Thermo Scientific UltiMate User manual

M1393R0
UltiMate
™
Capillary- and Nano HPLC Systems
Operational Qualification
and
Performance Qualification
Operating Instructions
P/N 163960
www.lcpackings.com

Page 2 of 60 M1393R0 OQ/PQ Operating Instructions
The material included in this manual is provided to assist authorized personnel in
performing operation qualification (OQ) and performance qualification (PQ) on the LC
Packings UltiMate Capillary and Nano HPLC system. It is assumed that the individual using
this manual has sufficient training in the use of analytical instrumentation and is aware of
the potential hazards including (but not limited to) electrical hazards, chemical solvent
hazards, exposure to UV radiation and the exposure to pressurized solvents.
All information in this manual is subject to change without notice and does not represent a
commitment on the part of LC Packings, BV.
© October 2004, LC Packings, BV - A Dionex Company. All rights reserved.
No part of this manual may be reproduced or transmitted in any form or by any means
without the written permission of LC Packings, BV.
The following are Registered Trademarks of LC Packings - A Dionex Company: Probot,
µCarrier, UltiMate, UltiChrom, FAMOS, Thermos, Switchos , Acurate, UZ-View,
UV-Booster, FluoBoost, Pepmap, µ-Dumper, µ-Fluidics, Fusica, Nano Series, Pico Series,
µ-Guard, µ-Precolumn, Nano-Precolumn.
Dionex™ is a Trademark of Dionex Corporation.
CHROMELEON®is a Registered Trademark of the Dionex Corporation.
Analyst® is a Registered Trademark of Applied Biosystems/MDS Sciex.
HyStar™is a Trademark of Bruker-Daltonics Group.
MassLynx™is a Trademark of Waters® Corporation.
Xcalibur® is a Registered Trademark of ThermoFinnigan.
Windows® is a Registered Trademark of Microsoft Corporation.
PEEK™ polymer is a Trademark of Victrex plc.
Other product names or company names mentioned herein may be the trademarks of their
respective owners.
Printed in the Netherlands.

OQ/PQ Operating Instructions M1393R0 Page 3 of 60
Warnings
The Danger sign, Warning sign and the Caution sign shown below are included in various
locations in this manual or in the manuals provided with the instruments which are to be
tested. These signs provide the following information:
DANGER Danger: The information in a danger statement relates to a procedure, practice condition or
action that if not done correctly or adhered to could lead to personal injury or loss of life.
WARNING Warning: The information in a warning statement relates to a procedure, practice,
condition or action that if not done correctly or adhered to could lead to severe injury
and/or damage or destruction to parts or all of the equipment.
CAUTION Caution: The information in a caution statement relates to a condition that could lead to
damage to equipment and/or lead to invalid analytical results.
Note: The information in a note statement relates to important information that should be
read and understood before continuing.
Safety Precautions
Note: The following precautions should be followed to minimize the possibility of personal
injury and/or damage to property.
Note: Make certain that you are familiar with the contents of this manual before working
on the system.

Page 4 of 60 M1393R0 OQ/PQ Operating Instructions
The operator should follow all safety precautions, warnings, etc provided with the
instruments, in addition, please note the items presented below:
1. All components of the system should be plugged into a common power line that is
directly connected to a true ground.
2. Repair or replace faulty power cords and all communication cables.
3. If a leak occurs, turn off power to the instrument and remedy the situation
immediately.
4. If the mobile phase includes volatile or flammable solvents, avoid open flames and
sparks.
5. Many organic solvents and buffers are toxic. Make certain that you know the
toxicological properties of all mobile phases that you are using.
6. The toxicological properties of many samples may not be well known. If you have
any doubt about a sample, treat it as if it contained a potentially harmful substance.
7. Wear protective eye goggles when handling mobile phases or operating the
instrument. An eye wash facility and a sink should be close to the unit. If any mobile
phase splash on the eyes or skin, wash the affected area and seek medical attention.
8. Dispose of all waste mobile phase in an environmentally safe manner that is
consistent with all local regulations. Do not allow flammable and/or toxic solvents to
accumulate. Follow a regulated, approved waste disposal program. Never dispose
flammable and/or toxic solvents through the municipal sewage system
9. Wear protective eye goggles when handling fused silica tubing (i.e. installation,
cutting etc.)
10. If a buffer is used as a part of the mobile phase, flush the system with several
volumes of a methanol/water (50/50) solution before it is shut down. This will
prevent salt buildup inside the unit.
11. Do not use the instrument in ways other than those indicated in the instructions
given in this manual.
WARNING Warning: The OQ/PQ kit (P/N163932) contains a chemical or chemicals known to the State
of California to cause cancer and/or birth defects or other reproductive harm. For additional
information, consult the product Material Safety Data Sheet (MSDS).

OQ/PQ Operating Instructions M1393R0 Page 5 of 60
Table of Contents
Warnings ................................................................................................. 3
Safety Precautions .................................................................................... 3
CHAPTER 1 Introduction .........................................................................7
1.1 The Purpose of OQ/PQ.................................................................... 7
1.2 Defining the Limits ......................................................................... 7
1.3 General Notes and Recommendations ............................................... 8
1.4 How this Manual is structured ......................................................... 8
1.5 For Additional Information ............................................................... 8
CHAPTER 2 Requirements for a Successful OQ/PQ .....................................9
2.1 Overview ...................................................................................... 9
2.2 Checks and Acceptance Limits....................................................... 10
CHAPTER 3 Process ............................................................................. 13
3.1 Required Materials ........................................................................ 13
3.2 Preparations ................................................................................ 15
3.2.1 CHROMELEON® Setup ................................................................ 15
3.2.2 System Setup ............................................................................ 18
3.3 Performing the Checks .................................................................. 21
3.4 Evaluating the Tests ..................................................................... 22
3.4.1 Repeating Tests ......................................................................... 22
CHAPTER 4 Test Procedures.................................................................. 23
4.1 Overview .................................................................................... 23
4.2 Lamp Intensity of the UV Detector ................................................. 24
4.2.1 Performing the Lamp Intensity Test .............................................. 24
4.3 Wavelength Check ....................................................................... 25
4.3.1 Performing the Test.................................................................... 25
4.4 Flow Cell Check ........................................................................... 26
4.4.1 Performing the Intensity Test of the Flow Cell ............................... 26
4.5 UltiMate Fluid Path Test ................................................................ 27
4.5.1 Performing the Test.................................................................... 27
4.6 Baseline Noise and Drift Test of the UV Detector and Oven Test........ 28
4.6.1 Drift and Noise........................................................................... 28
4.6.2 Theory of the Noise and Drift Calculation of CHROMELEON ............ 29
4.6.3 Oven accuracy........................................................................... 29

Table of Contents
Page 6 of 60 M1393R0 OQ/PQ Operating Instructions
4.6.4 Performing the UV Detector Drift and Noise Test and the Oven Test. 30
4.7 Linearity of the UV Detector...........................................................31
4.7.1 Performing the UV Detector Linearity Test ..................................... 32
4.8 Reproducibility of the Injection Volume ............................................ 33
4.8.1 Performing the Test .................................................................... 33
4.9 Linearity of the injection ................................................................ 34
4.9.1 Performing the Test .................................................................... 34
4.10 Gradient Accuracy ........................................................................ 35
4.10.1 Performing the Test .................................................................... 37
4.11 Switchos Fluid Path Check ............................................................. 38
4.11.1 Performing the test .....................................................................38
4.12 Switchos Flow Rate and Pressure Stability Test................................ 39
4.12.1 Performing the test .....................................................................39
4.13 Switchos Valve Position Check ....................................................... 40
4.13.1 Performing the Test .................................................................... 40
4.14 Completing and Printing the OQ/PQ Report ......................................41
4.14.1 Result of the Gradient Accuracy Test ............................................ 41
4.14.2 Completing the Cover Page and Printing ........................................ 42
CHAPTER 5 Troubleshooting ................................................................. 43
5.1 Overview ..................................................................................... 43
5.2 Sample and Mobile Phase Considerations......................................... 43
5.3 Probable Causes and Solutions ....................................................... 44
CHAPTER 6 CHROMELEON®Listings ...................................................... 45
6.1 Overview ..................................................................................... 45
6.2 Wavelength Check (NAN Configuration)........................................... 46
6.3 Noise and Drift Test (NAN Configuration).........................................47
6.4 UV Linearity (NAN Configuration).................................................... 49
6.5 Autosampler Reproducibility (NAN Configuration).............................. 51
6.6 Autosampler Injection Linearity (NAN Configuration) .........................53
6.7 Gradient Accuracy Test (NAN Configuration).................................... 55
6.8 Switchos Pump Test (NAN Configuration) ........................................ 57
6.9 Stop Flow .................................................................................... 59

OQ/PQ Operating Instructions M1393R0 Page 7 of 60
CHAPTER 1 Introduction
1.1 The Purpose of OQ/PQ
An increasing number of standards and official regulations require that the end user is able
to provide evidence that the instrumentation used for analytical work is working in a
satisfactory manner. In the same manner, quality management according to ISO 9000
(and similar standards) requires that the user monitor and document the ability of the
equipment to obtain valid data on a periodic basis. This manual provides a detailed series
of procedures to perform these Operational Qualification (OQ) and Performance
Qualification (PQ) protocols on the LC Packings UltiMate system components.
The validation procedures described herein are designed to demonstrate that the
instrument was working in an acceptable manner using the standards provided on the day
of validation. It is likely that the instrument performance will vary over time due to small
changes in the various components in the instrument and the validation protocol should be
performed on a periodic basis. The frequency of validation is dependent on the level of
usage of the system and the degree of tolerance that is acceptable for the system.
1.2 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:
•Operational Qualification (OQ) – The purpose of the Operational Qualification is to
demonstrate and document that an analytical system functions according to its
specifications when specific environmental conditions are taken into account. In this
specification, the supplier must define exactly the conditions that must be observed
for the measurement.
•Performance Qualification (PQ) – The purpose of the Performance Qualification is to
demonstrate and document that an analytical system is capable of accurately
measuring the concentration of one or more compounds in a standard sample.
To simplify the overall qualification protocol, the same procedures can be used for both OQ
and PQ, but the tolerances used for Performance Qualification are less restrictive than
those used for Operational Qualification.

Introduction
Page 8 of 60 M1393R0 OQ/PQ Operating Instructions
1.3 General Notes and Recommendations
•After the validation of the different modules, a cytochrome C separation should be
performed according to the LC Packings factory qualification protocol (as described in
the user’s manual).
•For Ultimate Dual Gradient system configurations, the gradient accuracy test
(Section 4.10 ) must be performed on both pumps.
•Channel C & D should only be tested on customer request.
•If the customer ordered a system with two configurations (e.g. a NAN configuration
and a CAP upgrade kit), the OQ/PQ should be performed for the system configuration
that the customer will be using on a routine basis.
•If required, test any other configuration according to the factory qualification protocol
(cytochrome C digest separation).
•After the instrument has been validated via the protocols described in this document,
the analyst should perform a validation of the assay with standards of the compounds
of interest.
1.4 How this Manual is structured
This manual describes the Operation and Performance Qualification for the LC Packings
UltiMate Systems in its different configurations (including the FAMOS Microautosampler
and the Switchos Advanced Microcolumn Switching Unit) and provides the following
information:
Chapter 2: Requirements for a Successful OQ/PQ provides an overview of the parameters
to be tested, a short description of the tests and lists the required acceptance limits for OQ
and the recommended limits for PQ.
Chapter 3: Process lists all required materials, standards and solvents that are necessary
to perform the OQ/PQ test procedures. In addition, it describes how to prepare
CHROMELEON and the UltiMate system to perform the tests.
Chapter 4: Test Procedures provides step-by-step instructions about how to perform and
evaluate the various OQ/PQ tests.
Chapter 5: Troubleshooting discusses how the operator can determine the cause of a
difficulty in the performing of the OQ/PQ.
Chapter 6: CHROMELEON
® Listings provides the listings of all CHROMELEON programs
used to perform the QQ/PQ test procedures on an UltiMate system in NAN configuration.
1.5 For Additional Information
For more detailed information about the operation, maintenance or troubleshooting of the
instruments of the UltiMate system or how to use the CHROMELEON software package,
please refer to the documentation provided with these products and to the online help of
CHROMELEON (F1 key).

OQ/PQ Operating Instructions M1393R0 Page 9 of 60
CHAPTER 2 Requirements for a Successful
OQ/PQ
2.1 Overview
The Operational Qualification (OQ) and Performance Qualification (PQ) procedures are
system-specific procedures. The procedures provided with this document apply for the
components of the LC Packings UltiMate Capillary HPLC system listed in TABLE 2-1:
TABLE 2-1 List of UltiMate System Components with available OQ/PQ Procedures
Instrument Version Option
UltiMate [Plus] Nano- and
Capillary HPLC System (b)
Standard / Inert - Flow Sensor
- Without UV Detector (a)
- Manual Injection Valve
UltiMate [Plus] Dual Gradient
Nano- and Capillary HPLC
System (a) (b)
Standard / Inert - Flow Sensor(s)
FAMOS Well Plate
Microautosampler
Standard / Inert - Sample Cooling
FAMOS Carousel
Microautosampler
Standard / Inert - Sample Cooling
Switchos II Advanced
Microcolumn Switching Unit
Standard / Inert N/A
Notes: (a) If no UV Detector is installed, an extra detector is required to perform the OQ/PQ procedures.
(b) For test procedures regarding the MIC versions of the UltiMate system, contact LCP.
Note: The OQ/PQ procedure are identical for LC Packings UltiMate and UltiMate Plus
Capillary- and Nano HPLC Systems. When the LC Packings UltiMate system is mentioned,
the reader should assume that the material applies to both systems.
Note: If the system does not include an UltiMate UV Detector, a standalone UltiMate UV
Detector is required to perform the OQ/PQ procedures.
The instruments should be controlled by CHROMELEON® 6.6 SP1 or higher (previous
versions will have compatibility problems with the CHROMELEON report file). All necessary
CHROMELEON programs and sequences are provided on the CD ROM ‘IQOQPQ on
UltiMate™(Plus) Systems’ (P/N 163935). If a different software package (e.g. Xcalibur™,
Analyst™, HyStar™, MassLynx™, UltiChrom™, etc.) is used to control the instruments, all
programs will need to be prepared manually. Some limitations may apply due to different
or limited control capabilities of these software packages.

Requirements for a Successful OQ/PQ
Page 10 of 60 M1393R0 OQ/PQ Operating Instructions
2.2 Checks and Acceptance Limits
TABLE 2-2 lists all OQ/PQ test procedures that are to be performed in the order they must
be performed. In most instances, it is necessary that a test is passed before the next test
in the overall is attempted. As an example, if the ‘Linearity of the UV Detector’ test is
failed (Section 4.7), the result of the ‘Gradient Accuracy’ test will be questionable (Section
4.10).
TABLE 2-2 List of OQ and PQ Test Procedures to be performed
Test Procedure Performed Section
Lamp Intensity of the UV Detector Manually 4.2
Wavelength Check CM Sequence 4.3
Flow Cell Check Manually 4.4
UltiMate Fluid Path Test Manually 4.5
Baseline Noise and Drift Test of the UV Detector CM Sequence 4.6
Linearity of the UV Detector CM Sequence 4.7
Reproducibility of the Injection Volume CM Sequence 4.8
Linearity of the injection CM Sequence 4.9
Gradient Accuracy CM Sequence 4.10
Switchos Fluid Path Check Manually 4.11
Switchos Flow Rate and Pressure Stability Test CM Sequence 4.12
Switchos Valve Position Check Manually 4.13
Note: According to GLP, a test procedure that failed needs to be repeated and all test
procedures following the one that failed must be repeated.
TABLE 2-3 presents an overview of the parameters to be tested and a short description of
the tests. In addition, it presents the required acceptance limits for OQ and the
recommended limits for PQ.
TABLE 2-3 Overview of the OQ and PQ Test Procedures and Limits
Limits (a)Instrument Parameter Description
OQ PQ
Wavelength
accuracy
External holmium filter is required. ± 2 nm ± 2 nm
Lamp Intensity
(Section 4.2)
The dummy flow cell is installed
and the lamp intensity is read
from the detector SIGNALS
screen.
0.4 <SIG <0.9
0.1 <REF <0.9
0.4 <SIG <0.9
0.1 <REF <0.9
Flow cell
transmittance
(Section 4.4)
(b)
The flow cell is installed and filled
with water. The transmittance of
this flow cell is indicated by the
‘SIG’ value and is read from the
detector SIGNALS screen.
> 15%
of reference
intensity
> 15%
of reference
intensity
Baseline drift
(Section 4.6)
< 4000 µAU/hr < 4000 µAU/hr
Baseline noise
(Section 4.6)
The drift and noise are recorded
for 21 minutes at 254 nm with a
flow cell (filled with mobile phase
A). < 50 µAU < 50 µAU
UltiMate
UV Detector
(Note: If the
system does
not include an
UltiMate UV
Detector, a
standalone
UltiMate UV
Detector is
required to
perform the
OQ/PQ test
procedures).
Oven test
(Section 4.6)
(d)
An external thermometer is used
to measure the oven temperature.
Accuracy
+/- 1°C
Accuracy
+/- 1°C

Requirements for a Successful OQ/PQ
OQ/PQ Operating Instructions M1393R0 Page 11 of 60
Linearity
(Section 4.7)
(c)
Injections of caffeine standards
covering the linear range of the
UV detector are injected and the
peak height is measured. The
regression coefficient of the
resulting calibration curve
indicates the linearity.
R ≥99.50%
peak height
R ≥99. 0%
peak height
Gradient
accuracy, step
gradient
(Section 4.10)
(c)
A step gradient is performed and
the UV trace recorded. The step
intensity indicates the gradient
accuracy.
Channel A: water
Channel B: water with 0.8 %
acetone (NAN configuration) or
0.3% acetone (CAP
configuration), respectively.
The range is
defined by the
0% and 100%
values.
A relative
deviation of 3%
is allowed for
each step (5%,
50%, 95%).
The range is
defined by the
0% and 100%
values.
A relative
deviation of 3%
is allowed for
each step (5%,
50%, 95%).
UltiMate
System
(Single and
Dual Gradient
Version)
Gradient
reproducibility
(Section 4.10)
(d)
A step gradient is programmed
and measured 3 times.
The reproducibility of the
proportioning is evaluated.
For each step,
the intensity of
the signal is
measured. The
maximum %RSD
allowed is 1%.
For each step,
the intensity of
the signal is
measured. The
maximum %RSD
allowed is 1%.
Reproducibility
of the injection
(Section 4.8)
(c) (d)
8 injections of a caffeine standard
are analyzed. The relative
standard deviation of the peak
height is calculated.
Peak height
RSD ≤1.5%
Peak height
RSD ≤1.5%
FAMOS
(Well Plate /
Carousel
Version) Linearity of the
injection
(Section 4.9)
(d)
Partial loop injections of a
caffeine standard are performed
(from 0.05µl to 0.5µl). The
regression coefficient of the
resulting calibration curve
indicates the linearity.
R ≥99.50%
(calculated via
peak area)
R ≥99.0%
(calculated via
peak area)
Switchos fluid
path test
(Section 4.11)
The flow rate is measured for all
the channels.
The flow rate is
at least
0.15 mL/min.
The flow rate is
at least
0.15 mL/min.
Valve position
test
(Section 4.13)
The position and is switching of
the valves is checked.
LED indication
must correspond
to the open
channel on the
valve.
LED indication
must correspond
to the open
channel on the
valve.
Switchos
Switchos flow
rate and
pressure
stability
(Section 4.12)
(d)
The Switchos pump is
programmed to deliver a flow rate
of 30 µl/min. The output is
connected to one of the valves
with a restriction connected to
port 2. During this program the
valve is switched from position 1-
2 to 10-1. The backpressure must
be stable and reduce to zero
without restriction.
Flow rate must
be 30 ±3 µL/min
Pressure must be
18 ±5 bars
when restriction
is in line and 0
bars otherwise.
Flow rate must
be 30 ±3 µL/min
Pressure must be
18 ±5 bars
when restriction
is in line and 0
bars otherwise.
Notes: a) OQ limits with optimum measuring conditions, recommended PQ limits.
b) The signal intensity should be ≥5% of the reference signal for a 30 mm UV Booster™
flow cell.
c) Up to maximum signal height of CAP = 250 mAU or NAN = 30 mAU.
d) All tests are performed by using the sequences provided with the IQ/OQ/PQ CD ROM.

Requirements for a Successful OQ/PQ
Page 12 of 60 M1393R0 OQ/PQ Operating Instructions
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OQ/PQ Operating Instructions M1393R0 Page 13 of 60
CHAPTER 3 Process
3.1 Required Materials
An OQ/PQ kit (P/N 163936) is available for performing the OQ/PQ on the UtliMate system,
an OQ/PQ kit (P/N 163936) is available. In addition, parts from the standard instruments
accessory kits are required. TABLE 3-1 list all accessories and standards, which are
provided with the OQ/PQ kit:
TABLE 3-1 Parts included in the OQ/PQ Kit
Item Description Part Number Note
UltiMate System - OQ/PQ Operating
Instructions
163960
CD ROM ‘IQOQPQ on UltiMate™ (Plus)
Systems’
163935
Set of 9 samples (flame sealed amber
ampoule)
163932 Do NOT Freeze!
Fused silica tubing I.D. 15 µm ± 3 µm/O.D.
375 µm ± 10 µm, 3,2 meters
163933 Restriction capillary, NAN
configurations only.
Fused silica tubing I.D. 30 µm ± 3 µm/O.D.
375 µm ± 10 µm, 2,5 meters
163934 Restriction capillary, CAP
configurations only.
Tubing set consisting of 130 µm I.D. x
50 cm PEEK tubing
160180
Microtight Union, includes 2 fittings and 1
gauge plug
161497 Used to connect the
restriction capillary.
PEEK sleeves, precision cut and polished for
connections with Microtight Union (380 µm
O.D.), 10 pieces
161405 Used to connect the
restriction capillary.
PEEK sleeves, precision cut and polished for
connections with Microtight Union (280 µm
O.D.), 10 pieces
161498 Used to connect the
restriction capillary.
1/16" Valco Ferrule and Nut, stainless steel,
10 pc. (for 10-port valve)
161103 Used with stainless steel
systems only.
PEEK sleeves, precision cut and polished for
connections with fused silica tubing
(360 µm O.D.), 5 each
160493 Used with stainless steel
systems only.
PEEK 1/16" Universal Fitting for Switchos,
INERT, 10 pc., long hex nut and ferrule with
groove
161007 Used with INERT systems
only.
Syringe adapter 160465 Flow rate (droplet) test.
Phillips-head screw M3 X 4 mm 163964 Used to install the
temperature probe.
Toothed Lock Washer 3,2 mm x 6 mm 163275 Used to install the
temperature probe.

Process
Page 14 of 60 M1393R0 OQ/PQ Operating Instructions
TABLE 3-2 and TABLE 3-3 list accessories and standards that are necessary to perform the
OQ/PQ test procedures (and which are not included in the OQ/PQ kit)
TABLE 3-2 Accessories required for the OQ/PQ
Item Description Part Number (a) Included in the Instrument’s
Accessory Kit
Accessories
UV Detector (b) 160008 or
162346 or
163653
No
Dummy flow cell 162053 Yes
UV flow cell 160015 (NAN)
160013 (CAP)
Yes
Calibrator Cartridge 160061 (NAN)
160059 (CAP)
Yes
Dual Calibrator Cartridge 161082 (NAN/NAN)
161084 (CAP/CAP)
161083 (CAP/NAN)
Yes
P600 Precision Thermometer
or equivalent
163961 No
Special Thermocouple for P600
Precision Thermometer
163962 No
Test Cell, Holmium Oxide Filter
for UltiMate™ UV-Detector
163963 No
Syringe adapter for Valco valve 160259 No
Syringe of 250 µL 163241 No
Connecting tubing 30 µm ID x
15 cm
160182 Yes
Notes (a) P/N applicable depends on the system configuration.
(b) If the system does not include a UV Detector, a standalone instrument is required to
perform the OQ/PQ procedures.
TABLE 3-3 Standards and Solvents for the OQ/PQ
Included in theItem Description P/N
Instrument’s
Accessory
Kit
OQ/PQ Kit
Caffeine in water standards:
0.25, 0.5, 1.0, 2.5, 5.0, 10.0,
20, 40, 60 µg/ml
163932 No Yes
Cytochrome C Digest Test
Sample
161089 Yes No
Acetone (HPLC Grade) N/A No No
Water (HPLC Gradient Grade) N/A No No
Formic Acid (HPLC Grade) N/A No No
Acetonitrile N/A No No

Process
OQ/PQ Operating Instructions M1393R0 Page 15 of 60
3.2 Preparations
The system components that are included in the system configuration have to be prepared
according to the following steps before starting the OQ/PQ procedure.
3.2.1 CHROMELEON® Setup
All CHROMELEON programs required to perform the OQ/PQ test procedures are provided
as a CHROMELEON backup file on the CD ROM ‘IQOQPQ on UltiMate™(Plus) Systems’
(P/N 163935). Two folders are provided, one is for the Nano LC (NAN) and capillary LC
(CAP) configurations. In addition, different CHROMELEON server configuration files are
available.
It is assumed that the service engineer has basic knowledge of the UltiMate system and
CHROMELEON software. Please refer for more detailed information about the installation
and usage of the UltiMate system and CHROMELEON to the manuals provided along with
the products. A detailed description of the tests is provided in Chapter 4.
3.2.1 A Restoring the Backup Files
Refer to the CHROMELEON on-line help (F1 key) for more information on how to restore
backup files.
To prepare the PC and the CHROMELEON software for the OQ/PQ test procedures:
a) Restore the CHROMELEON backup file from the CD ROM. All programs and sequences
are now available for the test procedures (FIGURE 3-1).
FIGURE 3-1 OQ/PQ Programs after Restoring
b) Copy the three server configuration files that are available on the same CD ROM into
the Chromel/Bin directory of the computer.

Process
Page 16 of 60 M1393R0 OQ/PQ Operating Instructions
c) Open the CHROMELEON Server Configuration. Load the server configuration file and
check the COM port settings. Modify the configuration if necessary.
3.2.1 B Modifying Sequences and Programs
It is recommended that you do not modify the sequences provided on the CD ROM
‘IQOQPQ on UltiMate™(Plus) Systems’ (P/N 163935) unless it is absolutely necessary.
Changing them may have an impact on the links with the report. However, it may be
necessary to make modifications to correspond to the available hardware. If changes are
made, take care that the sample names and numbers are not modified.
3.2.1.B.1 Choosing to perform OQ or PQ
The sequences required to perform the OQ/PQ on a Nano LC system have the extension
‘_NAN’ in their names (e.g. XQ_6_Gradient Formation Test_NAN.seq). Sequences for a
capillary LC system have the extension ‘_CAP’ (FIGURE 3-2).
FIGURE 3-2 Modifying the Sequence Names
All sequence names start with the prefix ‘XQ_’. Depending on the test that is to be
performed, change ‘XQ’ to ‘OQ’ or ‘PQ’ (FIGURE 3-2). This will select the right limits for
the report file. If you do not modify the name, the PQ limits will be selected.
3.2.1.B.2 Flow Sensor Support and CRP Value
All sequences are prepared to support the flow sensor option. If the system to be checked
does not include the flow sensor, all relevant programs must be adjusted:
To change the programs for a using a fixed CRP value, identify the ‘CRP’ or ‘CalibrateCRP’
command in the program and remove the semicolon from the ‘CRP’ line and place it on the
‘CalibrateCRP’ line as presented below:
•Flow Sensor Support:
;CRP = 625
CalibrateCRP When=BeforeFirstSample
•Using a fixed CRP Value:
CRP = 625
;CalibrateCRP When=BeforeFirstSample
3.2.1.B.3 FAMOS Cooling Option
All sequences are prepared to support the cooling option of the FAMOS. If the system to
be checked does not include the cooling option, all relevant programs must be adjusted:
To change the programs for instruments for which do not have this option installed,
identify the ‘Sampler_TempCtrl’ command and in the programs and add a semicolon to this
line and the 3 following lines as presented below:

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OQ/PQ Operating Instructions M1393R0 Page 17 of 60
•FAMOS with Cooling Option:
;Commands for FAMOS with cooling option
Sampler.TempCtrl = On
Sampler.Temperature.Nominal = 20.00
Sampler.Temperature.LowerLimit = 5.00
Sampler.Temperature.UpperLimit = 30.00
•FAMOS without Cooling Option:
;Commands for FAMOS with cooling option
;Sampler.TempCtrl = On
;Sampler.Temperature.Nominal = 20.00
;Sampler.Temperature.LowerLimit = 5.00
;Sampler.Temperature.UpperLimit = 30.00
3.2.1.B.4 Oven Support
During the warm up sequence of the UV detector noise and drift test (Section 4.6) the
oven accuracy is checked. If the current configuration does not include an oven, a different
program must be used:
Depending on the test configuration (e.g. the Switchos is included or not),
•If the system includes an oven, run the ‘warm up and oven test.pgm’ (FIGURE 3-3).
•If the system does not include an oven, run the ‘warm up.pgm’ (FIGURE 3-3).
FIGURE 3-3 Selecting the proper Warm-up Program
3.2.1.B.5 Miscellaneous
The sequences for the test 4.10 and 4.12 include a stop flow method which reduces the
time that the lamp is lit and minimizes solvent consumption. Select the status of these
stop flow samples to ‘single’ (activated) or ‘interrupted’ (deactivated) to correspond to the
test configuration (e.g. the Switchos is included or not).
3.2.1 C Report File
No changes should be made in the report file itself (PQ_OQ_LCP.rdf), except for the
spreadsheets ‘Specification’ and ‘Others tests’. These two spreadsheets allow entering of
some test details (i.e. the serial numbers of the instruments) and the results of tests that
were carried out manually (i.e. 4.2, 4.4, 4.5, 4.11, 4.13).

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3.2.2 System Setup
The OQ/PQ procedure is similar for the different system configurations (e.g. NAN or CAP
configuration). However, some parts are different and depend on the system configuration
being tested (e.g. column, calibrator, connecting tubing, UV flow cell). Make sure that the
system is configured properly. Please refer to the UltiMate user’s manual for more
information.
In addition, several test parameters used with the tests are different for the individual
system configurations (e.g. flow rate, CRP value and injection volume). Refer to Section
3.2.1 and Chapter 6 for more details about how to setup CHROMELEON.
Note: Make certain that enough COM ports are available. For the Dual Gradient system
with flow sensors, the FAMOS and the Switchos 9 COM ports are required (including the
one required for the precision thermometer).
3.2.2 A UltiMate System
To prepare the UltiMate system for the OQ/PQ test procedures 4.4 to 4.10:
a) Switch on the UV detector at least 1 hour before starting the tests to warm-up the
instrument.
b) Configure the system as required for the application (e.g. install the NAN calibrator
cartridge and NAN connection tubing for a proper NAN setup).
Note: If the system does not include a UV Detector, a standalone instrument is required to
perform the OQ/PQ procedures.
c) Connect the appropriate restriction tubing to the port 6 of injection valve of the FAMOS
Microautosampler (‘column’ port). Use P/N 163933 for the NAN configuration and P/N
163934 for the CAP configuration.
d) Use the Microtight union and appropriate PEEK sleeves to connect the restriction tubing
(360 µm O.D.) to the flow cell inlet (280 µm O.D.). Use P/N 161405 for the 360 µm
tubing and P/N 161498 for the 280 µm tubing (e.g. for the flow cell).
e) Prepare the solvents as presented in TABLE 3-4, and start helium sparging to degas the
solvents. Thoroughly purge all channels.
TABLE 3-4 Required Solvents
Channel Solvent
A 100% Water + 0.1% FA
B 99% Water + 0.1% FA + 0.8% acetone (NAN)
99.5% Water + 0.1% FA + 0.3% acetone (CAP)
C 100% ACN + 0.1% FA (a)
D 100% Water + 0.1% FA
Note a) The acetonitrile is used to perform a wash step during the FAMOS test procedure 4.8.
CAUTION Caution: Older revisions of the UltiMate system may be equipped with the
older type of the C and D solenoid valves (P/N160051). Due to their
limited resistance against strong organic solvents, do not expose these
valves to acetonitrile for a longer period than required to perform the wash
cycle of test 4.8 (e.g. not longer than 4 h).

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OQ/PQ Operating Instructions M1393R0 Page 19 of 60
It is recommended that you perform the wash step separately and to use channel A or B
on such systems. Alternatively, the system can be upgraded with the new valve type (P/N
162297). Refer to Service Information #036 for more details.
If an oven is included in the UltiMate system, install the temperature probe (P/N 163962 )
in the oven compartment using the supplied screw and washer (FIGURE 3-4) and connect
the thermometer to a free COM port of the computer.
Built-in
Temperature
Sensor
Temperature
Probe
FIGURE 3-4 Installing the Temperature Probe
Note: Make certain that the low on battery indicator on the thermometer is not lit, because
a weak battery may interrupt the sequence if serial communication is broken.

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Page 20 of 60 M1393R0 OQ/PQ Operating Instructions
3.2.2 B FAMOS Microautosampler
To prepare the FAMOS Microautosampler for the OQ/PQ tests:
a) Position the caffeine standards of the OQ/PQ kit in the FAMOS sample rack as
presented in TABLE 3-5.
TABLE 3-5 Sample Positions in the Autosampler Rack
Tray Position Sample
A2 0.25 µg/ml Caffeine (CAP system only)
A3 0.5 µg/ml Caffeine (CAP system only)
A4 1.0 µg/ml Caffeine
A5 2.5 µg/ml Caffeine
A6 5.0 µg/ml Caffeine
A7 10.0 µg/ml Caffeine
A8 20.0 µg/ml Caffeine
B1 40.0 µg/ml Caffeine (NAN system only)
B2 60.0 µg/ml Caffeine (NAN system only)
b) Use mobile phase A as the wash solvent (TABLE 3-4).
c) Degas the wash solvent. Helium sparging is strongly recommended.
d) Check that there is no air in the syringe and run a wash cycle on the instrument.
3.2.2 C Switchos Advanced Microcolumn Switching Unit
To prepare the Switchos Advanced Microcolumn Switching Unit for the OQ/PQ:
a) Prepare the Switchos with the following mobile phases
Solvent A: 0.1 % TFA in water
Solvent B: 0.1 % TFA in water
Solvent C: 0.1 % TFA in water
Solvent D: 0.1 % TFA in water.
b) Connect a trap-column connecting tubing 30 µm ID x 15 cm (P/N 160182) to port 2 of
switching valve A.
c) Start helium sparging to degas the solvents.
d) Thoroughly purge all channels as described in the user’s manual of the instrument.