Thermo Scientific UltiMate Installation Qualification/Operational Qualification Protocols And Instructions Download Page 1

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M1393R0

UltiMate

™

Capillary- and Nano HPLC Systems

Operational Qualification

and

Performance Qualification

Operating Instructions

P/N 163960

www.lcpackings.com

Summary of Contents for UltiMate

Page 1: ...M1393R0 UltiMate Capillary and Nano HPLC Systems Operational Qualification and Performance Qualification Operating Instructions P N 163960 www lcpackings com ...

Page 2: ...erved 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 D...

Page 3: ... 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 relate...

Page 4: ...perating 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 p...

Page 5: ...ations 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 2...

Page 6: ...rforming 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 O...

Page 7: ...n 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 def...

Page 8: ...ms 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...

Page 9: ...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 system...

Page 10: ...led 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 P...

Page 11: ...ak 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 th...

Page 12: ...Requirements for a Successful OQ PQ Page 12 of 60 M1393R0 OQ PQ Operating Instructions This page intentionally left blank ...

Page 13: ...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...

Page 14: ...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 re...

Page 15: ...vice 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 informat...

Page 16: ...ix 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 adj...

Page 17: ...ion 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 consumpt...

Page 18: ...AN 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 t...

Page 19: ...tails 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 b...

Page 20: ...0 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 followi...

Page 21: ...tchos Tests if applicable 4 11 Switchos Fluid Path Check 15 min 4 12 Switchos Flow Rate and Pressure Stability Test 15 min 4 13 Switchos Valve Position Check 10 min Notes a Additional UV detector required b The Gradient Accuracy Test must be performed for both gradient pumps Depending on the configuration an additional CAP flow is required c No FAMOS Microautosampler and no Switchos Unit included ...

Page 22: ...m tests 4 3 4 6 4 7 4 8 4 9 4 10 and 4 11 the pump and trap column pressure data is saved These traces can be opened and reviewed from the CHROMELEON program For the tests 4 7 4 8 and 4 9 it is essential to check the integration carefully Manual integration of peaks may be required 3 4 1 Repeating Tests It may be necessary to repeat one or several tests In this case refer to Chapter 5 Troubleshoot...

Page 23: ... 11 4 13 will not be performed The tests procedures 4 6 to 4 10 are using the same system setup as described in Section 3 2 2 A Testing of the UltiMate system and the FAMOS Microautosampler starts with a manual lamp check followed by the automated wavelength check After these tests are passed the condition of the flow cell and the fluid path are checked again manually From this point on the UltiMa...

Page 24: ...ensity Test The lamp intensity values are presented in the SIGNAL screen of the SETUP menu To check the lamp intensity a Set the wavelength to 240 nm b Install the dummy cell and access the SIGNAL screen on the SETUP menu FIGURE 4 1 UV Detector SIGNAL Screen c Monitor the signal value sig and the reference value ref Both the signal and the reference values should be in the limits defined in TABLE ...

Page 25: ...nd pull the filter holder item 2 FIGURE 4 2 out of the light path just pull it out by 5 mm 0 2 the filter holder remains in the filter body c Perform an autozero run Either use the F8 key of CHROMELEON or press the Autozero button of the UV detector d Place the filter back in its original position all the way in the filter body tighten the screw e Start the sequence XQ_1_Wavelength Check_NAN or _C...

Page 26: ...Detector SIGNAL Screen 4 4 1 Performing the Intensity Test of the Flow Cell To test the flow cell a Make certain that the flow cell to be tested is clean and flushed properly with mobile phase A b Set the wavelength to 240 nm c Access the SIGNAL screen on the SETUP menu d Monitor the Reference signal ref and the Sample signal sig e Compare the result with the signal intensity limits presented TABL...

Page 27: ...head and connect the 250 µl syringe P N 163241 the plunger should be removed using an adapter P N 160259 as presented in FIGURE 4 4 FIGURE 4 4 Placing the Syringe on the Solvent Inlet Line d Enter the purge screen of the micro pump set the flow rate to 0 0 ml min and select channel A The LED of channel A should be illuminated e Measure the flow rate for one minute The flow rate should be greater t...

Page 28: ...a considerable increase in noise if an old lamp with poor light intensity is used or if the flow cell is dirty To minimize any effect from the flow cell make sure that the measuring and ambient conditions are constant and that are no gas bubbles in the flow cell In addition it is very important that a new lamp has been burning for several hours In the detector environment avoid drafts and direct s...

Page 29: ... the regression line all data points are weighted with their corresponding step unless the step is equidistant Adding both values supplies the noise value The noise intensity read in the report is the average noise calculated using 20 segments Drift To compute the drift a regression line is drawn through all data points The slope of the regression line is the calculated drift Therefore to compute ...

Page 30: ...the system includes a flow sensor perform a manual calibration of the CRP value at this point e g use the F8 key and execute the CalibrateCRP command from the UltiMate_System Pump Pump_FlowSensor menu The CRP value should be close to 625 for NAN system or 50 for a CAP system respectively The CRP value should not vary by more than 10 e After equilibration start the sequence XQ_2_UV_Noise and Drift ...

Page 31: ...esented in TABLE 3 5 The resulting peak height is used for the calculation 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 Correlat...

Page 32: ...tem configurations TABLE 4 1 Experimental Conditions UV Detector Linearity Parameter Nano Configuration Capillary Configuration Detection wavelength 272 nm 272 nm Run time 8 min 4 min Flow rate 0 4 µL min 4 0 µL min CRP 625 50 Injection loop 1 0 µL 1 0 µL Injection type Partial Loop Fill 0 2 µL Partial Loop Fill 0 5 µL To perform the UV Detector linearity test a Setup the UltiMate system and the F...

Page 33: ...pump from the CHROMELEON control panel c After equilibration start the sequence XQ_4_Autosampler Reproducibility_NAN or _CAP respectively TABLE 4 2 Experimental Conditions Repro Injection Volume Parameter Nano Configuration Capillary Configuration Detection wavelength 272 nm 272 nm Run time 8 min 4 min Flow rate 0 4 µL min 4 0 µL min CRP 625 50 Injection loop 1 0 µL 1 0 µL Injection type Partial L...

Page 34: ...low delivery of the UltiMate Micropump from the CHROMELEN control panel c After equilibration start the sequence XQ_5_Autosampler Linearity_NAN or _CAP respectively TABLE 4 3 Experimental Conditions Repro Injection Volume Parameter Nano Configuration Capillary Configuration Detection wavelength 272 nm 272 nm Run time 8 min 4 min Flow rate 0 4 µL min 4 0 µL min CRP 625 50 Injection loop 1 0 µL 1 0 ...

Page 35: ...50 minutes Typical examples of the step gradient profiles are presented in FIGURE 4 10 NAN configuration and in FIGURE 4 11 CAP configuration Legend Step a 0 5 B step b 0 50 B step c 0 100 B step d 95 100 B step e step change time c d e a b FIGURE 4 10 Typical Step Gradient Profile NAN Configuration FIGURE 4 11 Typical Step Gradient Profile CAP Configuration The steps items a d FIGURE 4 10 are qua...

Page 36: ...velength nm 0 00 100 0 254 5 00 100 0 5 01 95 5 10 00 95 5 10 01 50 50 15 00 50 50 15 01 5 95 20 00 5 95 20 01 0 100 25 00 0 100 25 01 100 0 50 00 100 0 Experimental conditions for the step gradient test for nano and capillary LC systems are listed in TABLE 4 6 TABLE 4 6 Experimental Conditions for the Step Gradient Test Parameter Nano Configuration Capillary Configuration Solvents A HPLC Water B ...

Page 37: ...el B from the CHROMELEON panel and monitor the baseline until it is stable d After equilibration start the sequence XQ_6_ Gradient Formation Test_NAN or _CAP respectively Note If the maximum absorbance observed during this test is greater than the highest absorption monitored during the linearity test 4 7 dilute B or D with mobile phase A so that the signal remains on scale when this test is run e...

Page 38: ...r channels b Disconnect the solvent inlet line from the pump head and connect the 250 µl syringe P N 163241 the plunger should be removed using an adapter P N 160259 as presented in FIGURE 4 12 FIGURE 4 12 Placing the Syringe on the Solvent Inlet Line c Set the flow rate to 0 00 mL min and set the Switchos in LOCAL mode d Select solvent channel A by the SSV button on the rear The LED A should be i...

Page 39: ...s the solvent b The four channels should be well flushed using the Purge function of the pump c Connect a 30 µm I D tubing P N 160182 to port 2 of the Switchos valve A d Connect the Switchos output to the port 1 of the Switchos valve A using a 130 µm I D tubing P N 160180 e Connect port 10 to waste using the same type of tubing f Start the Switchos pump from the panel g After equilibration start t...

Page 40: ...n 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 to position 10 1 using the manual control button on the front panel c Connect a syringe adapter P N 160259 to port 1 of valve A d Flush with a 250 µl syringe water through port 1 into port 10 e Verify that the valve position corresponds to the LED ...

Page 41: ...t is not within the specifications only TABLE 4 7 Predefined Step Delay Time Step NAN CAP 5 17 0 18 0 min 8 0 9 0 min 50 27 0 28 0 min 13 0 14 0 min 95 37 0 38 0 min 18 0 19 0 min 100 47 0 48 0 min 23 0 24 0 min To change the delay time a Open the UV trace obtained for the third step gradient out of 3 b Measure the retention times corresponding to the middle of 5 50 95 and 100 steps c Select the r...

Page 42: ...for the report can not be completed automatically by CHROMELEON and require manual input e g customer information S N of the FAMOS Microautosampler To complete the Basic Tests page of the report a Open the report PQ_OQ_LCP rdf b Choose the Specifications sheet c Select the Instruments section and enter the missing information e g S N of the FAMOS flow cell thermometer etc d Select the Additional I...

Page 43: ...ination may also seriously damage the flow cell e g coating of the capillary walls After you have finished using the system flush the system with a water methanol or water acetonitrile mobile phase before shutting it down The solvents must be degassed via the He degassing technique described before If other techniques are used e g vacuum degassing the performance of the system will be seriously de...

Page 44: ... UV Detector 4 7 Signal is out of range Acetone concentration too high Dilute with mobile phase A Ghost peak before the caffeine peak Contaminated injection valve Extend wash step Sonicate rotor and stator in CAN for 10 min Caffeine peak shows some spikes improper integration Air in the syringe Check syringe speed and time constant Chapter 6 Reproducibility of the Injection Volume 4 8 Linearity of...

Page 45: ... UltiMate system configurations Please refer to Section 3 2 1 for modifications which may be necessary due to a different hardware configuration 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...

Page 46: ...terPressure LowerLimit 0 0 MasterPressure UpperLimit 400 0 ColumnPressure LowerLimit 0 0 ColumnPressure UpperLimit 200 0 TrapColumnPressure LowerLimit 0 0 TrapColumnPressure UpperLimit 200 0 InjectMode FullLoop LowDispersionMode Off UseAirSegment Off UseHeadSpace Off SyringeSpeed Low SyringeSpeedFactor 0 1 SampleHeight 4 FlushVolume 5 0 WashVolume 50 RinseBetweenReinjections Yes Data_Collection_Ra...

Page 47: ... 0 TrapColumnPressure LowerLimit 0 0 TrapColumnPressure UpperLimit 200 0 Settings for UV detector Data_Collection_Rate 2 UV_VIS_1 Wavelength 254 TimeConstant 2 0 FAMOS settings Wait Sampler Ready InjectMode Fullloop Commands for FAMOS with cooling option Sampler TempCtrl On Sampler Temperature Nominal 20 00 Sampler Temperature LowerLimit 5 00 Sampler Temperature UpperLimit 30 00 Virtual channel se...

Page 48: ...CHROMELEON Listings Page 48 of 60 M1393R0 OQ PQ Operating Instructions UV_VIS_1 AcqOn pump_pressure AcqOn column_pressure AcqOn 21 000 UV_VIS_1 AcqOff pump_pressure AcqOff column_pressure AcqOff End ...

Page 49: ... 0 TrapColumnPressure UpperLimit 200 0 Settings for UV detector Data_Collection_Rate 2 UV_VIS_1 Wavelength 272 TimeConstant 2 FAMOS settings Wait Sampler Ready InjectMode Partial LowDispersionMode Off UseAirSegment Off UseHeadSpace Off RinseBetweenReinjections Yes SyringeSpeed Low SyringeSpeedFactor 0 1 SampleHeight 9 FlushVolume 5 0 WashVolume 50 Commands for FAMOS with cooling option Sampler Tem...

Page 50: ...ons Pump D Equate D Pump Flow 0 4 B 0 C 0 D 0 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 End ...

Page 51: ... 0 0 Pump columnPressure UpperLimit 200 0 TrapColumnPressure LowerLimit 0 0 TrapColumnPressure UpperLimit 200 0 Settings for UV detector Data_Collection_Rate 2 UV_VIS_1 Wavelength 272 TimeConstant 0 5 FAMOS settings Wait Sampler Ready InjectMode Partial LowDispersionMode Off UseAirSegment Off UseHeadSpace Off RinseBetweenReinjections Yes SyringeSpeed Low SyringeSpeedFactor 0 1 SampleHeight 9 Flush...

Page 52: ... A 0 8 acetone Pump C Equate C Pump D Equate D Pump Flow 0 4 B 0 C 0 D 0 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 End ...

Page 53: ...e UpperLimit 200 0 TrapColumnPressure LowerLimit 0 0 TrapColumnPressure UpperLimit 200 0 Settings for UV detector Data_Collection_Rate 2 UV_VIS_1 Wavelength 272 TimeConstant 0 5 FAMOS settings Wait Sampler Ready InjectMode Partial LowDispersionMode Off UseAirSegment Off UseHeadSpace Off RinseBetweenReinjections Yes SyringeSpeed Low SyringeSpeedFactor 0 1 SampleHeight 9 FlushVolume 5 0 WashVolume 5...

Page 54: ...uctions Pump C Equate C Pump D Equate D Pump Flow 0 4 B 0 C 0 D 0 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 End ...

Page 55: ...UpperLimit 200 0 TrapColumnPressure LowerLimit 0 0 TrapColumnPressure UpperLimit 200 0 Settings for UV detector Data_Collection_Rate 2 UV_VIS_1 Wavelength 254 TimeConstant 0 5 FAMOS settings InjectMode Fullloop LowDispersionMode Off UseAirSegment Off UseHeadSpace Off RinseBetweenReinjections Yes SyringeSpeed Low SyringeSpeedFactor 0 1 SampleHeight 4 FlushVolume 5 0 WashVolume 50 Commands for FAMOS...

Page 56: ...400 B 100 C 0 D 0 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 100 C 0 D 0 7 000 B 100 7 010 B 0 30 000 UV_VIS_1 AcqOff Pump_pressure AcqOff Column_pressure AcqOff pump Flow 0 400 B 0 C 0 D 0 End ...

Page 57: ...tor Data_Collection_Rate 2 UV_VIS_1 Wavelength 254 TimeConstant 2 FAMOS settings InjectMode Fullloop LowDispersionMode Off UseAirSegment Off UseHeadSpace Off RinseBetweenReinjections Yes SyringeSpeed Low SyringeSpeedFactor 0 1 SampleHeight 4 FlushVolume 5 0 WashVolume 50 Commands for FAMOS with cooling option Sampler TempCtrl On Sampler Temperature Nominal 20 00 Sampler Temperature LowerLimit 5 00...

Page 58: ...oading_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 1_2 5 000 Valve_A Position 10_1 10 000 Valve_A Position 1_2 15 000 Trap_Column_Pressure AcqOff Loading_Pump Flow 0 030 End ...

Page 59: ...rapColumnPressure LowerLimit 0 0 TrapColumnPressure UpperLimit 200 0 Settings for UV detector Data_Collection_Rate 2 UV_VIS_1 Wavelength 254 TimeConstant 2 FAMOS settings InjectMode Fullloop LowDispersionMode Off UseAirSegment Off UseHeadSpace Off RinseBetweenReinjections Yes SyringeSpeed Low SyringeSpeedFactor 0 1 SampleHeight 4 FlushVolume 5 0 WashVolume 50 Commands for FAMOS with cooling option...

Page 60: ...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 0 C 0 D 0 Wait UV Ready and Pump Ready and Sampler Ready Inject pump Flow 0 4 B 0 C 0 D 0 4 000 pump Flow 0 000 Loading_Pump Flow 0 000 B 0 C 0 D 0 5 000 Lamp Off End ...

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