HPLC OQ PQ OperatingInstructions

Thermo Scientific Dionex
ChromeleonTM
Operational Qualification/
Performance Qualification
for HPLC Instruments
Operating Instructions
Version: 8.3
Date: May 2012
2012 Thermo Fisher Scientific Inc.
Doc.: HPLC_OQ_PQ_OperatingInstructions_V8_3.docx
Doc. No. 4828.3250A
OQ and PQ Operating Instructions
Contents
1 How to Use This Manual ................................................................................... 1
2 Introduction ....................................................................................................... 3
2.1 Defining the Limits .......................................................................................................................3
2.1.1 Operational Qualification (OQ) ...............................................................................................3
2.1.2 Performance Qualification (PQ) .............................................................................................3
2.1.3 System Suitability Check (SSC; also: System Suitability Test, SST).....................................3
2.2 Basic Requirements for Successful OQ and PQ ......................................................................4
3 Test Procedures ................................................................................................ 5

3.1 General Test Procedure ...............................................................................................................5
3.2 Test Procedure for Single Wavelength and VWD-3400RS Detectors .....................................7
3.3 Connecting and Configuring the System ..................................................................................8
3.3.1 System Connections ..............................................................................................................8
3.3.2 Configuration ........................................................................................................................12
3.4 Preparations ...............................................................................................................................15
3.4.1 Preparing the HPLC System ................................................................................................15
3.4.2 Checking the Fluidics ...........................................................................................................15
3.5 Preparations in Chromeleon .....................................................................................................16
3.5.1 Template Structure ...............................................................................................................16
3.5.2 Creating the Sequence Templates .......................................................................................17
3.5.3 Adapting the Report and Method .........................................................................................20
3.5.4 Device Names ......................................................................................................................21
3.6 Performing the Checks ..............................................................................................................22
3.7 Duration .......................................................................................................................................24
3.8 Evaluating the Test Sequences ................................................................................................25
3.9 Repeating Individual Checks ....................................................................................................25
4 Special Test Procedures for Individual Modules.......................................... 26

4.1 Introduction ................................................................................................................................26
4.2 Dionex VWD-3x00 Detectors: Noise and Drift with Dummy Flow Cells ................................26
4.3 Dionex Autosamplers: Sample Temperature Accuracy .........................................................27
4.3.1 Test Procedures ...................................................................................................................27
4.3.2 Connecting and Configuring the System ..............................................................................27
4.3.3 Performing the Check ...........................................................................................................28
4.3.4 Duration ................................................................................................................................29
4.4 Agilent G1321 Fluorescence Detector Linearity ..................................................................30
4.4.1 Introduction ...........................................................................................................................30
4.4.2 Preparing the System ...........................................................................................................30
4.4.3 Notes for Performing the Check ...........................................................................................30
4.5 Dionex Corona Detector ............................................................................................................31
4.5.1 Introduction ...........................................................................................................................31
4.5.2 Required Materials ...............................................................................................................31
4.5.3 Preparing the System ...........................................................................................................31
4.5.4 Notes for Performing the Check ...........................................................................................32
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5 Chromeleon 7 .................................................................................................. 33
5.1 Chromeleon 7 Terminology .......................................................................................................33
5.2 Creating the Sequences for the Qualification Checks ...........................................................33
5.3 Performing the Checks ..............................................................................................................38
5.4 Supported Instruments ..............................................................................................................39
5.5 Evaluating the Test Sequences ................................................................................................39
5.6 Selecting Special Test Procedures in Chromeleon 7 .............................................................40
6 Supported Instruments / Overview of the Checks ....................................... 41

6.1 Supported Instruments ..............................................................................................................41
6.2 Overview of the Checks .............................................................................................................44
6.2.1 Pumps ..................................................................................................................................44
6.2.2 Autosampler .........................................................................................................................48
6.2.3 Thermostatted Column Compartments and Column Ovens ................................................52
6.2.4 UV Detectors with Analytical Flow Cells ..............................................................................53
6.2.5 UV Detectors with Non-Analytical Flow Cells .......................................................................57
6.2.6 Fluorescence Detectors with Analytical Flow Cells ..............................................................60
6.2.7 Fluorescence Detectors with Non-Analytical Flow Cells ......................................................61
6.2.8 Refractive Index Detectors ...................................................................................................62
6.2.9 Evaporative Light Scattering Detectors ................................................................................62
6.2.10 Corona Detectors .................................................................................................................62
7 Procedures ...................................................................................................... 63
7.1 Baseline Noise, Drift, and Lamp Intensity of the UV Detector ...............................................63
7.1.1 Theory ..................................................................................................................................63
7.1.2 Performing and Evaluating the Check ..................................................................................63
7.2 Wavelength Accuracy of the UV Detector ...............................................................................64
7.2.2 Evaluation of the Check for UV Detectors ............................................................................64
7.2.3 Evaluation of the Check for Photodiode Array Detectors .....................................................64
7.2.4 Evaluation of the Check for Two-Channel Detectors ...........................................................65
7.2.5 Evaluation of the Check for Single Wavelength and VWD-3400RS Detectors ....................65
7.3 Linearity of the UV Detector ......................................................................................................65
7.3.1 Theory ..................................................................................................................................65
7.4 Precision of Injection Volume ...................................................................................................67
7.4.1 Theory ..................................................................................................................................67
7.5 Carry-Over by the Autosampler ................................................................................................68
7.5.1 Theory ..................................................................................................................................68
7.6 Linearity of Injection Volume ....................................................................................................69
7.6.1 Theory ..................................................................................................................................69
7.7 Sample Temperature Accuracy of Autosamplers ...................................................................70
7.7.1 Theory ..................................................................................................................................70
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7.8 Flow Precision ............................................................................................................................71

7.8.1 Theory ..................................................................................................................................71
7.8.2 Note on Performing and Evaluating the Check ....................................................................71
7.9 Solvent Composition of the Gradient Pump, Accuracy, Precision, and Ripple ..................72
7.9.1 Theory ..................................................................................................................................72
7.9.3 Performing the Checks for the Dionex P680 and UltiMate 3000 Pumps .............................73
7.9.4 Evaluating the Check ...........................................................................................................73
7.10 Temperature Accuracy of the Column Compartment ............................................................74
7.10.1 Theory ..................................................................................................................................74
7.11 Baseline Noise / Signal Height of the Fluorescence Detector ...............................................75
7.11.1 Theory ..................................................................................................................................75
7.11.3 Performing and Evaluating the Check for Dionex RF2000 and RF1002 Fluorescence
Detectors .............................................................................................................................................75
7.12 Wavelength Accuracy of the Fluorescence Detector .............................................................76
7.12.2 Performing and Evaluating the Check for Dionex RF2000 and RF1002 Fluorescence
Detectors .............................................................................................................................................76
7.12.3 Remarks on the Manufacturer Specification ........................................................................76
7.13 Linearity of the Fluorescence Detector....................................................................................77
7.13.1 Theory ..................................................................................................................................77
7.14 Baseline Noise and Drift of the RI Detector .............................................................................77
7.14.1 Theory ..................................................................................................................................77
7.15 Linearity of the RI Detector .......................................................................................................78
7.15.1 Theory ..................................................................................................................................78
7.16 Baseline Noise of the Evaporative Light Scattering Detector ...............................................78
7.16.1 Theory ..................................................................................................................................78
7.17 Baseline Noise/Signal Height/Drift/Spikes/Precision of the Corona Detector .....................79
7.17.1 Theory ..................................................................................................................................79
7.18 Signal Calibration of the Corona Detector ..............................................................................80
7.18.1 Theory ..................................................................................................................................80
8 Troubleshooting .............................................................................................. 81
8.1 General Notes .............................................................................................................................81
8.2 Failure of Individual Checks .....................................................................................................81
8.2.1 UV Detector ..........................................................................................................................81
8.2.2 Autosamplers........................................................................................................................82
8.2.3 Pump ....................................................................................................................................82
8.2.4 RF2000 Fluorescence Detector ...........................................................................................82
8.2.5 RI Detector ...........................................................................................................................83
8.2.6 ELS Detector ........................................................................................................................83
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8.2.7 Corona Detector ...................................................................................................................83
9 PGM Files ......................................................................................................... 85

9.1 Wavelength Accuracy of the Photodiode Array Detector ......................................................85
9.2 Baseline Noise, Drift, and Lamp Intensity of the UV Detector ...............................................88
9.3 Linearity of the UV Detector ......................................................................................................90
9.4 Precision of Injection Volume ...................................................................................................92
9.5 Carry-Over of the Autosampler and Linearity of the Injection Volume ................................94
9.6 Sample Temperature Accuracy ................................................................................................96
9.7 Solvent Composition of a Gradient Pump, Accuracy, Precision, and Ripple (Standard
Gradient) ................................................................................................................................................98
9.8 Temperature Accuracy of the Column Compartments for Automatic Data Acquisition ..101
9.9 Signal-to-Noise Ratio of the Fluorescence Detector ............................................................103
9.10 Wavelength Accuracy of the Fluorescence Detector (Emission) .......................................105
9.11 Wavelength Accuracy of the Fluorescence Detector (Extinction) ......................................107
9.12 Baseline Noise and Drift of the RI Detector ...........................................................................109
9.13 Linearity of the RI Detector .....................................................................................................111
9.14 Baseline Noise and Drift of the ELS Detector .......................................................................113
9.15 Baseline Noise, Signal Height, Spikes, and Drift of the Corona Detector ..........................115
9.16 Signal Height, Precision, and Signal Calibration of the Corona Detector ..........................117
10 Example Report ............................................................................................. 119
Contents: IV of IV HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012

1 How to Use This Manual

The layout of this manual is designed to provide quick reference to the sections of interest to the user.
However, we recommend that you review the manual thoroughly before starting Operational or
Performance Qualification in order to obtain full understanding of the procedure.
The descriptions throughout the manual often refert to instruments of the Thermo Scientific Dionex
TM
UltiMate 3000 series. In the following, all these instruments are named without the labelling "Thermo
Fisher Scientific" and partially also without "Dionex".
At various points throughout the manual, messages of particular importance are indicated by certain
symbols:
Note: Indicates general information to help obtain optimum performance of the instrument.
The information contained in this manual should not be construed as a commitment by Thermo Fisher
Scientific. Thermo Fisher Scientific assumes no responsibility for any errors that may appear in this
document. This document is believed to be complete and accurate at the time of publication. In no
event shall Thermo Fisher Scientific be liable for incidental or consequential damages in connection
with or arising from the use of this document. We appreciate your help in eliminating any errors that
may appear in this document.
Tip: The descriptions in this manual refer to sequence templates version 8.3 or later. For
sequence templates of a later version than 8.3, the Release Notes for all sequence
templates with a version later than 8.3 complement this manual. The version number
of the sequence template is indicated in the name of the report template.
The information contained in this document is subject to change without notice.

All rights reserved, including those for photomechanical reproduction and storage on electronic media.
No part of this publication may be copied or distributed, transmitted, transcribed, stored in a retrieval
system, or transmitted into any human or computer language, in any form or by any means, electronic,
mechanical, magnetic, manual, or otherwise, or disclosed to third parties without the express written
permission of Thermo Fisher Scientific Inc.
Trademarks
PEEK is a trademark of Victrex PLC.
All other trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries.
HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012 Page 1 of 119

Page 2 of 119 HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012

2 Introduction
The increasing number of standards and official regulations provide evidence that it is extremely
important to monitor the used instruments and to make sure that they work as intended if you want to
achieve reliable analytical results. To make the results transparent, quality management according to
ISO 9000 and following monitors and documents the quality of the equipment at different times.
This is the purpose of the Operational Qualification (OQ) and Performance Qualification (PQ)
procedures described in the sections below.
2.1 Defining the Limits

According to "The development and application of guidance on equipment qualification of analytical
instruments" of P. Bedson and M. Sargent [Accred. Qual. Assur. (1996) 1: 265 - 274] the following
definitions apply:
2.1.1 Operational Qualification (OQ)

The purpose of Operational Qualification is to prove and document that an analytical system functions
according to its operating specification while the specific environmental conditions are taken into
account.
In instrument specifications, suppliers must therefore define exactly the conditions that must be
observed. As conditions vary, for example, varying ambient temperatures, higher limits must be used.
Usually, Operational Qualification is only performed directly after a new device has been installed.
2.1.2 Performance Qualification (PQ)

The purpose of Performance Qualification is to prove and document that an analytical system
functions according to a specification that is suitable for the system's routine operation. As a system is
subject to wear when being operated, it may happen that the supplier's specification is no longer met.
This means that the procedures used for Performance Qualification are the same as those used for
Operational Qualification, but the tolerances are less strict. If required (for example, if stricter
requirements apply for routine analysis), users can adapt the limits. However, the adapted limits must
not be narrower than the OQ limits. Performance Qualification is usually performed after repair or
regular system service procedures have been performed.
Using the same procedures for OQ and PQ also simplifies the handling.
2.1.3 System Suitability Check (SSC; also: System Suitability Test, SST)
The purpose of SSC is to prove and document that the necessary limits are met for a specific
measuring application. The specific conditions required for that application, e.g., solvents, column
material, and temperature, must be taken into account. The check can be developed by the supplier
on request. However, it is not part of the test procedures below.
Do not use SSC limits that are more restrictive than the limits used for Performance Qualification.

2.2 Basic Requirements for Successful OQ and PQ

As described in section 1, OQ and PQ are instrument-specific test procedures. The procedures
described below apply to defined instruments (see section 6.1):
If a system includes more than one module (A + B) of the same module class (examples for module
classes: pump, autosampler, UV detector, FL detector, RI detector, etc.), remove module B from the
Server Configuration before running the OQ/PQ setups. The tests will match the remaining module A.
If you want to qualify a second module (B) of the same class, you must remove module (A) from the
Server Configuration temporarily, add module B, and run the OQ/PQ setup for module (B) to create
the templates.
When qualifying systems that include a Dionex FLM-3x00 Flow Manager, a NCS system or a NCP
pump, use the NANO_CAP_MIC_LC_Templates (not the HPLC_TEMPLATES).
These OQ and PQ procedures require Chromeleon 6.80 SR11 or 7.1 SR1.

3 Test Procedures
3.1 General Test Procedure
The following required materials and parts are provided in the Performance Qualification kit
(part no. 4832.5000A).
Part no. Description Quantity

All kits
709.7021 10 l sample loop (1/16) 1
754.ZU1M SST connecting union 0.5 mm ID, 1/16" OD 1
2200.5502 Single-part, hand-tight fitting 2
3323.0010 Standards kit (caffeine and pyrene) 1
709.6000.066 Finger-tight 33 mm fitting kit (5 sets with ferrule and fittings) 0.2
5035.3000 Restriction tubing (ID: 0.18 mm; length: 15 m) 1
754.ZU1M SST connecting union 0.5 mm ID, 1/16" OD 1
TM
2251.6001 PEEK tubing (ID: 0.25 mm; length: 0.10 m) 1
For a kit without sample loop, order part no. 4832.5010A.
The Standards kit (part no. 3323.0010) contains the seven required caffeine and pyrene standards.
The standard at position RA8 (or 8) contains water as solvent. Due to legal shipping restrictions, the
pyrene standard is shipped in solid form. Before you can use the standard, dissolve the solid pyrene in
1 mL of methanol (HPLC-grade). Complete the following steps:
Unscrew the cap from the 2 mL vial labeled 3 g Pyrene.
Add about 1 mL of methanol (HPLC-grade), which is about half the vial volume.
Screw the cap onto the vial. Make sure that the cap seals tight.
Shake the vial for about 10 seconds to dissolve the solid pyrene.
Place the vial at the appropriate position in the autosampler.
Note: The pyrene standard is used for checking the wavelength accuracy of all UV detectors (except
the Dionex VWD-3100 and VWD-3400 detectors, the AD25 detector, and any other supported single
wavelength detectors; see section 3.2). Concentration deviations of 30% do not affect the test
results.
Sample Position Substance Concentration Checks

Dionex Any
(1)
Sampler Sampler
RA1 1 Pyrene in 3 g/mL Wavelength accuracy of a UV
methanol detector (see section 3.2 for a
list of excluded detectors)
RA2 2 Caffeine in water 10 g/mL Linearity of injection volume (for
sample loop volumes > 50 l)
Detector linearity
Carry-over by the autosampler


Dionex Any
(1)
Sampler Sampler
RA3 3 Caffeine in water 60 g/mL Wavelength accuracy (VWD-
3400RS, single wavelength
detectors)
Precision of injection volume
and flow (Gina 50, ACC-3000
and WPS-3000 with sample loop
volumes > 200 l)
Linearity of injection volume (for
sample loop volumes < 50 l)
Detector linearity
RA4 4 Caffeine in water 140 g/mL Precision of injection volume
and flow (without Gina 50)
Detector linearity
RA5 5 Caffeine in water 220 g/mL Precision of injection volume
and flow (typically for smaple
loop volume 25 L)
Detector linearity
RA6 6 Caffeine in water 300 g/mL Detector linearity
RA7 7 Caffeine in water 2000 g/mL Carry-over by the autosampler
RA8 8 Water (solvent) Carry-over by the autosampler
(1)
Dionex autosamplers: ASI-100(T), WPS-3000(T)SL / PL, WPS-3000TBPL Analytical, WPS-
3000T(B)FC AnalyticalWPS-3000(T)(X)RS, WPS-3000(T)PL RS and ACC-3000(T)
In order to qualify the flow precision of a Dionex DGP pump in combination with a Dionex WPS-
3000T(B)FC Analytical autosampler, you must position the following additional standard in the
carousel (concentration depends on the autosampler configuration) for the second pump (test
sequence: "XQ_INJECTOR_FLOW_REPRO_P680DGP_LEFT".)
Sample Position Substance Concentration WPS-3000T(B)FC Analytical

Configuration
RC1 Caffeine in water 140 g/mL Standard configuration (sample loop

volume: 50 l)
60 g/mL Standard configuration (sample loop
volume: 250 l)
The kit with part no. 3325.0010 contains the five required standards for qualification of an RI detector.

(2)
ASI 100 WPS-3000xx Any
Sampler
RA9 RB1 9 Glycerin in water 5 mg/mL RI detector linearity
(2)
xx: WPS-3000(T)SL / PL, WPS-3000TBPL Analytical, WPS-3000T(B)FC Analytical, WPS-
3000(T)(X)RS, WPS-3000(T)PL RS and ACC-3000(T)

In addition, the following solvents are required:
Solvent Quantity Checks

Methanol (HPLC grade) Approx. Wavelength accuracy of a UV detector (see section
Channel A 100 mL 3.2 for a list of excluded detectors)
Water (HPLC grade) Approx. All checks except the wavelength accuracy of a UV
Channel A 600 detector (see section 3.2 for a list of excluded
1200 mL detectors)
Water (HPLC grade) with ca. 300 mL Gradient accuracy, gradient precision, and ripple
0.1% Vol. acetone
Channel B
For qualifying a column compartment, a calibrated thermometer is required. The thermometer is

provided in the Column Thermostat PQ kit (part no. 5705.0050A). The temperature sensor is also
available as spare part, part no. 6705.0060.
For qualifying the column compartment of the ACC-3000(T), a flexible temperature sensor is required
in addition to the Column Thermostat PQ kit. The temperature sensor is available as Temperature
Sensor Type K for Thermometer P600/P700, part no. 6820.0010.
3.2 Test Procedure for Single Wavelength and VWD-3400RS

Detectors
As some important steps in the test procedure for single wavelength detectors and Dionex VWD-
3400RS detectors differ from the steps described in section 3.1, the test procedure is described here.
The Standards kit (part no. 3323.0010) includes seven caffeine and pyrene standards. However, as
caffeine is used for the wavelength accuracy check, you do not have to prepare (that is, dissolve) the
pyrene standard. Sample position RA1 (or 1) is not used.
In addition, the solvent used for the wavelength accuracy check is water (not methanol). Therefore,
only the following solvents are required:
Solvent Quantity Checks

Water (HPLC grade) Approx. 700 All
Channel A 1300 mL
Water (HPLC grade) with ca. 300 mL Gradient accuracy, gradient precision, and ripple
0.1% Vol. acetone
Channel B

3.3 Connecting and Configuring the System

The steps below describe the fluid connections of the HPLC system and all configuration settings
required for OQ and PQ in Chromeleon (Server Configuration Program) or on the instrument. Perform
all steps for each module in the system.
3.3.1 System Connections

System
Remove the column from the system. Thoroughly rinse all fluid components of the autosampler and
injection valve with water at both positions of the motorized switching valve of the autosampler.
Also rinse the pump thoroughly with water. Only then, connect the pressure regulator or restriction
tubing from the Performance Qualification kit to the injection valve and the UV detector, using the
fitting screws shipped with the kit, and using the 33 mm finger-tight fitting for the injection valve and
the single-part finger-tight fitting for the UV detector. Install the PEEK tubing from the Performance
Qualification kit between the injection valve and the restriction tubing, using the union fittings from
the kit if necessary ( Figure 1).
Injection valve
1 Fitting, finger-tight, 33 mm
(709.6000.066)
2 PEEK tubing, approx.
10 cm (2252.6001)
3 Single-part fitting, finger-
tight (2200.5502)
4 Union (2261.0102)
Thermostatted
column 5 Restriction tubing
(5035.3000)
Detector
Figure 1: Restriction tubing installed between injection valve and detector

If the system includes several detectors that are connected in series, connect the restriction tubing
to the detector that was connected to the column.
Qualifying a Dionex dual gradient pump
To qualify both pumps of a Dionex dual gradient pump with the same autosampler, restriction
tubing, and detector, you have to use an external motorized switching valve, such as, a valve in
the Dionex TCC-100, TCC-3100, TCC-3200(B) or TCC-3000RS/SD. (For a list of the supported
valves, see section 3.5.4). For information about the fluid connections, see the images below.

Case a: 6 port/2-position valve
to the waste
to the waste
Pump unit (L = left, R =

right)
Autosampler
Restriction tubing
to the waste
Detector
Figure 2: Fluid connection for testing the Dionex dual gradient pump, using a 6-
port/2-position valve
The valve is in position A or 1, depending on the valve type
to the waste
to the waste

right)
Autosampler
Restriction tubing
Detector
to the waste
The valve is in position B or 2, depending on the valve type


Case b: 10-port/2-position valve: right)
to the waste Autosampler
Restriction tubing
Detector
to the waste
to the waste
The valve is in position A
right)
Autosampler
to the waste
Restriction tubing
Detector
to the waste
to the waste
The valve is in position B
Setting up Agilent systems that are controlled by the Agilent Instrument Control Framework
(ICF)
As described above under "System", the motor switching valve of the column oven is not required
to qualify Agilent systems. However, if an Agilent system includes a column oven with motor
switching valve, the created instrument methods will automatically set the valve position depending
on the valve type (corresponds to the first position in the Instrument Method Editor window). For
this case, make sure that the valve is not switched dry.
Manual injection valve
Verify that the injection valve is fitted with a 10 l sample loop.
Autosampler
Position the standards as shown in the tables on page 5 and the following pages.
For the Thermo Scientific Accela autosampler, BT1 (bottle) is determined as solvent
reservoir.

Column compartment
When qualifying the column compartments, the temperature sensor of the thermometer must be
securely attached to the heating block.
When qualifying the column compartments TCC-100 and TCC-3x00(SC/RS) the temperature
sensor must be installed as shown in Figure 6.
Position of the sensor tip.
Figure 6: TCC-100/TCC-3x00(SD/RS) - Position of the temperature sensor
When qualifying the Dionex ACC-3000(T) column compartment, install the temperature sensor
as shown in Figure 7. Be sure to use the type K temperature sensor (and not the sensor from
the Column Thermostat PQ Kit). Install the temperature sensor behind the left capillary clip,
from a vertical point of view in the center of the oven, and 2 cm away from the right edge of
the heat-conductive pad.
Position of the sensor tip behind the capillary clip
Figure 7: ACC-3000(T) Position of the temperature sensor
When qualifying the column compartment of the Thermo Scientific Accela autosampler the
temperature sensor of the thermometer must be positioned within the oven close to the internal
temperature sensors.
When qualifying the supported Shimadzu column compartments, loosen a fastening screw, insert
the sensor between the screw and the metal block, and carefully retighten the screw.

3.3.2 Configuration
Agilent Instrument Control Framework (ICF)
The OQ/PQ templates are can be used to qualify "pure" Agilent systems (that include Agilent
modules only).
RF2000 fluorescence detector
Before you connect the RF2000 fluorescence detector with Chromeleon, set the ZWAVE
parameter to 1. You can set this value only on the instrument. It is not possible to set the value
from Chromeleon. Complete the following steps:
Disable the keyboard interlock by simultaneously pressing <Shift> and <CE>.
Press <func> repeatedly until the ZWAVE command appears on the display.
Press <1> on the number keypad and confirm with <Enter>.
Enable remote operation. To do so, press <func>, until the RS232 command appears on the
display. Confirm with <Enter>, and then press <func>. On the display, the reading is
CONNECT. Confirm with <Enter>. You can now connect the instrument with Chromeleon.
AD25 UV detector
On the Signals page for the AD25 in the Server Configuration program, change the unit to mAU
(instead of AU) and the factor to 1000 (instead of 1.00).
WPS-3000(T)PL and WPS-3000(T)PLRS autosamplers
For successful qualification of the Dionex WPS-3000(T)PL and WPS-3000(T)PLRS autosamplers,
make sure that the Upgrade Kit for a 250 l syringe is installed.
WPS-3000T(B)FC Analytical autosampler
The "WPS-3000TFC/WPS-3000TBFC" check box must be activated in the Server Configuration on
the Options page of the autosampler:
WPS-3000TBPL Analytical autosampler
In order to ensure a successful qualification, the Dionex WPS-3000TBPL Analytical autosampler
must be equipped with the Standard or Large Volume configuration. In addition, the WPS-
3000TBPL Analytical check box must be activated in the Server Configuration on the Options page
of the autosampler:
Figure 8: WPS-3000TB PL Analytical configuration

Dionex (UltiMate 3000) autosamplers with user-defined sample loop volume

The sample loop volume must be at least 20 l. In addition, you must set the sample loop volume
in the Server Configuration program to a value predefined by Chromeleon (see table).
Autosampler User-defined sample loop Sample loop volume

volume setting
WPS-3000(T)PL / 20 39 l 20 l
WPS-3000(T)PLRS 40 99 l 50 l
100 124 l 100 l
> 125 l 125 l
WPS-3000(T)SL / 20 39 l 20 l
WPS-3000(T)RS 40 129 l Micro
> 130 l Analytical
With 250 l injection volume kit 344 l 344 l
ACC-3000(T) 21 49 l 20 l
51 199 l 50 l
> 200 l 200 l
Qualifying the column compartment

Option A: Using the Column Thermostat PQ kit:
Thermometer P500/600:
Connect the thermometer to a free COM port on the Chromeleon server PC. Install the
Dostmann Thermometer P500/P600 driver in the Chromeleon Server Configuration program.
On the General page, select the COM port to which the thermometer is connected. In addition,
install a virtual channel (Device name: VirtualChannels_01; signal Name: TemperatureOVEN).
Thermometer P700:
Double-click CDM 2.06.00.exe (or a higher version) and install the virtual COM port driver to the
Chromeleon server PC. An additional COM port is made available in the Windows Device
Manager. The CDM 2.06.00.exe driver is on the Chromeleon Service Release DVD in the folder
Drivers\USB Virtual COM Port. Connect the thermometer to a free USB port on the Chromeleon
server PC. Re-start the Chromeleon server to update the settings. Add the Dostmann
Thermometer P500/P600 driver to the timebase in the Chromeleon Server Configuration
program. On the configuration page, select the new COM port that has been created by the
CDM driver. In addition, install a virtual channel (Device Name: VirtualChannels_01,
Signalname: TemperatureOVEN).
Tip: When changing the temperature sensor, you may have to adapt the
calibration values and sensor type setting of the thermometer. To do so, follow
the Operating Instructions shipped with the instrument. Otherwise, the
thermometer may show the wrong temperature. This is especially important
when qualifying the column compartment of the ACC-3000(T), which is
qualified using a type K temperature sensor.
Option B: Automatic data acquisition as analog signal:

In the Chromeleon Server Configuration program, install the analog output of the external
thermometer (Device: Integrator Driver) as an analog channel named TemperatureOVEN.

Option C: Manual data acquisition:

In the Chromeleon Server Configuration program, install the STH_manual device. The driver is
available under Generic on the Manufacturers list. Verify that the driver is in Demo Mode. In
addition, install a virtual channel (Device Name: VirtualChannels_01, Signal Name:
TemperatureOVEN). During the chromatographic run, you can then enter the temperature
indicated on the external thermometer on the OQ_PQ_STH_manual control panel. Option C
does not support qualification of the Agilent, Shimadzu, and Waters column compartments.
Tip: Sequence templates created with Chromeleon < 6.50 can be used in
Chromeleon 6.50 or later only after you have deleted the
STH_manual.connect line from the COLUMN_OVEN program file. If you
created new sequence templates from the Chromeleon 6.50 or later master
templates, you do not need to adapt the program file manually.
Tip: Option C (qualifying the column compartment with manual data acquisition) is
not supported for Agilent, Shimadzu, and Waters instruments.

3.4 Preparations
3.4.1 Preparing the HPLC System

To prepare the HPLC system for OQ or PQ, follow the steps below. Perform all steps for the
instruments in your system, observing the correct order.
UV detector
Turn on the detector lamp at least six hours before you start the check.
For detectors that have an additional Vis lamp, it is sufficient to turn on the UV lamp.
Refractive index detector
When you use an RI detector, turn on the instrument at least one hour before you start the check.
Rinse the reference cell and the sample cell at a flow rate of 1.0 mL/min (mobile phase: water). If
you check the wavelength accuracy of the UV detector using methanol (all detectors except the
Dionex VWD-3x00 detectors and all single wavelength detectors), disconnect the fluid components
of the RI detector from the HPLC system after you have rinsed the cells with water.
Fluorescence detector
For detectors with a continuously burning lamp (for example, Dionex RF2000): Turn on the detector
lamp approximately 30 minutes before you start the check.
For detectors with a flash lamp (for example, Dionex UltiMate 3000 FLD-3x00): Make sure that the
detector is sufficiently equilibrated (for example, the flow cell temperature).
Evaporative light scattering detector
Turn on the detector lamp approximately 30 minutes before you start the check.
TM
Corona detector
To qualify a Corona detector, follow the instructions in section 0. A supported UV detector is
required to qualify the other system modules. When qualifying the other system modules, you must
separate the fluidics of the Corona detector from the HPLC system.
Autosampler
Before you start the check, rinse the autosampler thoroughly with water. To do so, inject 250 l of
water at least five times. (If the allowed maximum injection volume of the autosampler is smaller,
inject five times the largest possible volume). Make sure that the fluid components and the syringe
are free of air bubbles. (Note: Although methanol is used as solvent for the first OQ and PQ check,
rinse the autosampler with water, as water is the solvent for all successive checks. Automatically
rinsing the system after the wavelength accuracy check ensures that the fluid system is sufficiently
prepared.)
Pump
When qualifying an RI detector, rinse the entire HPLC system with water. If you want to check the
wavelength accuracy for the UV detector, disconnect the fluid components of the RI detector from
the HPLC system before you rinse channel A with methanol. When qualifying Dionex VWD-
3400RS detectors and all single wavelength detectors, or if the wavelength accuracy is not
checked, use water to rinse channel A. In this special case, you need not disconnect the fluid
components of the RI detector from the HPLC system. For gradient pumps, use water + 0.1 % Vol.
acetone to rinse channel B. For a ternary high-pressure gradient system, use water to rinse
channel C.
3.4.2 Checking the Fluidics

Injection valve or autosampler
Verify that there are no pressure fluctuations when the valve switches from Load to Inject and vice
versa. Pressure fluctuations indicate system leakage or contamination. Eliminate any leaks and
contamination before you start the check.

3.5 Preparations in Chromeleon
3.5.1 Template Structure

Running Operational Qualification or Performance Qualification in Chromeleon comprises several
steps:
A wizard generates sequence templates from the master sequences of the Chromeleon CD, providing
only sequences that match the timebase. In addition, the wizard adapts the programs automatically to
the devices installed in the timebase ( Figure 9, step 1). For each check that is performed on the
same system, a separate copy of the sequence template is created ( section 3.6 and Figure 9, step
2). OQ/PQ is then performed with the sequences of the copied template ( section 3.6). In this way,
you may need to adapt the sequence templates only once to the device configuration to be checked.
(a)
(b)
Figure 9: Performing OQ/PQ: (a) Step 1: OQ/PQ Setup; (b) Step 2: Instruments OQ/PQ
The PQ_OQ directory on the Chromeleon CD has the following subdirectories ( Figure 10):
AS_AP_TEMPLATES, ED_TEMPLATES, HPLC_TEMPLATES, IC_TEMPLATES and
NANO_CAP_MIC_LC_TEMPLATES, Demo, and Reports.
The HPLC_TEMPLATES directory contains all master sequences required for OQ or PQ of a common
HPLC configuration. This directory has a SPECIAL HPLC TEMPLATES subdirectory for special
checks ( section 4). When creating the sequence templates, the wizard provides only those
sequences that match the timebase. For IC and BioLC systems, the wizard provides the sequences
from the IC_TEMPLATES directory. for systems with an electrochemical detector, the sequences from
the ED_TEMPLATES directory are provided, and for nano, cap, and micro systems, the wizard
provides the sequences from the NANO_CAP_MIC_LC_TEMPLATES directory. For HPLC systems,
the wizard provides the sequences from the HPLC_TEMPLATES directory. The Dionex PDA-100 and
PDA-3000 detectors are included in the master sequences of the IC_TEMPLATES and
HPLC_TEMPLATES directories. These OQ/PQ operating instructions refer only to the sequences of
the HPLC_TEMPLATES directory.

Figure 10: PQ_OQ directory structure on the CM_CD
Tip: The sequences in the HPLC_TEMPLATES directory do not support the qualification of
systems that include a Dionex FLM-3x00 Flow Manager, a Dionex NCS-3500RS
system, or a Dionex NCP-3200RS pump. Sequences for qualifying these systems are
available in the NANO_CAP_LC_TEMPLATES directory.
3.5.2 Creating the Sequence Templates

To install the sequences required for your system, follow the steps below:
Insert the Chromeleon CD or verify that you can access the PQ_OQ directory.
In the Browser, open the Qualification menu.
(a)
Figure 11: (a) Selecting OQ or PQ setup

In the menu, click "OQ Setup" or "PQ Setup". A wizard guides you through copying of the
sequences. Click Next > to go to the next step.
Figure 12: OQ/PQ setup wizard welcome page
Select the timebase for which you want to perform OQ or PQ and enter the name of the computer
on which the timebase is installed.
Figure 13: Selecting a timebase

Select PQ_OQ as the source directory of the master sequences.
Figure 14: Selecting the source directory
Select a unique name under which the sequence directory that contains all sequence templates
for this instrument is saved.
Figure 15: Selecting the storage location

A list of sequences is displayed. The list is adapted to the instrument configuration of the selected
timebase as defined in the Chromeleon Server Configuration program.
Figure 16: List of sequences for the timebase

When Chromeleon cannot automatically determine the pump type or the pump variant, you can select
the sequences as required. This applies to the Dionex P680 and UltiMate 3000 pumps. For the low-
pressure micro pumps, for example, the sequence must be selected according to the volume of the
installed mixing chamber (STD_GRAD sequence for the standard mixing chamber, Micro_Grad
sequence when the MicroFlow Kit is installed, and LONG_GRAD sequence when a mixing chamber
extension is installed). In all other cases, the selection is read-only. Select the sequences required for
the checks that you want to perform (see section 6). The selected sequences are automatically copied
to the corresponding datasource. When installation is complete, the report opens on the Specification
page.
Tip: If you use the TSP UV1000 UV detector, you can select the sequences only if the UV
lamp is installed in the detector.
3.5.3 Adapting the Report and Method

Disable the write protection (on the Edit menu, click Layout Mode), and then enter the
batch number, expiration date, and actual concentration of the standard
Names of customer and tester
Name of the item that is used to generate the backpressure [default: capillary (L: 15 m; ID:
0.18 mm)]
For all devices listed in the table below, the instrument names and limits recommended by Thermo
Fisher Scientific are automatically entered into the report only when you open the report after the
Warm up sample. The information is not yet entered when the sequence is copied.

Do not fill in the report for the supported devices (see below). The limits are listed from line 174
on. Change the limits only if you do not want to use the limits recommended by Thermo Fisher
Scientific.
Tip: When you use a Dionex UVD 170S, UVD 340S, UVD 170U or UVD 340U detector
with a non-analytical flow cell, you have to enter the specifications listed in section 4.2
or 6.2.5 manually into the report. The reason is that automatic recognition of flow cells
is not supported or not implemented.
The serial number is entered automatically for the following devices: all devices of the Agilent
1100/12x0 series, all devices of the Dionex UltiMate 3000 series, all P680 and P580 pumps, the
Dionex ASI-100 autosampler, the Dionex PDA-100, AD25, UVD 340U, and UVD 170U detectors,
and the supported Shimadzu devices. For all other devices, enter the serial number in column K
from line 141 on. (The fields have a yellow background.) To delete the value in the related box, on
the Edit menu, click Clear Values. This removes the Chromeleon variable from the cell and clears
the audit.xxx entry for the cell on the status bar. When qualifying instruments that are not listed in
the table below, enter the model name in column H (cells with a yellow background), deleting the
existing audit.xxx entry as before. From line 159 on, enter the limits in the column with the related
model name.
Enable the write protection (on the Edit menu, click Layout Mode), and then enter the
SAVE the report. To do so, click Save Report Definition on the Workspace menu.
To check the linearity of the UV detector, adapt the amounts in the QNT file of the sequence to the
actual amounts of the used standards.
3.5.4 Device Names

You can use user-defined device names and channel names (as defined in the Chromeleon Server
Configuration Program) for all devices except the thermometer and virtual channels required for
qualifying thermostatted column compartments (see table below). All other device names may differ
from the defaults. The PGM files in the sequences that were created as described above are
automatically adapted to the used device and channel names.
Instrument Name
Devices names of the pump's eluent channels %A, (%B), (%C), (%D)
Device name of the external thermometer Thermometer
Signal name of the external thermometer TemperatureOVEN
Device name of the virtual channel VirtualChannels_01
Tips:
When you start the batch, the following warnings may appear:
{SOLVENT_CHANGE (91)} SOLVENT_CHANGE (91): Warning P0001: The program start time is undefined.
{SOLVENT_CHANGE - Sampler} Missing inject command.
{Pump} Eluent %A changed from Methanol to Water. Is this correct?
{OQ_COLUMN_OVEN (64) TemperatureOven} Setting of property Average overrides channel type default.
For systems that are controlled by the Agilent Control Framework, the following warning may occur
(repeatedly):
{GRADIENT - LCSystem} Automatically resolving method inconsistencies. Inconsistencies: Parameters for Thermostat are set,
but not supported in current configuration.
Sequence templates created with Chromeleon < 6.50 can be used in Chromeleon 6.50 or later for
qualifying a thermostatted column compartment only after you have deleted the STH_manual.connect
line from the COLUMN_OVEN program file.

3.6 Performing the Checks

Create a copy of the template (see section 3.5.2). To do so:
1. In the Browser, open the Qualification menu.
2. In the menu, click Instruments OQ or Instruments PQ. A wizard guides you through copying of the
sequences. Click Next > to go to the next step.
3. Select the timebase for which you want to perform OQ or PQ and enter the name of the computer
on which the timebase is installed.
4. Select the source directory of the template to be used.
Each directory in the OQPQ_6_7_Draft_Tests folder contains a number of instrument-specific

sequence templates (see 3.5.2), as the figure shows for the Templates_6_6 directory. Click
Browse to select the OQPQ_6_7_Draft_Tests directory. A list of directories with the instrument-
specific sequence templates is shown:

Figure 17: Selecting a template directory

5. Enter a unique name for saving the copy (default: template name + date).
6. A list of all sequences of the corresponding template is displayed. Click to select the sequences
required for the checks (see section 6). After the sequences have been copied, the batch list of
the corresponding timebase is automatically opened. Start the batch to run the sequences.
The batch list contains the checks in the following order:

1. Fluid preparation of the system (Warm up sequence).
2. Temperature accuracy of the column compartment for manual data acquisition (Column Oven
sequence)
3. Wavelength accuracy of the UV detector (Wavelength sequence)
4. Baseline noise, drift, and lamp intensity of the UV detector (UV Noise Drift sequence)
5. Precision of injection volume and flow (Injector Flow Repro sequence and Injector Flow
Repro_P680DGP_Left sequence (optional))
6. Linearity of the UV detector (UV Linearity sequence)
7. Linearity of the injection volume (Sampler Lin CO sequence)
8. Carry-over by the autosampler (Sampler Lin CO sequence)
9. Baseline noise, signal height, and wavelength accuracy of the fluorescence detector
(Fluorescence or Fluores_V2 sequence)
10. Baseline noise and drift of the RI detector (RI_Noise_Drift sequence)
11. Linearity of the RI detector (RI_Linearity sequence)
12. Baseline noise of the evaporative light scattering detector (ELS_Noise sequence)
13. Solvent composition of gradient pumps: accuracy, precision, and ripple (STD_GRAD,
MICRO_GRAD, or LONG_GRAD sequences, or STD_GRAD_P680DGP_Left or
LONG_GRAD_P680DGP_Left sequences)
14. Solvent composition for ternary high-pressure gradient pumps: accuracy, precision, and ripple
between channels C and B (Tern_Grad_C_B sequence)

15. Solvent composition for quaternary low-pressure gradient pumps: accuracy, precision, and
ripple between channels C and B (Quad_Grad_C_D sequence)
16. Temperature accuracy of the column compartments for automatic data acquisition (Column
Oven sequence)
17. Resetting the solvent flow rate to 0.05 mL/min (Stop sequence)
Tip: When you use a manual injection valve, make sure that no air is injected with the
samples. Always inject at least five times the sample loop volume, that is, at least
50 l.
3.7 Duration
If the column compartment and the non-UV detectors are not included in the check, the entire check
takes approximately 3.5 hours. The additional duration for the other checks is as follows:
2 more hours when checking Dionex P680 or UltiMate 3000 pumps with mixing chamber extension,
an UltiMate 3000 LPG-3400M(B) pump, or an UltiMate 3000 LPG-3400BM pump (LONG_GRAD
sequence instead of the STD_GRAD or MICRO_GRAD sequence)
2 more hours when checking Dionex P680 DGP or UltiMate 3000 DGP pumps (standard
configuration)
4 more hours when checking Dionex P680 DGP or UltiMate 3000 DGP micro pumps, or pumps
with mixing chamber extension
3 more hours when checking the thermostatted column compartment
1.5 more hours when checking an RI detector
0.5 more hours when checking an ELS detector
1 more hour when checking a fluorescence detector
2 more hours when checking a ternary high-pressure gradient system (channels C and B)
After the wavelength accuracy of the UV detector has been checked, that is, after approximately 15
min. or 3 h 15 min., you are prompted to change the solvent for channel A from methanol to water. If
necessary, connect the fluid components of the RI detector to the system. If an autosampler is
installed, OQ/PQ will then run automatically.
Note: For Agilent systems that are controlled via the Agilent Instrument Control Framework
(ICF), the procedure for changing the eluent takes several samples. Check the Audit Trail for log
commands that describe the necessary manual tasks. After the "Solvent change step 1" sample,
the batch is aborted. You are asked to change the eluent to channel A and restart the batch. No
more manual tasks are required afterward.
Exemption: It is not necessary to change the solvent manually when qualifying systems with a
Dionex VWD-3400RS or a single wavelength detector as UV detector.

3.8 Evaluating the Test Sequences

To evaluate the detector linearity, enter the actual concentrations for the used standards into the
amount columns of the QNT file.
The master sequences on the Chromeleon CD and thus, all copies made from it for OQ and PQ are
linked to the corresponding report. Do not change this report (except certain sheets, see section
3.5.3). The report contains many references between data sheets. If you insert or delete lines and
columns, these references will be lost and the calculations will be wrong.
To ensure that the data is correctly read and processed in the report, print the report as Batch Report
from the Browser. Select the sequence for which you want to print the report. Verify that no sample
is selected! On the File menu, select Batch Report, and then click OK to start printing.
3.9 Repeating Individual Checks

It may be necessary to repeat one or several checks. In this case, refer to section 8. This section
provides possible causes for the failure. According to GLP, you have to repeat all checks following the
one that failed. The reason is that almost all checks require that the previous check be passed
successfully.
Example: If the UV detector linearity check fails, the results regarding the linearity of the injection
volume are questionable because the detector linearity is a prerequisite for checking the injection
volume.

4 Special Test Procedures for Individual Modules

4.1 Introduction
This section describes test procedures that fundamentally differ from the procedures described in
section 3. These are special procedures that can only be used for certain instruments. In addition, all
test sequences must be run one after the other, as the tests require different system configurations.
The test procedures described in sections 3 and 7 serve as a basis for the descriptions below, and this
section focuses on the differences in particular. When the test steps are identical, you will find a
reference to these sections.
The sequence templates for these tests are available in the SPECIAL_HPLC_TEMPLATES directory
(see Figure 10). Start the OQ/PQ Setup from this directory as described in section 3.5.2.
4.2 Dionex VWD-3x00 Detectors: Noise and Drift with Dummy Flow
Cells
For qualifying the Dionex VWD-3100 and VWD-3400(RS) detectors with the dummy flow cell, two
sequences are offered and can be added manually to the batch list.
UV_NOISE_DRIFT_VWD3x00: measuring noise and drift with a wavelength of 254 nm
UV_NOISE_VWD3X00_230nm: measuring noise with a wavelength of 230 nm.
These sequences can be used only for the above detectors, and the test procedure requires that the
flow cell be changed twice. If you want to carry out both tests consecutively, you do not need to
change the flow cell between the sequences (however, a manual confirmation of the message dialogs
is required).
The following table shows the drift and noise limits for dummy flow cells depending on the measured
wavelength. The specifications have to be entered into the report manually.
Instrument Parameter Description Limits(1)

OQ PQ
VWD-3100 / Baseline Noise Measuring wavelength: 254 nm 0.010 mAU 0.020 mAU
VWD-3400RS Drift Measured with the dummy flow cell 0.2 mAU/h 0.2 mAU/h
(dummy flow cell) that is shipped with the detector
(without fluidics).
Baseline Noise Measuring wavelength: 230 nm 0.004 mAU 0.008 mAU
Measured with the dummy flow cell
that is shipped with the detector
(without fluidics).
(1)
OQ limits with optimum measuring conditions, recommended PQ limits.

4.3 Dionex Autosamplers: Sample Temperature Accuracy

This section describes how the sample temperature accuracy is determined for the following Dionex
autosamplers: WPS-3000TRS, WPS-3000TXRS, WPS-3000TPL, WPS-3000TBPL Analytical, WPS-
3000T(B)FC Analytical, WPS-3000TSL, WPS-3000TPL RS, ACC-3000T, and the Thermo Scientific
Accela autosamplers. For this test, only the autosampler is required. The other modules of the HPLC
system are not required.
4.3.1 Test Procedures

The following table lists the materials required for performing the test.
Part No. Description Quantity

6820.0010 Type K temperature sensor for P600/P700 thermometers 1
5705.0050A Column Thermostat PQ Kit 1
In addition, a standard glass vial (1.8 mL) is required. Fill the vial with water (do not seal)
4.3.2 Connecting and Configuring the System

System Connections
Connect the type K temperature sensor to the thermometer and make the necessary settings
(sensor type and calibration values) as described in the Operating Instructions for the
thermometer.
Dionex autosamplers: Fill an open standard glass vial (1.8 mL) with water and place it at
sample position RC8.
Thermo Scientific Accela autosampler: Fill two open standard glass vials (1.8 mL) with water
and place one at sample position C1 and the other at sample position A8.
Configuration
Thermometer P500/P600:
Connect the thermometer to a free COM port on the Chromeleon Server PC. Install the Dostmann
Thermometer P500/P600 driver in the Chromeleon Server Configuration program. On the General
page, select the COM port to which the thermometer is connected. In addition, install a virtual
channel (Device Name: VirtualChannels_01, Signal Name: TemperatureOVEN).
Thermometer P700:
Double-click CDM 2.06.00.exe (or a higher version) and install the virtual COM port driver to the
Chromeleon server PC. An additional COM port is made available in the Windows Device
Manager. The CDM 2.06.00.exe driver is on the Chromeleon Service Release DVD in the folder
Drivers\USB Virtual COM Port. Connect the thermometer to a free USB port on the Chromeleon
server PC. Re-start the Chromeleon server to update the settings. Add the driver Dostmann
Thermometer P500/P600 to the timebase in the Chromeleon Server Configuration program. On the
configuration page, select the new COM port that has been created by the CDM driver. In addition,
register a virtual channel (Device Name: VirtualChannels_01, Signalname: TemperatureOVEN).
Preparation in Chromeleon
To qualify the sample temperature accuracy, select the following sequence:
SAMPLER_TEMP_ACC.

4.3.3 Performing the Check

Start the SAMPLER_TEMP_ACC sequence. The sample temperature is set to 10C (15C for the
ACC-3000T, 30 C for the Thermo Scientific Accela autosampler), and the automatic carousel rotation
is stopped if applicable (also see note below). A message box prompts you to position the temperature
sensor as shown below:
Insert the temperature sensor into the vial from above at a right angle until the tip touches the
vial bottom.
Figure 18: Temperature sensor inserted into the vial of a Dionex autosampler (example)
Dionex autosampler: Rotate the carousel until the carousel cover closes completely.
Thermo Scientific Accela autosampler: Close the autosampler door carefully.
Figure 19: Carousel cover closed of a Dionex autosampler (example)
When the nominal temperature is reached, the external thermometer is used to record the sample
temperature over a period of 60 minutes. At the end of the check, you are prompted to remove the
temperature sensor. Afterward, the carousel rotation is restarted.

Tip: Do not perform any autosampler commands during the test. Movements of the needle
arm or carousel may damage the thermometer or autosampler.
Note: For Dionex instruments with a firmware version 4.07: If the check is interrupted,
the carousel rotation is not automatically turned on afterward. In this case, repeat the
check or use the "Tray_Rotation_On" program to turn it on again. Simply add the
program to the batch (object type: Program) and start the batch.
For Dionex instruments with firmware version < 4.07: These instruments do not
support carousel rotation. When you issue a command that relates to carousel
rotation, the following error message is displayed in the Audit trail:
[Error] 13:50:25 0.000 {Sampler} Unknown property. Perform a driver and/or firmware
update.
4.3.4 Duration
The test takes approximately 75 minutes.

4.4 Agilent G1321 Fluorescence Detector Linearity
4.4.1 Introduction
For determining the linearity of the Agilent series G1321 fluorescence detector, you must select the
"FLUORES_LINEARITY" sequence.
In addition to the detector to be qualified, this test requires an autosampler and an HPLC pump.
4.4.2 Preparing the System
To determine the linearity of the Agilent 1100/12x0 series G1321 fluorescence detector, you need the
standards listed in the table below.
Sample Substance Concentration

Position [mg / 100 mL]
15 Acetonitrile / water 90:10 (v/v) -
16 Anthracene in acetonitrile 0.5
In addition, a chromatographic column, for example, Dionex Acclaim 120 (C18, 5 m, ID: 4.6 mm,
length: 100 mm) or similar is required. This column can be ordered under part no. 059147.
Furthermore, approx. 200 mL of eluent acetonitrile / water 90:10 (v/v) are needed.
4.4.3 Notes for Performing the Check

In the first sample of the FLUORES_LINEARITY sequence, Chromeleon prompts the user to
exchange the eluent and to install the column. In the next sample, the system is prepared for the
measurement. Therefore, manual equilibration can be omitted.
At the end of the sequence, the eluent remains in the system.
Note on eluent change: For Agilent systems that are controlled via the Agilent Instrument
Control Framework (ICF), the procedure for changing the eluent takes several samples. Check
the Audit Trail for log commands that describe the necessary manual tasks. After the "Solvent
change step 1" sample, the batch is aborted. You are asked to change the eluent on channel A
and restart the batch (the system still includes the restriction tubing from the general qualification
tests performed earlier). After the "Solvent change step 3" sample, the batch is aborted again.
You are asked to install the required column and restart the batch.

4.5 Dionex Corona Detector
4.5.1 Introduction
In order to fully qualify the detector, select the CORONA_NOISE_DRIFT_SNR and
CORONA_RESP_CALIB sequences.
In addition to the detector to be qualified, this test requires an autosampler and an HPLC pump.
Qualification is performed by using the digital signal of the detector (USB port, unit: pA).
Note: Before qualifying the detector, make sure that the other modules in the system (for
example, the pump and autosampler) have been qualified successfully. Qualification of the other
system modules requires a supported UV detector. In this case, note the information on Corona
detectors in section 3.4.1.
4.5.2 Required Materials
Part number 70-6565 includes the standard kit that is required for qualification of Corona detectors. It
includes vials with five different caffeine solutions. You must fill the solutions into 1.8 mL autosampler
vials and position them in the autosampler as shown in the table below.
Sample Substance Concentration

Position [mg / mL]
Dionex Sampler Any Sampler
RD1 25 Caffeine in water 25
RD2 26 125
RD3 27 250
RD4 28 500
In addition, the following materials are required:
Shiseido column holder, 20 mm for 2 and 4 mm ID pre-column cartridges
(part number: 88-12414)
Shiseido C18 pre-column, 20 x 4.0 mm, 3 m (part number: 88-12307)
Eluent A: water / methanol 80:20 (v/v), degassed - approx. 500 mL
4.5.3 Preparing the System
The system is not automatically equilibrated. All the steps below must be completed manually.
Ensure that the gas flow is on for a sufficient period of time (> 5 min - see Operating Instructions)
and that the gas pressure with within the required range of 35 psi 1 psi while the pump flow is still
turned off.
Change the eluent from channel A [water/methanol 80:20 (v/v)] and purge the system for at least
5 min at a flow rate of 5 mL/min.
Make sure that the capillary at the column outlet is not connected to the detector at this point, but
goes directly to the waste. Install the required column and equilibrate the system at a flow rate of
1 mL/min for at least 15 minutes.
Connect the capillary from the column outlet to the detector inlet and equilibrate the system for
another 30 minutes.
Finally, start the batch with the two qualification sequences.

4.5.4 Notes for Performing the Check

The pump flow and gas flow must be turned off manually after qualification. Note that the pump flow
should be turned off approx. 15 minutes before the gas flow (also see related description in the
Operating Instructions).
The qualification (running the sequence templates) takes about 55 minutes.

5 Chromeleon 7
5.1 Chromeleon 7 Terminology
Tip: Please note that Chromeleon 7 terminology is different from the terminology used in
Chromeleon 6.80. For details, refer to the 'Glossary - Chromeleon 7.0,' which is
available in the Documents folder of your Chromeleon 7 installation.
5.2 Creating the Sequences for the Qualification Checks

For performing OQ and PQ checks in Chromeleon 7, it is not required to create and copy the
sequence templates from a Chromeleon CD. An Instrument Qualification Wizard automatically
performs these steps for you. The wizard creates the sequences to be run. No instrument-specific
sequence templates are created.
To start the wizard, in the "Tools" menu of the Chromeleon Console, click "Instrument
Qualification".
Figure 20: Starting the Instrument Qualification Wizard
Select the qualification type: Installation (qualification of the installation), Operational (qualification
in the working environment), or Performance Qualification (qualification during routine operation).

Figure 21: Selecting the qualification type
Select the instrument that you want to qualify.
Figure 22: Selecting the instrument

Click to connect the selected instrument to Chromeleon. The Instrument Connection

dialog box shows the connection process.
Figure 23: Connecting the instrument
On the third wizard page, you can select special test procedures for each module
(see also 5.6 and 4). This option is available only for the following modules:
VWD-3100
VWD-3400RS
Corona (all supported models)
WPS-3000T (+ Dostmann thermometer with channel TemperatureOVEN)
ACC-3000T (+ Dostmann thermometer with channel TemperatureOVEN)
Accela Autosampler (+ Dostmann thermometer with channel TemperatureOVEN)
Agilent G1321A/B
Figure 24: Selecting special tests

If the selected instrument does not include one of the above modules, the wizard page is skipped.

A list of sequences (tests) is displayed. The list is adapted to the instrument configuration of the
selected instrument as defined in the Chromeleon Instrument Configuration Manager.
Figure 25: List of sequences for the selected instrument
Select the sequences that you need for the tests that you want to perform. Mandatory tests, such
as "Warm up", are shown in the list, but the selection cannot be changed.
On the last wizard page, select a unique name under which the OQ and/or PQ sequence directory
for this instrument is saved.

Figure 26: Selecting the storage location

5.3 Performing the Checks

When the wizard has been completed, the selected sequences are created and are automatically
added to the instrument queue. A progress window shows which steps have been performed:
Figure 27: Progress during sequence creation
Finally, the Instrument View dialog box open, showing the Queue tab. As soon as you start the
queue, Chromeleon runs the sequences.
Figure 28: Sequence queue

5.4 Supported Instruments

In general, all modules and instruments listed under 6.1 are supported, if their driver is shipped with
the CM7 version in question.
The device and channel names that are used in the instrument methods of the OQ/PQ sequences can
be selected by the user as in CM6.x0 OQ/PQ. Only the additional modules required for the
qualification of thermostatted column compartments and column ovens require defined names (see
section 3.5.4).
Tip: To make the qualification sequence available for supported column thermostats or
column compartsments when using the Dostmann thermometer, the signal name must
be "TemperatureOven" (also see section 3.3.2 - "Qualifying the column
compartment").
5.5 Evaluating the Test Sequences

The qualification sequences are saved under the path that was selected in the wizard (see 5.2). The
sequences also include a report template.
To edit the report, open the report and remove the protection from the SPECIFICATION sheet. Enter
the following information:
Names of customer and tester
Sample information such as batch number, expiration date, and actual concentration of the
standard.
Name of the item that is used to generate the backpressure [default: capillary (L: 15 m; ID:
0.18 mm)]
Do not change any of the other report sheets. The report contains many references between data
sheets. If you insert or delete lines and columns, these references will be lost and the calculations will
be wrong.
To make sure that Chromeleon reads and processes the data in the report correctly, always print the
report from the Chromeleon Console.
In the Data category, right-click the sequence for which you want to print the report, and then click
"Print Report".

As an alternative, click "Print" on the Sequence Editor toolbar to print the report.
5.6 Selecting Special Test Procedures in Chromeleon 7

In CM7, sequence templates for these tests must not be downloaded from the Chromeleon CD - they
can be selected directly in the wizard.
A wizard page for special tests is displayed for the following modules (also see 4 and 5.2):
VWD- 3100
VWD-3400RS
Corona (all supported models)
WPS-3000T (+ Dostmann thermometer with channel TemperatureOVEN)

ACC-3000T (+ Dostmann thermometer with channel TemperatureOVEN)
Accela autosampler (+ Dostmann thermometer with channel TemperatureOVEN)
Agilent G1321A/B
Tip: The test for determining the sample temperature accuracy for the WPS, ACC-3000T,
or Accela autosampler is available only if the instrument configuration includes a
Dostmann thermometer with a TemperatureOVEN temperature channel.
The tests are prepared and performed as described in 4. Exception: CM7 includes a new driver for the
Dostmann thermometer that allows you to record temperature data directly as a signal channel. It is no
longer required to set up a virtual channel. All you need to do is go to the "Signals" tab page in the
driver configuration and change the signal name to TemperatureOVEN:
Figure 29: Configuring the Dostmann thermometer

6 Supported Instruments / Overview of the Checks

6.1 Supported Instruments
The procedures described below apply to the following instruments:
Instrument Supported Models

Pumps Thermo Scientific Dionex ISO-3100A (UltiMate 3000)
Thermo Scientific Dionex ISO-3100SD (UltiMate 3000)
Thermo Scientific Dionex ISO-3100BM (UltiMate 3000)
Thermo Scientific Dionex LPG-3400A(B) (UltiMate 3000)
Thermo Scientific Dionex LPG-3400XRS (UltiMate 3000)
Thermo Scientific Dionex LPG-3400RS (UltiMate 3000)
Thermo Scientific Dionex LPG-3400SD (UltiMate 3000)
Thermo Scientific Dionex LPG-3400M(B) (UltiMate 3000)
Thermo Scientific Dionex LPG-3400BM (UltiMate 3000)
Thermo Scientific Dionex DGP-3600A(B) (UltiMate 3000)
Thermo Scientific Dionex DGP-3600RS (UltiMate 3000)
Thermo Scientific Dionex DGP-3600SD (UltiMate 3000)
Thermo Scientific Dionex DGP-3600M(B) (UltiMate 3000)
Thermo Scientific Dionex HPG-3200A (UltiMate 3000)
Thermo Scientific Dionex HPG-3200M (UltiMate 3000)
Thermo Scientific Dionex HPG-3200RS (UltiMate 3000)
Thermo Scientific Dionex HPG-3200SD (UltiMate 3000)
Thermo Scientific Dionex HPG-3400M (UltiMate 3000)
Thermo Scientific Dionex HPG 3400RS (UltiMate 3000)
Thermo Scientific Dionex HPG 3400SD (UltiMate 3000)
Dionex P680
Dionex P580
Dionex M300
Thermo Scientific Accela Pump
(1,2)
Agilent 1100/12x0 series G1310A
(1)
Agilent 1100/12x0 series G1310B
(1,2)
(1)
(1)
Agilent 1100/12x0 series G1311C
(1)
(1,2)
(1,2)
(1)
(1)
(1)
Pump of the Waters Alliance 2690 Separation Module
TSP P2000
TSP P4000
Shimadzu LC-2010 Pump
Shimadzu LC-10Ai
Shimadzu LC-10AD
Shimadzu LC-10ADvp
Shimadzu LC-10AT
Shimadzu LC-10ATvp
Shimadzu LC-20AD
Shimadzu LC-20ADXR
Shimadzu LC-20AT


Autosampler Thermo Scientific Dionex ACC-3000(T) (UltiMate 3000)
Thermo Scientific Dionex OAS-3300TXRS (UltiMate 3000)
Thermo Scientific Dionex WPS-3000TXRS (UltiMate 3000)
Thermo Scientific Dionex WPS-3000(T)RS (UltiMate 3000)
Thermo Scientific Dionex WPS-3000(T)SL (UltiMate 3000)
Thermo Scientific Dionex WPS-3000(T)PL (UltiMate 3000)
Thermo Scientific Dionex WPS-3000TBPL Analytical (UltiMate 3000)
Thermo Scientific Dionex WPS-3000T(B)FC Analytical (UltiMate 3000)
Dionex ASI 100
Dionex GINA 50
Thermo Scientific Accela Autosampler
Thermo Scientifc Accela Open Autosampler
(1,2)
(1,2)
(1,2)
(1,2)
(1,2)
(1,2)
(1)
Agilent 1100/12x0 series G1367D
(1)
Agilent 1100/12x0 series G1367E
(1)
(1)
Autosampler of the Waters Alliance 2690 Separation Module
Waters WISP 717plus
TSP AS3000/AS3500
Shimadzu LC-2010 Autosampler
Shimadzu SIL-10A
Shimadzu SIL-10Ai
Shimadzu SIL-10AF
Shimadzu SIL-HTA
Shimadzu SIL-HTC
Shimadzu SIL-10ADvp
Shimadzu SIL-20AHT
Shimadzu SIL-20ACHT
Shimadzu SIL-20AXR
Shimadzu SIL-20ACXR
Thermostatted Column Thermo Scientific Dionex ACC-3000(T) (UltiMate 3000)
Compartments: Thermo Scientific Dionex TCC-3000RS (UltiMate 3000)
Thermo Scientific Dionex TCC-3000SD (UltiMate 3000)
Thermo Scientific Dionex TCC-3000 (UltiMate 3000)
Thermo Scientific Dionex TCC-3100 (UltiMate 3000)
Thermo Scientific Dionex TCC-3200(B) (UltiMate 3000)
Dionex STH 585
Dionex TCC-100
Thermo Scientific Accela Autosampler (column thermostat)
(1,2)
(1,2)
(1)
Column thermostat of the Waters Alliance 2690 Separation Module
TSP AS3000/AS3500 optional
Shimadzu LC-2010 column thermostat
Shimadzu CTO-10A
Shimadzu CTO-10Avp
Shimadzu CTO-10AC
Shimadzu CTO-10ACvp
Shimadzu CTO-10ASvp
Shimadzu CTO-20A
Shimadzu CTO-20AC


UV Detectors Thermo Scientific Dionex DAD-3000(RS) (UltiMate 3000)
Thermo Scientific Dionex MWD-3000(RS) (UltiMate 3000)
Thermo Scientific Dionex VWD-3100 (UltiMate 3000)
Thermo Scientific Dionex VWD-3400RS (UltiMate 3000)
Thermo Scientific Dionex PDA-3000 (UltiMate 3000)
Dionex PDA-100
Dionex PDA-100U
Dionex AD25
Dionex UVD 340U
Dionex UVD 170U
Dionex UVD 340S
Dionex UVD 170S
Thermo Scientific Accela PDA
(1,2)
(1,2)
(1,2)
(1,2)
(1)
(1)
(1,2)
(1,2)
(1,2)
(1)
(1)
Agilent 1100/12x0 series G1314E
(1)
Agilent 1100/12x0 series G1314F
(1,2)
(1,2)
(1,2)
(1,2)
Waters PDA996 Diode Array Detector
Waters PDA2996 Diode Array Detector
Waters 2487 Dual Lambda Absorbance Detector
TSP UV1000 Single Lambda Detector
TSP UV2000 Dual Lambda Detector
TSP UV3000 (analog and digital data recording)
TSP UV6000 PDA
Shimadzu LC-2010 SPD
Shimadzu SPD-10A
Shimadzu SPD-10Avp
Shimadzu SPD-10AV
Shimadzu SPD-10AVvp
Shimadzu SPD-20A
Shimadzu SPD-20AV
Fluorescence Detectors Dionex RF2000
Dionex RF1002
Thermo Scientific Dionex FLD-3100 (UltiMate 3000)
Thermo Scientific Dionex FLD-3400RS (UltiMate 3000)
(1,2)
(1)
Refractive Index Detectors Shodex RI-101
(1,2)
Evaporative Light Polymer Laboratories ELS2100
Scattering Detectors Polymer Laboratories ELS 2100 Ice
Varian 380-LC ELS detector
Varian 385-LC ELS detector


Corona Detectors Thermo Scientific Dionex Corona
Thermo Scientific Dionex Corona plus
Thermo Scientific Dionex Corona ultra
Thermo Scientific Dionex Corona ultra RS
(1)
Supported for control via Agilent Instrument Control Framework (ICF).
(2)
Supported for control via 1100/1200 HPLC system driver.
6.2 Overview of the Checks

The following tables provide an overview of the parameters to be checked and list the recommended
PQ limits for each HPLC module.
6.2.1 Pumps
The used test procedures are described in detail in sections 7.8 and 7.9.
Instrument Parameter Nominal values and conditions Limits(1)

OQ PQ
ISO-3100A / Flow Precision Flow rate: 0.3 mL/min RSD RSD
ISO-3100SD / Determined using the retention time 0.05 % or 0.1 % or
ISO-3100BM / precision (standard deviation and SD SD
LPG-3400A(B) / relative standard deviation) of 0.01 min 0.02 min
LPG-3400XRS / caffeine. The greater value is the
LPG-3400RS / valid limit.
LPG-3400SD /
LPG-3400M(B) /
LPG-3400BM /
DGP-3600A(B) /
DGP-3600RS /
DGP-3600SD /
DGP-3600M(B) /
DGP-3600BM /
HPG-3200A /
HPG-3200RS /
HPG-3200SD /
HPG-3200M /
HPG-3400A /
HPG-3400M /
HPG-3400RS /
HPG-3400SD /
P680 and P580
with analytical
pump heads
LPG-3400A(B) / Gradient Step gradient channel A/B HPG: HPG:
DGP-3600A(B) / Accuracy Steps: 1, 50, 99% channel B 0.2 % 0.5 %
HPG-3200A / Flow rate: 2 mL/min LPG/DGP: LPG/DGP:
HPG-3200M / 1.0 % 2.0 %
HPG-3400A / Gradient SD 0.2 % SD 0.5 %
HPG-3400M / Precision
P580 (HPG und Ripple 0.5 % 0.5 %
LPG) and
P680 (HPG, LPG
and DGP: all mixing
chamber variants
with analytical
pump heads


OQ PQ
LPG-3400M(B) / Gradient Step gradient channel A/B 2.0 % 3.0 %
DGP-3600M(B) Accuracy Steps: 10, 50, 90% channel B
Flow rate: 1 mL/min
Gradient SD 0.5 % SD 0.5 %
Precision
Ripple 0.5 % 0.5 %
HPG-3200RS / Gradient Step gradient channel A/B HPG: HPG:
HPG-3200SD / Accuracy Steps: 1, 50, 99% channel B 0.2 % 0.5 %
HPG-3400RS / Flow rate: 2 mL/min LPG/DGP: LPG/DGP:
HPG-3400SD / 1.0 % 2.0 %
LPG-3400RS /
LPG-3400SD / Gradient SD 0.15% SD 0.5%
DGP-3600RS / Precision
DGP-3600SD Ripple 0.5 % 0.5 %
LPG-3400BM / Gradient Step gradient channel A/B 1.0 % 2.0 %
DGP-3600BM Accuracy Steps: 10, 50, 90% channel B
Flow rate: 1 mL/min
Precision
Ripple 0.5 % 0.5 %
LPG-3400XRS Gradient Step gradient channel A/B: 1.0 % 2.0 %
Accuracy Steps: 10, 50, 90% channel B
Gradient Flow rate: 1mL/min SD 0.2 % SD 0.5 %
Precision
Ripple 0.5 % 0.5 %
Thermo Scientific Flow Precision Flow rate: 0.3 mL/min RSD RSD
Accela Pump Determined using the retention time 0.05 % or 0.1 % or
precision (standard deviation and SD SD
relative standard deviation) of 0.01 min 0.02 min
caffeine. The greater value is the
valid limit.
Gradient Step gradient channel A/B and C/D: 2,0 % 2,0 %
Gradient Flow rate: 1 mL/min SD 0,5 % SD 0,5 %
Precision
Ripple 0,5 % 0,5 %
Agilent 1100/12x0: Flow Precision Flow rate: 0.3 mL/min RSD RSD
G1310A/B Determined using the retention time 0.07 % or 0.07 % or
G1311A/B/C precision (standard deviation and SD SD
G5611A relative standard deviation) of 0.02 min 0.02 min
G1312A/B/C caffeine. The greater value is the
valid limit.
Agilent 1100/12x0: Gradient Step gradient channel A/B G1311 / G1311 /
G1311A/B/C Accuracy Steps: 1, 50, 99% channel B G5611: G5611:
G5611A Flow rate: 2 mL/min 1.5 % 1.5 %
G1312A/B G1312: G1312:
Step gradient channel C/D 0.7 % 0.7 %
Gradient (G1311A/B/C only): SD 0.5 % SD 0.5 %
Precision Steps: 1, 50, 99% channel D
Flow rate: 2 mL/min
Ripple 0.5 % 0.5 %


OQ PQ
Agilent 1290: Flow Precision Flow rate: 0.3 mL/min RSD RSD
G4220A/B Determined using the retention time 0.07 % or 0.07 % or
valid limit.
Gradient Step gradient channel A/B 0.7 % 0.7 %
Flow rate: 2 mL/min
Precision
Ripple 0.5 % 0.5 %
Pump module of Flow Precision Flow rate: 0.3 mL/min RSD RSD
the Waters Alliance Determined using the retention time 4.0 % or 4.0% or
2690 Separation precision (standard deviation and SD SD
Module relative standard deviation) of 0.1 min 0.1 min
valid limit.
Gradient Flow rate: 2 mL/min SD 0.5 % SD 0.5 %
Precision
Ripple 0.5 % 0.5 %
P2000(2) Flow Precision Flow rate: 0.3 mL/min RSD RSD
P4000 Determined using the retention time 1.5 % or 2.0% or
valid limit.
Accuracy Step: 50% channel B
Flow rate: 2 mL/min

Precision
Ripple 0.5 % 0.5 %
P4000 Gradient Step gradient channel C/D 1.0 % 2.0 %
Accuracy Step: 50% channel D
Flow rate: 2 mL/min
Precision
Ripple 0.5 % 0.5 %


OQ PQ
Shimadzu Flow Precision Flow rate: 0.3 mL/min RSD RSD
LC-2010 Determined using the retention time 0.075 % or 0.15 % or
LC-10Ai precision (standard deviation and SD SD
LC-10AD relative standard deviation) of 0.02 min 0.04 min
LC-10ADvp caffeine. The greater value is the
LC-10AT valid limit.
LC-10ATvp Gradient Step gradient channel A/B 1.0 % 2.0 %
LC-20AD(XR) accuracy Steps: 1, 50, 99% channel B
LC-20AT Gradient Flow rate: 2 mL/min SD 0.5 % SD 0.5 %
precision
Ripple 0.5 % 0.5 %
(1)
(2)
To determine the gradient accuracy and the gradient precision for the TSP P2000 pump, the
solvent composition must be as follows: 0, 50, and 100% of solvent B. This is because the
pump does not support a gradient program with more than 9 steps.

6.2.2 Autosampler
The used test procedures are described in detail in sections 7.4 to 7.7.

OQ PQ
ACC-3000(T) Precision of Injection volume: 5 l RSD RSD
Injection (20 and 50 l sample loop) 0.5 % 1.0 %
Volume Injection volume: 20 l
(200 l sample loop)
Linearity of Inject volume range: 1 10 l (20 l r r
Injection sample loop) 99.95 % 99.90 %
Volume Inject volume range: 5 25 l (50 l RSD RSD
sample loop) 1.0 % 1.0 %
Inject volume range: 5 80 l (200
l sample loop)
Carry-Over Injection volume: 10 l 0.02 % 0.02 %
(20 and 50 l sample loop)
Injection volume: 20 l
(200 l sample loop)
Temperature Temperature: 15C 2 C 4 C
Accuracy
OAS-3300TXRS Precision of Injection volume: 10 l RSD RSD
Injection 0.5 % 1.0 %
Volume
Linearity of Injection volume range: 4 12 l r 99.99 % r 99.99 %
Injection RSD RSD
Volume 1.0 % 1.0 %
WPS-3000(T)RS Precision of Injection volume: 5 l (analytical) RSD RSD
(Analytical version Injection Injection volume: 2 l (micro + XRS) 0.3 % 0.5 %
and micro option, Volume Injection volume: 10 l (250 l kit)
and 250 l injection
volume kit)
WPS-3000TXRS Linearity of Inject volume range: 5 90 l r r
Injection (analytical) 99.99 % 99.90 %
Volume Inject volume range: 1 20 l (micro RSD RSD
+ XRS) 0.5 % 1.0 %
Inject volume range: 10 160 l
(250 l kit)
Carry-Over Injection volume: 10 l (micro + 0.01 % 0.01 %
XRS)
Injection volume: 20 l (other
variants)
Accuracy


OQ PQ
WPS-3000(T)SL Precision of Injection volume: 5 l (analytical) RSD RSD
(Analytical version Injection Injection volume: 2 l (micro) 0.3 % 0.5 %
and micro option, Volume Injection volume: 10 l (250 l kit)
and 250 l injection Linearity of Inject volume range: 5 90 l r r
volume kit) Injection (analytical) 99.99 % 99.90 %
Volume Inject volume range: 1 20 l RSD RSD
(micro) 0.5 % 1.0 %
Inject volume range: 10 160 l
(250 l kit)
Carry-Over Injection volume: 10 l (micro) 0.01 % 0.01 %
variants)
Accuracy
WPS-3000(T)PL / Precision of Injection volume: 5 l RSD RSD
WPS-3000(T)PLRS Injection 0.3 % 0.5 %
(only with upgrade Volume
kit for 250 l Linearity of Inject volume range: 1 12 l (20 l r r
syringe) Injection sample loop) 99.99 % 99.90 %
Volume Inject volume range: 1 20 l (50 l RSD RSD
sample loop) 0.5 % 1.0 %
l sample loop)
l sample loop)
Carry-Over Injection volume: 10 l (20 and 50 l 0.05 % 0.05 %
sample loops)
variants)
Accuracy
WPS-3000TBPL Precision of Injection volume: 5 l (standard) RSD RSD
Analytical Injection Injection volume: 20 l (lage 0.3 % 0.5 %
(Standard and Volume volume)
Large Volume Linearity of Inject volume range: 5 25 l r r
configuration) Injection (standard) 99.99 % 99.90 %
Volume Inject volume range: 20 140 l RSD RSD
WPS-3000T(B)FC (large volume) 0.5 % 1.0 %
Analytical Carry-Over Injection volume: 10 l (standard) TBPL: TBPL:
(Standard and Injection volume: 20 l (large 0.03 % 0.05 %
Large Volume volume) T(B)FC: T(B)FC:
configuration) 0.05 % 0.10 %
Accuracy
Thermo Scientific Precision of Injection volume: 10 l RSD RSD
Accela Injection 1.0 % 2.0 %
Autosampler Volume
(25 L sample loop) Linearity of Injection volume rang: 2.5 12.5 l r 99.95 % r 99.90 %
Injection RSD RSD
Volume 1.0 % 1.0 %
Temperature Temperature: 30 C 2 C 4 C
accuracy


OQ PQ
ASI 100 Precision of Injection volume: 5 l RSD RSD
(250 l syringe) Injection 0.3 % 0.5 %
Volume
Linearity of Inject volume range: 5 80 l r r
Injection 99.99 % 99.90 %
Volume RSD RSD
0.5 % 1.0 %
Gina 50 Precision of Injection volume: 10 l RSD RSD
Volume
Linearity of Injection volume range: 10 80 l r 99.99 % r 99.90 %
Injection RSD RSD
Volume 0.5 % 1.0 %
Agilent 1100/12x0: Precision of Injection volume: 5 l RSD RSD
G1313A Injection 1.0 % 1.0 %
G1329A/B Volume
G1367A/B/C/D Linearity of Inject volume range: 5 80 l r r
Injection Inject volume range: 5 40 l 99.90 % 99.90 %
Volume (G1367D only) RSD RSD
1.0 % 1.0 %
Agilent 12x0: Precision of Injection volume: 5 l RSD RSD
G1367E Injection 0.5 % 0.5 %
G5667A Volume
G4226A

Injection Inject volume range: 1 20 l 99.90 % 99.90 %
Volume (G4226A only) RSD RSD
1.0 % 1.0 %
Sampler module of Precision of Injection volume: 5 l RSD RSD
the Waters Alliance Injection 1.0 % 1.0 %
2690 Separation Volume
Module Linearity of Inject volume range: 5 80 l r r
Injection 99.90 % 99.90 %
Volume RSD RSD
1.0 % 1.0 %
Waters WISP Precision of Injection volume: 5 l RSD RSD
717plus Injection 1.0 % 1.0 %
autosampler Volume
Injection 99.90 % 99.90 %
Volume RSD RSD
1.0 % 1.0 %
TSP AS3000/3500 Precision of Injection volume: 5 l RSD RSD
Volume
Injection 99.90 % 99.90 %
Volume RSD RSD
1.5 % 1.5 %


OQ PQ
Shimadzu Precision of Injection volume: 5 l RSD RSD
SIL-10A Injection 1.0 % 2.0 %
SIL-10Ai Volume
SIL-10AF

Injection 99.90 % 99.90 %
Volume RSD RSD
1.0 % 1.0 %
Shimadzu Precision of Injection volume: 5 l RSD RSD
LC-2010 Injection 0.3 % 0.5 %
SIL-10HTA Volume
SIL-10HTC Linearity of Inject volume range: 5 50 l r r
SIL-10ADvp Injection (SIL-10ADvp, SIL-20A(C)XR) 99.90 % 99.90 %
SIL-20A(C)HT Volume Inject volume range: 5 80 l RSD RSD
SIL-20A(C)XR (Other autosamplers) 1.0 % 1.0 %
(1)

6.2.3 Thermostatted Column Compartments and Column Ovens

The used test procedure is described in detail in section 7.10.

OQ PQ
(3)
Column Temperature OQ: Measured at : 35, 40, 50 C 2 C 3 C
(3)
compartment Accuracy PQ: Measured at : 35, 40, 45 C
module of the
ACC-3000(T)
autosampler
TCC-3000RS Temperature OQ: Measured at: 10, 30, 60, 105 C 1 C 2 C
Accuracy PQ: Measured at: 10, 30, 60, 90 C
TCC-3000SD Temperature OQ: Measured at (firmware < 1.30): 1 C 2 C
Accuracy 10, 30, 50, 65 C
OQ: Measured at (firmware 1.30):
10, 30, 50, 80 C
PQ: Measured at: 10, 30, 45, 60 C
TCC-3000 / Temperature OQ: Measured at: 10, 30, 60, 80 C 1 C 2 C
TCC-3100 / Accuracy PQ: Measured at: 15, 30, 45, 60 C
TCC-3200(B) /
TCC-100
STH 585 Temperature OQ: Measured at: 5, 20, 60, 85 C 1 C 2 C
Accuracy PQ: Measured at: 15, 30, 45, 60 C
(5)
Column Temperature OQ: Measured at : 30 C 2 C 3 C
(5)
compartment Accuracy PQ: Measured at : 30 C
module of the
Thermo Scientific
Accela
autosampler
(4)
Agilent Temperature OQ: Measured at : 5, 20, 60, 80 C 2 C 2 C
(4)
1100/12x0: Accuracy PQ: Measured at : 15, 30, 45, 60 C
G1316A
G1316B
(2)
Agilent 1290: Temperature OQ: Measured at : 20, 60, 95 C 2 C 2 C
(2)
G1316C Accuracy PQ: Measured at : 30, 50, 75 C
(2.3)
Column Temperature OQ: Measured at : 35, 45, 55 C 1 C 2
(2.3)
compartment Accuracy PQ: Measured at : 35, 45, 55 C
module of the
Waters Alliance
2690 Separation
Module
Column oven of Temperature OQ: Measured at: 20, 40, 60, 80 C 2 C 3 C
the TSP Accuracy PQ: Measured at: 25, 35, 45, 60 C
AS3000/AS3500
autosamplers
(2)
Shimadzu Temperature OQ: Measured at : 20, 40, 60 C 3 C 3 C
(2)
thermostatted Accuracy PQ: Measured at : 25, 35, 50 C
column
compartment
LC-2010
CTO-10ASvp
(2.3)
(2.3)
column
compartment
- CTO-10A
- CTO-10Avp
- CTO-20A


OQ PQ
(2)
(2)
column
compartment
- CTO-10AC
- CTO-10ACvp
- CTO-20AC
(1)
(2)
It is not possible to set the temperature on the column compartment module when the retention
time is negative. The first measurement reading is 10 minutes after the sample has been
started. At this time, equilibration of the column compartment may not be complete. Therefore,
the same temperature is set for the second measuring point. The column compartment module
has passed the check even if the target temperature is reached only for the second measuring
point.
(3)
According to the specification of the column compartment module, only target temperatures
above ambient are permitted. That is why measuring points below 35C are not evaluated.
(4)
Qualification of the first measuring point is omitted for the above-mentioned Agilent column
(2)
thermostats when controlled by Agilent ICF (also see footnote ).
(5)
According to the specification of the column compartment module, entering a target temperature
is only permitted during a sample. That is why temperature precision is evaluated at one
measuring point.
6.2.4 UV Detectors with Analytical Flow Cells


OQ PQ
VWD-3100 / Baseline Noise Measuring wavelength: 254 nm. 0.025 mAU 0.050 mAU
VWD-3400RS Drift 0.3 mAU/h 0.3 mAU/h
(analytical flow cell) Lamp Intensity Measuring wavelength: 230 nm. > 50 % > 40 %
(2)
Wavelength Nominal wavelength: 272.5 nm 2.0 nm 2.0 nm
Accuracy (maximum of caffeine)
Linearity Absorption range: up to 2.5 AU r r
99.97 % 99.90 %
RSD 3 % RSD 5 %
UVD 340S / Baseline Noise Measuring wavelength: 254 nm. 0.03 mAU 0.05 mAU
UVD 170S / Drift 0.8 mAU/h 2.0 mAU/h
UVD 340U / Lamp Intensity > 500000 > 400000
UVD 170U counts/s counts/s
(analytical flow cell)
(2)
Wavelength Nominal wavelength 1: 272.1 nm 0.75 nm 0.75 nm
Accuracy (UVD-340x only)
Nominal wavelength 2: 333.3 nm
(maxima of pyrene)
99.98 % 99.90 %
RSD 5 % RSD 5 %


OQ PQ
DAD-3000(RS) / Baseline Noise Measuring wavelength: 254 nm. 0.03 mAU 0.10 mAU
MWD-3000(RS) Drift 1.0 mAU/h 1.0 mAU/h
6 6
(analytical flow cell) Lamp Intensity > 2 x 10 > 1 x 10
(2)
counts/s counts/s
Accuracy (DAD only)
(maxima of pyrene)
99.95 % 99.90 %
RSD 5 % RSD 5 %
6 6
DAD-3000(RS) / Lamp Intensity Measuring wavelength: 254 nm. > 1 x 10 > 0.5 x 10
MWD-3000(RS) counts/s counts/s
Hardware Revision
Optics: 0
(analytical flow cell)
(2)
PDA-3000 / Baseline Noise Measuring wavelength: 254 nm. 0.03 mAU 0.10 mAU
PDA 100 / Drift 1.0 mAU/h 1.0 mAU/h
PDA 100U Lamp Intensity >13 x 10
6
>10 x 10
6
counts/s counts/s
Accuracy Nominal wavelength 2: 333.3 nm
(maxima of pyrene)
99.90 % 99.90 %
RSD 5 % RSD 5 %
Thermo Scientific Baseline Noise Measuring wavelength: 254 nm 0.30 mAU 0.50 mAU
Accela PDA Drift 2.0 mAU/h 2.0 mAU/h
Lamp Intensity Not Not
(Analytical flow cell checked checked
with 1 cm path) Wavelength Nominal wavelength 1: 272.1 nm 2.0 nm 2.0 nm
(maximum of pyrene)
Linearity Absorption range: up to 1.5 AU r 99.95 % r 99.90 %
RSD 5 % RSD 5 %
AD25 Baseline Noise Measuring wavelength: 254 nm. 0.03 mAU 0.04 mAU
Drift 0.2 mAU/h 0.2 mAU/h
checked checked
99.90 % 99.90 %
RSD 5 % RSD 5 %


OQ PQ
Agilent 1100/12x0: Drift Measuring wavelength: 254 nm. 5.0 mAU/h 5.0 mAU/h
G1314A/B/C/D/E/F Lamp Intensity Not Not
G1315A/B/C/D checked checked
G1365A/B/C/D
Absorption range: up to 2.5 AU 99.90 % 99.90 %
(G1314D/E/F only) RSD 5 % RSD 5 %
G1315A/B/C/D Baseline Noise Measuring wavelength: 254 nm. 0.05 mAU 0.05 mAU
G1365A/B/C/D Wavelength Nominal wavelength 1: 272.1 nm 2.0 nm 2.0 nm
Accuracy (DAD only)
(maxima of pyrene)
G1314A/B/C/D/E/F Baseline Noise Measuring wavelength: 254 nm. G1314A-E: G1314A-E:
0.04 mAU 0.04 mAU
G1314F: G1314F:
0.05 mAU 0.05 mAU
Agilent 1290: Baseline Noise Measuring wavelength: 254 nm. 0.03 mAU 0.05 mAU
G4212A/B
checked checked
(maxima of pyrene)
99.90 % 99.90 %
RSD 5 % RSD 5 %
Waters PDA996 Baseline Noise Measuring wavelength: 254 nm. 0.10 mAU 0.10 AU
Waters PDA2996
checked checked
(maxima of pyrene)
Linearity Absorption range: up to 1.5 AU r 99.90% r 99.90%
RSD 5% RSD 5%
Waters 2487 Dual Baseline Noise Measuring wavelength: 254 nm. 0.05 mAU 0.05 mAU
Lambda Drift 0.5 mAU/h 0.5 mAU/h
Absorbance Lamp Intensity Not Not
Detector checked checked
Wavelength Nominal wavelength: 239 nm 1.0 nm 1.0 nm
Accuracy (maximum of pyrene)
RSD 5% RSD 5%
TSP UV1000 Baseline Noise Measuring wavelength: 254 nm. 0.50 mAU 0.10 mAU
checked checked
Wavelength Not checked Not Not
Accuracy checked checked
RSD 5% RSD 5%


OQ PQ
checked checked
Wavelength Nominal wavelength: 239 nm 1.0 nm 1.0 nm
RSD 5% RSD 5%
checked checked
RSD 5% RSD 5%
checked checked
(maxima of pyrene)
RSD 5% RSD 5%
Shimadzu Baseline Noise Measuring wavelength: 254 nm. 0.05 mAU 0.10 mAU
LC-2010 SPD Drift 0.8 mAU/h 2.0 mAU/h
SPD-10A(V) Lamp Intensity Not Not
SPD-10A(V)vp checked checked
SPD-20A(V) Wavelength Nominal wavelength: 333.3 nm 1.0 nm 1.0 nm
RSD 5% RSD 5%
(1)
(2) Depending on the detector type, when qualifying a detector with a non-analytical flow cell, such
as, a micro, nano, or dummy flow cell, you may have to enter the corresponding specifications
manually into the report. The reason is that automatic recognition of micro and nano flow cells is
not always supported. For information about the limits for non-analytical flow cells, refer to the
table in section 6.2.5.
(3)
The lamp intensity is measured only for controlled detectors.

6.2.5 UV Detectors with Non-Analytical Flow Cells

Depending on the detector type, when qualifying a detector with a non-analytical flow cell, such as, a
micro, nano, or dummy flow cell, you may have to enter the corresponding specifications manually into
the report. The reason is that automatic recognition of micro and nano flow cells is not always possible
or not supported. For information about the limits for non-analytical flow cells, refer to the table below.
Tip: When qualifying a detector of the UltiMate 3000 series (VWD-3x00 or DAD/MWD-
3000), the flow cell type is recognized automatically, and the specifications are
entered automatically in the report.
When qualifying a Dionex detector of the Summit series (UVD), you have to enter
the corresponding specifications manually.

OQ PQ
(micro flow cell) Lamp Intensity Measuring wavelength: 230 nm > 50 % > 40 %
99.95 % 99.90 %
RSD 3 % RSD 5 %
(semi-micro flow Lamp Intensity Measuring wavelength: 230 nm > 50 % > 40 %
cell) Wavelength Nominal wavelength: 272.5 nm 2.0 nm 2.0 nm
99.95 % 99.90 %
RSD 3 % RSD 5 %
(3)
(3)
(3)
(3)
(micro flow cell)
(maxima of pyrene)
99.98 % 99.90 %
RSD 5 % RSD 5 %
(3)
(3)
(3)
(3)
(nano flow cell)
(maxima of pyrene)
Linearity Absorption range: up to 1.0 AU at r r
8 L 99.90 % 99.90 %
RSD 5 % RSD 5 %


OQ PQ
6 6
(semi-analytical Lamp Intensity > 2 x 10 > 1 x 10
flow cell) counts/s counts/s
Accuracy (DAD only)
(maxima of pyrene)
99.95 % 99.90 %
RSD 5 % RSD 5 %
6 6
DAD-3000(RS) / Lamp Intensity Measuring wavelength: 254 nm. > 1 x 10 > 0.5 x 10
Hardware
Revision Optics: 0
(semi-analytical
flow cell)
(semi-micro flow Lamp Intensity > 1 x 10
6
> 0.5 x 10
6
cell) counts/s counts/s

Accuracy (DAD only)
(maxima of pyrene)
99.95 % 99.90 %
RSD 5 % RSD 5 %
6 6
DAD-3000(RS) / Lamp Intensity Measuring wavelength: 254 nm. > 0.5 x 10 > 0.25x10
Hardware
Revision Optics: 0
(semi-micro flow
cell)
6 6
(semipreparative Lamp Intensity > 3 x 10 > 1.5 x 10
flow cell) counts/s counts/s
Accuracy (DAD only)
(maxima of pyrene)
(2)
99.95 % 99.90 %
RSD 5 % RSD 5 %


OQ PQ
6
DAD-3000(RS) / Lamp Intensity Measuring wavelength: 254 nm. > 1.5 x 10 >
6
MWD-3000(RS) counts/s 0.75 x 10
Hardware counts/s
Revision Optics: 0
(semipreparative
flow cell)
(1)
(2)
This test cannot be performed with the Standards kit (part no. 3323.0010), which is part of the
Performance Qualification kits (part no. 4832.5000A or 4832.5010A). This test must be
performed with standards that ensure that the peak height of the sample with the highest
concentration does not exceed 1.5 AU. This would be outside the linear range of the detector.
(3)
The specifications have to be entered manually in the report.

6.2.6 Fluorescence Detectors with Analytical Flow Cells

The used test procedures are described in detail in sections 7.11, 7.12, and 7.13.

OQ PQ
RF2000 Baseline Noise Excitation wavelength: 350 nm; 0.30 mV 0.30 mV
emission wavelength: 397 nm
Signal Excitation wavelength: 350 nm; > 40 mV > 40 mV
Minimum Emission wavelength range:
Signal 450 - 397 nm < 80 mV < 80 mV
Maximum
Wavelength Excitation wavelength: 350 nm 10 nm 10 nm
(2)
Accuracy Emission wavelength range:
380 - 410 nm (step: 1 nm)
Nominal emission wavelength:
397 nm (maximum)
RF1002 Baseline Noise Excitation wavelength: 350 nm; 0.60 mV 0.60 mV
emission wavelength: 397 nm
Signal Excitation wavelength: 350 nm; > 40 mV > 40 mV
Minimum Emission wavelength range:
Signal 450 - 397 nm < 80 mV < 80 mV
Maximum
Wavelength Excitation wavelength: 350 nm 10 nm 10 nm
(2)
Accuracy Emission wavelength range:
380 - 410 nm (step: 1 nm)
Nominal emission wavelength:
397 nm (maximum)
FLD-3100 / Signal-to-Noise Excitation wavelength 350 nm; ASTM: ASTM:
FLD-3400RS Ratio emission wavelength 397 nm for the 550 225
(analytical flow cell) first 20 minutes and 450 nm for Dark Dark
another 20 minutes. Signal: Signal:
2100 1100
Wavelength Emission wavelength 397 nm. 3 nm 3 nm
Accuracy Nominal excitation wavelength:
Excitation 350 nm
(maximum of the Raman signal of
water)
Wavelength Excitation wavelength: 350 nm. 3 nm 3 nm
Accuracy Nominal emission wavelength:
Emission 397 nm
water)


OQ PQ
Agilent 1100/12x0 Signal-to-Noise Excitation wavelength 350 nm; Dark Dark
G1321A Ratio emission wavelength 397 nm for the Signal: Signal:
(standard flow cell) first 20 minutes and 450 nm for 500 200
another 20 minutes.
G1321B Signal-to-Noise Excitation wavelength 350 nm; ASTM: ASTM:
Ratio emission wavelength 397 nm for the 500 200
first 20 minutes and 450 nm for Dark Dark
2000 1000
G1321A/B: Wavelength Emission wavelength 397 nm. 3 nm 3 nm
Excitation 350 nm
water)
Wavelength Excitation wavelength: 350 nm. 3 nm 3 nm
Emission 397 nm
water)
Linearity Excitation wavelength: 250 nm. r 99.8 % r 99.0 %
Emission wavelength: 400 nm RSD1.5% RSD 3 %
Absorption range: up to approx. Offset: Offset:
10 LU 1.5 % 3 %
(1)
(2)
The manufacturer specification of 2 nm for the excitation and emission wavelengths can be
checked only by using a special flow cell and a mercury lamp. For OQ and PQ, the instrument
should preferably be checked with the components used for the measurements.
6.2.7 Fluorescence Detectors with Non-Analytical Flow Cells

The test procedures applied are described in detail in sections 7.11, 7.12 and 7.13.
Tip: When qualifying a detector of the Dionex UltiMate 3000 series (FLD-3100 or FLD-
3400RS), the flow cell type is recognized automatically, and the specifications are
entered automatically in the report.

OQ PQ
FLD-3100 / Signal-to-Noise Excitation wavelength 350 nm; ASTM: ASTM:
FLD-3400RS Ratio Emission wavelength 397 nm for the 225 110
(micro flow cell) first 20 minutes and 450 nm for Dunkel Dunkel
1025 500
Wavelength Emission wavelength 397 nm. 3 nm 3 nm
Excitation 350 nm
water)
Wavelength Excitation wavelength 350 nm. 3 nm 3 nm
Emission 397 nm
water)
(1)

6.2.8 Refractive Index Detectors


OQ PQ
Shodex RI-101 Baseline Noise Temperature: 35C 50 nRIU 50 nRIU
Agilent 1100/1200 Drift 500 nRIU/ 2500 nRIU/h
G1362A h
Linearity Signal range: up to approx. r > 99.9% r > 99.9%
500 RIU
(1)
6.2.9 Evaporative Light Scattering Detectors

The used test procedure is described in detail in section 7.10.

OQ PQ
Polymer Baseline noise Evaporator temperature: 90C 0.3 mV 0.3 mV
Laboratories Carrier gas flow: 1.6 SLM
(2)
ELS 2100 / @4.1 bar
ELS 2100 Ice
Varian 380/385-LC
ELS detector
(1)
(2)
SLM: Standard liter per minute.
6.2.10 Corona Detectors


OQ PQ
Dionex Baseline noise Filter: None (Corona, Corona plus) 0.040 pA 0.040 pA
Corona Height of Filter: Corona (Corona ultra) 0.200 pA 0.200 pA
Corona plus spikes Filter: 4 (Corona ultra RS)
Corona ultra (RS) Drift 0.040 pA/ 0.040 pA/
min min
Signal-to-Noise caffeine concentration: 25 g/ml 10 10
Ratio
Precision RSD RSD
(height) 10.0 % 10.0 %
2 2
Signal Signal range: up to approx. 40 pA r r
calibration 99.90 % 99.90 %
(1) OQ limits with optimum measuring conditions, recommended PQ limits.

7 Procedures
7.1 Baseline Noise, Drift, and Lamp Intensity of the UV Detector
7.1.1 Theory
Drift and baseline noise are important factors for UV detectors. Increased baseline noise considerably
reduces the sensitivity, as it is not possible to distinguish between low-level signals and noise. With
increased drift, it is more difficult to integrate the signals correctly because the less stable the baseline
is, the more inaccurate is integration.
The baseline noise of the detector mainly depends on the condition of the lamp. There is a
considerable increase in noise if an old lamp with poor light intensity is used. This is also true when
the flow cell is dirty. In addition, make sure that the measuring and ambient conditions are constant
and that the flow cell is free from gas bubbles.
To measure the drift of a UV detector, also make sure that the measuring and ambient conditions are
constant. In addition, it is very important that the lamp has been turned on for several hours. In the
detector environment, avoid drafts and direct sunlight.
Figure 30: Lamp drift directly after the lamp has been turned on (bottom chromatogram)
and after it has been turned on for six hours (top chromatogram)
The lamp intensity decreases while the lamp is in operation. In addition, lamps age when turned on
and off frequently.
7.1.2 Performing and Evaluating the Check

The checks for noise, drift, and lamp intensity are included in the UV_Noise_Drift sequence. For those
checks, water is pumped through the cell at a flow rate of 1 mL/min. The UV signal is recorded at
254 nm.
If the lamp intensity can be determined, it is read directly from Chromeleon (wavelength: = 254 nm).
However, for the Dionex VWD-3100 and VWD-3400RS detectors, the lamp intensity is determined at a
wavelength of = 230 nm. The results are no absolute measured physical quantities such as luminous
density or luminous flux). That is why deviations of 5 % from lamp to lamp and from detector to
detector are quite normal. Therefore, to evaluate the data compare them to previously determined
values.

To calculate noise, the measuring signal is split into 20 intervals of 1 minute each. For each interval,
Chromeleon calculates a regression based on measured values, using the method of least squares.
Parallel to the regression line, two lines are drawn through the values with maximum distance from this
regression line. The noise is the distances of these lines. The calculated values are averaged for all 20
intervals to establish the final value. To calculate the drift, Chromeleon calculates a regression line
from all data points within a range of 1 to 21 minutes based on the method of least squares. The slope
of the regression line is the calculated drift.
7.2 Wavelength Accuracy of the UV Detector

The UV_Wavelength sequence is used to determine the wavelength accuracy of UV detectors without
PDA option.
For photodiode array detectors, use the DAD_Wavelength sequence. For single wavelength detectors
and the Dionex VWD-3400RS detector, use the Wavelength_Single sequence. Separate sequences
are available for the following detectors: TSP UV 2000 detector, and the Shimadzu LC-2010 SPD,
SPD-10A(V), and SPD-10A(V)vp detectors. The Wizard uses the appropriate sequence automatically
when the corresponding detector is installed.
Wavelength accuracy is determined using pyrene in methanol (c = 3 g/mL) at a flow rate of 1 mL/min.
However, for single wavelength detectors and the Dionex VWD-3400RS detector, the wavelength
accuracy is determined using caffeine in water (c = 60 g/mL) at a flow rate of 1 mL/min. As water is
used as solvent, not methanol, it is not necessary to change the solvent manually.
7.2.2 Evaluation of the Check for UV Detectors

The signals are recorded at 331 nm, 333 nm, and 335 nm. A parabola is calculated from the signal
heights of the pyrene signal and the wavelengths. The parabola maximum is determined and
compared to the theoretical value of the spectral maximum of pyrene (333.3 nm).
7.2.3 Evaluation of the Check for Photodiode Array Detectors

The UV spectrum for pyrene is recorded between 250 nm and 350 nm. Chromeleon determines the
spectral maxima between 250 nm and 290 nm and between 330 nm and 350 nm and compares them
to their theoretical values (272.1 nm and 333.3 nm).
Pyrene 100%
60,0
%
271.8 333.2
50,0
40,0
317.8
30,0
20,0
10,0
0,0
nm
-10,0
249,4 260,0 270,0 280,0 290,0 300,0 310,0 320,0 330,0 340,0 348,2
Figure 31: UV Spectrum of pyrene in methanol

7.2.4 Evaluation of the Check for Two-Channel Detectors

For the TSP UV2000 and the Waters 2487 detectors, the signals are recorded at 235 nm,
240 nm, and 245 nm. A parabola is calculated from the signal heights of the pyrene signal and
the wavelengths. The maximum of the parabola is determined and compared to the theoretical
value of the spectral maximum for pyrene (239.4 nm).
For the Shimadzu detectors, the signals are recorded at 331 nm, 333 nm, and 335 nm. A
parabola is calculated from the signal heights of the pyrene signal and the wavelengths. The
maximum of the parabola is determined and compared to the theoretical value of the spectral
maximum for pyrene (333.3 nm).
7.2.5 Evaluation of the Check for Single Wavelength and VWD-3400RS

Detectors
For the Dionex VWD-3100, VWD-3400, AD25 detectors and the Agilent G1314 detectors of the
1100/12x0 series, the signals are recorded at 270 nm, 272 nm, and 274 nm. A parabola is calculated
from the signal heights of the caffeine signal and the wavelengths. The maximum of the parabola is
determined and compared to the theoretical value of the spectral maximum of caffeine (272.5 nm).
7.3 Linearity of the UV Detector

7.3.1 Theory
The linearity of a detector mainly depends on the optical and electronic systems. With electronic
systems, non-linearity is caused by dark current and dark current drift. Dark measurements can be
used to compensate the influence of these factors. However, as the light intensity decreases due to
lamp ageing or absorption of the eluent or sample, the influence of the dark current on the linearity
increases. The influence of the eluent is insignificant in this case, as water is used for the test
procedure . The influence of the sample is fully used in this test procedure to determine the detector
linearity. Consider that the resulting deviations of the linear behavior are only important with extremely
high absorption (> 1.5 AU).
250
200
Peak Area / Peakflche [mAU*min]
150
100
50
0
0 50 100 150 200 250 300 350
Concentration/ Konzentration [ppm]
Figure 32: Linearity of the detector signal depending on the peak area


The UV_LINEARITY sequence is used to measure the detector linearity. The detector linearity is
determined at 272 nm using five different caffeine standards (set concentrations: 10 g/mL, 60 g/mL,
140 g/mL, 220 g/mL, and 300 g/mL, dissolved in water; the actual concentrations are entered into
the QNT file and taken into account). Water is used as solvent. The flow rate is 1 mL/min.
Concentration and peak area are represented in a graph. The regression coefficient and the relative
standard deviation of this line indicate the linearity.
Tip: Depending on which injection module you use, it may happen that the peak height of
the sample with the highest concentration exceeds 1500 mAU. This is usually not
within the linearity range of UV detectors. Thus, the limits for the regression coefficient
and the relative standard deviation may not be met. In this case, reduce the injection
volume for all samples used for the linearity check so that the peak height of the
sample with the highest concentration is in the linearity range of the tested detector,
that is, usually below 1500 mAU. As an exception, a linearity range of up to 2500 mAU
is specified for the supported Shimadzu detectors and the Dionex VWD-3100 and
VWD 3400RS detectors with an analytical flow cell installed. The Dionex VWD-3100
and VWD-3400RS detectors with a semi-micro or micro flow cell installed have a
specified linearity range of up to 1700 mAU. If an autosampler is used when the
Dionex PDA-100 or PDA-3000 detectors are tested, only 8 l of sample will be
injected by default. However, when qualifying the Dionex VWD-3100 and VWD-
3400RS detectors using an autosampler, 13 l of sample will be injected.

7.4 Precision of Injection Volume

7.4.1 Theory
The precision of the injection volume is mainly influenced by the quality of the autosampler syringe
and the syringe volume that has been adjusted to the injection volume. In addition, the mechanics for
the syringe movement is a decisive factor for the accuracy and precision of the injection volume.
Especially when you use a manual injection valve, verify that there are no air bubbles in the sample.
If a manual injection valve is used, inject at least five times the sample loop volume; that is, inject at
least 50 l.
Varying injection volumes affect the peak areas even if the same standard is injected.

With the Injector_Flow_Repro sequence, a caffeine standard (solvent: water at a flow rate of
0.3 mL/min; wavelength: 272 nm) is injected six or ten times. The autosampler type determines the
injection volume, and the standard to be used (see the table).
The relative standard deviation of the peak areas of the ten injections indicates the precision of the
injection volume.
Autosampler Standard Injection

used* volume
Other Standard 4 5 l
Dionex Gina 50 Standard 3 10 l
Dionex ACC-3000(T)
- Sample loop volume: 200 l Standard 3 20 l
Dionex OAS-3300TXRS Standard 3 10 l
Dionex WPS-3000(T)SL
- Micro Standard 5 2 l
- with 250 l injection volume kit Standard 3 10 l
Dionex WPS-3000TBPL / WPS-
3000T(B)FC Analytical
- Large Volume Configuration Standard 3 20 l
Dionex WPS-3000(T)RS Micro option Standard 5 2 l
Dionex WPS-3000TXRS Standard 5 2 l
Thermo Scientific Accela Autosampler Standard 3 10 l
*) Also see section 3.1.

7.5 Carry-Over by the Autosampler

7.5.1 Theory
After a highly concentrated sample, a sample containing only solvent is injected. Ideally, only the
signal for the solvent is displayed in the chromatogram. However, if a signal for the sample is
displayed, this indicates the carry-over by the autosampler. As the highly concentrated sample
exceeds the linearity range of the detector, a reference sample with a considerably lower
concentration is also injected.

The carry-over by the autosampler is measured with samples 6 to 9 of the Sampler_Lin_CO sequence
(solvent: water at a flow rate of 1.0 mL/min, wavelength: 272 nm). Samples 6 and 9 contain water
(same vial), sample 7 contains a solution of caffeine in water; the concentration is 10 g/mL (standard
2 - reference sample), and sample 8 contains a solution of caffeine in water; the concentration is
2000 g/mL (standard 7). The carry-over (CO in [%]) is calculated as follows:
AreaWater ,corr AreaWater ,CarryOver AreaWater

CO = =
AreaConc:2000 g / ml c
AreaRe ference HighConcentratedSample
cRe ference
AreaWater ,CarryOver AreaWater cRe ference
=
AreaRe ference cConc 2000 g / ml
Areawater ,corr : Area of the caffeine peak in the water sample (sample 9 sample 6)
AreaConc:2000g/mL : Peak area of the highly concentrated caffeine sample (sample 8)
Areawater ,CarryOver: Peak area of the water injection (sample 9: solvent and caffeine peaks)
after the carry-over sample (sample 8)
Areawater : Peak area of the water injection (sample 6: solvent peak) before the
carry-over sample (sample 8)
AreaReference : Peak area of the reference sample (sample 7)
cReference : Caffeine concentration of the reference solution (conc.: 10 g/mL)
cConc:2000g/ml : Caffeine concentration of the carry-over solution (conc.: 2000 g/mL)

7.6 Linearity of Injection Volume

7.6.1 Theory
The linearity of the injection volume and its precision depend on the quality of the syringe and the
syringe volume that has been adjusted to the injection volume. Besides, the quality of the autosampler
mechanics also affects the result.
Select the concentration of the standard, which is injected in different volumes, in such a way that the
detector works in the linear range for all injections; usually between 10 mAU and 1000 mAU.

With the Sampler_Lin_CO sequence, a caffeine standard (solvent: water at a flow rate of 1 mL/min,
wavelength: 272 nm) is injected five times. The autosampler type determines the injection volume and
the standard (see table). The peak area and injection volume are represented in a graph and the
regression line is determined. The correlation coefficient and the standard deviation of this line
indicate the linearity.
Autosampler Standard used* Injection volume

Other Standard 2 5, 10, 20, 40, and 80 l
Agilent G1367D Standard 2 5, 10, 20, 30, and 40 l
Agilent G4226A Standard 3 1, 5, 10, 15, and 20 l
Shimadzu SIL-10A / SIL-10Ai / SIL- Standard 2 5, 10, 20, 40, and 50 l
10AF / SIL-10ADvp / SIL-20A(C)XR
Dionex Gina 50 Standard 2 10, 20, 40, 60, and 80 l
Dionex ACC-3000(T)
- Sample loop volume: 20 l Standard 3 1, 3, 5, 7, and 10 l
Dionex OAS-3300TXRS Standard 3 4, 6, 8, 10, and 12 l
Dionex WPS-3000(T)SL / WPS-
3000(T)RS
- Analytical Standard 2 5, 10, 20, 40, and 90 l
- Micro Standard 3 1, 5, 10, 15, and 20 l
- with 250 l injection volume kit Standard 2 10, 20, 40, 80, and 160 l
Dionex WPS-3000(T)PL /
WPS-3000(T)PLRS
Dionex WPS-3000TBPL / WPS-
3000T(B)FC Analytical
- Standard configuration Standard 3 5, 10, 15, 20, and 25 l
- Large Volume configuration Standard 2 20, 50, 80, 110, and 140 l
Dionex WPS-3000(T)RS Micro option Standard 3 1, 5, 10, 15, and 20 l
Dionex WPS-3000TXRS Standard 3 1, 5, 10, 15, and 20 l
Thermo Scientific Accela Autosampler Standard 3 2.5, 5, 7.5, 10, and 12.5 l
*) Also see section 3.1.

40
35
30
Area / Flche [mAU*min]

25
20
15
10
0
0 20 40 60 80 100
Injection Volume / Injektionsvolumen [l]
Figure 33: Linearity of injection volume
7.7 Sample Temperature Accuracy of Autosamplers

7.7.1 Theory
The sample temperature accuracy mainly depends on the cooling and heating accuracy of the
autosampler, the insulation of the sample compartment, and the thermal transfer from the carousel to
the vial.

The sample temperature accuracy is determined with the help of an external thermometer. The
temperature sensor is placed in a standard vial (1.8 mL) filled with water. The carousel cover must be
closed during the test. The autosampler temperature is set to a nominal temperature (e.g., 10C),
depending on the autosampler type. When the nominal temperature is reached, the sample (water)
temperature is recorded over a period of 30 minutes. Within the 30 minutes, the sample temperature
reaches a stable value. The temperature accuracy is the temperature difference between the sample
temperature and the nominal autosampler temperature.

7.8 Flow Precision

7.8.1 Theory
The flow precision can be determined very exactly by weighing out which quantity of solvent is
delivered over a specific period. For statistic evaluation of the results, repeat this measurement
several times. However, this requires a lot of work: The measuring time must be at least five minutes if
it is not electronically linked to the weighing process. Otherwise, inaccuracies in the timing affect the
results. An additional disadvantage is that the procedure cannot be automated and that the used
scales must be very exact.
As an alternative, the flow precision can be determined by injecting the same sample standard
multiple times. The flow precision primarily affects the precision of the retention time. This method is
used during automated OQ and PQ.
7.8.2 Note on Performing and Evaluating the Check

The precision of the flow and the precision of the injection volume are established with the
Injector_Flow_Repro sequence. Standard 4 is injected ten times, using an injection volume of 5 l for
each injection (deviations see the table in section 7.4.2).
The relative standard deviation (RSD) or the standard deviation (SD) of the retention times of the ten
injections indicates the flow precision. The larger of the values is the valid limit.
The expression The greater value is the valid limit refers to comparable values. This means that
either the RSD must be converted to an SD value or vice versa. This conversion takes the absolute
retention time tR of the peak of the interest into account
For the pumps of the UltiMate 3000 series with analytical pump head, the values are RSD 0.05% or
SD 0.01 min. We assume that caffeine elutes at about 1.5 min.
including
RSD= SD / t R or SD= RSD t R :
RSD 0.05 % corresponds to SD 0.00075 min
SD 0.01 min corresponds to RSD 0.67 %
This means that SD 0.01 min is greater than RSD 0.05%. The test is passed when the measured
result for SD is below or equal to 0.01 min.

7.9 Solvent Composition of the Gradient Pump, Accuracy,

Precision, and Ripple
7.9.1 Theory
If the gradient pump composes the solvent inaccurately, this will mainly effect the retention times. To
keep the measuring effort low, different compositions are checked based on the ASTM instructions.
Use 100% water for solvent A. Solvent B is a mixture of water and acetone (0.1% Vol.). Acetone is
highly absorbing in the range of = 265 nm. The gradient can be observed in a chromatogram. There
are no injections required.
Figure 34: Theoretical (broken line) and real gradients (STD_GRAD standard sequence for
gradient pumps)

Except for the TSP P2000 Pump, the following solvent compositions (in %B) are mixed: 0, 1, 50, 99,
and 100. The solvent composition (in %B) for the TSP P2000 Pump is as follows: 0, 50, and 100%.
This is because the pump does not support gradient programs with more than 9 steps.
For the described arrangement and non-changing solvent composition, the ripple is indicated by the
signal noise.
To qualify a gradient pump (except the Dionex UltiMate 3000 pumps and the P680), the STD_GRAD
sequence is required.
For the ternary high-pressure gradient systems from Shimadzu, the Wizard automatically selects
TERN_GRAD_C_B sequence. This sequence is used to determine the accuracy and the precision of
the gradient and the ripple between the solvent channels C and B.
For the quaternary high-pressure gradient systems from Agilent and the TSP P4000 pump, the Wizard
automatically selects QUAD_GRAD_C_D sequence. This sequence is used to determine the accuracy
and the precision of the gradient and the ripple between the solvent channels C and D.

7.9.3 Performing the Checks for the Dionex P680 and UltiMate 3000 Pumps
Five sequences are provided for qualifying the Dionex LPG-3400A(B), LPG-3400RS, LPG-3400SD,
DPG-3600A(B), HPG-3200A, HPG-3200M, HPG-3200RS, HPG-3200SD ,HPG-3400A, HPG-3400M,
HPG-3400RS, HPG-3400SD, and P680 pumps: STD_GRAD, MICRO_GRAD, LONG_GRAD,
STD_GRAD_P680DGP_Left, and LONG_GRAD_P680DGP_Left.
The MICRO_GRAD sequence is used to qualify a pump with MicroFlow kit installed (e.g., an HPG-
3200M or HPG-3400M). The gradient program corresponds to the program of the STD_GRAD
sequence. However, the mixing volume of a pump with MicroFlow kit is lower than the volume of a
pump with standard mixing chamber, and therefore the detector signal is detected earlier. This fact is
considered for the evaluation of the check.
The LONG_GRAD sequence is used if a mixing chamber extension (600 l or 1200 l for UltiMate
3000 pumps, or 1250 l for the P680 pump) is installed. A higher mixing chamber volume increases
the equilibration time of the gradient. The gradient program has been adapted accordingly and
evaluation of the check considers this.
For DGP pumps (P680, UltiMate 3000 DGP-3600A(B), UltiMate 3000 DGP-3600RS and UltiMate
3000 DGP-3600SD), qualification is performed for both pump units. The sequences STD_GRAD and
LONG_GRAD are used to check the right pump unit. The sequences STD_GRAD_P680DGP_Left
and LONG_GRAD_P680DGP_Left are used to check the left pump unit.
For the UltiMate 3000 LPG-3400M(B), LPG-3400BM, DGP-3600M(B) and DGP-3600BM micro
pumps, only the LONG_GRAD and LONG_GRAD_P680DGP_Left sequences are available.
7.9.4 Evaluating the Check

To facilitate the comparison, the absorption values are converted and expressed as %B. To
compensate the detector drift, the absorption of the pure solvent A is measured at the beginning and
at the end of the gradient. These values are the basis for the regression line that is used to correct the
baseline of the entire chromatogram.
To define the gradient accuracy, the measured step height is compared to the height that must
theoretically result from the solvent composition.
To define the precision, three gradients are recorded. The standard deviations of the step heights
indicate the precision.
The ripple is determined for all steps. A one-minute interval is defined for each step. For each interval,
Chromeleon uses the data to calculate a regression line, based on the method of least squares.
Parallel to the regression line, two lines are drawn through the measured minimum and maximum
value. The noise values in relation to the absorption signal when 100% eluent is pumped is an
indication for the ripple in [%].

7.10 Temperature Accuracy of the Column Compartment

7.10.1 Theory
Depending on the type of application, temperature fluctuations of the solvent and especially of the
column can result in considerable retention time fluctuations. In addition to the precision of the
temperature achieved with the column compartment, the accuracy is important as well. Only high
accuracy allows transferring applications to different systems.

Four measuring points are used to check the temperature accuracy of the column compartment. The
check is performed with the Column_Oven sequence. An external, calibrated thermometer is used to
measure the achieved temperature.
The achieved temperatures are compared to the set values. The difference indicates the temperature
accuracy.
Exceptions:
It is not possible to set the temperature on certain column compartment modules (Waters Alliance
2690 Separation Module, Shimadzu and Agilent G1316) when the retention time is negative. The
first measurement reading is 10 minutes after the sample has been started. At this time,
equilibration of the column compartment may not be complete. Therefore, the same temperature
is set for the second measuring point (50 minutes). The column compartment module has passed
the check even if the target temperature is reached only for the second measuring point. This
means that evaluation is performed for three measuring points only.
Due to its small temperature range, evaluation for the Dionex ACC-3000(T) is performed for three
measuring points only.
With column compartments of the Thermo Scientific Accela Autosampler the set temperature can
only be entered once during a sample. This means that qualification is performed for one
measuring point only.
Tip: With the column compartment of the Waters Alliance 2690 Separation Module, the set
temperature can be changed during a sample only when the autosampler is injecting.
That is why 1 l of water is injected for qualifying the column compartment of the
Waters Alliance 2690 Separation Module.

7.11 Baseline Noise / Signal Height of the Fluorescence Detector

7.11.1 Theory
reduces the sensitivity, as it is not possible to distinguish between low-level signals and noise.
considerable increase in noise if an old lamp with poor light intensity is used. This is also true when
the flow cell is dirty. In addition, make sure that the measuring and ambient conditions are constant
and that the flow cell is free from gas bubbles.
In addition to the absolute value of the baseline noise, the signal height to noise ratio is important. The
signal height mainly depends on the condition of the lamp and the flow cell. A contaminated flow cell
may result in a higher fluorescence signal.

The FLUORES_V2 sequence is used to determine the signal to noise ratio (SNR). Water is pumped
through the flow cell at a flow rate of 1 mL/min. The excitation wavelength is 350 nm. The emission
signal is recorded for 20 min at an emission wavelength of 397 nm (Raman signal of water) and for
another 20 min at 450 nm (dark current).
The signal to noise ratio (SNR) is evaluated as follows:
a) Noise evaluation at 450 nm - SNR(Dark Current)
Average Signal Value 397 nm Average Signal Value 450 nm

SNR(Dark Current) =
Noise 450 nm
b) According to ASTM with noise evaluation at 397 nm (only for FLD-3100 and FLD-3400RS)
Average Signal Value 397 nm Average Signal Value450 nm

SNR(ASTM) =
Noise397 nm
To determine the noise, the measuring signal is split into 40 intervals of 30 seconds each. For each
interval, Chromeleon calculates a regression line, based on the method of least squares. The noise
value is the distance between two parallel lines and the regression line through the lowest and highest
values. For the calculated values, the 40 interval values are averaged.
7.11.3 Performing and Evaluating the Check for Dionex RF2000 and RF1002
Fluorescence Detectors
The Fluorescence sequence is used to determine the noise and the signal height. When testing the
Dionex RF2000, make sure that the detector's ZWAVE parameter is set to 1 ( 3.3.2). Water is
pumped through the flow cell at a flow rate of 1 mL/min. The excitation wavelength is 350 nm; the
emission wavelength is 397 nm.
To determine the noise, the measuring signal is split into 30 intervals of 30 seconds each. For each
interval, Chromeleon calculates a regression line, based on the method of least squares. The noise
value is the distance between two parallel lines and the regression line through the lowest and highest
values. For the calculated values, the 30 interval values are averaged.

7.12 Wavelength Accuracy of the Fluorescence Detector

The FLUORES_V2 sequence is used to determine the wavelength accuracy (emission and excitation)
by using spectra. Water is pumped through the flow cell at a flow rate of 1 mL/min. The emission
spectrum is recorded in the range around 397 nm (excitation wavelength: 350 nm). The excitation
spectrum is recorded in the range around 350 nm (emission wavelength: 397 nm). The relative signal
maximum of both spectra is determined and compared to the theoretical maximum.
7.12.2 Performing and Evaluating the Check for Dionex RF2000 and RF1002
Fluorescence Detectors
The Fluorescence sequence is used to determine the wavelength accuracy of the emission spectrum.
Water is pumped through the flow cell at a flow rate of 1 mL/min. For an excitation wavelength of
350 nm, the emission wavelength changes in 1 nm increments from 380 nm to 410 nm. The relative
signal maximum is compared to the theoretical maximum.
Figure 35: Chromatogram for defining the relative maximum of the emission spectra
between 380 nm and 410 nm
7.12.3 Remarks on the Manufacturer Specification

It is only possible to check the manufacturer specification of 2 nm for the excitation and the emission
wavelengths by using a special flow cell and a mercury lamp. For OQ and PQ, the instrument should
preferably be checked with the components used for the measurements.

7.13 Linearity of the Fluorescence Detector

7.13.1 Theory
The linearity of a fluorescence detector mainly depends on the optical and electronic systems, the
sample concentration and the eluent. With electronic systems, non-linearity is caused by dark current
and dark current drift. Dark measurements can be used to compensate the influence of these factors.
Contamination in the flow cell or optics and extremely high sample concentrations or the eluent may
cause stray light that influences the detector linearity. In addition, adsorption from the sample on the
cell walls can be found with very low sample concentrations. This effect also influences detector
linearity.
The influence of the sample in a suitable concentration range is fully used in this test procedure to
determine the detector linearity. Consider that the resulting deviations of the linear behavior are only
important with extremely high or very low sample concentrations. Therefore, the test results reflect the
influence of the detector itself on linearity.

The FLUORES_LINEARITY sequence is used to measure the detector linearity. The detector linearity
is determined using seven anthracene standards with acetonitrile / water 90:10 (v/v) as eluent. The
flow rate is 1 mL/min. The set concentrations are: 0.5 mg/100 mL, 0.4 mg/100 mL, 0.3 mg/100 mL,
0.2 mg/100 mL, 0.1 mg/100 mL, 0.05 mg/100 mL, 0.005 mg/100 mL, dissolved in acetonitrile. The
actual concentrations are entered into the QNT file and are taken into account. Concentration and
peak area are represented in a graph. The regression coefficient, the relative standard deviation, and
the relative y axis intercept (relative to the peak area of the sample with the highest concentration) of
the graph indicate the linearity.
7.14 Baseline Noise and Drift of the RI Detector

7.14.1 Theory
reduces the sensitivity, as it is not possible to distinguish between low-level signals and noise. With
increased drift, it is more difficult to integrate the signals correctly because the less stable the baseline
is, the more inaccurate is integration.
considerable increase in noise if an old lamp with poor light intensity is used. The noise also increases
when the reference cell or flow cell is dirty. In addition, make sure that the measuring and ambient
conditions are constant and that the flow cell is free from gas bubbles.
To measure the drift of a RI detector, make sure that the measuring and ambient conditions are
constant. In addition, it is very important that the lamp has been burning for several hours and that the
flow cell has been rinsed sufficiently

The RI_NOISE_DRIFT sequence includes both the checks of the noise and the drift. Water is pumped
through the sample cell at a flow rate of 1 mL/min; the reference cell, too, has been rinsed with water
before. The RI signal is recorded at a temperature of 35 C.
To calculate drift and noise, the measuring signal is split into 20 intervals of 1 minute each. For each
interval, Chromeleon calculates a regression based on measured values, using the method of least
squares. The slop of this curve corresponds to the drift of the measuring signal, the value of the slope
is the value of the drift. Parallel to the regression line, two lines are drawn through the smallest and
largest values. The noise is the distance of these lines. The calculated values are averaged for all 20
intervals to establish the final value.

7.15 Linearity of the RI Detector

7.15.1 Theory
The linearity of a detector mainly depends on the optical and electronic systems. With electronic
systems, non-linearity is caused by dark current and dark current drift. Dark measurements can be
used to compensate the influence of these factors. However, as the light intensity decreases due to
lamp ageing or refraction by the sample, the influence of the dark current on the linearity increases.
The influence of the eluent is insignificant in this case, as the water used during the test procedure is
present in the sample flow cell as well as the reference cell. The influence of the sample is fully used
in this test procedure to determine the detector linearity. Consider that the resulting deviations of the
linear behavior are only important at extremely high sample concentrations due to the strong refraction
of the light beam (signals > 600 RIU).

The RI_LINEARITY sequence is used to measure the detector linearity. The detector linearity is
determined using five glycerin standards (set concentrations: 5 mg/mL, 10 mg/mL, 15 mg/mL,
25 mg/mL, and 35 mg/mL, dissolved in water; the actual concentrations are entered into the QNT file
and taken into account). Water is used as solvent. The flow rate is 1 mL/min. Concentration and peak
area are represented in a graph. The regression coefficient of this line indicates the linearity.
7.16 Baseline Noise of the Evaporative Light Scattering Detector

7.16.1 Theory
Baseline noise is an important specification for a detector. Increased baseline noise considerably
reduces the detection sensitivity, as it is not possible to distinguish between small signals and noise.
considerable increase in noise if an old lamp with poor light intensity is used. The evaporator
temperature and carrier gas flow also affect the noise. Therefore, make sure that the measuring and
ambient conditions are kept constant.

The ELS_NOISE sequence is used for the noise test. Water is pumped through the detector at a flow
rate of 1 mL/min. The conditions for recording the ELS signal are as follows
Nebulizer temperature: 50C
Evaporator temperature: 90C
Carrier gas flow: 1.6 SLM@4.1 bar
Chromeleon calculates a regression based on measured values, using the method of least squares.
Parallel to the regression line, two lines are drawn through the smallest and largest values. The noise
is the distance of these lines. The calculated values are averaged for all 20 intervals to establish the
final value.

7.17 Baseline Noise/Signal Height/Drift/Spikes/Precision of the

Corona Detector
7.17.1 Theory
Baseline noise is an important specification for a detector. Increased baseline noise considerably
reduces the detection sensitivity, as it is not possible to distinguish between small signals and noise.
In addition to the absolute value of the baseline noise, the signal height to noise ratio is important.
The causes that may influence these specifications are diverse and are described in detail in the
Operating Instructions for the instrument.

Use the CORONA_NOISE_DRIFT_SNR sequence to run the following checks:
1. Noise
2. Height of spikes
3. Drift
4. Signal-to-Noise Ratio
5. Precision of height
For all checks, an eluent of water/methanol 80:20 (v/v) is pumped through the detector at a flow rate of
1 mL/min. For checks 4 and 5, six additional caffeine injections of 10 L each are performed. The
standard concentration of the caffeine solution is 25 g/ml. The settings for recording the detector
signal are as follows
Filter: None (Corona, Corona plus)
Corona (Corona ultra)
4 (Corona ultra RS)
Nebulizer temperature: off (Corona, Corona plus)
25 C (Corona ultra (RS))
Data collection rate: 10 Hz
Gain: 100 pA
Offset 0%
Chromeleon calculates a regression line, based on the method of least squares. Parallel to the
regression line, two lines are drawn through the smallest and largest values. The noise is the distance
of these lines. The calculated values are averaged for all 20 intervals to establish the final value.
To calculate the height of the largest spike, the measuring signal is also split into 20 intervals of 1
minute each. For each interval, Chromeleon calculates the signal average value, the minimum, and
the maximum signal. The height of the positive spikes within an interval is calculated according to the
following formula:
Spike Height
Int. X
( )
= Signal Maximum Int. X Signal AverageInt. X-1 + Signal AverageInt. X+1 / 2
The height of the negative spikes within an interval is calculated according to the above formula,
however, the signal minimum is used instead of the signal maximum. The absolute greatest height of
a spike of all intervals corresponds to the height of the largest spike. The signal average values of the
first and last interval outside of the measuring signal are extrapolated.

To calculate the drift, Chromeleon calculates a regression line from all data points within a range of 2
to 22 minutes based on the method of least squares. The slope of the regression line is the calculated
drift.
The signal-to-noise ratio (SNR) is calculated as follows:

Peak Height Average
SNR =
Signal noise
The relative standard deviation of the peak heights of the six injections indicates the precision of the
peak height.
7.18 Signal Calibration of the Corona Detector

7.18.1 Theory
The mass of the analyte and the corresponding detector response (peak area) are proportional to the
square root. Therefore, the calibration function used here is a quadratic regression.

The CORONA_Resp_Calib sequence is used to measure the detector linearity. The detector linearity
is determined using four different caffeine standards (set concentrations: 25 g/mL, 125 g/mL, 250
g/mL, and 500 g/mL, dissolved in water). The actual concentrations are entered into the QNT file
and are taken into account. User water / methanol 80:20 (v/v) as eluent. The flow rate is 1 mL/min.
Concentration and peak area are represented in a graph, and the quadratic regression determined.
The coefficient of determination of this quadratic regression indicates the calibration.

8 Troubleshooting
8.1 General Notes
A system pressure that is well above 130 bar for a flow rate of 1 mL/min (solvent: water) after the
restriction tubing has been connected indicates that a capillary is contaminated. Inspect and
exchange the capillaries (including the restriction tubing) to ensure that OQ and PQ are correctly
performed.
If problems occur during the checks that cannot be solved observing the notes below, also refer
to the respective sections in the instruments' manuals.
8.2 Failure of Individual Checks

8.2.1 UV Detector
Test Cause Remedial Action

Wavelength accuracy Spectrum calibration was not First, disconnect and then,
successful during connect in reconnect the detector in
Chromeleon. Chromeleon.
Increased drift See below.
Baseline noise The solvent is contaminated. Exchange the solvent.
The lamp is too old. Replace the lamp.
There are air bubbles in the flow Prime the flow cell.
cell.
Drift The detector is not yet warmed Allow the detector sufficient time
up. to warm up.
The system is not equilibrated. Rinse the system.
The lamp is defective. Replace the lamp.
There are fluctuations in the If necessary, close the windows
ambient temperature. and shield the instrument from
the air conditioning system.
There is a draft. If necessary, close the windows
and shield the instrument from
the air conditioning system.
Lamp Intensity The lamp is too old. Replace the lamp.
The flow cell is incorrectly Install the flow cell correctly.
installed.
Detector linearity The lamp is too old. Replace the lamp.
The concentration of standards Use new standards.
is not correct.
The peak height of the sample Reduce the injection volume for
with the highest concentration is all samples used for the detector
not in the linearity range linearity check so that the peak
specified for the detector; that is, height of the sample with the
usually > 1500 mAU (for highest concentration is in the
Shimadzu detectors and Dionex linearity range of the detector;
VWD-3100 and VWD-3400RS that is, usually < 1500 mAU.
detectors > 2500 mAU).

8.2.2 Autosamplers

Precision of Injection Volume The autosampler draws air from Either there is too little sample
the vial. volume in vial or the value set for
the Needle Depth parameter is
too low.
There are air bubbles in the Prime the syringe.
syringe.
The autosampler is leaking. Autosampler Manual
The injection valve is leaking. Autosampler Manual
Linearity of injection volume The detector linearity check See above.
failed.
The syringe is old. Replace the syringe.
8.2.3 Pump

Flow precision The autosampler draws air from Either there is too little sample
the vial. volume in vial or the value set for
the Needle Depth parameter is
too low.
There are air bubbles in the Prime the syringe.
syringe.
The autosampler is leaking. Autosampler Manual
The injection valve is leaking. Autosampler Manual
Gradient accuracy There is air in the system. Prime the system.
The composition of solvent B is Make sure that the solvent
not correct. composition is correct.
Gradient precision There is air in the system. Prime the system.
Ripple There is air in the system. Prime the system.
8.2.4 RF2000 Fluorescence Detector

Wavelength accuracy The Raman peak of water is not On the instrument, set the
visible because the instrument ZWAVE parameter to 1 (
performs an Autozero whenever section 3.4.1).
the wavelength is changed.

8.2.5 RI Detector

Baseline noise There are air bubbles in the flow Rinse the sample and reference
cell. cells for up to one hour, using
degassed water (flow rate:
1 mL/min). Repeatedly press the
Purge key. If necessary, repeat
the procedure with methanol.
Drift The solvent is contaminated. Use new solvent.
There are fluctuations in the Position the detector at a
ambient temperature. location with few temperature
fluctuations.
There are air bubbles in the flow Rinse the sample and reference
cell. cells with degassed water (see
above).
Detector linearity The concentration of the Use fresh standards.
standard is not correct.
8.2.6 ELS Detector

Baseline noise The pump pulsation is too high. Purge the pump and all channels
if necessary.
Baseline spikes The gas supply is contaminated. Replace the gas supply.
8.2.7 Corona Detector
The possible causes for errors are diverse and are described in detail and using many examples in the
Operating Instructions for the instrument. Therefore, this manual does not give a list of errors.


9 PGM Files
As described in section 3.5, the OQ or PQ Setup Wizard adapts the program files to the device names
of the instruments installed on the timebase. This section lists the program files of the following Dionex
HPLC instruments:
Instrument Used device and channel names

Pump: LPG-3400RS DX_PumpModule
DX_Pump
DX_Pump_Pressure
Autosampler: WPS-3000TRS DX_Sampler
Thermostatted column DX_ColumnOven
compartment: TCC-3000RS
UV detector: DAD-3000RS DX_UV
DX_UV_VIS_x (x: 1, 2, 3)
RI detector: RI-101 DX_RI
DX_RI_1
FL detector: FLD-3400RS DX_FLD
DX_Emission_1
ELS detector: Poly. Lab. ELS-2100 DX_ELSD
DX_ELSD_1
Corona detector: Corona ultra DX_CAD
DX_CAD_1
9.1 Wavelength Accuracy of the Photodiode Array Detector

;===============================================================
; Wavelength Accuracy for PDA detectors
; --------------------------------------------------------------
; PGM-Version: Jan 2011
;
; Pressure regulator: Pressure: 103+/-25 bar, Flow: 0.3-2 ml/min
; (Or SST-Restriction capillary)
; Eluent: Methanol, degassed via online degasser
;
; HPLC-System:
; ------------
; Pump specific settings
; Pump: Dionex LPG-3400(A/BM/RS/SD) Pump (UltiMate 3000)
; Sampler specific settings

; Sampler: Dionex WPS-3000(T)RS Autosampler (UltiMate 3000)
;
; Detector specific settings
; Detector: Dionex DAD-3000 Detector (UltiMate 3000)
;
; Sample: Pyrene 3g/ml
;
;============================================================================
; Macros used for pump modules
WriteReportMacro Name="PumpModule", Value="DX_PumpModule"
; Pump
WriteReportMacro Name="Pump", Value="DX_Pump"
WriteReportMacro Name="Pump_Pressure", Value="DX_Pump_Pressure"
; Pump DGP
WriteReportMacro Name="PumpLeft", Value="$PumpLeft"
WriteReportMacro Name="Pump_Pressure_LeftBlock", Value="$Pump_Pressure_LeftBlock"
WriteReportMacro Name="PumpRight", Value="$PumpRight"

WriteReportMacro Name="Pump_Pressure_RightBlock",
Value="$Pump_Pressure_RightBlock"
; Macros used for inject devices

WriteReportMacro Name="Sampler", Value="DX_Sampler"
; Macros used for column compartements / ovens

; ColumnOven without autosampler, e.g. TCC-3000
WriteReportMacro Name="ColumnOven", Value="DX_ColumnOven"
WriteReportMacro Name="ColumnOven_Temp", Value="ColumnOven_Temp"
; Macros used for detectors

WriteReportMacro Name="UV", Value="DX_UV"
WriteReportMacro Name="UV_VIS_1", Value="DX_UV_VIS_1"
WriteReportMacro Name="FLD", Value="DX_FLD"

WriteReportMacro Name="Emission_1", Value="DX_Emission_1"
WriteReportMacro Name="Emission_2", Value="Emission_2"
WriteReportMacro Name="Emission", Value="$Emission"

; Dionex RF-2000
WriteReportMacro Name="RI", Value="DX_RI"

WriteReportMacro Name="RI_1", Value="DX_RI_1"
WriteReportMacro Name="ELSD", Value="DX_ELSD"

WriteReportMacro Name="ELS_1", Value="DX_ELS_1"
; Macros used for HPLC_Systems

WriteReportMacro Name="HPLC_System", Value="$HPLC_System"
; Macros used for Valves

WriteReportMacro Name="Valve", Value="$Valve"
;==================================================================
DX_Sampler.AcquireExclusiveAccess
DX_Pump.Pressure.LowerLimit = 10 [bar]
DX_Pump.Pressure.UpperLimit = 300 [bar]
DX_Pump.%A.Equate = "Methanol"

DX_Pump.Flow = 1.000
DX_Pump.%B = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0
DX_Pump_Pressure.Step = 0.01 [s]
DX_Pump_Pressure.Average = Off
3DFIELD.MaxWavelength = 350.0
3DFIELD.MinWavelength = 250.0
DX_UV_VIS_1.Wavelength = 331
; Settings specific for the Dionex DAD-3000, DAD-3000RS

DX_UV.Data_Collection_Rate = 10 [Hz]
DX_UV.ResponseTime = 2 [s]

; Settings specific for the Dionex DAD-3000 and DAD-3000RS
DX_UV.UV_Lamp = On
DX_UV.Visible_Lamp = Off
3DFIELD.BunchWidth = 1.0 [nm]
3DFIELD.RefWavelength = 400.0 [nm]
3DFIELD.RefBandwidth = 1.0 [nm]

DX_UV_VIS_1.Bandwidth = 1.0 [nm]

DX_UV_VIS_1.RefWavelength = Off
DX_UV_VIS_1.RefBandwidth = 1.0 [nm]
; Autosampler specific settings

DX_Sampler.DrawSpeed = 5000 [nl/s]
DX_Sampler.DrawDelay = 3000 [ms]
DX_Sampler.DispSpeed = 20000 [nl/s]
DX_Sampler.DispenseDelay = 0 [ms]
DX_Sampler.WasteSpeed = 32000 [nl/s]
DX_Sampler.WashSpeed = 20000 [nl/s]
DX_Sampler.SampleHeight = 2.000 [mm]
DX_Sampler.InjectWash = NoWash
DX_Sampler.InjectMode = Normal
DX_Sampler.PunctureOffset = 0000 [m]
0.000
DX_UV.Autozero
; Settings specific for complete systems

Wait DX_PumpModule.Ready
Wait DX_Sampler.Ready
; Column Oven specific settings

Wait DX_ColumnOven.Ready
Wait DX_UV.Ready
DX_Sampler.Inject
3DFIELD.AcqOn
DX_UV_VIS_1.AcqOn
DX_UV_VIS_2.AcqOn
DX_UV_VIS_3.AcqOn
DX_Pump_Pressure.AcqOn
1.500 3DFIELD.AcqOff
DX_UV_VIS_1.AcqOff
DX_UV_VIS_2.AcqOff
DX_UV_VIS_3.AcqOff
DX_Pump_Pressure.AcqOff
DX_Sampler.ReleaseExclusiveAccess
End

9.2 Baseline Noise, Drift, and Lamp Intensity of the UV Detector

;===========================================================================
; Noise and Drift for UV Detectors
; --------------------------------------------------------------------------
;
; Eluent A : Water (HPLC quality)
;
; Solvent degassed via online degasser
;
; HPLC-System:
; ------------
;

;
;
;============================================================================
; Pump
; Pump DGP





; Dionex RF-2000




;==================================================================
; Agilent ICF specific LC system settings
DX_Pump.%A.Equate = "Water"
DX_Pump.%B = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0


; Settings specific for the Dionex DAD-3000(RS) / MWD-3000(RS)


DX_UV.UV_Lamp = On
0.000
DX_UV.Autozero


Wait DX_UV.Ready
DX_Sampler.Inject
DX_UV_VIS_1.AcqOn
21.000 DX_UV_VIS_1.AcqOff
22.000 Log DX_UV.UVLampIntensity
Protocol "The lamp intensity is given at 254 nm in [counts/s]"
End

9.3 Linearity of the UV Detector

;================================================================
; UV Detector Linearity
; ---------------------------------------------------------------
;
;
;
; HPLC-System:
; ------------
;; Pump specific settings

;
;
; Samples: Caffeine 10 g/ml, 60 g/ml, 140 g/ml, 220 g/ml, 300 g/ml
;
;==================================================================
; Pump
; Pump DGP





; Dionex RF-2000





;==================================================================

DX_Pump.%B = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0


DX_UV.ResponseTime = 0.2 [s]

DX_UV.UV_Lamp = On

0.000
DX_UV.Autozero


Wait DX_UV.Ready
DX_Sampler.Inject
DX_UV_VIS_1.AcqOn
End

9.4 Precision of Injection Volume

;================================================================
; Injector and Flow Reproducibility
; ---------------------------------------
;
;
;
; HPLC-System:
; ------------
;

;
; Sample: Caffeine 140 g/ml
;==================================================================
; Pump
; Pump DGP





; Dionex RF-2000





;==================================================================
DX_Pump.%B = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0




DX_UV.UV_Lamp = On

0.000
DX_UV.Autozero


Wait DX_UV.Ready
DX_Sampler.Inject
DX_UV_VIS_1.AcqOn
End

9.5 Carry-Over of the Autosampler and Linearity of the Injection

Volume
;================================================================
; Injector Linearity and Carry over
; ---------------------------------------------------------------
;
;
;
; HPLC-System:
; ------------
;

;
;
; Sample: Caffeine 10 g/ml (Linearity and Carry Over)
; Caffeine 2000 g/ml (Carry over)
; Water (Carry over)
;==================================================================
; Pump
; Pump DGP





; Dionex RF-2000





;==================================================================
DX_Pump.%B = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0




DX_UV.UV_Lamp = On

0.000
DX_UV.Autozero


Wait DX_UV.Ready
DX_Sampler.Inject
DX_UV_VIS_1.AcqOn
1.500
DX_UV_VIS_1.AcqOff
End

9.6 Sample Temperature Accuracy

;================================================================
; Autosampler Temperature Accuracy
; ---------------------------------------------------------------
;
; HPLC-System:
; ------------

;
;
; Sample: Water (Vial uncapped)
;==================================================================
; Pump
; Pump DGP





; Dionex RF-2000




;==================================================================

; Virtual channel settings

VirtualChannels_01.SamplingStep = 1.00
TemperatureOVEN.Type = Fixed
TemperatureOVEN.Formula Formula=Temperature_1
; Sampler settings
DX_Sampler.TempCtrl = On
DX_Sampler.ReadyTempDelta = 0.5 [C]
DX_Sampler.Temperature.Nominal = 10.0 [C]
0.000
Message"Make sure that the thermometer is located correctly in the vial (refer to
the manual for details)."
Message"Caution: To avoid damage to the system, do not perform sampler commands
while the test is running."
Wait DX_Sampler.TemperatureReady
DX_Sampler.Inject
TemperatureOven.AcqOn
30.000 TemperatureOven.AcqOff
Message"The test is now complete. Please remove the thermometer."
log DX_Sampler.Temperature.Value
End

9.7 Solvent Composition of a Gradient Pump, Accuracy, Precision,

and Ripple (Standard Gradient)
;================================================================
; Pump Gradient (Standard conditions)
; ---------------------------------------------------------------
;
; Eluent B : Water + 0.1 % Acetone (both HPLC quality)
;
; Solvents degassed via online degasser
;
; HPLC-System:
; ------------
;

;
;
;==================================================================
; Pump
; Pump DGP





; Dionex RF-2000




;==================================================================

-2.000 DX_Pump.Flow = 2.000
DX_Pump.%B.Value = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0
DX_Pump.%B.Equate = "Water + 0.1 % Acetone"
0.000

; Settings specific for the Dionex DAD-3000 and DAD-3000RS

DX_UV.UV_Lamp = On
0.000
DX_UV.Autozero


Wait DX_UV.Ready
DX_Sampler.Inject
DX_UV_VIS_1.AcqOn
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0
1.000 DX_Pump.%B.Value = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0

DX_Pump.%C = 0.0
DX_Pump.%D = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0
15.000 DX_Pump.%B.Value = 100.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0
DX_Pump.Flow = 2.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0
End

9.8 Temperature Accuracy of the Column Compartments for

Automatic Data Acquisition
; ===============================================================
; Temperature Accuracy of Column Thermostat
; ---------------------------------------------------------------
; PGM-Version: Nov 2010
;
; This program file is used to determinate the temperature accuracy
; of the column thermostat. The temperature is measured with an
; external thermometer.
; The data are acquired with a virtual channel.
; The property "Temperature_1" of the controlled thermometer is assigned
; to the channel "TemperatureOVEN".
;
; HPLC-System:
; ------------
;
; Settings specific for the Dionex TCC-3x00
; Column Thermostat: TCC-3x00
;
;==================================================================
; Pump
; Pump DGP





; Dionex RF-2000





;==================================================================
-35.00 DX_ColumnOven.Temperature.Nominal = 10.00

DX_ColumnOven.ReadyTempDelta = 4.0
DX_ColumnOven.EquilibrationTime = 0.5
VirtualChannels_01.SamplingStep = 1.00
TemperatureOVEN.Type = Fixed
Thermometer.Connect
TemperatureOVEN.Formula Formula=Temperature_1
TemperatureOVEN.Step= 0.01
TemperatureOVEN.Average= On
0.000
DX_UV.Autozero

; Column thermostat specific settings

Wait DX_ColumnOven.Ready, Timeout=120.00
Wait DX_UV.Ready
DX_Sampler.Inject
ColumnOven_TEMP.Step = 1
ColumnOven_TEMP.Average = On
ColumnOven_TEMP.AcqOn
TemperatureOVEN.AcqOn
0.000 DX_ColumnOven.Temperature.Nominal = 10.00
130.10 TemperatureOVEN.AcqOff
ColumnOven_TEMP.AcqOff
DX_ColumnOven.Temperature.Nominal = 25.00
End

9.9 Signal-to-Noise Ratio of the Fluorescence Detector

;================================================================
; SNR for Fluorescence Detectors
; ---------------------------------------
;
;
;
; HPLC-System:
; ------------

;
;
; Fluorescence detector specific settings
; Detector: Dionex FLD-3400RS Fluorescence Detector (UltiMate 3000)
;
;==================================================================
; Pump
; Pump DGP





; Dionex RF-2000





;==================================================================

DX_Pump.%B = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0


DX_Emission_1.ExWavelength = 350 [nm]
DX_Emission_1.EmWavelength = 450 [nm]
DX_FLD.BaselineBehavior = Free
; Settings specific for the Dionex UltiMate 3000 Fluorescence Detectors

FLD_FlowCell.Temperature = 35.00 [C]
FLD_Flowcell.ReadyTempDelta = 0.50 [C]
Delay 5
Wait FLD_FlowCell.TemperatureReady
DX_FLD.Data_Collection_Rate = 20 [Hz]
DX_FLD.ResponseTime = 8 [s]
DX_FLD.LampMode = HighPower
DX_Emission_1.Sensitivity = 6
0.000


Wait DX_UV.Ready
Wait DX_FLD.Ready
DX_Sampler.Inject
DX_Emission_1.AcqOn
1.000
23.000
45.000 DX_Emission_1.AcqOff
End

9.10 Wavelength Accuracy of the Fluorescence Detector (Emission)

;================================================================
; Wavelength accuracy for fluorescence detector
; Version 2 uses real scans of the water spectra
; Emission Spectrum
; ---------------------------------------------------------------
;
;
;
; HPLC-System:
; ------------
;; Pump specific settings

;
;
;
;==================================================================
; Pump
; Pump DGP





; Dionex RF-2000




;==================================================================
DX_Pump.%B = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0


Delay 5
DX_FLD.ScanStartExWavelength = 350 [nm]
DX_FLD.ScanStartEmWavelength = 385 [nm]
DX_FLD.ScanSpeed = Medium
DX_FLD.ScanSensitivity = 5
DX_FLD.Data_Collection_Rate = 20
DX_FLD.ResponseTime = 8

DX_FLD.ScanEndEmWavelength = 430 [nm]

0.000


Wait DX_UV.Ready
Wait DX_FLD.Ready
DX_Sampler.Inject
DX_Emission_1.AcqOn
1.000
DX_FLD.ScanEmission
End

9.11 Wavelength Accuracy of the Fluorescence Detector

(Extinction)
;================================================================
; Wavelength accuracy for fluorescence detector
; Version 2 uses real scans of the water spectra
; Excitation Spectrum
; ---------------------------------------------------------------
;
;
;
; HPLC-System:
; ------------

;
;
;
;==================================================================
; Pump
; Pump DGP





; Dionex RF-2000




;==================================================================
DX_Pump.%B = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0


Delay 5
DX_FLD.ScanStartEmWavelength = 397 [nm]
DX_FLD.ScanSpeed = Medium
DX_FLD.ScanSensitivity = 5
DX_FLD.Data_Collection_Rate = 20
DX_FLD.ResponseTime = 8

DX_FLD.ScanStartExWavelength = 330 [nm]
DX_FLD.ScanEndExWavelength = 370 [nm]

0.000


Wait DX_UV.Ready
Wait DX_FLD.Ready
DX_Sampler.Inject
DX_Emission_1.AcqOn
1.000
DX_FLD.ScanExcitation
End

9.12 Baseline Noise and Drift of the RI Detector

;===========================================================================
; Noise and Drift for RI Detctors
; --------------------------------------------------------------------------
;
; Solvent A : Water (HPLC quality)
;
;
; HPLC-System:
; ------------

;
;
; RI-Detector specific settings
; Detector: Shodex RI-101
;
;============================================================================
; Pump
; Pump DGP





; Dionex RF-2000





;==================================================================
DX_Pump.%B = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0

DX_RI_1.Step = 1.50
DX_RI_1.Average = On

; Settings specific for the Shodex RI-101
DX_RI.Temperature.Nominal = 35
DX_RI.Purge = off
DX_RI.Rise_Time = 0.50
DX_RI.Polarity = Minus
DX_RI.Baseline_Shift = 0
-40.000
DX_RI.Autozero



Wait DX_RI.Ready
Wait DX_UV.Ready
DX_RI.Purge = on
-39.500 DX_RI.Purge = off
-39.000 DX_RI.Purge = On
-38.500 DX_RI.Purge = Off
-38.000 DX_RI.Purge = On
-20.000 DX_RI.Purge = Off
-3.000 DX_RI.Autozero
0.000
DX_Sampler.Inject
DX_RI_1.AcqOn
22.000 DX_RI_1.AcqOff
End

9.13 Linearity of the RI Detector

;================================================================
; RI Detector Linearity
; ---------------------------------------------------------------
; PGM-Version Jan 2011
;
;
;
; HPLC-System:
; ------------

;
;
; RI-Detector specific settings
; Detector: Shodex RI-101
;
; Samples: Glycerine 5 mg/ml, 10 mg/ml, 15 mg/ml, 25 mg/ml, 35 mg/ml
;
;==================================================================
; Pump
; Pump DGP





; Dionex RF-2000





;==================================================================
DX_Pump.%B = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0

DX_RI_1.Step = 0.2

; Settings specific for the Shodex RI-101
DX_RI.Temperature.Nominal = 35
DX_RI.Purge = off
DX_RI.Recorder_Range = 512.00
DX_RI.Integrator_Range = 500
DX_RI.Rise_Time = 0.50
DX_RI.Polarity = plus
DX_RI.Baseline_Shift = 0

0.000
DX_RI.Autozero


Wait DX_RI.Ready
Wait DX_UV.Ready
DX_Sampler.Inject
DX_RI_1.AcqOn
2.000 DX_RI_1.Acqoff
End

9.14 Baseline Noise and Drift of the ELS Detector

;===========================================================================
; Noise for ELS Detctors
; --------------------------------------------------------------------------
;
;
;
; HPLC-System:
; ------------

;
;
; ELS-Detector specific settings
; Detector: Polymer Laboratories ELS 2100 Detector
;============================================================================
; Pump
; Pump DGP





; Dionex RF-2000





;==================================================================
DX_Pump.%B = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0
DX_ELS_1.Average = On
DX_ELS_1.Step = 1.0 [s]

DX_ELSD.Standby = NoStandby
DX_ELSD.LightSourceIntensity = 90 [%]
DX_ELSD.PMTGain = 1.0
DX_ELSD.SmoothWidth = 30
DX_ELSD.CarrierFlow = 1.60 [slm]
DX_ELSD.EvaporatorTemperature = 90 [C]
DX_ELSD.NebuliserTemperature = 50 [C]
0.000
DX_ELSD.Autozero


Wait DX_ELSD.Ready
DX_Sampler.Inject
DX_ELS_1.AcqOn
21.000 DX_ELS_1.AcqOff
End

9.15 Baseline Noise, Signal Height, Spikes, and Drift of the Corona
Detector
;===========================================================================
; Noise and Drift for Corona Detectors
; --------------------------------------------------------------------------
; PGM-Version Jun 2011
;
; Column Holder: Shiseido Guard Holder, 20 mm for 2.0 and 4.0 ID cartridges
; Column: Shiseido 20 x 4.0 mm C18 MG 3 m guard column
; Eluent A : Water/Methanol (80/20 Vol.%) (HPLC quality)
;
; Solvent degassed manually or via online degasser
;
; HPLC-System:
; ------------

;
;
; Detector: Dionex Corona ultra Detector
;============================================================================

; Pump
; Pump DGP





; Dionex RF-2000


WriteReportMacro Name="CAD", Value="DX_CAD"

WriteReportMacro Name="CAD_1", Value="DX_CAD_1"


;==================================================================
DX_Pump.%B = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0
DX_RI_1.Step = 1.50

; Settings specific for the Corona ultra
DX_CAD.Nebulizer_TempCtrl = On
DX_CAD.Nebulizer_TemperatureNominal = 25 [C]
DX_CAD.Data_Collection_Rate = 10 [Hz]
DX_CAD_1.FilterConstant = Corona
DX_CAD_1.GainRange = 100pA
DX_CAD_1.Offset = 0 [%]

DX_CAD.Autozero


Wait DX_CAD.Ready
Wait DX_CAD.Nebulizer_WaitForTempReady
Wait DX_UV.Ready
0.000
DX_Sampler.Inject
DX_CAD_1.AcqOn
22.000 DX_CAD_1.AcqOff
End

9.16 Signal Height, Precision, and Signal Calibration of the Corona

Detector
;================================================================
; Corona Detector Response Calibration and Signal-to-Noise
; ---------------------------------------------------------------
; PGM-Version Jun 2011
;
; Column Holder: Shiseido Guard Holder, 20 mm for 2.0 and 4.0 ID cartridges
; Column: Shiseido 20 x 4.0 mm C18 MG 3 m guard column
; Eluent A : Water/Methanol (80/20 Vol.%) (HPLC quality)
;
; Solvent degassed manually or via online degasser
;
; HPLC-System:
; ------------
; Detector: Dionex Corona ultra Detector
;
; Samples (Linearity): Caffeine 5 g/ml, 25 g/ml, 125 g/ml, 250 g/ml, 500 g/ml
; Sample (Signal-to-noise): Caffeine 5 g/ml (OQ)
; Sample (Signal-to-noise): Caffeine 25 g/ml (PQ)
;==================================================================

; Pump
; Pump DGP





; Dionex RF-2000



WriteReportMacro Name="CAD", Value="DX_CAD"

WriteReportMacro Name="CAD_1", Value="DX_CAD_1"


;==================================================================
DX_Pump.%B = 0.0
DX_Pump.%C = 0.0
DX_Pump.%D = 0.0

DX_RI_1.Step = 0.2

; Settings specific for the Corona ultra
DX_CAD.Nebulizer_TempCtrl = On
DX_CAD.Nebulizer_TemperatureNominal = 25 [C]
DX_CAD.Data_Collection_Rate = 10 [Hz]
DX_CAD_1.FilterConstant = Corona
DX_CAD_1.GainRange = 100pA
DX_CAD_1.Offset = 0 [%]

0.000
DX_CAD.Autozero
Wait DX_CAD.Ready
Wait DX_CAD.Nebulizer_WaitForTempReady
Wait DX_UV.Ready
DX_Sampler.Inject
DX_CAD_1.AcqOn
2.500 DX_CAD_1.Acqoff
End

10 Example Report
For an OQ example report, refer to the following pages.
The report was generated for the following system configuration:
P680 LPG
ASI 100
UVD 340S
STH 585
RI-101
RF 2000.

Smp: Warm up Runtime: 14.04.2004 14:36:35 Page 1 of 4
Seq: DQ_PQ_OQ_FOQ\DEMO_for_Manual\HPLC_OQ_DEMO_RUNS\OQ_WARM_UP
Operational Qualification
Instruments
Instrument Name Model Supplier's Name Serial Number
Pump P680 LPG DIONEX 1920401
Autosampler ASI-100 DIONEX 1860410
Column Oven STH 585 DIONEX 1850409
UV Detector Other DIONEX 1830402
Fluorescence Detector RF2000 DIONEX C20954571971US
RI Detector RI-101 Shodex 126739
ELS Detector Other DIONEX not available
Corona Detector Other DIONEX not available

Chromeleon Datasystem V. 6.80 SR11d Build 3290 Thermo Scientific 12
Additional Information
Customer: Customer's Name
Operator: Operator's Name
Operator's Jobtitle
Execution Date: Apr-14-04

Period between two Qualifications: 6 months
Next Qualification: Okt-04
______________________________ ______________________________
Reviewer's signature // Date Operator's signature // Date
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / SPECIFICATION

Version 6.80 SR11d Build 3290 Printed: 25.4.2012 2:22 PM
Accessories - General Tests

Accessories Name Lot No. Exp. Date
Back Pressure Device Capillary (L:15 m; ID:0,18 mm)
Sample 1 Pyrene in Methanol 3 g/ml CPS0057 Mrz-11
Sample 2 Caffeine in Water 10 g/ml CPS0057 Mrz-11
Sample 8 Water (HPLC-Grade)
Sample 9 Glycerine in Water 5 mg/ml HC997837 Mai-12
Solvent A Water (HPLC-Grade)
Solvent A for Wavelength Accuracy Methanol (HPLC-Grade)
Solvent B for Gradient Water + 0.1 % Acetone
Thermometer SN: 43077
Temperature Sensor SN: 111988
______________________________ ______________________________

Additional Accessories - Special Tests

Accessories Name Lot No. Exp. Date
Column C18,120A,5m,100mm,ID:4.6mm
Column Holder Shiseido, 20 mm for 2.0 & 4.0 ID cart.
Column Shiseido C18, 3 m, 20 x 4.0 mm
Sample 14 Water (Uncapped Vial)

Sample 15 Acetonitrile/Water 90:10
Sample 16 Anthracene in ACN 5 g/ml
Sample 21 Anthracene in ACN 0.5 g/ml
Sample 22 Anthracene in ACN 0.05 g/ml
Sample 23 Caffeine in Water 25 g/ml 763291 Sep-12
Solvent A for FLD Linearity Acetonitrile/Water 90:10
Solvent A for Corona Tests Water/Methanol 80:20
Solvent C for Gradient Water (HPLC-Grade)
Solvent D for Gradient Water + 0.1 % Acetone
______________________________ ______________________________

Limits
Criterion Adjusted Limits Limits with Optimized Conditions

Noise (UV) 0.030 mAU 0.030 mAU
Drift (UV) 0.80 mAU/h 0.80 mAU/h
Lamp Intensity (UV) 500000 counts/s 500000 counts/s
Detector Wavelength Accuracy +/- 0.75 nm +/- 0.75 nm
Detector Lin. - Corr. (UV) 99.980 % 99.980 %
Detector Lin. - RSD (UV) 5.000 % RSD 5.000 % RSD
Injector Precision - Area 0.300 % RSD 0.300 % RSD
Flow Precision - Ret. Time 0.0100 min SD 0.0100 min SD
0.050 % RSD 0.050 % RSD
Carry Over (Area) 0.100 % 0.100 %
Injector Linearity - Corr. 99.99000 % 99.99000 %
Injector Linearity 0.500 % RSD 0.500 % RSD
Temperature of Injector n.a. n.a.
Gradient Accuracy 1.000 % 1.000 %
Gradient Precision 0.500 % SD 0.500 % SD
Pump Ripple 0.500 % 0.500 %
Temperature of Column Oven +/- 1.0 C +/- 1.0 C
Noise (RF) 0.30 mV 0.30 mV
Signal (RF) min 40.00 mV 40.00 mV
Signal (RF) max 80.00 mV 80.00 mV
SNR ASTM (FL) n.a. n.a.
SNR Dark Current (FL) n.a. n.a.
Wavelength Accuracy (FL) +/- 10 nm +/- 10 nm
Detector Lin. - Corr. (FL) n.a. n.a.
Detector Lin. - RSD (FL) n.a. n.a.
Detector Lin. - Offset (FL) n.a. n.a.
Noise (RI) 50.0 nRIU 50.0 nRIU
Drift (RI) 500.0 nRIU/h 500.0 nRIU/h
Detector Lin. - Corr. (RI) 99.900 % 99.900 %
Noise (ELS) 0.3 mV 0.3 mV
Noise (Corona) 0.040 pA 0.040 pA
Random Spikes (Corona) 0.200 pA 0.200 pA
Drift (Corona) 0.040 pA/min 0.040 pA/min
SNR (Corona) 10 10
Precision (Corona) 10.0 % RSD 10.0 % RSD
Det.Calib. - Coef. of Det. (Corona) 99.90 % 99.90 %
______________________________ ______________________________

Smp: Detector wavelength accuracy_DAD Runtime: 13.04.2004 19:32:27 Page 1 of 2
Seq: DQ_PQ_OQ_FOQ\DEMO_for_Manual\HPLC_OQ_DEMO_RUNS\OQ_DAD_WAVELENGTH
Wavelength Accuracy of the DAD
Instruments and Fluidics

Accessories Name
Sample 1 Pyrene in Methanol 3 g/ml
Solvent A for Wavelength Accuracy Methanol (HPLC-Grade)

Operator's Jobtitle

Next Qualification: Oct-12
Limits, Values and Test Results
Limit Obs. Value Result

Wavelength Accuracy at 272,1 nm +/- 0.75 nm -0.22 nm Test passed
Wavelength Accuracy at 333,3 nm +/- 0.75 nm -0.02 nm Test passed
______________________________ ______________________________
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / DET_WAVELENGTH_DAD

Smp: Detector wavelength accuracy_DAD Runtime: 13.04.2004 19:32:27 Page 2 of 2
Seq: DQ_PQ_OQ_FOQ\DEMO_for_Manual\HPLC_OQ_DEMO_RUNS\OQ_DAD_WAVELENGTH
Data for the Wavelength Accuracy Test (DAD)
Observed Wavelength Expected Abs.Critical Calculated

Testpoint Result
[nm] Wavelength [nm] Deviation[nm] Deviation[nm]
Pyrene Maximum 1 271.88 272.10 0.75 -0.22 ok

Pyrene Maximum 2 333.28 333.30 0.75 -0.02 ok
Pyrene-Spectrum for the Wavelength Accuracy Test (DAD)
Pyrene 100% at 0.31 min

60.0
%
271.9
50.0 333.3
40.0
317.9
30.0 261.0
20.0
251.1
10.0
0.0
nm
-10.0
250 260 270 280 290 300 310 320 330 340 350
______________________________ ______________________________
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / DET_WAVELENGTH_DAD

Smp: Detector noise drift and lamp intensity Runtime: 13.04.2004 23:12:48 Page 1 of 3
Seq: DQ_PQ_OQ_FOQ\DEMO_for_Manual\HPLC_OQ_DEMO_RUNS\OQ_UV_NOISE_DRIFT
UV Detector Noise and Drift

Accessories Name

Operator's Jobtitle


Limit Observed Value Result
Noise (UV) 0.030 mAU 0.021 mAU Test passed
Drift (UV) 0.80 mAU/h 0.44 mAU/h Test passed

Lamp Intensity (UV) 500000 counts/s n.a. counts/s no data
Remark: Noise and drift are measured dynamically with a floated cell. The limits are different from
published specifications, because they are valid for static conditions only (empty cell).
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / DET_NOISE_AND_DRIFT

Data for Detector Noise Test
Segment No. Noise [mAU]
1 0.024
2 0.018
3 0.021
4 0.019
5 0.024
6 0.027
7 0.020
8 0.024
9 0.020
10 0.017
11 0.023
12 0.016
13 0.026
14 0.016
15 0.018
16 0.022
17 0.022
18 0.018
19 0.025
20 0.026
Average: 0.021 mAU
Limit: 0.030 mAU
Result: ok
______________________________ ______________________________

Chart for Noise Test
Detector Noise
0.10
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
0 2 4 6 8 10 12 14 16 18 20
Segment No.
______________________________ ______________________________

Smp: Injector and flow reproducibility_10 Runtime: 14.04.2004 00:20:19 Page 1 of 4
Seq: DQ_PQ_OQ_FOQ\DEMO_for_Manual\HPLC_OQ_DEMO_RUNS\OQ_INJECTOR_FLOW_REPRO
Injector and Flow Precision

Accessories Name
Sample 4 Caffeine in Water 140 g/ml

Operator's Jobtitle


Injector Precision - Area 0.300 % RSD 0.188 % RSD Test passed
0.050 % RSD 0.104 % RSD
Flow Precision - Ret. Time OR Test passed
0.0100 min SD 0.0011 min SD
______________________________ ______________________________
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / INJ_REPRO_AND_RET_REPRO

Data for Injector and Flow Precision Test: Volume 5.0 l
Sample Name Ret.Time Area
min mAU*min
Caffeine Caffeine
UV_VIS_1 UV_VIS_1
Injector and flow reproducibility_1 1.0067 93.036

Average: 1.0097 92.833
RSD: 0.104 % 0.188 %
RSD Limit: 0.050 % 0.300 %
SD: 0.0011
SD Limit: 0.0100
Result: ok ok
______________________________ ______________________________

Charts for Injector and Flow Precision Test
Area % Deviation from Mean Value
1.00
0.60
0.20
-0.20
1 2 3 4 5 6 7 8 9 10
-0.60
-1.00
Sample No.
Retention Time % Deviation from Mean Value
1.00
0.75
0.50
0.25
0.00
-0.25 1 2 3 4 5 6 7 8 9 10
-0.50
-0.75
-1.00
Sample No.
______________________________ ______________________________

Chromatogram Overlay
400 1 - OQ_INJECTOR_FLOW_REPRO #11 UV_VIS_1

mAU WVL:272 nm
1 - Caffeine - 1.010
1
-50
mAU WVL:272 nm
2
-50
mAU WVL:272 nm
3
-50
mAU WVL:272 nm
4
-50
mAU WVL:272 nm
5
-50
mAU WVL:272 nm
6
-50
mAU WVL:272 nm
7
-50
mAU WVL:272 nm
8
-50
mAU WVL:272 nm
9
-50
mAU WVL:272 nm
10 min
-50
0.00 0.50 1.00 1.50 2.00 2.50 3.00
______________________________ ______________________________

Smp: Detector linearity_5 Runtime: 14.04.2004 00:33:46 Page 1 of 2
Seq: DQ_PQ_OQ_FOQ\DEMO_for_Manual\HPLC_OQ_DEMO_RUNS\OQ_UV_LINEARITY
UV Detector Linearity

Accessories Name

Operator's Jobtitle


Detector Lin. - Corr. (UV) 99.980 % 99.999 % Test passed
Detector Lin. - RSD (UV) 5.000 % RSD 0.422 % RSD Test passed
______________________________ ______________________________
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / DET_LINEARITY

Smp: Detector linearity_5 Runtime: 14.04.2004 00:33:46 Page 2 of 2
Seq: DQ_PQ_OQ_FOQ\DEMO_for_Manual\HPLC_OQ_DEMO_RUNS\OQ_UV_LINEARITY
Data for Detector Linearity
Sample Name Amount Area

ppm mAU*min
Caffeine Caffeine
$UV_VIS_1 UV_VIS_1
Detector linearity_1 10.0 4.258
Calibration Curve
140 Caffeine External UV_VIS_1

Area [mAU*min]
100
50
0 ppm
0 50 100 150 200 250 310
Cal.Type Number of Points Offset Slope
UV_VIS_1 UV_VIS_1 UV_VIS_1 UV_VIS_1
Lin 5 0.000 0.417
Correlation Coefficient RSD

99.999 % 0.422 %
Limit: 99.980 % 5.000 %
Result: ok ok
______________________________ ______________________________
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / DET_LINEARITY

Smp: RI-Detector noise drift Runtime: 31.01.2003 16:10:23 Page 1 of 3
Seq: DQ_PQ_OQ_FOQ\DEMO_for_Manual\HPLC_OQ_DEMO_RUNS\OQ_RI_NOISE_DRIFT
RI Detector Noise and Drift

Accessories Name

Operator's Jobtitle


Noise (RI) 50.0 nRIU 26.6 nRIU Test passed
Drift (RI) 500.0 nRIU/h 321.8 nRIU/h Test passed
Remark: Noise and drift are measured dynamically with a floated cell. The limits are different from
published specifications, because they are valid for static conditions only (empty cell).
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / RI_NOISE_AND_DRIFT

Data for Detector Noise and Drift Test
Segment No. Noise [nRIU] Amount of Drift [nRIU/h]
1 25.3 173.299
2 26.3 231.224
3 36.8 282.414
4 44.8 839.815
5 27.2 4.386
6 30.3 214.676
7 24.7 204.259
8 19.0 953.907
9 30.5 494.897
10 22.8 344.469
11 29.8 47.102
12 23.4 923.453
13 21.6 4.237
14 19.8 221.655
15 25.2 181.431
16 22.7 3.639
17 23.7 185.468
18 25.5 545.040
19 23.4 163.138
20 28.5 417.141
Average: 26.6 nRIU 321.8 nRIU/h
Limit: 50.0 nRIU 500.0 nRIU/h
Result: ok ok
______________________________ ______________________________

Charts for Noise and Drift Test
Detector Noise
75.00
60.00
45.00
30.00
15.00
0.00
0 2 4 6 8 10 12 14 16 18 20
Segment No.
Amount of Drift
1000.00
800.00
600.00
400.00
200.00
0.00
0 2 4 6 8 10 12 14 16 18
Segment No.
______________________________ ______________________________

Smp: RI_Detector linearity_5 Runtime: 24.02.2003 09:45:59 Page 1 of 2
Seq: DQ_PQ_OQ_FOQ\DEMO_for_Manual\HPLC_OQ_DEMO_RUNS\OQ_RI_LINEARITY
RI Detector Linearity

Accessories Name
Sample 9 Glycerine in Water 5 mg/ml

Operator's Jobtitle


Detector Lin. - Corr. (RI) 99.900 % 99.999 % Test passed
______________________________ ______________________________
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / RI_LINEARITY

Smp: RI_Detector linearity_5 Runtime: 24.02.2003 09:45:59 Page 2 of 2
Seq: DQ_PQ_OQ_FOQ\DEMO_for_Manual\HPLC_OQ_DEMO_RUNS\OQ_RI_LINEARITY
Data for Detector Linearity
Sample Name Amount Area Height

RIU*min RIU
Glycerine Glycerine Glycerine
$RI_1 RI_1 RI_1
RI_Detector linearity_1 5.00 6.536 77.6
45.0 Glycerine External RI_1

Area [RIU*min]
30.0
20.0
10.0
0.0
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0
RI RI RI RI
LOff 5 0.926 1.131
Correlation Coefficient
99.999 %
Limit: 99.900 %
Result: ok
______________________________ ______________________________
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / RI_LINEARITY

Smp: Injector linearity_5 Runtime: 14.04.2004 00:43:39 Page 1 of 2
Seq: DQ_PQ_OQ_FOQ\DEMO_for_Manual\HPLC_OQ_DEMO_RUNS\OQ_SAMPLER_LIN_CO
Injector Linearity

Accessories Name

Operator's Jobtitle


Injector Linearity - Corr. 99.99000 % 99.99979 % Test passed
Injector Linearity 0.500 % RSD 0.238 % RSD Test passed
______________________________ ______________________________
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / INJ_LINEARITY

Smp: Injector linearity_5 Runtime: 14.04.2004 00:43:39 Page 2 of 2
Calibration Curve
40.0 Caffeine External UV_VIS_1
Area [mAU*min]
30.0
20.0
10.0
l
0.0
0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0
Data for Injector Linearity Test

Sample Name Ret.Time Inj.Vol. Area
min l mAU*min
Caffeine Caffeine
UV_VIS_1 UV_VIS_1
Injector linearity_1 0.31 5.0 2.029

$UV_VIS_1 UV_VIS_1 UV_VIS_1 UV_VIS_1
n.a. 5 -0.184 0.447
Correlation Coefficient RSD
99.99979 % 0.238 %
Limit: 99.99000 % 0.500 %
Result: ok ok
______________________________ ______________________________
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / INJ_LINEARITY

Smp: Inject solvent Runtime: 26.01.2007 15:30:06 Page 1 of 2
Injector Carry Over

Accessories Name
Sample 8 Water (HPLC-Grade)

Operator's Jobtitle


Carry Over (Area) 0.100 % < 0.005 % Test passed
______________________________ ______________________________
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / INJ_CARRY_OVER

Smp: Inject solvent Runtime: 26.01.2007 15:30:06 Page 2 of 2
Chromatogram for Carry Over Test

OQ_SAMPLER_LIN_CO #9 Inject solvent UV_VIS_1
3.00
mAU WVL:272 nm
2.50
2.00
1.50
1.00
0.50 1 - 0.200
3 - 1.150
-0.00
min
-0.50
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.25
Data for Carry Over Test
Sample Name Ret.Time Area
min mAU*min
Caffeine Caffeine
UV_VIS_1 UV_VIS_1
Inject solvent_Reference 0.58 0.132
Carry over_Reference 0.55 4.842
Carry over 0.53 447.221
Inject solvent 0.57 0.093
Carry over: < 0.005 %
Limit: 0.100 %
Result: ok
______________________________ ______________________________
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / INJ_CARRY_OVER

Smp: Fluorescence_Detector_Noise Runtime: 30.05.2001 00:36:00 Page 1 of 3
Seq: DQ_PQ_OQ_FOQ\DEMO_for_Manual\HPLC_OQ_DEMO_RUNS\OQ_FLUORESCENCE
Fluorescence Detector Noise

Accessories Name

Operator's Jobtitle


Noise (RF) 0.30 mV 0.10 mV Test passed
Signal (RF) min 40.00 mV 42.97 mV Test passed
Signal (RF) max 80.00 mV 43.10 mV Test passed
______________________________ ______________________________
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / RF_DET_NOISE

Data for RF Detector Noise
Segment No. Noise [mV]
1 0.118
2 0.109
3 0.099
4 0.074
5 0.101
6 0.109
7 0.123
8 0.069
9 0.089
10 0.066
11 0.073
12 0.097
13 0.149
14 0.080
15 0.076
16 0.074
17 0.151
18 0.052
19 0.129
20 0.110
21 0.091
22 0.112
23 0.109
24 0.079
25 0.128
26 0.113
27 0.101
28 0.113
29 0.108
30 0.073
Average: 0.10 mV
Limit: 0.30 mV
Result: ok
______________________________ ______________________________

Charts for RF Detector Noise Test
RF Detector Noise
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0 5 10 15 20 25 30
Segment No.
______________________________ ______________________________

Smp: Fluorescence_Detector_Wavelength Runtime: 30.05.2001 00:54:06 Page 1 of 2
Fluorescence Detector Wavelength Accuracy

Accessories Name

Operator's Jobtitle


Limit Obs. Deviation Result
Wavelength Accuracy (FL) +/- 10 nm 0 nm Test passed
______________________________ ______________________________
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / RF_DET_WAVE

Smp: Fluorescence_Detector_Wavelength Runtime: 30.05.2001 00:54:06 Page 2 of 2
Data for the Wavelength Accuracy Test (Fluorescence Detector)
Obs. Wavelength Exp. Wavelength Deviation Limit
397 nm 397 nm 0 nm +/- 10 nm
Chromatogram of Wavelength Accuracy Test (Fluorescence Detector)
The emission wavelength is changed from 380 nm to 410 nm in steps of 1 nm per 15 sec.
The maximum of the emission spectrum is determinated as maximum of the signal
of this chromatogram.
40.0 OQ_FLUORESCENCE #2 Fluorescence_Detector_Wavelength Emission

mV EM:380 nm
30.0
20.0
10.0
-5.0 min
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
______________________________ ______________________________
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / RF_DET_WAVE

Smp: STD Gradient_1 Runtime: 14.04.2004 10:10:40 Page 1 of 3
Seq: DQ_PQ_OQ_FOQ\DEMO_for_Manual\HPLC_OQ_DEMO_RUNS\OQ_STD_GRAD
Step Accuracy: STD Gradient_1
Accessories Name

Operator's Jobtitle

Limits and Test Results

Limit Observed max. Deviation Result of all Steps
Step Accuracy 1.000 % 0.275 % Test passed
Step Ripple 0.500 % 0.025 % Test passed
______________________________ ______________________________
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / PUMP_GRADIENT

Chromatogram of STD Gradient_1
OQ_STD_GRAD #2 STD Gradient_1 UV_VIS_1

250
mAU WVL:265 nm
200
150
100
50
min
-50
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0
Flow [ml/min]: 2.000
______________________________ ______________________________

Data of STD Gradient_1
Expected Calculated Abs. Critical Calculated

Observed Value [mAU] Result
Value [%] Value [%] Deviation [%] Deviation [%]
0.00 0.00 0.000 1.000 0.000 ok
2.40 1.00 1.118 1.000 0.118 ok
106.65 50.00 49.725 1.000 -0.275 ok
212.37 99.00 99.017 1.000 0.017 ok
214.48 100.00 100.000 1.000 0.000 ok
Ripple of STD Gradient_1
Ripple Calculated Critical

Step [%] Result
[mAU] Ripple [%] Ripple [%]
1.00 0.007 0.003 0.500 ok
50.00 0.018 0.008 0.500 ok
99.00 0.053 0.025 0.500 ok
______________________________ ______________________________

Accessories Name

Operator's Jobtitle


______________________________ ______________________________


250
mAU WVL:265 nm
200
150
100
50
min
-50
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0
______________________________ ______________________________


0.00 0.00 0.000 1.000 0.000 ok
2.47 1.00 1.150 1.000 0.150 ok
106.78 50.00 49.788 1.000 -0.212 ok
212.30 99.00 98.991 1.000 -0.009 ok
214.46 100.00 100.000 1.000 0.000 ok

Step [%] Result
1.00 0.033 0.015 0.500 ok
50.00 0.036 0.017 0.500 ok
99.00 0.012 0.006 0.500 ok
______________________________ ______________________________

Accessories Name

Operator's Jobtitle


______________________________ ______________________________


250
mAU WVL:265 nm
200
150
100
50
min
-50
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0
______________________________ ______________________________


0.00 0.00 0.000 1.000 0.000 ok
2.50 1.00 1.163 1.000 0.163 ok
106.87 50.00 49.826 1.000 -0.174 ok
212.29 99.00 98.976 1.000 -0.024 ok
214.48 100.00 100.000 1.000 0.000 ok

Step [%] Result
1.00 0.012 0.006 0.500 ok
50.00 0.023 0.011 0.500 ok
99.00 0.017 0.008 0.500 ok
______________________________ ______________________________

Precision of the Stand. Gradient
Accessories Name

Operator's Jobtitle

Limit Observed max. SD Result of all Steps

Gradient Precision 0.500 % 0.051 % Test passed
______________________________ ______________________________
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / PUMP_GRADIENT_REPRO

Overlay of three Gradients

1 - OQ_STD_GRAD #4 STD Gradient_3 UV_VIS_1
250
mAU WVL:265 nm
200
150
100
50
3
2
0 1
min
-50
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0
Data of three Gradients

Calculated Calculated Calculated
Value Value Value Critical Calculated
Expected Value [%] Result
Gradient_1 Gradient_2 Gradient_3 SD [%] SD [%]
[%] [%] [%]
0.00 0.00 0.00 0.00 0.50 0.000 ok
1.00 1.12 1.15 1.16 0.50 0.023 ok
50.00 49.72 49.79 49.83 0.50 0.051 ok
99.00 99.02 98.99 98.98 0.50 0.020 ok
100.00 100.00 100.00 100.00 0.50 0.000 ok
______________________________ ______________________________
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / PUMP_GRADIENT_REPRO

Smp: Oven Temperature Runtime: 14.04.2004 11:29:06 Page 1 of 2
Seq: DQ_PQ_OQ_FOQ\DEMO_for_Manual\HPLC_OQ_DEMO_RUNS\OQ_COLUMN_OVEN
Temperature Accuracy of the Column Oven

Accessories
Thermometer SN: 43077
Temperature Sensor SN: 111988

Operator's Jobtitle

Limit Obs. max. Deviation Result

Temperature of Column
+/- 1.0 C 0.4 C Test passed
Oven
______________________________ ______________________________
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / COLUMN OVEN
Smp: Oven Temperature Runtime: 14.04.2004 11:29:06 Page 2 of 2
Seq: DQ_PQ_OQ_FOQ\DEMO_for_Manual\HPLC_OQ_DEMO_RUNS\OQ_COLUMN_OVEN
Data for Temperature Accuracy
Setpoint Measured Deviation Result
Temperature Temperature
[C] [C] [C]
80 80.4 0.4 ok
60 60.2 0.2 ok
30 30.3 0.3 ok
10 10.1 0.1 ok
Obs. max. Deviation 0.4 ok
Limit: +/- 1.0
______________________________ ______________________________
Chromeleon (c) 2012 Thermo Fisher Scientific Inc. PQ_OQ_Report_8_3 / COLUMN OVEN

HPLC OQ PQ OperatingInstructions

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HPLC OQ PQ OperatingInstructions

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Thermo Scientific Dionex

2012 Thermo Fisher Scientific Inc.

3 Test Procedures ................................................................................................ 5

4 Special Test Procedures for Individual Modules.......................................... 26

6 Supported Instruments / Overview of the Checks ....................................... 41

Contents: II of IV HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012

7.8 Flow Precision ............................................................................................................................71

8.2.7 Corona Detector ...................................................................................................................83

9 PGM Files ......................................................................................................... 85

10 Example Report ............................................................................................. 119

Contents: IV of IV HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012

1 How to Use This Manual

The information contained in this document is subject to change without notice.

HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012 Page 1 of 119

Page 2 of 119 HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012

2.1 Defining the Limits

2.1.1 Operational Qualification (OQ)

2.1.2 Performance Qualification (PQ)

HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012 Page 3 of 119

2.2 Basic Requirements for Successful OQ and PQ

Page 4 of 119 HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012

Part no. Description Quantity

Sample Position Substance Concentration Checks

HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012 Page 5 of 119

Sample Position Substance Concentration Checks

Sample Position Substance Concentration WPS-3000T(B)FC Analytical

RC1 Caffeine in water 140 g/mL Standard configuration (sample loop

Sample Position Substance Concentration Checks

Page 6 of 119 HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012

In addition, the following solvents are required:

Solvent Quantity Checks

For qualifying a column compartment, a calibrated thermometer is required. The thermometer is

3.2 Test Procedure for Single Wavelength and VWD-3400RS

Solvent Quantity Checks

HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012 Page 7 of 119

3.3 Connecting and Configuring the System

3.3.1 System Connections

Figure 1: Restriction tubing installed between injection valve and detector

Page 8 of 119 HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012

Case a: 6 port/2-position valve

Pump unit (L = left, R =

Pump unit (L = left, R =

HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012 Page 9 of 119

Pump unit (L = left, R =

Page 10 of 119 HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012

Position of the sensor tip.

Figure 6: TCC-100/TCC-3x00(SD/RS) - Position of the temperature sensor

Position of the sensor tip behind the capillary clip

Figure 7: ACC-3000(T) Position of the temperature sensor

HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012 Page 11 of 119

Figure 8: WPS-3000TB PL Analytical configuration

Page 12 of 119 HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012

Dionex (UltiMate 3000) autosamplers with user-defined sample loop volume

Autosampler User-defined sample loop Sample loop volume

Qualifying the column compartment

Option B: Automatic data acquisition as analog signal:

HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012 Page 13 of 119

Option C: Manual data acquisition:

Page 14 of 119 HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012

3.4.1 Preparing the HPLC System

3.4.2 Checking the Fluidics

HPLC_OQ_PQ_OperatingInstructions_V8_3.docx Version: 8.3 of May 2012 Page 15 of 119

3.5 Preparations in Chromeleon

3.5.1 Template Structure