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Thermo Scientific UltiMate Installation Qualification/Operational Qualification Protocols And Instructions

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

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  • Page 1 UltiMate ™ Capillary- and Nano HPLC Systems Operational Qualification Performance Qualification Operating Instructions P/N 163960 www.lcpackings.com M1393R0...
  • Page 2 (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.

  • Page 3: Warnings

    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: The information in a danger statement relates to a procedure, practice condition or DANGER action that if not done correctly or adhered to could lead to personal injury or loss of life.
  • Page 4 The operator should follow all safety precautions, warnings, etc provided with the instruments, in addition, please note the items presented below: All components of the system should be plugged into a common power line that is directly connected to a true ground. Repair or replace faulty power cords and all communication cables.

  • Page 5: Table Of Contents

    Flow Cell Check ................26 4.4.1 Performing the Intensity Test of the Flow Cell ....... 26 UltiMate Fluid Path Test ..............27 4.5.1 Performing the Test ..............27 Baseline Noise and Drift Test of the UV Detector and Oven Test..28 4.6.1 Drift and Noise................
  • Page 6 Table of Contents 4.6.4 Performing the UV Detector Drift and Noise Test and the Oven Test . 30 Linearity of the UV Detector............31 4.7.1 Performing the UV Detector Linearity Test ........32 Reproducibility of the Injection Volume ..........33 4.8.1 Performing the Test ..............

  • Page 7: Introduction

    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.

  • Page 8: General Notes And Recommendations

    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. For Additional Information For more detailed information about the operation, maintenance or troubleshooting of the...

  • Page 9: Requirements For A Successful Oq/Pq

    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.

  • Page 10: Checks And Acceptance Limits

    Lamp Intensity of the UV Detector CM Sequence Wavelength Check Manually Flow Cell Check Manually UltiMate Fluid Path Test CM Sequence Baseline Noise and Drift Test of the UV Detector CM Sequence Linearity of the UV Detector CM Sequence Reproducibility of the Injection Volume...
  • Page 11 UV detector are injected and the peak height is measured. The regression coefficient of the resulting calibration curve indicates the linearity. The range is UltiMate Gradient A step gradient is performed and The range is System accuracy, step the UV trace recorded. The step...
  • Page 12 Requirements for a Successful OQ/PQ [This page intentionally left blank] Page 12 of 60 OQ/PQ Operating Instructions M1393R0...

  • Page 13: Process

    Parts included in the OQ/PQ Kit Item Description Part Number Note UltiMate System - OQ/PQ Operating 163960 Instructions CD ROM ‘IQOQPQ on UltiMate™ (Plus) 163935 Systems’ Set of 9 samples (flame sealed amber 163932 Do NOT Freeze! ampoule) Fused silica tubing I.D. 15 µm ± 3 µm/O.D.
  • Page 14 P600 Precision Thermometer 163961 or equivalent Special Thermocouple for P600 163962 Precision Thermometer Test Cell, Holmium Oxide Filter 163963 for UltiMate™ UV-Detector Syringe adapter for Valco valve 160259 Syringe of 250 µL 163241 Connecting tubing 30 µm ID x 160182 15 cm Notes P/N applicable depends on the system configuration.

  • Page 15: Preparations

    (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.
  • Page 16 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.
  • Page 17 Process • FAMOS with Cooling Option: ;Commands for FAMOS with cooling option Sampler.TempCtrl = 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 = ;Sampler.Temperature.Nominal = 20.00 ;Sampler.Temperature.LowerLimit = 5.00 ;Sampler.Temperature.UpperLimit = 30.00...

  • Page 18: System Setup

    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.
  • Page 19 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.
  • Page 20 Process 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 0.25 µg/ml Caffeine...

  • Page 21: Performing The Checks

    (d) Includes a wash cycle of the FAMOS injection valve. (e) If included in the configuration. TABLE 3-6 indicates the tests for the OQ and PQ procedure for different UltiMate system configurations. The time required to complete a test procedure is also indicated. The total time required for checking an entire system (e.g.

  • Page 22: Evaluating The Tests

    According to GLP, a test which failed and all tests following the one which failed need to be repeated. Exception: there is no need to repeat UltiMate and FAMOS related tests if a Switchos test fails. The reason is that almost all checks require that the previous check be passed successfully.

  • Page 23: Test Procedures

    (again manually). From this point on the UltiMate and FAMOS the tests procedures 4.6 to 4.10 are automated. These automated tests are performed with the same system setup (Section 3.2.2 A).

  • Page 24: Lamp Intensity Of The Uv Detector

    If the lamp intensity is below the acceptance criteria, baseline instability problems may occur. For more information refer to the UltiMate service manual. The absolute intensity of the lamp is not measured in this test, and deviations from lamp to lamp and from detector to detector are quite normal.

  • Page 25: Wavelength Check

    Test Procedures Wavelength Check The wavelength accuracy is measured by evaluating local maxima of absorbance on a holmium filter in the UV range. These results are compared with the ones provided on the certificate of the material. 4.3.1 Performing the Test a) Install the holmium filter (FIGURE 4-2) in the UV detector.

  • Page 26: Flow Cell Check

    Test Procedures Flow Cell Check The transmittance of the UV flow cell is important for proper detection sensitivity and baseline stability. The transmittance of the flow cell is read from the SIGNAL screen of the Setup menu of the UV detector (FIGURE 4-3). SIGNAL: sig: 0.1795 ♦...

  • Page 27: Ultimate Fluid Path Test

    The UltiMate fluid path test is used to check the solenoids and the solvent delivery. 4.5.1 Performing the Test a) Fill each solvent bottle until the fluid level corresponds to the top of the UltiMate housing with the solvents specified in TABLE 3-4. b) Degas all solvents properly and purge all solvent channels.

  • Page 28: Baseline Noise And Drift Test Of The Uv Detector And Oven Test

    Note: This test must also be performed for any UV Detector that used for the OQ/PQ, even if it’s not part of the UltiMate system. Drift and baseline noise are important parameter for the UltiMate UV Detector. Increased baseline noise reduces the sensitivity considerably, as it is not possible to distinguish between low-level signals and noise.

  • Page 29: Theory Of The Noise And Drift Calculation Of Chromeleon

    4.6.3 Oven accuracy The temperature in the Ultimate oven is monitored by the P600 Precision Thermometer (P/N 163961). The readout of the built-in sensor is compared with the read-out of the thermometer.

  • Page 30: Performing The Uv Detector Drift And Noise Test And The Oven Test

    Section 3.2.2 A. c) Set the pump to the appropriate flow rate. Use 0.4 µL/min for a NAN version and 4 µL/min for CAP configuration and start the flow delivery of the UltiMate Micropump from the CHROMELEON control panel.

  • Page 31: Linearity Of The Uv Detector

    An example of an injection (0.2 µL) of a caffeine standard of 20 µg/mL onto a Nano UltiMate system is shown in FIGURE 4-7. FIGURE 4-7 UV Trace of the Injection of Caffeine onto a Nano UltiMate System An example of a calibration curve is shown in FIGURE 4-8. FIGURE 4-8 Example of a Calibration Curve - Nano LC system, 99.93 % Correlation Coefficient...

  • Page 32: Performing The Uv Detector Linearity Test

    Setup the UltiMate system and the FAMOS Microautosampler as discussed in Sections 3.2.2 A and 3.2.2 B. b) Start the flow delivery of the UltiMate Micropump from the CHROMELEON control panel. c) After equilibration, start the sequence ‘XQ_3_UV_Linearity_NAN’ (or ‘_CAP’, respectively).

  • Page 33: Reproducibility Of The Injection Volume

    Setup the UltiMate system and the FAMOS Microautosampler as discussed in Sections 3.2.2 A and 3.2.2 B. b) Start the flow delivery of the UltiMate Micropump from the CHROMELEON control panel. c) After equilibration start the sequence ‘XQ_4_Autosampler Reproducibility_NAN’ (or ‘_CAP’, respectively).

  • Page 34: Linearity Of The Injection

    Setup the UltiMate system and the FAMOS Microautosampler as discussed in Sections 3.2.2 A and 3.2.2 B. b) Start the flow delivery of the UltiMate Micropump from the CHROMELEN control panel. c) After equilibration start the sequence ‘XQ_5_Autosampler Linearity_NAN’ (or ‘_CAP’, respectively).

  • Page 35: Gradient Accuracy

    Test Procedures 4.10 Gradient Accuracy The gradient accuracy is tested by performing a step gradient. Water with a trace of acetone is mixed with pure water. Detection of the acetone is performed at 254 nm. Different gradient programs are used for the NAN and the CAP configurations (the NAN program lasts 80 minutes while the CAP program is 50 minutes).
  • Page 36 Test Procedures The programs used for the gradient accuracy tests are listed in TABLE 4-4 and TABLE 4-5. TABLE 4-4 Program for the Gradient Accuracy Test on a NAN Configuration Time Wavelength [min] [nm] 0.00 10.00 “ 10.01 “ 20.00 “...

  • Page 37: 4.10.1 Performing The Test

    Verify that the results meet the acceptance criteria (TABLE 2-3). f) Repeat steps (a) and (h) for the Gradient Pump 2 of the UltiMate Dual Gradient system (if applicable).

  • Page 38: Switchos Fluid Path Check

    Test Procedures 4.11 Switchos Fluid Path Check The Switchos path test is to check if the solvent selection valve close and open properly and if the resistance of the flow path is within the specifications. 4.11.1 Performing the test To determine if all flow paths are within the specifications: a) Prepare the Switchos with the mobile phases listed in Section 4.12.1 and degas them properly.

  • Page 39: Switchos Flow Rate And Pressure Stability Test

    Test Procedures 4.12 Switchos Flow Rate and Pressure Stability Test The Switchos is used for pre-concentration experiments. A stable loading flow is essential for the sample transport to the trap column. The flow rate and pressure stability are checked with a CHROMELEON program. A typical pressure profile for this test is presented in FIGURE 4-13.

  • Page 40: Switchos Valve Position Check

    Test Procedures 4.13 Switchos Valve Position Check The proper functioning of the switching valves is checked in local control mode. The local/remote switch can be found on the back panel of the Switchos. 4.13.1 Performing the Test a) Put the Switchos in LOCAL mode. b) Switch valve A and valve B from position ‘1-2’...

  • Page 41: Completing And Printing The Oq/Pq Report

    Test Procedures 4.14 Completing and Printing the OQ/PQ Report 4.14.1 Result of the Gradient Accuracy Test The observed delay time may be slightly different than the expected delay time indicated in (TABLE 4-7) because of small differences in the tubing size due to the manufacturing process.

  • Page 42: 4.14.2 Completing The Cover Page And Printing

    Test Procedures FIGURE 4-15 The Properties Report Column Box f) Check that the segment that is used for calculation is in the middle of the step. g) Modify the values in the ‘Formula’ field (FIGURE 4-15). Each segment used for the calculation is defined by a start and a stop time (e.g.

  • Page 43: Troubleshooting

    (1:1). If crystalline materials are deposited in the pump head, irreversible damage to seals and or the piston may result; this will dramatically shorten the life of these components. Caution: Older revisions of the UltiMate system may be equipped with the older type of the CAUTION C and D solenoid valves (P/N160051).

  • Page 44: Probable Causes And Solutions

    See above (4.2). See above (4.2). • • (4.4) low. Dirty flow cell Clean/replace. • • UltiMate Fluid Path Test Flow rate too low. Solvent filter Check/replace. • • (4.5) clogged. Baseline Noise and Drift Drift too high. Old lamp.

  • Page 45: Chromeleon ® Listings

    FIGURE 6-1 Available CHROMELEON Sequences for OQ/PQ The following sections lists the CHROMELEON programs used to run a UltiMate system in NAN configuration with a flow sensor installed in conjunction with a FAMOS Well Plate Microautosampler (cooling option installed) and a Switchos Advanced Microcolumn Switching Unit.

  • Page 46: Wavelength Check (Nan Configuration)

    CHROMELEON Listings Wavelength Check (NAN Configuration) Sequence: XQ_1_Wavelength Check_NAN ;Program file for wavelength check ;System must be setup for Nano LC experiments ;Program version 08/10/2004 Sampler.TempCtrl = Oven.TempCtrl = pump.%A.Equate = "100% water + 0.1% FA" %B.Equate = "Mob. phase A + 0.8% acetone" %C.Equate = "%C"...

  • Page 47: Noise And Drift Test (Nan Configuration)

    30.00 ;Virtual channel settings pump_pressure.Formula Formula=masterpressure.value pump_pressure.Type = Analog column_pressure.Formula Formula=pump.columnpressure.value column_pressure.Type = Analog ;Ultimate pump settings Pump.%A.Equate = "100% water + 0.1% FA" Pump.%B.Equate = "Mob. phase A + 0.8% acetone" Pump.%C.Equate = "%C" Pump.%D.Equate = "%D" Pump.Flow =...
  • Page 48 CHROMELEON Listings UV_VIS_1.AcqOn pump_pressure.AcqOn column_pressure.AcqOn 21.000 UV_VIS_1.AcqOff pump_pressure.AcqOff column_pressure.AcqOff Page 48 of 60 OQ/PQ Operating Instructions M1393R0...

  • Page 49: Uv Linearity (Nan Configuration)

    ;Virtual channel settings pump_pressure.Formula Formula=masterpressure.value pump_pressure.Type = Analog column_pressure.Formula Formula=pump.columnpressure.value column_pressure.Type = Analog ;Ultimate pump settings Pump.%A.Equate = "100% water + 0.1% FA" Pump.%B.Equate = "Mob. phase A + 0.8% acetone" Pump.%C.Equate = "%C" OQ/PQ Operating Instructions Page 49 of 60...
  • Page 50 CHROMELEON Listings Pump.%D.Equate = "%D" Pump.Flow = %B = %C = %D = 0.000 UV.Autozero Wait Sampler.Ready and UV.Ready and Pump.Ready and pump_flowsensor.ready Inject UV_VIS_1.AcqOn pump_pressure.AcqOn column_pressure.AcqOn 8.00 UV_VIS_1.AcqOff pump_pressure.AcqOff column_pressure.AcqOff Page 50 of 60 OQ/PQ Operating Instructions M1393R0...

  • Page 51: Autosampler Reproducibility (Nan Configuration)

    ;Sampler.Temperature.Nominal = 20.00 ;Sampler.Temperature.LowerLimit = 5.00 ;Sampler.Temperature.UpperLimit = 30.00 ;Virtual channel settings pump_pressure.Formula Formula=masterpressure.value pump_pressure.Type = Analog column_pressure.Formula Formula=pump.columnpressure.value column_pressure.Type = Analog ;Ultimate pump settings Pump.%A.Equate = "100% water + 0.1% FA" OQ/PQ Operating Instructions Page 51 of 60 M1393R0...
  • Page 52 CHROMELEON Listings Pump.%B.Equate = "Mob. phase A + 0.8% acetone" Pump.%C.Equate = "%C" Pump.%D.Equate = "%D" Pump.Flow = %B = %C = %D = 0.000 UV.Autozero Wait Sampler.Ready and UV.Ready and Pump.Ready and pump_flowsensor.ready Inject UV_VIS_1.AcqOn Pump_pressure.AcqOn Column_pressure.AcqOn 8.00 UV_VIS_1.AcqOff Pump_pressure.AcqOff Column_pressure.AcqOff Page 52 of 60...

  • Page 53: Autosampler Injection Linearity (Nan Configuration)

    30.00 ;Virtual channel settings pump_pressure.Formula Formula=masterpressure.value pump_pressure.Type = Analog column_pressure.Formula Formula=pump.columnpressure.value column_pressure.Type = Analog ;Ultimate pump settings Pump.%A.Equate = "100% water + 0.1% FA" Pump.%B.Equate = "Mob. phase A + 0.8% acetone" OQ/PQ Operating Instructions Page 53 of 60 M1393R0...
  • Page 54 CHROMELEON Listings Pump.%C.Equate = "%C" Pump.%D.Equate = "%D" Pump.Flow = %B = %C = %D = 0.000 UV.Autozero Wait Sampler.Ready and UV.Ready and Pump.Ready Inject UV_VIS_1.AcqOn pump_pressure.AcqOn column_pressure.AcqOn 10.000 UV_VIS_1.AcqOff pump_pressure.AcqOff column_pressure.AcqOff Page 54 of 60 OQ/PQ Operating Instructions M1393R0...

  • Page 55: Gradient Accuracy Test (Nan Configuration)

    ;Virtual channel settings pump_pressure.Formula Formula=masterpressure.value pump_pressure.Type = Analog column_pressure.Formula Formula=pump.columnpressure.value column_pressure.Type = Analog ;Ultimate pump settings pump.%A.Equate = "100% water + 0.1% FA" %B.Equate = "Mob. phase A + 0.8% acetone" %C.Equate = "%C" OQ/PQ Operating Instructions Page 55 of 60...
  • Page 56 CHROMELEON Listings %D.Equate = "%D" 0.000 UV.Autozero pump.Flow = 0.400 %B = %C = %D = Wait UV.Ready and Pump.Ready and Pump_FlowSensor.Ready and Sampler.Ready Sampler.Inject UV_VIS_1.AcqOn Pump_pressure.AcqOn Column_pressure.AcqOn pump.Flow = 0.400 %B = %C = %D = 7.000 %B = 7.010 %B = 30.000...

  • Page 57: Switchos Pump Test (Nan Configuration)

    CHROMELEON Listings Switchos Pump Test (NAN Configuration) Sequence: XQ_7_Switchos Pump Test_NAN Program: ;Program file for switchos pump stability test ;Program version 08/10/2004 ;Settings for restrictor (320cm, 15um ID) Diameter = 75um Length = 15cm StationaryPhase = C18_3um_100A ;CRP = CalibrateCRP When=BeforeFirstSample ;ParkPercentage = Disabled...
  • Page 58 CHROMELEON Listings ;Switchos pump settings Loading_Pump.%A.Equate = "Pure water + 0.1% FA" 0.000 Loading_Pump.Flow = 0.030 Wait Loading_Pump.Ready inject Trap_Column_Pressure.AcqOn Loading_Pump.Flow = 0.030 0.000 Valve_A.Position = 5.000 Valve_A.Position = 10_1 10.000 Valve_A.Position = 15.000 Trap_Column_Pressure.AcqOff Loading_Pump.Flow = 0.030 Page 58 of 60 OQ/PQ Operating Instructions M1393R0...

  • Page 59: Stop Flow

    ;Sampler.Temperature.Nominal = 20.00 ;Sampler.Temperature.LowerLimit = 5.00 ;Sampler.Temperature.UpperLimit = 30.00 ;Virtual channel settings pump_pressure.Formula Formula=masterpressure.value pump_pressure.Type = Analog column_pressure.Formula Formula=pump.columnpressure.value column_pressure.Type = Analog ;loading pump settings TrapColumnPressure.LowerLimit = TrapColumnPressure.UpperLimit = 400.0 ;Ultimate pump settings OQ/PQ Operating Instructions Page 59 of 60 M1393R0...
  • Page 60 CHROMELEON Listings pump.%A.Equate = "100% water + 0.1% FA" pump.%B.Equate = "Mob. phase A + 0.8% acetone" pump.%C.Equate = "%C" pump.%D.Equate = "%D" 0.000 UV.Autozero %B = %C = %D = Wait UV.Ready and Pump.Ready and Sampler.Ready Inject pump.Flow = %B = %C = %D =...

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