms that a duplicate user name cannot be registered.
(Procedure) 1) Run [User Administration] from LCsolution Launcher. Login as the “OQ_USER_2” test
user.
2) Open the [Add] screen from [Users] and enter the user ID “OQ_USER_1”. Set any user
name, password, and authority and click [OK].
3) Confirm that a message indicates that “This user ID is already registered.” and confirm
that the duplicate user registration is not possible.
(Criteria) A message indicates that “This user ID is already registered.”

Duplicate user registration is not possible.
Test 2-10 Software function checks

Checks the LCsolution peak integration, peak identification from retention times, quantitative calculations using
peak areas and peak heights, column performance parameter calculations for each Pharmacopoeia, and the
output of data information, calibration curve information, and set parameters.
Preparations Also conduct Procedure 2 if the inspected computer is used to register data in the CLASS-
Agent Manager database.
(Procedure 1) Obtain an LCsolution Install CD-ROM (LCsolution Ver.1.02, or later).
1) Run \English\LCsolution\CheckData\Setup.exe for the CD-ROM drive containing the

install CD-ROM. Copy the following files to the hard disk according to the screen
instructions.
File name Size Copy destination
Area100.lcb 15KB C: \Labsolutions\LCsolution\CheckData
Area100.lcm 77KB C: \Labsolutions\LCsolution\CheckData
Area100.lcr 92KB C: \Labsolutions\LCsolution\CheckData
Background.lcd 200KB C: \Labsolutions\LCsolution\CheckData
Demo_Data-001.lcd 293KB C: \Labsolutions\LCsolution\CheckData
Demo_Data-IT01.lcd 226KB C: \Labsolutions\LCsolution\CheckData
External.lcb 24KB C: \Labsolutions\LCsolution\CheckData
External.lcm 88KB C: \Labsolutions\LCsolution\CheckData
External.lcr 133KB C: \Labsolutions\LCsolution\CheckData
Integration.lcb 23KB C: \Labsolutions\LCsolution\CheckData
Integration.lcr 96KB C: \Labsolutions\LCsolution\CheckData
Integration_Baseline.lcm 54KB C: \Labsolutions\LCsolution\CheckData
Integration_Tailing.lcm 45KB C: \Labsolutions\LCsolution\CheckData
Integration_Vertical.lcm 54KB C: \Labsolutions\LCsolution\CheckData
Internal.lcb 13KB C: \Labsolutions\LCsolution\CheckData
Internal.lcm 77KB C: \Labsolutions\LCsolution\CheckData
Internal.lcr 94KB C: \Labsolutions\LCsolution\CheckData
(Caution)
(1) The copy destination folder C:\LabSolutions\LCsolution is the folder where
LCsolution is installed.
(2) This data is overwritten each time the function check is conducted. If previous
data remains, deleted the existing data before conducting this procedure.
(3) Always check the date and time of the data used with Explorer.
(Procedure 2) 2) Select [Postrun] from the LCsolution Launcher. Login as the “OQ_USER_2” test user.
* Conduct only 3) Select [Tools] -> [Agent Registration Setting] and make the settings required for
if data is automatic registration to the Agent Manager database. Note the existing settings. Make
registered to the settings such that the data from Manual Registration and data from Postrun Analysis of
Agent Manager data collected by Instrument 1 is registered to the usually used database. Also make
database settings so that data files, method files, batch files, and report format files are saved as
compressed files.
If the PDF file output function is used, set the PDF file registration as the Compression
Files. Also, set the PDF file to be deleted after data registration.
* After completing Test 5, revert these Agent Manager registration settings to their
previous values.
Page 60 of 97 Rev. 3.12

Test 2-10-1
(Item) Peak integration
(Outline) Checks the peak integration functions.
(Procedure) 1) Use the LCsolution “Batch Table” screen from [Postrun Analysis] to open the batch file
“Integration.lcb” that was copied from the CD-ROM.
2) Run the batch table. Click [Start] to run the batch table and print the reports. Attach the
three printed data reports. Check the results of peak integration.
(Criteria) Peak tailing is conducted in the “Demo_Data-IT01.lcd” report.

Peak vertical splitting is conducted in the “Demo_Data-IT02.lcd” report.
Peak baseline processing is conducted in the “Demo_Data-IT03.lcd” report.
Test 2-10-2
(Item) Peak area percentage method
(Outline) Checks the quantitation calculation functions using the peak area percentage method.
(Procedure) 1) Use the LCsolution “Batch Table” screen from [Postrun Analysis] to open the batch file
“Area100.lcb” that was copied from the CD-ROM.
2) Run the batch table. Click [Start] to run the batch table and print the report. Attach the
printed report. Confirm that the values in the report match the values shown below.
ID# Name Ret.Time Area Conc.

Units
(Compound name) (Retention time) (Concentration)
1 Methyl_paraben 2.636 74904 27.75 %
2 Ethyl_paraben 3.216 82688 30.63 %
3 Propyl_paraben 3.926 54166 20.07 %
4 Butyl_paraben 4.619 58179 21.55 %
ID# Separation Theoretical Resolution Tailing

k'
Plates Factor
1 0.000 4151 1.028 0.000 1.213
2 1.435 6795 1.474 3.631 1.180
3 1.370 12725 2.020 4.805 1.183
4 1.264 20994 2.553 5.200 1.195
(Criteria) The results match.
Test 2-10-3
(Item) Internal standard method
(Outline) Checks the quantitation calculation functions using the internal standard method.
(Procedure) 1) Use the LCsolution “Batch Table” screen from [Postrun Analysis] screen to open the
batch file “Internal.lcb” that was copied from the CD-ROM.
2) Run the batch table. Click [Start] to run the batch table and print the report. Attach
the printed report. Confirm that the values in the report match the values shown
below.
Name Ret.Time Height Conc.

ID# Height Units
(Compound name) (Retention time) Ratio (Concentration)
1 Methyl_paraben 2.636 26191 1.00 0.000 mg/L
2 Ethyl_paraben 3.217 30303 1.16 0.994 mg/L
3 Propyl_paraben 3.927 22908 0.87 1.007 mg/L
4 Butyl_paraben 4.620 26640 1.02 1.001 mg/L
Theoretical
ID# Separation k' Resolution Tailing Factor
Plates
1 0.000 4919 1.027 0.000 1.212
2 1.435 8136 1.474 3.976 1.186
3 1.371 16041 2.021 5.345 1.179
4 1.264 26539 2.554 5.850 1.195
Test 2-10-4
(Item) External standard method
(Outline) Checks the quantitation calculation functions using the external standard method.
(Procedure) 1) Use the LCsolution “Batch Table” screen from [Postrun Analysis] to open the batch
file “External.lcb” that was copied from the CD-ROM.
2) Run the batch table. Click [Start] to run the batch table and print the report. Attach
the printed report. Confirm that the values in the report match the values shown
below. Also confirm that the sample information, calibration curve information, and
set parameters are printed in the report.
ID Name Ret.Time Conc.

Area Units
(Compound name) (Retention time) (Concentration)
1 Methyl_paraben 2.631 224575 3.021 mg/L
2 Ethyl_paraben 3.211 246947 3.011 mg/L
3 Propyl_paraben 3.921 162870 3.016 mg/L
4 Butyl_paraben 4.614 174120 3.014 mg/L
ID Separation Theoretical Plates k' Resolution Tailing Factor

1 0.000 4886 1.024 0.000 1.208
2 1.436 8167 1.470 3.972 1.181
3 1.371 16016 2.016 5.348 1.180
4 1.264 26360 2.549 5.849 1.191

Operator name (Acquired by), analysis date (Data Acquired), processed date
(Data Processed), and name of data file (Data File Name) used are printed as
sample information.
Calibration curve information is printed.
Unit Parameters and Peak Integration, peak Identification, and Quantitative
parameters and Compound Table are printed.
Page 62 of 97 Rev. 3.12

Operational Qualification CLASS-VP Inspection Procedure
4.11.2 CLASS-VP
1. Introduction
Workstation CLASS-VP. Refer to the Instruction Manual for details about the operation procedure and to
・User Registration of Performer
Before conducting this Operational Qualification, the system administrator must complete user registration
for the Performer who conducts the work (OQ User). Account registration of the Performer is also necessary
to use Windows and Shimadzu application software. Disable the Performer's user registration when all
operations are complete.
・Windows Account Registration

Register two accounts in Windows. Create one account for the analyst who will belong to the group having
normal authority, and the other for the individual who will have administrator authority. The account
pertaining to the analyst authority group is used for checking Shimadzu Corporation application software,
including CLASS-VP software, while the account pertaining to the administrator authority group is used for
checking the computer etc.
・CLASS-VP Account Registration

A user with System Administrator starts the CLASS-VP System Administration and logs in. Register the
CLASS-VP Users as shown in the table below. If the OQ user is already created, check that the authority
shown below is set for the user.
＜OQ User Registration＞
Login ID User name Password CLASS-Agent Manager CLASS-VP
Administration Administration
OQ_USER_1 OQ_Operator ******* Operator, but with From “Properties” in
additional “Approve” “Add/Delete Users”,
Permission for Agent Privilege level : 3
Manager Permitted to use :
Offline Processing
Instrument1
OQ_USER_2 OQ_Approver ******* Administrator From “Properties” in
“Add/Delete Users”,
Privilege level : 2
Permitted to use :
Offline Processing
Instrument1
* The administrator should issue the initial user password and inform the user of it.
　　　
Test 1-1
(Item) Computer boot-up test
(Outline) Checks that the computer boots up and runs normally. Self test at the time of computer startup
checks for memory errors.
(Procedure) 1) Turn on the PC.

2) Check that Windows starts up normally.
(Criteria) Windows starts up normally.
Test 1-2
(Item) Time and date check
(Outline) Checks that the PC time and date are correct.
(Procedure) 1) Open the Windows Control Panel.

2) Double-click [Date/Time] in the Control Panel to display [Date/Time Properties].
3) Check that the date and time are correct. Check the time against an accurate clock, such as
the telephone time service.
(Criteria) Date and time are correct.
Test 1-3
(Item) Hard disk drive test
(Outline) Checks that the hard disk drive operates correctly.
(Procedure) The user using this test must login to Windows with Administrator authorization. Moreover,
during procedure execution of 6) of the following, do not open and close a file.
1) All open windows are closed.

2) Open the Explorer.
3) Right-click of the hard disk drive used and select [Properties].
4) [Tools] tab is clicked.
5) It clicks [Check Now…] under “Error-checking”.
6) Check is not put into “Check disk options”, and [start] is clicked.
(Criteria) “Disk Check Complete.” is displayed.
Page 64 of 97 Rev. 3.12

Test 1-4
(Item) Print driver check
(Outline) Checks the print driver test page and the version number.
(Procedure) 1) Select [Printers] ([Printers and FAX] for Windows XP) from the Windows Control Panel to
display the printers used.
2) Right-click on the printer used and select [Properties].
3) Click the [Print Test Page] button in the [General] tab.
4) Check that the printed test page is correct and record the print driver version number
printed on it.
(Criteria) Test page printed correctly.

The driver version number matches the controlled version number.
Test 1-5
(Item) Network connection test
This inspection is required only for network connected PCs.
(Outline) Checks that the network connection is normal between the PC and the reference computer
such as the server computer.
(Procedure) 1) Run the Command Prompt and enter “C: ∖>PING***.***.***.***”

(substituting the IP address of the server computer or other reference computer for
“***.***.***.***”).
2) Data communication is conducted with the designated computer and the results are
displayed.
3) Check that no packets were lost.
4) Maximize the screen. Hold down the [Alt] key and press [PrtSc] to copy the screen.
5) Paste into the Windows Paint accessory and print.
(Criteria) No packets lost.
Test 2-1
(Item) Software version check
(Outline) Checks that the software is not changed to an inappropriate version.
(Procedure) 1) Check the version number printed in the program alteration check results from Test 2-2.
2) Check that the version number matches the controlled version number.
(Criteria) The version number matches the controlled version number.
Test 2-2
(Item) Program alteration check
(Outline) Conducts the program alteration check.
(Procedure) 1) Select the [Start] button of the Windows, [Programs], [Chromatography] and [CLASS-VP
Alteration Test] in this order to display the [Check the Program Files] window.
2) Click the [Execute] button on the [Check the Program Files] window.
3) The files are compared, and the alteration check result is displayed.
Click the [Browse], print the alteration check result, then attach it.
(Criteria) Total “Pass” is displayed for every item.
Page 66 of 97 Rev. 3.12

Operational Qualification SPD-M20A Inspection Procedure
4.12 Photodiode Array Detector
4.12.1 SPD-M20A
1. Introduction
Photodiode Array Detector SPD-M20Avp. Refer to the Instruction Manual for details about the operation
procedure and to clarify uncertain points.
Test 1
(Procedure) 1) Turn on the SPD-M20Avp unit and the system controller.

<For LCsolution>
2) Start up the PC and click the Windows [Start] button. Select [Programs] ->
[LabSolutions] -> [LCsolution] -> [Unit Maintenance Tools] to run [LC Tool
Launcher].
3) Select the application program [PDA Utilities] in [LC Tool Launcher], select the
connected unit number, a nd run PDA Utilities.
4) Run [Unit Information] in [PDA Utilities] and check the displayed ROM version
number.
<For CLASS-VP>
2) Start up the PC and click the Windows [Start] button.
3) Next, start up the CLASS-VP, select [File] -> [Configure], and click [PDA] icon.
4) Select [SPD-M20A] for Detector Configuration window -> [Device Information].
5) Check the displayed ROM version number.

Test 2
(Outline) Checks the wavelength accuracy using the emission lines of a mercury lamp and
deuterium lamp.
<For LCsolution>
2) Run [Wavelength Check] from [PDA Utilities] that was started in Test 1.
<For CLASS-VP>
2) Start up the PC and click the Windows [Start] button, select [Programs] ->
[Chromatography], and run [Wavelength Check] from [PDA Utility].
<Common>
3) Select “D2/Ho” from the [Wavelength Check] menu.
The wavelength check runs automatically and the results are displayed on the screen.
Print the results and attach the hardcopy printout.
The D2 lamp check is conducted at wavelengths of 486.0nm and 656.1nm.
4) Select “Hg” from the [Wavelength Check] menu. Replace the flow cell with a
dummy cell. Mount the mercury lamp holder and the mercury lamp.
5) Click [OK] on the screen. The wavelength check (slit 1.2nm and 8nm) runs
automatically and the results are displayed on the screen. Print the results and attach
the hardcopy printout.The Hg lamp check is conducted at wavelengths of 253.7 nm,
404.7 nm, 435.8 nm, and 546.1 nm.
Page 68 of 97 Rev. 3.12

(Criteria) All evaluation results displayed as “PASS”.

(Wavelength displacement  1.0 nm at 253.7 nm, 404.7 nm 435.8 nm, 546.1 nm, 486.0
nm, and 656.1nm)

SPD-M20A uses the 486.0 nm and 656.1 nm emission lines of a deuterium lamp (D 2
lamp) and the 253.7nm, 404.7nm, 435.8nm, and 546.1nm emission lines of a mercury
lamp (Hg lamp) for wavelength calibration. Wavelength calibration using the emission
lines of a deuterium lamp (D 2 lamp) and mercury lamp (Hg lamp) is prescribed in the
JIS standards, as follows:
JIS K0115 RULES FOR ABSORBANCE ANALYSIS

Measure the light intensity of a low-pressure mercury lamp or deuterium
discharge tube to determine the wavelengths exhibiting the maximum intensity,
and compare with the emission line wavelengths to calibrate the wavelength scale.
Use the mercury lamp designated by Shimadzu for the wavelength accuracy test. The
test evaluation is based on a direct comparison of the emission line (natural physical
constant) unique to the lamp. The following document prescribes the use of natural
physical constants for traceability.
The use of calibration standards such as natural physical constants, derived
standards, standard reference materials, or internal standards which are not
traceable to national standards, will require adequate documentation to describe
the validity of the calibration standards used.”
Test 3
(Procedure) 1) The brightness of the D2 and W lamp are measured automatically during Test 2
Wavelength Accuracy Test, above.
2) Read and record the maximum voltage value (slit 1.2nm : 200 to 260 nm and 400 to
800 nm) for each lamp from the results of Test 2 Wavelength Accuracy Test.
(Criteria) Maximum intensity of each lamp satisfies the criteria below:

Maximum intensity (200 to 260 nm) ≧ 3.5 V
Test 4
(Procedure) 1) Start LCsolution and run [System Check].

2) Set the check level to “Normal” and start the system check.
3) Read the lighting time for each lamp (units: hours).
(Criteria) D2 TIME≦ 2, 000h

W TIME≦ 2, 000h
Page 70 of 97 Rev. 3.12

Operational Qualification SPD-M10Avp Inspection Procedure
4.12.2 SPD-M10Avp
1. Introduction
Photodiode Array Detector SPD-M10Avp. Refer to the Instruction Manual for details about the operation
procedure and to clarify uncertain points.
Test 1
(Procedure) 1) Turn on the SPD-M10Avp unit and the PC to start up Windows.

<For LCsolution>
2) Click the Windows [Start]. Select [Programs] -> [LabSolutions] -> [LCsolution] ->
[Unit Maintenance Tools] to run [LC Tool Launcher].
3) Select the application program [PDA Utilities] in [LC Tool Launcher], select the
connected unit number, and run PDA Utilities.
4) Run [Unit Information] in [PDA Utilities] and check the displayed ROM version
number.
<For CLASS-VP>
2) Start up the CLASS-VP, select [File] -> [Configure], and click [PDA] icon.
3) Select [SPD-M20A] for Detector Configuration window -> [Device Information].
4) Check the displayed ROM version number.

Test 2
(Outline) Checks the wavelength accuracy using the emission lines of a mercury lamp and
deuterium lamp.

<For LCsolution>
2) Run [Wavelength Check] from [PDA Utilities] that was started in Test 1.
<For CLASS-VP>
2) Start up the PC and click the Windows [Start] button, select [Programs] ->
[Chromatography], and run [Wavelength Check] from [PDA Utility].
<Common>
3) Select “D2/Ho” from the [Wavelength Check] menu.
The wavelength check runs automatically and the results are displayed on the screen.
Print the results and attach the hardcopy printout.
The D2 lamp check is conducted at wavelengths of 486.0nm and 656.1nm.
4) Select “Hg” from the [Wavelength Check] menu. Replace the flow cell with a
dummy cell. Mount the mercury lamp holder and the mercury lamp.
5) Click [OK] on the screen. The wavelength check runs automatically and the results
are displayed on the screen. Print the results and attach the hardcopy printout.
The Hg lamp check is conducted at wavelengths of 253.7 nm, 404.7 nm, 435.8 nm,
and 546.1 nm.
(Criteria) All evaluation results displayed as “PASS”.
(Wavelength displacement within  1.0 nm at 253.7 nm, 404.7 nm 435.8 nm, 546.1
nm, 486.0 nm, and 656.1nm)
Page 72 of 97 Rev. 3.12


SPD-M10Avp uses the 486.0 nm and 656.1 nm emission lines of a deuterium lamp (D 2
lamp) and the 253.7nm, 404.7nm, 435.8nm, and 546.1nm emission lines of a mercury
lamp (Hg lamp) for wavelength calibration. Wavelength calibration using the emission
lines of a deuterium lamp (D 2 lamp) and mercury lamp (Hg lamp) is prescribed in the
JIS standards, as follows:
JIS K0115 RULES FOR ABSORBANCE ANALYSIS

Measure the light intensity of a low-pressure mercury lamp or deuterium
discharge tube to determine the wavelengths exhibiting the maximum intensity,
and compare with the emission line wavelengths to calibrate the wavelength scale.
Use the mercury lamp designated by Shimadzu for the wavelength accuracy test. The
test evaluation is based on a direct comparison of the emission line (natural physical
constant) unique to the lamp. The following document prescribes the use of natural
physical constants for traceability.
The use of calibration standards such as natural physical constants, derived
standards, standard reference materials, or internal standards which are not
traceable to national standards, will require adequate documentation to describe
the validity of the calibration standards used.”
Test 3
(Procedure) 1) The brightness of the D2 and W lamp are measured automatically during Test 2
Wavelength Accuracy Test, above.
2) Read and record the maximum voltage value (200 to 260 nm and 400 to 800 nm) for
each lamp from the results of Test 2 Wavelength Accuracy Test.
(Criteria) Maximum intensity of each lamp satisfies the criteria below:

Test 4
(Procedure) 1) Start LCsolution and run [System Check].

2) Set the check level to “Normal” and start the system check.
3) Read the lighting time for each lamp (units: hours).
(Criteria) D2 TIME ≦ 2000h

W TIME ≦ 2000h
Page 74 of 97 Rev. 3.12

Operational Qualification LC/SPD System Inspection Procedure
4.13 System
4.13.1 System with UV-VIS Detector
1. Introduction
with UV-VIS Detector.
Test 1
(Outline) Checks the concentration accuracy of the gradient system.
(Procedure) [Low-pressure Gradient System]
1) Connect a resistor tube (4 m x 0.1 mm ID) between the mixer (1.7 mL) outlet and
the detector.
2) Measure out 20 mL of caffeine solution (250 mg/L) and add water to make the total
volume 500 mL.
3) Set 500 mL of water as Liquid A, and the caffeine solution prepared at step 2) above
as Liquid B, C and D, as the mobile phase.
4) Degas the mobile phase.
5) Set the parameters as follows:
Pump system Mode LP.GE
T.Flow 2.000mL/min
Detector Wavelength 272nm
Response 3 (0.5sec)
AUX RANGE 2 (1.0AU/V)
Data-processing Full scale 1024mV(ATTEN=10) or 1024mAU
unit (LCsolution automatically sets the intensity
axis.
For CLASS-VP, Normalize is possible.)
6) Open the drain valve and purge for 5 minutes using the following conditions:
T.Flow : 9.000 ｍ L/min
B.Conc : 25％
C.Conc : 25％
D.Conc : 25％
7) Change T.Flow to 3.000 mL/min. Close the drain valve and continue pumping until
the baseline stabilizes.
8) Change the conditions as follows and continue pumping until the baseline stabilizes.
B.Conc : 0％
C.Conc : 0％
D.Conc : 0％
9) After zeroing the baseline, conduct the gradient performance test described in
Additional Information. For Chromatopac, run the time program to print the Level
every 30 seconds.
10) For LCsolution, move the cursor over the measured chromatogram and read the
signal levels at the concentrations
0%(B.Conc=0), 10% (B.Conc=10), 50% (B.Conc=50), 90%(B.Conc=90), and
100% (B.Conc=100).
(Measure the signal level at the point where the baseline stabilizes.)
11) Calculate the actual concentrations, as follows:
10% actual concentration = (B.Conc 10 level) – (B.Conc 0 level) x 100
(B.Conc 100 level) – (B.Conc 0 level)
Similarly, calculate the 50% and 90% actual concentrations.
12) Repeat the gradient performance test for Liquids A and C and Liquids A and D.
[High-pressure Gradient System]

1) Connect a resistor tube (4 m x 0.1 mm ID) between the mixer (1.7 mL) outlet
and the detector.
2) Measure out 20 mL of caffeine solution (250 mg/L) and add water to make the
total volume 500 mL.
3) Set 500 mL of water as Liquid A, and the caffeine solution prepared at step 2)
above as Liquid B, as the mobile phase.
Pump system Mode B.GE
T.Flow 2.000mL/min
Detector Wavelength 272nm
Response 3 (0.5sec)
Data-processing Full scale 1024mV(ATTEN=10) or 1024mAU
unit (LCsolution automatically sets the intensity
axis.
For CLASS-VP, Normalize is possible.)
6) Open the drain valve and purge for 5 minutes using T.Flow = 3.000 mL/min,
B.Conc=100%.
7) Close the drain valve, set B.Conc to 0% and repeat the purging.
8) Change T.Flow to 3.000 mL/min. and B.Conc to 50% and continue pumping
until the baseline stabilizes.
9) Change T.Flow to 2.000 mL/min. and B.Conc to 0% and continue pumping
until the baseline stabilizes.
After zeroing the baseline, conduct the gradient performance test described in
Additional Information. For Chromatopac, run the time program to print the Level
every 30 seconds.
10) For LCsolution, move the cursor over the measured chromatogram and read
the signal levels at the concentrations
0%(B.Conc=0), 10% (B.Conc=10), 50% (B.Conc=50), 90%(B.Conc=90), and
100% (B.Conc=100).

10% actual concentration = (B.Conc 10 level) – (B.Conc 0 level) x 100
* For a high-pressure gradient system with three pumps, replace Liquid B (pump B)
with Liquid C (pump C) and repeat steps 1) to 10) to compile more data.
(Criteria) Set value  1.0%

Set value  2.0%[for Low-pressure Gradient System]
Page 76 of 97 Rev. 3.12

<Additional (Gradient program)

Information> Create and run the gradient program below.
TIME FUNC VALUE
5.00 B.Conc 0.0
5.01 B.Conc 10.0
10.00 B.Conc 10.0
10.01 B.Conc 50.0
15.00 B.Conc 50.0
15.01 B.Conc 90.0
20.00 B.Conc 90.0
20.01 B.Conc 100.0
25.00 B.Conc 100.0
25.01 B.Conc 0.0
30.00 Stop
(Chromatopac time program to print out the levels)

Create the BASIC program shown below.
It prints the input level on the chart at 30-second intervals
BASIC PROGRAM
10 LPRINT LEVEL (channel number);
20 WAIT 30
30 GOTO 10
40 END
Test 2
(Item) Noise/drift test
(Outline) Checks that the noise and drift values meet the criteria.
(Procedure) 1) Connect a resistor tube (4 m x 0.1 mm ID) between the autosampler outlet piping and
the detector.
2) Insert the On-line Degasser (IN) suction tube into degassed methanol and purge the
pump for 10 minutes at 2.000 mL/min.
3) Set the pump flow rate to 1.000 mL/min.
[Drift test]
4) Set the detector wavelength to 250 nm, response to 10 (2 s), and AUX RANGE to 2
(1.0 AU/V).
5) Set the data-processing unit recording fullscale to 4 mV (4 mAU) FS.
6) After the lamp lights, wait one hour for it to stabilize.
7) Record the baseline for one hour and record the amount of change over the hour as
the drift.
[Noise test]
8) Change the fullscale to 0.5 mV (1 mV) (0.5 mAU (1.0 mAU)) and record the
baseline for 15 minutes. Record the maximum noise amplitude.
9) For SPD-20AV or 10AVvp, also conduct measurements at 600 nm wavelength.

10) Replace the lamp with a W lamp.
11) Change the wavelength to 600 nm and repeat the measurements in steps 4) to 8).
Note: Noise measurement time can be applied to 45min-60min(15min) of Drift
measurement time.
(Criteria) Drift ≦ 6.0 x 10-4 AU/h　

Noise ≦ 2.0 x 10-5 AU
Test 3
(Item) UV-VIS repeatability test
(Outline) Checks the system repeatability of peak area and retention time for different injected
sample volumes.
(Procedure) 1) The following items are required:

a) Mobile phase
[For isocratic system] Water : Methanol = 3:2
[For gradient system] Liquid A: water, Liquid B: Methanol
Use degassed HPLC-grade methanol and water that passes through the on-line
degasser.
b) Column Shim-pack VP-ODS or LUNA C18(2)
150mm X 4.6mmID
c) Sample Caffeine solution (20mg/L)
d) Methanol and isopropanol for washing
A column equivalent to the product specified above can also be used.
2) Wash the flow channels with a suitable solvent. At this stage, use a 1.6C coupling
with no column connected.
3) Pump the mobile phase and then replace the coupling with the column.
4) Set the unit parameters as follows:
[Isocratic System]
[LC parameter]
Pump A.Flow : 1.000 mL/min
P.Max : 20 MPa
Time program 5.00 : Stop
Detector Wavelength : 272nm
AUX RANGE : 2 (1.0AU/V)
Response : 3 (0.5sec)
Column oven Temp : 40℃
Autosampler Injected volume : 5L, 10L and 50L
Analysis time : 5 min
Cooler temp. : － (OFF)
[Gradient System]
[LC parameter]
Pump T.Flow : 1.000 mL/min
B.Conc : 40 %
P.Max : 20 MPa
Time program 5.00 Stop
Detector Wavelength : 272 nm
AUX RANGE : 2 (1.0AU/V)
Autosampler Injected volume : 5 L, 10 L and 50 L
Use default settings for the other parameters.
Page 78 of 97 Rev. 3.12

5) Set the following values for the data-processing unit.

WIDTH :5
SLOPE : 1000
DRIFT :0
T.DBL : 1000
Minimum Area　: 100000
ATTEN : 10　
SPEED :5
STOP.TM : 5 min
(LCsolution )
Intensity Range : Normalize
(LCsolution automatically sets the intensity axis.)
(CLASS-VP)
*Detector Configuration
Y-Axis Units :mAU
Y-Axis Multiplier :0.001
*Data Graph Properties
Ymin :-100
Ymax : 1000
Units :mAU
(“Normalized” is possible. Set Full scale properly.)
Note: The SLOPE value can be changed as the optimal SLOPE value depends on the
mobile phase and column status.
6) Start the LC System.

not leak.
8) Wait until the baseline stabilizes. Inject 10 L mobile phase and check that no peak
is detected on the chromatogram. If using a manual injector, inject mobile phase
equivalent to several times the loop volume.
9) Inject 10 L test sample. If using a manual injector, inject test sample equivalent to
several times the loop volume. Repeat the analysis several times.
10) Calculate the coefficient of variance (CV%) for the retention time and peak area over
the six analyses.
11) If the autosampler is connected, similarly perform six continuous measurements
using 5μL and 50μL injections, and calculate the retention time and peak area
variation coefficients (CV%).
* For a high-pressure gradient system with three pumps, replace Liquid B (pump B) with
Liquid C (pump C) and repeat steps 1) to 10) to compile more data.
Step 11) is not performed for Solution C (Pump C).
(Criteria) Peak area CV％ ≦ 1.0%

Retention time CV％ ≦ 0.5%
* Refer to Additional Information for details about the calculation of CV%.
<Additional (Expression to Calculate CV％)

Information>
n : number of analyses
X1 … Xn : retention time of each peak (peak area)
X mean value
SD : standard deviation
CV% : coefficient of variation
C V ％  (SD / X )  100
値
n
Xi  X 
2
SD  i 1
n  1
X  ( X 1  X 2... Xn) / n
n ：分析回数
X 1, ... , Xn ：各ピークの保持時間
（ピーク面積）
X ：平均値
SD ：標準偏差
C V ％：変動係数
Page 80 of 97 Rev. 3.12

Operational Qualification LC/PDA System Inspection Procedure
4.13.2 System with Photodiode Array Detector
1. Introduction
with Photodiode Array Detector.
Test 1
(Outline) Checks the concentration accuracy of the gradient system.

(Procedure) [Low-pressure Gradient System]
1) Connect a resistor tube (4 m x 0.1 mm ID) between the mixer (1.7 mL) outlet and the
detector.
volume 500 mL.
as Liquid B, C and D, as the mobile phase.
Pump system Mode LP.GE
T.Flow 2.000mL/min
Detector Sampling 0.32sec
Analysis time 30min
Time constant 0.32sec
Lamp D2＆W
Start wavelength 190nm
End wavelength 800nm
Response 3 (0.5sec)
Data processing unit Full scale LCsolution : Auto setting
CLASS-VP: Normalize
Multi chromato LCsolution:
272nm, Band width ±4nm,
Ref. correction OFF
CLASS-VP:
272nm, Band width 8nm
6) Open the drain valve and purge for 5 minutes using the following conditions:
T.Flow : 9.000mL/min
B.Conc : 25％
C.Conc : 25％
D.Conc : 25％
7) Change T.Flow to 3.000 mL/min. Close the drain valve and continue pumping until
the baseline stabilizes.
8) Change the conditions as follows and continue pumping until the baseline stabilizes.
B.Conc : 0％
C.Conc : 0％
D.Conc : 0％
9) Conduct zero-point adjustment and start the gradient program shown in Additional
Information.
10) Move the cursor over the measured chromatogram and read the signal levels at the
concentrations
0%(B.Conc=0), 10% (B.Conc=10), 50% (B.Conc=50), 90%(B.Conc=90), and 100%
(B.Conc=100).
10% actual concentration = (B.Conc 10 level) – (B.Conc 0 level) ×100
12) Repeat the gradient performance test for Liquids A and C, and Liquids A and D.
[High-pressure Gradient System]

1) Connect a resistor tube (4 m x 0.1 mm ID) between the mixer (1.7 mL) outlet and the
detector.
volume 500 mL.
as Liquid B, as the mobile phase.
Pump system Mode B.GE
T.Flow 2.000mL/min
Detector Sampling 0.32sec
Analysis time 30min
Time constant 0.32sec
Lamp D2＆W
Start wavelength 190nm
End wavelength 800nm
Response 3 (0.5sec)
Data processing unit Full scale LCsolution : Auto setting
CLASS-VP: Normalize
Multi chromato 272nm, Band width±4nm, Ref. correction
OFF
6) Open the drain valve and purge for 5 minutes using T.Flow=3.000mL/min,
B.Conc=100%.
7) Close the drain valve, set B.Conc to 0% and repeat the purging.
8) Change T.Flow to 3.000 mL/min. and B.Conc to 50% and continue pumping until the
baseline stabilizes.
9) Change T.Flow to 2.000 mL/min. and B.Conc to 0% and continue pumping until the
baseline stabilizes. Conduct zero-point adjustment and start the gradient program
shown in Additional Information. For Chromatopac, run the time program to print the
Level every 30 seconds.
10) Move the cursor over the measured chromatogram and read the signal levels at the
concentrations
0%(B.Conc=0), 10% (B.Conc=10), 50% (B.Conc=50), 90%(B.Conc=90), and 100%
(B.Conc=100).
10% actual concentration = (B.Conc 10 level) – (B.Conc 0 level) ×100
(Criteria) Set value  1.0%
Page 82 of 97 Rev. 3.12

<Additional (Gradient program)

Information> Create and run the gradient program below.
TIME FUNC VALUE
5.00 B.Conc 0.0
5.01 B.Conc 10.0
10.00 B.Conc 10.0
10.01 B.Conc 50.0
15.00 B.Conc 50.0
15.01 B.Conc 90.0
20.00 B.Conc 90.0
20.01 B.Conc 100.0
25.00 B.Conc 100.0
25.01 B.Conc 0.0
30.00 Stop
Test 2
(Outline) Checks that the noise and drift values meet the criteria.
(Procedure) 1) Connect a resistor tube (4 m x 0.1 mm ID) between the autosampler outlet piping and
the detector.
2) Insert the on-line degasser (IN) suction tube into degassed methanol and purge the
pump for 10 minutes at 2.000 mL/min.
3) Set the pump flow rate to 1.000 mL/min.
[Drift test]
1) Turn on the detector and start the workstation.
Sampling : 2.00sec interval

Analysis time : 60min
Time constant : 2.00sec
Lamps : D2 &W
Start wavelength : 190nm
End wavelength : 800nm
3) Set the multi-chromatogram with the data-processing parameters, as follows:

(LCsolution)
# Enabled Display Wavelength: 250 nm Bandwidth: 4 nm
　　　No Ref. Correction
(CLASS-VP)
# Enabled Display Wavelength: 250 nm Bandwidth: 8 nm
# Enabled Display Wavelength: 600 nm Bandwidth: 8 nm

Pump Settings
[For Isocratic System]
A.Flow : 1.000mL/min
[For low-pressure Gradient System]
Flow : 1.000mL/min
B.Conc : 0％
C.Conc : 0％
D.Conc : 0％
[For High-pressure Gradient System]
Flow : 1.000mL/min
B.Conc : 0%
5) Start pumping. Wait at least 60 minutes after the D 2 and W lamps light for the
baseline to stabilize. Confirm that the baseline is stable and conduct a single analysis.
Set the vial number to -1 before starting the analysis.
6) Set the analysis data scale to 10 mAU fullscale. (Automatic setting is possible. Adjust
full-scale as appropriate.)
7) When the analysis is complete, print the chromatogram as a report and read the drift
value.
[Noise Test]
1)　 With settings 1) to 4) made for the Drift Test unchanged, set the analysis time to 15
minutes and start a single analysis. Set the vial number to -1 before starting the
analysis.
2) Set the analysis data scale to 1 mAU full scale.
3) When the analysis is complete, print the chromatogram as a report and read the noise
value.
(Criteria) Drift (250 nm) ≦ 2.0 x 10-3 AU/h

(600 nm) ≦ 2.0 x 10-3 AU/h
Noise (250nm) ≦ 5.0 x 10-5 AU
(600nm) ≦ 5.0 x 10-5 AU
Page 84 of 97 Rev. 3.12

Test 3
(Item) PDA repeatability test
(Outline) Checks the system repeatability of peak area and retention time for different injected sample
volumes.

a) Mobile phase
[For isocratic system] Water : Methanol = 3:2
[For gradient system] Liquid A: water, Liquid B: Methanol
Use degassed HPLC-grade methanol and water that passes through the on-line
degasser.
b) Column Shim-pack VP-ODS or LUNA C18(2)
150mm X 4.6mmID
d) Methanol and isopropanol for washing
2) Wash the flow channels with a suitable solvent. At this stage, use a 1.6C coupling with
no column connected.
3） Pump the mobile phase and then replace the coupling with the column.
4） Set the autosampler unit parameters as follows:
Sample cooler temperature : OFF
(Only if a sample cooler is supplied.)
Sampling : 0.32sec interval
Analysis time : 5min
Time constant : 0.32 sec
Lamps : D2 &W
Start wavelength : 190 nm
End wavelength : 800 nm
6) Set the multi-chromatogram with the data-processing parameters, as follows:

(LCsolution)
　　　No Ref. Correction
(CLASS-VP)
# Enabled Display Wavelength: 272 nm Bandwidth: 8nm
　　　
(Procedure) 7) Set the unit parameters as follows:

Pump Settings
A.Flow : 1.000 mL/min
P.Max : 20 MPa
Time Program Settings
5.00 Stop
Oven Settings
Temp. : 40℃
Autosampler Settings
Injected volume　 : 5L, 10L and 50L
[For Gradient System]

Pump Settings
Flow : 1.000 mL/min

B.Conc : 40 %
P.Max : 20 Mpa
Time Program Settings
5.00 Stop
Oven Settings
Temp. : 40℃
Autosampler Settings
Injected volume　 : 5L, 10L and 50L
8) Set the peak integration parameters as follows:

WIDTH :5
SLOPE : 1000
DRIFT :0
T.DBL : 1000
Minimum Area　　 : 100000
(For LCsolution, set Normalize for Intensity.)
(For CLASS-VP, “Normalized” is possible. Set Full scale properly.)
Note: The SLOPE value can be changed as the optimal SLOPE value depends on
the mobile phase and column status.
Page 86 of 97 Rev. 3.12


10) Check that mobile phase flows from the detector outlet and that the flow channels do not
leak.
11) Wait until the baseline stabilizes. Inject 10 L mobile phase and check that no peak is
detected on the chromatogram. If using a manual injector, inject mobile phase
12) Inject 10 L test sample. Repeat the analysis several times.
the six analyses.
14) If the autosampler is connected, similarly perform six continuous measurements using
5μL and 50μL injections, and calculate the retention time and peak area variation
coefficients (CV%).
However, step 14) is not performed for Solution C (Pump C).
(Criteria) Peak area CV% ≦ 1.0% each

Retention time CV% ≦ 0.5% each
<Additional (Expression to Calculate CV%)

Information> CV %  ( SD / X )  100
  Xi  X 
n
2
i 1
SD 
n 1
X  ( X 1  X 2  ...  Xn) / n
n ：分析回数
n X 1,..., Xn: number

：各ピークの保持時間
of analyses （ピーク面積）
X Xn ：平均値
X1 … : retention time of each peak (peak area)
SD ：標準偏差
X mean value
CV % ：変動係数
Operational Qualification LC/RF System Inspection Procedure
4.13.3 System with Spectrofluorometric Detector
1. Introduction
with Spectrofluorometric Detector.
Test 1
(Item) RF repeatability test
sample volumes.

a) Mobile phase
[For isocratic system] Water: acetonitrile = 1: 4
[For gradient system] Liquid A: water, Liquid B: acetonitrile
Use degassed HPLC-grade acetonitrile and water that passes through the
on-line degasser.
b)　 Column Shim-pack VP-ODS or LUNA C18(2)
　　　150mm X 4.6mmID
c)　Sample acridine, anthracene (10mg/L acetonitrile)
d)　Acetonitrile and isopropanol for washing
2) Wash the flow channels with a suitable solvent. At this stage, use a 1.6C coupling
with no column connected.
[LC parameter]
Pump A.Flow 　: 1.000mL/min
P.Max : 20 MPa
Detector Excitation wavelength : 360nm
Fluorescence wavelength : 450nm
Sensitivity : 2 (medium)
Gain : 1 (x1)
LAMP : 1 (ON)
RATIO :1
Autosampler Injected volume : 10μL
Analysis time　 :7
Cooler temp. : - (OFF)
Page 88 of 97 Rev. 3.12

[Low-pressure Gradient System]

[LC parameter]
B.Conc : 80 %
P.Max : 20 MPa
Time program 5.00 B.Conc 100
6.00 B.Conc 100
6.01 B.Conc 50
12.00 Stop
Detector Excitation wavelength : 360 nm
Fluorescence wavelength : 450 nm
Sensitivity : 2 (middle)
Gain : 1 (x1)
LAMP : 1 (ON)
RATIO :1
Response : 2 (0.5 s)
Cooler temp. : －(OFF)
[For High-pressure Gradient System]
[LC parameter]
B.Conc : 80 %
P.Max : 20 MPa
Time program 7.00 Stop
Detector Excitation wavelength : 360 nm
Fluorescence wavelength : 450 nm
Sensitivity : 2 (middle)
Gain : 1 (x1)
LAMP : 1 (ON)
RATIO :1
RESPONSE : 2 (0.5 s)
Cooler temp. : －(OFF)
5) Set the following values for the data-processing unit.

WIDTH :5
SLOPE : 500
DRIFT : 0 　[For isocratic system]
　　　　　　　　　　　　 : 500 [For gradient system]
T.DBL :0
Minimun.Area : 100000
ATTEN　　: 10
(LCsolution automatically sets the intensity axis.)
SPEED :5
STOP.TM : 7min
(PC Workstation)
LCsolution : Auto setting
CLASS-VP: Normalize
Note: The SLOPE value can be changed as the optimal SLOPE value depends on the
mobile phase and column status. If the sensitivity goes out of range, change the
SENS and Gain settings such that the peak height does not exceed full scale.

not leak.
8) Wait until the baseline stabilizes. Inject 10 L mobile phase and check that no peak
is detected on the chromatogram. If using a manual injector, inject mobile phase
9) Inject 10 L test sample. If using a manual injector, inject test sample equivalent to
several times the loop volume. Repeat the analysis several times.
10) Calculate the coefficient of variance (CV%) for the retention time and peak area
over the six analyses.
(Criteria) Peak area acridine CV％ ≦ 5.0％ each

anthracene CV％ ≦ 2.0％ each
Retention time acridine and anthracene CV％ ≦ 0.5％
* Room temperature fluctuations 2C max. during measurements.

Page 90 of 97 Rev. 3.12


Information>
C V ％  ( SD / X )  100
値
n
 Xi  X 
2
SD  i 1
n 1
X  ( X 1  X 2 . . . Xn) / n
nn : number of analyses
：分析回数
X1
X 1,…
.. . , Xn
Xn : retention time of each peak
：各ピークの保持時間 (peak area)
XX ：平均値
mean value
SD ：標準偏差
CV％：変動係数
Operational Qualification LC/RID System Inspection Procedure
4.13.4 System with Refractive Index Detector
1. Introduction
with Refractive Index Detector.
Test 1
(Outline) Measures the noise and drift while the flow cell is filled with water.
Note: Room temperature fluctuations 2C max. during this test.
(Preparation) (1) The following items and equipment are required:
(a) Water (distilled or ion-replacement)
Use water that has been placed in the measurement room temperature for several
hours in a container with its lid loosely attached. The refractive index of water is
dependent on ambient temperature and the amount of dissolved gases. Use air-
saturated distilled or ion-replacement water at room temperature for this test.
(b) Syringe
(c) Plugs (for inlet and outlet ports) x 2
(2) Turn on the detector. Set the controlled temperature to 40C and leave for at least two
hours with the power turned on. If room temperature exceeds 28C, set the controlled
temperature to (room temperature + 12C)
(Setting Procedure) Press the [func] key several times.
Enter “40.0” for <CELL TEMP>.
Press the [CE] key to return to the initial screen.
(Procedure) 1) Use the syringe to inject water into the cell.
During this process, turn RFLOW ON to replace the reference cell with water.
Ensure that no bubbles enter the flow channels.
2) Insert the plugs in the inlet and outlet ports.
3) User SCL or LCsolution to set the detector parameters as follows:
Parameter Set Value
Response : 5 (1.5 s)
Polarity : 1 (＋)
Cell controlled temp. : 40.00℃ (for room temperature below 28C)
Measurement mode : 0 (analysis mode)
AUX RANGE : 4 (2.5 x 10-4 RIU/V)
(For LCsolution, set intensity units to RIU.)
Page 92 of 97 Rev. 3.12

(Procedure) [Drift Test]

4) Drift Measurements
Record the baseline for 30 minutes.
(When the unit stabilizes, measure the baseline fluctuation and record it as the drift.)
<Setting when using Chromatopac>

ATTEN 4 (Chromatopac fullscale: 4 x 10-6 RIU)
<Setting when using LCsolution>
Make any setting that achieves 4 x 10-6 RIU fullscale.
(Can be set automatically. Adjust full-scale as appropriate.)
[Noise Test]
5) Noise Measurements
Record the baseline for 15 minutes.
<Setting when using Chromatopac>

ATTEN 0 (Chromatopac full scale: 0.25 x 10-6 RIU)
<Setting when using LCsolution>
Make any setting that achieves 0.25 x 10-6 RIU full scale.
Record the noise evaluation using the following evaluation method:

<Noise Evaluation Method>
Divide up the 15 minutes' data into 30 x 30-second increments.
Measure the noise amplitude in each increment.
Calculate the noise as the average noise amplitude over the 30 increments. (Reference:
ASTM E1303-89)
(Criteria) Drift: ≦ 5.0 x 10-7 RIU/h

Noise: ≦ 1.0 x 10-8 RIU
Test 2
(Item) RID repeatability test
sample volumes.

a) Mobile phase　　　Water: Methanol =3: 2
Use degassed HPLC-grade methanol and water that passes through
the on-line degasser.
b) Column Shim-pack VP-ODS or LUNA C18(2) 150mm x 4.6mmID
d) Acetonitrile and isopropanol for washing
2) Wash the flow channels with a suitable solvent. At this stage, use a 1.6C coupling with
no column connected.
[LC parameter]
Pump　　　 A.Flow : 1.000 mL/min
P.Max : 20 MPa
Time program 5.00 : Stop
Detector　　 AUX RANGE : 2 (1 x 10-3 RIU/V)
(For LCsolution, set intensity units to RIU.)
Response　　　　　　: 5 (1.5 s)
Cell controlled temp.: 40C or (room temperature +12C),
whichever is higher
Column Oven Temp 　: 40℃
Cooler temp. : - (OFF)
[Data-processing unit parameters]
If using C-R7A plus/C-R7Ae plus, C-R8A, or LCsolution, set the following values:
　RID-10A connected channels
WIDTH　　　　 : 5
SLOPE　　　　　: 200
DRIFT 　　　 : 0
Minimun.Area : 5000
ATTEN　　　　　: 6 (LCsolution automatically sets the intensity axis.)
STOP TM　　　 : 5
(PC Workstation)
LCsolution : Auto setting
CLASS-VP: Normalize
Note: Use Integration ON/OFF (LCsolution) as appropriate to avoid detecting peaks

near t0.
Other parameters are identical to Test 1.
5)Wait until the baseline stabilizes. Inject 10 L mobile phase and check that no peak is
detected on the chromatogram. If using a manual injector, inject mobile phase
6) Inject 10 L of caffeine solution (250 mg/L). If using a manual injector, inject test
sample equivalent to several times the loop volume. Repeat the analysis several times.
the six analyses.
Page 94 of 97 Rev. 3.12

(Criteria) Peak area CV% ≦ 2.0%

Retention time CV% ≦ 0.5%

Information> C V ％  ( SD / X )  100
値
n
 Xi  X 
2
SD  i 1
n 1
X  ( X 1  X 2 . . . Xn) / n
nn : number of analyses
：分析回数
X1
X 1…
, .. . ,Xn
Xn : retention time of each peak
：各ピークの保持時間 (peak area)
XX mean value
：平均値
SD ：標準偏差
CV％：変動係数
Operational Qualification Change List
5. Change List
When changing or deleting the contents of this document, or adding new information to this document, record
change list number, page number, reason, date, and name of the Performer in the Change List below, and obtain
the approval of the Reviewer and the Manager.
Change No: Enter the change number assigned in sequence from 1 onwards in accordance with “1.3
Entries and Revisions”.
Page: Enter the page number in which the change, deletion or addition was made.
Reason: Enter the reason for the change, deletion or addition.
Date: Enter the date on which the change, deletion or addition was made.
Change
Page Reason Date Performer Reviewer Manager
No.
Page 96 of 97 Rev. 3.12


Zeml-2010b Eng LC Oqp Qa

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ZEML-2010B

SHIMADZU HPLC SYSTEM

Operational Qualification Protocol

・ Performer Signature Date

・ Reviewer Signature Date

・ Manager Signature Date

Page 2 of 97 Rev. 3.12

6. This document is comprised of 1. Definitions, 2. Preliminary Checks, 3. Operational Qualification

1.3 Entries and Revisions

2. Rounding of numeric values

3. Deletion and correction of mistakenly filled items

4. Changing or deleting steps, procedures or acceptance criteria

Page 4 of 97 Rev. 3.12

7. In the event of a Fail

Additional Information Significant Figures

<What are Significant Figures?>

<Calculating Significant Figures>

<Format for Clearly Stating the Number of Significant Digits>

Page 6 of 97 Rev. 3.12

2.2 Performer Identification & Approval

2.3 Planning Items to be Evaluated in Operational Qualification

2.4 Calibration Certification and Instruments Used

* Thermometer and Recorder

* Resistance temperature sensor

* Standard reagents for performance inspection

Page 8 of 97 Rev. 3.12

(2) Items to be prepared by the customer

2.5 Other Tools Used

(1) Items prepared by the customer

(2) Items prepared by the Shimadzu Service Company

2.6 Before Inspections

Page 10 of 97 Rev. 3.12

3. Operational Qualification Outline

3.1 Outline of Inspection Procedures for Components

2. On-line Degasser DGU-20A3/20A5/DGU-3A/4A

3. Low Pressure Gradient Flow Control Valve (For LC-20AD/20AT)/FCV-10AL

5. UV-VIS Detector SPD-20A/20AV

6. Spectrofluorometric Detector RF-10AXL/10AXL Super

7. Refractive Index Detector RID-10A

8. Column Oven CTO-20A/20AC/10Avp/10ACvp/10ASvp

Page 12 of 97 Rev. 3.12

9. System Controller CBM-20A/20Alite

10. Autosampler SIL-20A/20AC/10ADvp

Page 14 of 97 Rev. 3.12

11. Data Processing Unit LCsolution

12. Data Processing Unit CLASS-VP

Page 16 of 97 Rev. 3.12

13. Photodiode Array Detector SPD-M20A/M10Avp

3.2 Outline of System Inspection Procedures

2. System with Photodiode Array Detector

3. System with Spectrofluorometric Detector

4. System with Refractive Index Detector

Page 18 of 97 Rev. 3.12

4. Operational Qualification Protocol

(Outline) Checks the display and LED operation.

(Procedure) 1) Turn on the power.

(Criteria) All display dots and key panel LEDs light.

(Outline) Checks that ROM and RAM memory operates normally.

(Criteria) Display shows “RAM OK/ROM OK”.

(Outline) Checks the firmware version.

(Criteria) The version number is displayed.

(Outline) Checks the time program functions.

　　　　　　　　　　　　　　　　　　　　ZEML-2010B

(Criteria) LC-20AD　　Pulsation ≦ 0.20 MPa (2.0kgf/cm2)