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UltiMate - OQ/PQ Operating Instructions

1. e Pa FIGURE 4 2 The Holmium Filter for the UV Detector OQ PQ Operating Instructions M1393RO Page 25 of 60 Test Procedures 4 4 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 absorbtion ref 0 3429 FIGURE 4 3 UV 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 TABLE 2 3 and fill in the appropriate field in the Other Tests page of the QO PQ report Page 26 of 60 M1393RO OQ PQ Operating Instructions Test Procedures 4 5 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 c gt Degas all solvents properly and purge all solvent channels Disconnect the solvent inlet line
2. OQ PQ Operating Instructions M1393RO Page 17 of 60 Process CAUTION 3 2 2 System Setup The OQ PO 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 injec
3. 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 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 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 OQ PQ Operating Instructions M1393RO Page 43 of 60 Troubleshooting 5 3 Probable Causes and Solutions Test Procedure Problem No enough COM ports Probable Cause Solution Control Switchos All available valves by event outputs i Id Replace Lamp Intensity of the UV Intensity values too e Old lamp e Replace low e Wrong wavelength e Check change Detector 4 2 e Integration time too e Check adjust low Wavelength Check 4 3 Failed Filter not in the optical path Check filter position Flow Cell Check Intensity values too See above 4 2 See above 4 2 4 4 low e Dirty flow cell e Clean replace UltiMate Fluid Path Te
4. d height is calculated Version Linearity of the Partial loop injections of a R gt 99 50 R gt 99 0 injection caffeine standard are performed calculated via calculated via Section 4 9 from 0 05ul to 0 5ul The peak area peak area d regression coefficient of the resulting calibration curve indicates the linearity Switchos Switchos fluid The flow rate is measured for all The flow rate is The flow rate is path test the channels at least at least Section 4 11 0 15 mL min 0 15 mL min Valve position The position and is switching of LED indication LED indication test the valves is checked must correspond must correspond Section 4 13 to the open to the open channel on the channel on the valve valve Switchos flow The Switchos pump is Flow rate must Flow rate must rate and programmed to deliver a flow rate be 30 3 uL min be 30 3 uL min pressure of 30 ul min The output is Pressure must be Pressure must be stability connected to one of the valves 18 5 bars 18 5 bars Section 4 12 with a restriction connected to when restriction when restriction d port 2 During this program the is in line and O is in line and O valve is switched from position 1 bars otherwise bars otherwise 2 to 10 1 The backpressure must be stable and reduce to zero without restriction Notes a OQ limits with optimum measuring conditions recommended PQ limits b The signal intensity should be gt 5 of the reference signal for a 3
5. 3 Wavelength Cheek eee ee ce viet ties kee ee eee ot 25 4 3 1 Performing the TeSt aiserpana esaeen Wap Save ete ee ake be Poe 25 4 4 Flow Cell Chek sess c0se ceee seed eedsieececsevel eek cee Ei ara riua ae aarp ti i EEEn 26 4 4 1 Performing the Intensity Test of the Flow Cell cccceceeeeeeeeneneees 26 4 5 UltiMate Fluid Path Test isinin eer a e i a e 27 4 5 1 Performing the TeSt s aoaaaansnnnnnnnnnnnnnnnnnnnnnnnnnrnnnnrrnnnnnnnnnnnrnnnnns 27 4 6 Baseline Noise and Drift Test of the UV Detector and Oven Test 28 4 6 1 Drift and NOS Crirxi eee iin OA Ai ee EAE 28 4 6 2 Theory of the Noise and Drift Calculation of CHROMELEON 29 40 3 OVEN atura CY cniin ea E AE alee Pas cha REE RE REE TAREA AD AEEA 29 OQ PQ Operating Instructions M1393RO Page 5 of 60 Table of Contents 4 6 4 Performing the UV Detector Drift and Noise Test and the Oven Test 30 4 7 Linearity of the UV DeteCtor cccccccceeee eee eeeeee eee eeeeae eae eaeeaeeaneneeaes 31 4 7 1 Performing the UV Detector Linearity T St ccececeeeneneeneeeeenees 32 4 8 Reproducibility of the Injection Volume 2 cceeeeeeeee eens eaeeeeeeaees 33 4 8 1 Performing the TeSt nriran eann arein iaa i teen eee e E o iaa 33 4 9 Linearity Of the injection sssssssressrrsssrrssrrrerrrrsrrnrrrrnrrrurrernerereerene 34 4 9 1 Performing the Test n ren seet iee niir A EAE ia 34 4 10 Gradient ACCUraACY sereine eari e E EE EEEa aa en Tea 35 4 10 1
6. Formula masterpressure value pump _pressure Type Analog column_pressure Formula Formula pump columnpressure value column_pressure Type Analog loading pump settings TrapColumnPressure LowerLimit 0 0 TrapColumnPressure UpperLimit 400 0 Ultimate pump settings OQ PQ Operating Instructions M1393RO Page 59 of 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 0 C 0 D 0 Wait U Inject pump Flow B C D V Ready and Pump Ready and Sampler Ready 4 oo0oo0oo 4 000 pump Flow 0 000 Loading_Pump Flow 0 000 B 0 C 0 D 0 5 000 Lamp Off End Page 60 of 60 M1393RO OQ PQ Operating Instructions
7. OQ PQ Linearity Injections of caffeine standards R gt 99 50 R gt 99 0 Section 4 7 covering the linear range of the peak height peak height c UV detector are injected and the peak height is measured The regression coefficient of the resulting calibration curve indicates the linearity UltiMate Gradient A step gradient is performed and The range is The range is System accuracy step the UV trace recorded The step defined by the defined by the gradient intensity indicates the gradient 0 and 100 0 and 100 Single and Section 4 10 accuracy values values Dual Gradient c Channel A water A relative A relative Version Channel B water with 0 8 deviation of 3 deviation of 3 acetone NAN configuration or is allowed for is allowed for 0 3 acetone CAP each step 5 each step 5 configuration respectively 50 95 50 95 Gradient A step gradient is programmed For each step For each step reproducibility and measured 3 times the intensity of the intensity of Section 4 10 The reproducibility of the the signal is the signal is d proportioning is evaluated measured The measured The maximum RSD maximum RSD allowed is 1 allowed is 1 FAMOS Reproducibility 8 injections of a caffeine standard Peak height Peak height of the injection are analyzed The relative RSD lt 1 5 RSD lt 1 5 Well Plate Section 4 8 standard deviation of the peak Carousel c
8. 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 Name Title Timebase fay XQ 1_Wavelength Check_CAP_seq ULT_FMS 2 UV Noise and Drift Oven Test_CAP_ seq ULT_FMS fal XQ_3 _UV Linearity _CAP seq ULT_FMS tf XQ_4 Autosampler Reproducibility CAP seq ULT_FMS fal XQ_5 Autosampler Linearity _CAP seq ULT_FMS ta XQ Gradient Formation Test_CAP seq ULT_FMS Eh PQ OQ_LCP rdf 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
9. 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 e Flow Sensor Support CRP 625 CalibrateCRP When BeforeFirstSample e 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 Page 16 of 60 M1393RO OQ PQ Operating Instructions Process e 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 e 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 c
10. e CAP configuration 0 25 0 5 1 0 2 0 5 0 10 0 and 20 0 pg mL e NAN configuration 1 0 2 0 5 0 10 0 20 0 40 0 and 60 0 g mL Install the samples in their proper positions in the FAMOS autosampler rack TABLE 3 5 TABLE 2 1 presents the conditions for the linearity test for the two different system 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 uL min 4 0 L min CRP 625 50 Injection loop 1 0 uL 1 0 uL Injection type Partial Loop Fill 0 2 uL Partial Loop Fill 0 5 uL To perform the UV Detector linearity test a 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 To evaluate the result of the UV Detector linearity Test a Use the quantization file caffeine qnt b Check the integration and correct if necessary c Verify that the correlation coefficient meets the acceptance criteria TABLE 2 3 Page 32 of 60 M1393RO OQ PQ Operating Instructions Test Procedures 4 8 Reproducibility of the Injection Volume For the injection reproducibility 8 consecutive partial loop injections of
11. pump_pressure AcqOff column_pressure AcqOff End M1393RO OQ PQ Operating Instructions 6 4 CHROMELEON Listings UV Linearity NAN Configuration Sequence XQ_3_UV Linearity NAN Wait Program file for UV linearity test Sampler Ready System must be setup for Nano LC experiments Program version 08 10 2004 Settings for restrictor 320cm 15um ID Diameter 75um Length 15cm StationaryPhase C18 3um_100A CRP 625 CalibrateCRP When BeforeFirstSample ParkPercentage Disabled Pump limits settings MasterPressure LowerLimit 0 0 MasterPressure UpperLimit 400 0 Pump columnPressure LowerLimit 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 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 TempCtrl On 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 colum
12. restriction capillary PEEK sleeves precision cut and polished for 161405 Used to connect the connections with Microtight Union 380 wm restriction capillary O D 10 pieces PEEK sleeves precision cut and polished for 161498 Used to connect the connections with Microtight Union 280 um restriction capillary O D 10 pieces 1 16 Valco Ferrule and Nut stainless steel 161103 Used with stainless steel 10 pc for 10 port valve systems only PEEK sleeves precision cut and polished for 160493 Used with stainless steel connections with fused silica tubing systems only 360 um O D 5 each PEEK 1 16 Universal Fitting for Switchos 161007 Used with INERT systems INERT 10 pc long hex nut and ferrule with only groove Syringe adapter 160465 Flow rate droplet test Phillips head screw M3 X 4mm 163964 Used to install the temperature probe Toothed Lock Washer 3 2 mm x 6 mm 163275 Used to install the M1393RO temperature probe Page 13 of 60 Process 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 No 162346 or 163653 Dummy flow cell 162053 Yes UV flow cell 160015 NAN Yes 16
13. seek medical attention 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 Wear protective eye goggles when handling fused silica tubing i e installation cutting etc 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 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 Page 4 of 60 M1393RO OQ PQ Operating Instructions Table of Contents Warming S cerar e ea ding ee e aia segue i EIRE ie cape cen Sae a A oa EAE a raah 3 Safety Precautions aa a wince a ade atten A a Ea Ea E EEEE 3 CHAPTER 1 INTPOMUCTION sniene uneren a E nE E AE R E EEE ENE ENM ee 7 1 1 The Purpose of QQ PO emro reir oa a a a a ai 7 1 2 Defining the Limits cccceeecee riain a a a E a a EE 7 1 3 General Notes and Recommendations cceeee
14. teens teens ee eeeeeeaene es 8 1 4 How this Manual iS structured ccccecceeeeceee eee eeeeeeeeeeaeeeeeeneaeeaeeeeas 8 1 5 For Additional Information ccccccceeceeeeeeeee eee eeeeeaeeeeeeeeeeeaeaneaeeneens 8 CHAPTER 2 Requirements for a Successful OO PO cccccceceeeeeeeeeeeeeees 9 2 1 OVGIVIOW E i gicesh T A steed ie tued couse task ova A EAT 9 2 2 Checks and Acceptance Limits ceeeeeee eee eee ea eee eaeeeeaeaeeeeaeas 10 CHAPTER 3 Processed va esccatshvwles Seguicias Vadavel a a e i 13 3 1 Required Materials ccceceeeeeee eee ee eee eeee eee ee eee eaeeeeaea eee eaeaenaeaeaes 13 3 2 Preparations cs icce vis cgevaves sive yeas stdves bstedecckt os voedee sd yesbe T 15 3 2 1 CHROMELEON Setup uu cccccccccccccceccecccecntecusecetssenssesesssenssensnesnsnss 15 32 2 System SCtUD sh iia eee A ieee ee re ti eee es 18 3 3 Performing the CheckS ccccceeee eee ee eee ee ee ee teen eee ease eee ea ene eae teenage 21 3 4 Evaluating the TeSts cececceeecee eee ee eee neta eee a eda ene naeaeeeeaeaes 22 3 4 1 Repeating Tests cstes begets bevesceeaede ta sincint tabs AAA a Raa Aaii 22 CHAPTER 4 Test Procedures marren iaaa EKE EEEE EEE N ENEE 23 4 1 OVeTVIEW oraraa ea a A duel a a Ar a Ar a ales E aaa 23 4 2 Lamp Intensity of the UV Detector ssssssersssrrrssrrerrrssrrrnsrrrnsrrennrs 24 4 2 1 Performing the Lamp Intensity T St ccccicsscencenecnceneenecnesaeenennenates 24 4
15. the QO PQ report re Note If the values are not within the specifications install a new lamp and perform the test again If the values are still not within the specifications check adjust the Integration Time setting Re 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 Page 24 of 60 M1393RO OQ PQ Operating Instructions Test Procedures 4 3 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 Install the holmium filter FIGURE 4 2 in the UV detector Loosen the knurled screw item 1 FIGURE 4 2 half a turn and 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 Perform an autozero run Either use the F8 key of CHROMELEON or press the Autozero button of the UV detector a b c d Place the filter back in its original position all the way in the filter body tighten the screw Start the sequence XQ_1_Wavelength Check_NAN or _CAP respectively f Check the results in the CHROMELEON report Verify that the reference values correspond to the values listed in the certificate provided with the holmium filter
16. 0 04 a a j p 25 0 T T T T T 00 25 50 75 100 125 150 175 200 25 min T T T T 250 275 30 0 325 35 0 375 400 FIGURE 4 6 Typical Result of the Oven Test OQ PQ Operating Instructions M1393RO Page 29 of 60 Test Procedures 4 6 4 Performing the UV Detector Drift and Noise Test and the Oven Test To perform the Drift and Noise Test Install the flow cell and let the baseline stabilize a b Install the appropriate connection tubing between the Ultimate and the FAMOS injection valve and connect the restriction capillary that is provided to the outlet of the flow cell as described in Section 3 2 2 A Set the pump to the appropriate flow rate Use 0 4 uL min for a NAN version and 4 uL min for CAP configuration and start the flow delivery of the UltiMate Micropump from the CHROMELEON control panel If 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 c d e After equilibration start the sequence XQ _ 2 UV_ Noise and Drift Oven Test NAN or CAP respectively f Make certain to run the appropriate Warm up program before Section 3 2 1 B 3 g Verify that all p
17. 0 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 I1Q OQ PQ CD ROM OQ PQ Operating Instructions M1393RO Page 11 of 60 Requirements for a Successful OO PO This page intentionally left blank Page 12 of 60 M1393RO OQ PQ Operating Instructions CHAPTER 3 Process OQ PQ Operating Instructions 3 1 Required Materials An OQ PQ kit P N 163936 is available for performing the OQ PQ on the UtliMate system an OQ PO 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 PO Kit Item Description Part Number Note UltiMate System OQ PQ Operating 163960 Instructions CD ROM IQOQPO on UltiMate Plus 163935 Systems Set of 9 samples flame sealed amber 163932 Do NOT Freeze ampoule Fused silica tubing I D 15 wm 3 ym O D 163933 Restriction capillary NAN 375 um 10 um 3 2 meters configurations only Fused silica tubing D 30 wm 3 um O D 163934 Restriction capillary CAP 375 um 10 um 2 5 meters configurations only Tubing set consisting of 130 wm I D x 160180 50 cm PEEK tubing Microtight Union includes 2 fittings and 1 161497 Used to connect the gauge plug
18. 0013 CAP Calibrator Cartridge 160061 NAN Yes 160059 CAP Dual Calibrator Cartridge 161082 NAN NAN Yes 161084 CAP CAP 161083 CAP NAN P600 Precision Thermometer 163961 No or equivalent Special Thermocouple for P600 163962 No Precision Thermometer Test Cell Holmium Oxide Filter 163963 No for UltiMate UV Detector Syringe adapter for Valco valve 160259 No Syringe of 250 uL 163241 No Connecting tubing 30 um ID x 160182 Yes 15 cm 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 Item Description P N Included in the Instrument s Accessory OQ PO Kit Kit Caffeine in water standards 163932 No Yes 0 25 0 5 1 0 2 5 5 0 10 0 20 40 60 ug ml Cytochrome C Digest Test 161089 Yes No Sample 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 Page 14 of 60 M1393RO OQ PQ Operating Instructions Process 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 backu
19. 004 Settings for restrictor 320cm 15um ID Diameter 75um Length 15cm StationaryPhase C18 3um_100A CRP 625 CalibrateCRP When BeforeFirstSample ParkPercentage Disabled Pump limits settings MasterPressure LowerLimit 0 0 MasterPressure UpperLimit 400 0 Pump columnPressure LowerLimit 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 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 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 0 4 B 0 C 0 D 0 0 000 Wait UV Ready and Pump Ready and Sampler Ready and pump_flowsensor ready Inject OQ PQ Operating Instructions M1393RO Page 47 of 60 CHROMELEON Listings 21 000 Page 48 of 60 UV_VIS_1 AcqOn pump_pressure AcqOn column_pressure AcqOn UV_VIS_1 AcqOff
20. 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 Off Oven TempCtrl Off pump A Equate 100 water 0 1 FA B Equate Mob phase A 0 8 acetone C Equate C D Equate D Diameter 75um Length 15cm StationaryPhase C18 3um_100A No automatic CRP calibration MasterPressure 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 Rate 2 pump Flow 0 000 B 0 C 0 D 0 Settings for UV detector Data_Collection Rate 2 UV_VIS_1 Wavelength 254 TimeConstant 2 0 Wait UV Ready and Pump Ready and Pump_FlowSensor Ready and Sampler Ready Inject Wavelength Check UV_VIS_1 AcqOn UV_VIS_1 AcqOff End M1393RO OQ PQ Operating Instructions CHROMELEON Listings 6 3 Noise and Drift Test NAN Configuration Sequence XQ 2 UV Noise and Drift Oven Test NAN Wait Sampler Ready Program file for drift and noise test System must be setup for Nano LC experiments Program version 08 10 2
21. 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 Once all UltiMate system and FAMOS tests are performed the Switchos unit will be tested if included in the configuration OQ PQ Operating Instructions M1393RO Page 23 of 60 Test Procedures 4 2 Lamp Intensity of the UV Detector The intensity of the UV lamp must be sufficient for correct detection sensitivity and baseline stability 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 The basic function of this test is to ensure that the output of the lamp is within an acceptable range 4 2 1 Performing the Lamp Intensity 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 SIGNAL sig 0 3485 absorbtion ref 0 1729 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 2 3 d Fill in the appropriate field in the Other Tests page of
22. IS_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 50 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 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 M1393RO Page 51 of 60 CHROMELEON Listings 0 000 8 00 Page 52 of 60 Pump B Equate Pump C Equate Pump D Equate Pump Flow B C D UV Autozero Wait Inject UV_VIS_1 AcqOn Pump_pressure AcqOn Column_pressure AcqOn UV_VIS_1 AcqOff Pump_pressure AcqOff Column_pressure AcqOff End M1393RO Mob phase A 0 8 acetone C D 0 4 0 0 0 Sampler Ready and UV Ready and Pump Ready and pump_flowsensor ready OQ PQ Operating Instructions CHROMELEON Listings 6 6 Autosampler Injection Linearity NAN Configuration Sequence XQ 5 Autosampler Linearity NAN Wait Sampler Ready Program file for injection lineari
23. LT_FMS pan 12 Oct 04 13 56 07 15 KB a XQ_7_Switchos Pump Test_NAN ULT_FMS_CAP v1 a XQ_1_Wavelength Check_CAP XQ_2_UV Noise and Drift Oven Test_CAP a XQ_3_UV Linearity_CAP a XQ_4_Autosampler Reproducibiity_CAP XQ_5_Autosampler Linearity_CAP a XQ_6_Gradient Formation Test_CAP ULT_FMS_NAN v1 XQ_1_Wavelength Check_NAN XQ_2 UV Noise and Drift Oven Test_NAN a XQ_3_UV Linearity_NAN XQ_4_Autosampler Reproducibiity_NAN a XQ_5_Autosampler Linearity_NAN a XQ_6_Gradient Formation Test_NAN ULT_FMS_SW_CAP v1 a XQ_1_Wavelength Check_CAP XQ_2_UV Noise and Drift Oven test_CAP XQ_3_UV Linearity_CAP a XQ_4_Autosampler Reproducibiity_CAP a XQ_5_Autosampler Linearity_CAP XQ_6_Gradient Formation Test_CAP fa XQ_7_Switchos Pump test_CAP ULT_FMS_SW_NAN v1 XQ_1_Wavelength Check_NAN a XQ_2 UV Noise and Drift Oven Test_NAN a XQ_3_UV Linearity_NAN a XQ_4_Autosampler Reproducibiity_NAN a XQ_5_Autosampler Linearity_NAN fa XQ_6_Gradient Formation Test_NAN XQ_7_Switchos Pump Test_NAN 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 OQ PQ Operating Instructions M1393RO Page 45 of 60 CHROMELEON Listings 6 2 0 000 1 00 Page 46 of 60 Wavelength
24. OMELEON calculates the results of the Noise and Drift test as follows e Signal Noise All data points recorded during a 60 seconds segment of a chromatogram form the basis for determining the noise value CHROMELEON calculates a regression line using the method of least squares then determines the maximum distance of two data points above and below the line When calculating 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 e 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 the drift always select a baseline range in which no peaks occur 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 Chromeleon Guillaume Laptop iQO0QPQ_UltiMate full test Test 06_ Ult_Fam_Cap draft 9 oQ_2_UV Noise and drift_CAP 1 system warm up and oven te Ta Fle edt vew Workspace Qualification window Hep 2x Ose 8s R id DEKE TOR Bim FRR lt 1 1 00 2 UV Noise and drift CAP 1 os AAE Rete ORRAG System warm up and oven test 60 0 i serere ee 45 04 i l 35 04 3
25. PLC System b Without UV Detector a Manual Injection Valve UltiMate Plus Dual Gradient Standard Inert Flow Sensor s Nano and Capillary HPLC System a b FAMOS Well Plate Standard Inert Sample Cooling Microautosampler FAMOS Carousel Standard Inert Sample Cooling Microautosampler Switchos Il Advanced Standard Inert N A Microcolumn Switching Unit 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 eS 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 me 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 du
26. Performing the TeSt ds enpa a a Ee E R 37 4 11 Switchos Fluid Path Check ssssssssssssssssssenrrnsrnsrrnrisrrrsrrerrerrrrrrenrent 38 4 11 1 Performing th t St apsiitirriiiti eena ae ratat ni ire 38 4 12 Switchos Flow Rate and Pressure Stability TeSt ccccceeeeeeneene eee 39 412 1 Performing th testy 0 2o oe teed eid Sede Bd ee a end eva EEEE 39 4 13 Switchos Valve Position Check cccceeeeeeeee estes ee ee eee eaeaeee teens taeas 40 4 13 1 Performing the TCSt 1cccceccccnceene ence ence e ene eee e neta nee e eden dent need anaes 40 4 14 Completing and Printing the OQ PO Report ceceeeeeee teens neta ees 41 4 14 1 Result of the Gradient Accuracy TOSt cciccccssccnecnteneenenateateneenenates 41 4 14 2 Completing the Cover Page and Printing 1 2 1 1ccccececeneneeneneeeeneeenenees 42 CHAPTER 5 TroubleShOoting ccccee nipa paea eae ene ete nate eae 43 5 1 OVGEIVIOW EEEE E cee i vedere stcattbew deed vole T E 43 5 2 Sample and Mobile Phase Considerations ccccceeeeeeeneeeeeneeaeeaeees 43 5 3 Probable Causes and Solutions ccceceeeeeee eee ee eee e tena eee naeaeeeeaees 44 CHAPTER 6 CHROMELEON LISTINGS fers noes arenneren AE ENN toad ieee ni aban ts 45 6 1 OVERVIEW erra e AALE E A AE A E ESRAS 45 6 2 Wavelength Check NAN Configuration essssssseesererrrrerrrrerreen 46 6 3 Noise and Drift Test NAN Configuration ssseeseeeeseeeereeeere ennn 47 6 4 UV Line
27. The step delay item e FIGURE 4 10 represents response time of the gradient formation and indicates that the volume of the tubings are correct OQ PQ Operating Instructions M1393RO Page 35 of 60 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 A B Wavelength min nm 0 00 100 0 254 10 00 100 0 10 01 95 5 20 00 95 5 20 01 50 50 x 30 00 50 50 30 01 5 95 40 00 5 95 i 40 01 0 100 H 50 00 0 100 K 50 01 100 0 j 80 00 100 0 i TABLE 4 5 Program for the Gradient Accuracy Test on a CAP Configuration Time A B Wavelength min nm 0 00 100 0 254 5 00 100 0 A 5 01 95 5 i 10 00 95 5 10 01 50 50 F 15 00 50 50 ji 15 01 5 95 20 00 5 95 f 20 01 0 100 25 00 0 100 4 25 01 100 0 7 50 00 100 0 g 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 HPLC Water with Acetone C 100 ACN 0 1 FA a D 100 Water 0 1 FA Acetone concentration of 0 8 0 3 Solvent B Detection wavelength 254 nm 254 nm Run time 80 min 50 min Flow ra
28. UltiMate Capillary and Nano HPLC Systems Operational Qualification and Performance Qualification Now sold under the Thermo Scientific brand Thermo SCIENTIFIC Operating Instructions P N 163960 LC PACKINGS www lcpackings com M1393RO 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 uCarrier UltiMate UltiChrom FAMOS Thermos Switchos Acurate UZ View UV Booster FluoBoost Pepmap u Dumper y Fluidics Fusica Nano Series Pico Series u Guard y Precolumn Nano Precolumn Dionex is a Trademark of Dionex Corporation CHROMELEON i
29. a standard solution of caffeine 20 0 ug ml for NAN and 5 0 ug ml for CAP are performed The peak area is used for the calculation of the injection reproducibility 4 8 1 Performing the Test a 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 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 uL min 4 0 uL min CRP 625 50 Injection loop 1 0 uL 1 0 uL Injection type Partial Loop Fill 0 2 uL Partial Loop Fill 0 5 uL To evaluate the result of the Reproducibility of the Injection Volumes Test a Use the quantization file caffeine qnt b Check the integration and correct if necessary c Calculate the correlation coefficient d Verify that the correlation coefficient meet the acceptance criteria TABLE 2 3 OQ PQ Operating Instructions M1393RO Page 33 of 60 Test Procedures 4 9 Linearity of the injection The injection linearity is measured by injecting different amounts of the same caffeine standard The resulting peak area is used for the calculations An example of a calibration curve i
30. about instrument specific troubleshooting e g what do if an instrument performs not within its specifications is provided in the documentation shipped with the instruments and in the service manuals available for the instruments 5 2 Sample and Mobile Phase Considerations To optimize performance of the system we recommend that all samples and mobile phases are free of particulate matter Samples and mobile phases should be filtered through a 0 22 um membrane filter The filter should be checked to ensure that extractable materials are not present cauTioN Caution It is strongly recommend that only bottled HPLC grade water and HPLC grade CAUTION solvent be used for these tests If water from water purification systems is used polymeric contamination may seriously damage the flow cell This is especially true if sample pre concentration or 2D separations are performed This polymeric contamination 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 degraded and the performance specifications will not be obtained Note The pump head of the Micropump should be backflushed with iso propanol water
31. 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 sunlight The detector baseline noise and drift test is performed at the following conditions e Flow cell installed and filled with mobile phase A e Al 254 nm A2 A3 A4 O nm e Time Constant 2 sec e Data Acquisition Rate 1 Hz e Data Acquisition Length 20 min FIGURE 4 5 shows a typical result from the noise and drift test fF Chromeleon Guillaume Laptop IQ0QPQ_UltiMate full tost Test 06_ Ult_Fam_Cap draft 9 0Q_2_UV Noite and drift_CAP 2 Drift and noise test UV_VIS Eg n Fle Edt vew Workspace Qualification Window Help 3x OSU S Lo R KHER DO PRA DEER TMB Mi mH aoe 0 100 4 102 2 UV Noise and drift_ CAP 2 Drift_and noise test uv visi mAU WVL 254 nm 0 000 MAN ham 0 100 ha mo a Ww WA Wy 0 200 v oat Winn pi vas y a te 0 300 wM WMA V 0 4004 a eo ov Varia 0 500 Win M WAN Eua 0 800 os 0 700 f T T T T T T T T T m 0 0 20 40 60 8 0 10 0 12 0 14 0 16 0 18 0 210 11 0 min 0 18 mAU Zoom Gulbume FIGURE 4 5 Typical UV Detector Noise and Drift Result 254 nm Page 28 of 60 M1393RO OQ PQ Operating Instructions Test Procedures 4 6 2 Theory of the Noise and Drift Calculation of CHROMELEON CHR
32. arameters meet the acceptance criteria TABLE 2 3 Discard data if appropriate Section 4 14 es Note When a new lamp is installed optimum performance is obtained after the lamp has been used for approximately 24 hours If this test performed on a new lamp the noise and drift levels will be larger than the specified values Page 30 of 60 M1393RO OQ PQ Operating Instructions Test Procedures 4 7 Linearity of the UV Detector The detector linearity is measured by injecting the different caffeine standards of the OQ PQ kit P N 16395 as presented in TABLE 3 5 The resulting peak height is used for the calculation An example of an injection 0 2 uL of a caffeine standard of 20 wg mL onto a Nano UltiMate system is shown in FIGURE 4 7 OTST a ae eee oe 100 200 00 400 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 Extemai 400r Heigmt mau EEE FIGURE 4 8 Example of a Calibration Curve Nano LC system 99 93 Correlation Coefficient OQ PQ Operating Instructions M1393RO Page 31 of 60 Test Procedures 4 7 1 Performing the UV Detector Linearity Test Depending on the system configuration the UV Detector linearity NAN or UV Detector linearity CAP sequence is used to perform the detector linearity test The detector linearity is determined at 272 nm using caffeine standard samples with different concentrations
33. arity NAN Configuration ssssesssssssessrrrsrrrrsrrrrrrrrrrrrerrrseree 49 6 5 Autosampler Reproducibility NAN Configuration ccccceeeeneeeeeeees 51 6 6 Autosampler Injection Linearity NAN Configuration ccceeeeeeeee es 53 6 7 Gradient Accuracy Test NAN Configuration ccccccceeeeeeeeeeeneeaeees 55 6 8 Switchos Pump Test NAN Configuration eeeeeee estes ee eeeeeeaees 57 6 9 SLOP OW sachet do tec hnta neti ea hetdeehtendwat daca edit satdeato cement hea leee 59 Page 6 of 60 M1393RO OQ PQ Operating Instructions mrs 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 c
34. cifications a Prepare the Switchos with the mobile phases listed in Section 4 12 1 and degas them properly Purge all four channels b Disconnect the solvent inlet line from the pump head and connect the 250 ul 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 Set the flow rate to 0 00 mL min and set the Switchos in LOCAL mode Select solvent channel A by the SSV button on the rear The LED A should be illuminated c d e Measure the flow rate from the inlet tubing for one minute f Repeat steps d and e for each solvent line g Verify that the results meet the acceptance criteria TABLE 2 3 and fill in the appropriate field in the Other tests page of the report Page 38 of 60 M1393RO OQ PQ Operating Instructions 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 n 7 P 100 114 120 130 14 150 FIGURE 4 13 Typical Pressure Profile of the Switchos Loading Pump 4 12 1 Performing the test a b c d Open the Helium shut off valve on the bottle cap as
35. e 53 of 60 CHROMELEON Listings 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 54 of 60 M1393RO OQ PQ Operating Instructions CHROMELEON Listings 6 7 Gradient Accuracy Test NAN Configuration Sequence XQ_6 Gradient Formation Test_NAN Wait Sampler Ready Program file for gradient formation test System must be setup for Nano LC experiments The time constant must be set to 0 5 instead of 2 0 Program version 08 10 2004 Settings for restrictor 320cm 15um ID Diameter 75um Length 15cm StationaryPhase C18 3um_100A CRP 625 CalibrateCRP When BeforeFirstSample ParkPercentage Disabled Pump limits settings MasterPressure LowerLimit 0 0 MasterPressure UpperLimit 400 0 ColumnPressure LowerLimit 0 0 ColumnPressure 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 Co
36. e to different or limited control capabilities of these software packages OQ PQ Operating Instructions M1393RO Page 9 of 60 Requirements for a Successful OO PO 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 Test Procedure List of OQ and PQ Test Procedures to be performed Performed Section Lamp Intensity of the UV Detector Manually 4 2 Wavelength Check CM Sequence 4 3 Flow Cell Check UltiMate Fluid Path Test Manually Manually 4 4 Baseline Noise and Drift Test of the UV Detector CM Sequence Linearity of the UV Detector CM Sequence Reproducibility of the Injection Volume Linearity of the injection CM Sequence CM Sequence Gradient Accuracy CM Sequence Switchos Fluid Path Check Manually Switchos Flow Rate and Pressure Stability Test Switchos Valve Position Check CM Sequence Manually 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 t
37. est of the UV Detector d Oven accuracy e 4 7 Linearity of the UV NAN 64 min e e Detector CAP 32 min 4 8 Reproducibility of the NAN 72 min e e Injection Volume CAP 36 min 4 9 Linearity of the injection NAN 56 min CAP 28 min 4 10 Gradient Accuracy NAN 4 5 h o CAP 3h b Switchos Tests if applicable 4 11 Switchos Fluid Path Check 15 min e 4 12 Switchos Flow Rate and 15 min Pressure Stability Test 4 13 Switchos Valve Position 10 min e Check 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 in the system configuration 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 UltiMate FAMOS and Switchos is approximately 13 hours UltiMate Dual Gradient systems require that the test 4 10 must be performed for both gradient pumps For the UltiMate Dual Gradient system the entire OQ PO requires approximately 17 hours For the OO and PQ of UltiMate system configurations which does not inc
38. fferent gradient programs are used for the NAN and the CAP configurations the NAN program lasts 80 minutes while the CAP program is 50 minutes Typical examples of the step gradient profiles are presented in FIGURE 4 10 NAN configuration and in FIGURE 4 11 CAP configuration A Chiromelecn Guillaume Laptop iQOGPQ_ UNUR tem testi Test 17_ UR Fam Nan draft VQ 6 Gradient Formation tert_NAW WY Wan low preisire grad pl fe Gi Vew Workspace Quaticacon wadon Fab Su a Sew he teh DPRA ia W ie age nk BEF os 0 AXOS Gradent fommaton test NAN Nan ow pressure gaden 2 VIS JTA WVL 254 Legend Step 2 d a 0 5 B step fe b 0 50 B step f c 0 100 B step d 95 100 B step e step change time D ee eee c 7 b e pea a a ee tee ke a mm Gara FIGURE 4 10 Typical Step Gradient Profile NAN Configuration Ai Chromeleon Guillaume Laptop OP E A test Test 06_ Fam Cap draft PALS Gradient formation test purp CAP 3 Cap low pronor AE ol fle ts Vew Workspace Qualification Window Hep a a Ogre bre Daa ad a SAE AAL be GY OMBEL BBA 120 XOS _Gradent formation test pump CAP ap iow pressure gasent 2 ima 10 J a 704 40 304 10 _ pei 450 14 5 man 30 may Zoom FIGURE 4 11 Typical Step Gradient Profile CAP Configuration The steps items a d FIGURE 4 10 are quantified by measuring the UV absorbance difference
39. from the pump head and connect the 250 pl syringe P N 163241 the plunger should be removed using an adapter P N 160259 as presented in FIGURE 4 4 P Fi N E T gt 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 than 0 15 mL min f Repeat steps d e for solvent channels B C and D g Switch off the pump The flow should stop immediately h gt Verify the result with the limits presented in TABLE 2 3 and fill in the appropriate field in the Other Tests page of the QO PQ report OQ PQ Operating Instructions M1393RO Page 27 of 60 Test Procedures 4 6 Baseline Noise and Drift Test of the UV Detector and Oven Test 4 6 1 Drift and Noise es 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 The baseline noise of the detector mainly depends on the lamp There is a considerable increase in noise if an old lamp with poor light intensity is used or if the flow cell is dirty To minimize
40. hanges 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 e 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 e 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 OQ PQ Operating Instructions M1393RO Page 7 of 60 Introduction 1 3 General Notes and Recommendations e After the validation of the differe
41. he 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 OO and PO Test Procedures and Limits Instrument Parameter Description Limits a oQ PQ UltiMate Wavelength External holmium filter is required 2 nm 2 nm UV Detector accuracy Lamp Intensity The dummy flow cell is installed 0 4 lt SIG lt 0 9 0 4 lt SIG lt 0 9 Section 4 2 and the lamp intensity is read 0 1 lt REF lt 0 9 0 1 lt REF lt 0 9 Note If the from the detector SIGNALS system does screen not include an Flow cell The flow cell is installed and filled gt 15 gt 15 UltiMate UV transmittance with water The transmittance of of reference of reference Detector a Section 4 4 this flow cell is indicated by the intensity intensity standalone b SIG value and is read from the UltiMate UV detector SIGNALS screen Detector is Baseline drift The drift and noise are recorded lt 4000 pAU hr lt 4000 pAU hr required to Section 4 6 for 21 minutes at 254 nm with a es Baseline A ae cell filled with mobile phase lt 50 AU lt 50 HAU procedures Section 4 6 Oven test An external thermometer is used Accuracy Accuracy Section 4 6 to measure the oven temperature 1 C 1 C d Page 10 of 60 M1393RO OQ PQ Operating Instructions Requirements for a Successful
42. ift Oven Test CAP Ba XQ_3_UV Unearity_CAP a XQ_4_Autosampler Reproducbilty_CAP Ga XQ_S_Autosampler LinearRy_CAP Ta XQ_6_Gradient Formation Test CAP GB ULT_FMS_NAN v1 Gi xQ_1_Wavelength Check _NAN Ga XQ_2 UV Nose and Drift Oven Test_NAN A Last Updat 19 Oct 04 19 Oct 04 2 PQ ULT_FMS pan Ba XQ_3_UV Unearty_NAN amp xQ utosampler Reproducbity_NAN G XQ_5_Autosarnpler LinearRy_NAN Aa XQ_6_Gradient Formation Test_NAN ULT_FMS_SW_CAP v1 s xQ_1_Wavelength Check _CAP Gi XQ_2_UV Nowe and Drift Oven test_CAP Ea XQ_3_UV Linearty_CAP a XQ_4_Autosampler Reproducibitty_CAP a XQ_S_Autosampier Linearty_CAP Ga XQ_6_Gradient Formation Test_CAP Ta XQ_7_Swatchos Pump test_CAP ULT_FMS_SW_NAN v1 i XQ_1_Wavelength Check_NAN Ga XQ_2 UV Nose and Orft Oven Test_NAN 2_3_UV Unearty_NAN 4_Autosampler Reproduchiity_NAN utosampler Linearty_NAN a XQ_6_Gradient Formation Test_NAN Ta XQ_7_Swatchos Pump Test_NAN 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 OQ PQ Operating Instructions M1393RO Page 15 of 60 Process 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
43. is chapter describes problems due to which a check may have failed 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 Example If the UV detector linearity check fails the results regarding the linearity of the injection volume are questionable because the detector linearity detector is a basic requirement for checking the injection volume Page 22 of 60 M1393RO OQ PQ Operating Instructions Test Procedures 4 1 Overview This section provides step by step instructions about how to perform and evaluate the various OQ PQ tests All tests which must be performed and the order in which they must be performed are presented in TABLE 3 6 Tests which are not applicable for the current system configuration will be skipped e g if the Switchos is missing in the current configuration the tests 4 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 UltiMate and
44. isabled Pump limits settings MasterPressure LowerLimit 0 0 MasterPressure UpperLimit 400 0 ColumnPressure LowerLimit 0 0 ColumnPressure 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 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 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 TrapColumnPressure LowerLimit 0 0 Loading Pump TrapColumnPressure UpperLimit 400 0 Trap_Column_Pressure Formula Formula Loading_ Pump TrapColumnPressure value Trap_Column_Pressure Type Analog OQ PQ Operating Instructions M1393RO Page 57 of 60 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 Positi
45. lace Switchos Valve Position Check 4 13 Both position LEDs are illuminated at the same time Improper initialization or faulty controller See Service Information 043 Page 44 of 60 M1393RO OQ PQ Operating Instructions owns CHROMELEON Listings 6 1 Overview The CD ROM IQOQPO on UltiMate Plus Systems Version 1 provides ready to use CHROMELEON programs and sequences FIGURE 6 1 which will allow easy control of the standard UltiMate system configurations Please refer to Section 3 2 1 for modifications which may be necessary due to a different hardware configuration Chromeleon Guillaume Laptop IQOQPQ_UltiMate template OQPQ v1 Browser Jee f Fle Edit View Workspace Qualification Batch Tools Window Help 8 x De YE amp G amp 3 O0QPQ vI a Name 4 Last Update Size DGU_FMS_SW_NAN CAP v1 C DGU_FMS_SW_NAN CAP v1 19 Oct 04 16 07 52 XQ_1_Wavelength Check_NAN ULT_FMS_CAP v1 19 Oct 04 14 58 50 a XQ_2 UV Noise and Drift Oven Test_NAN ULT_FMS_NAN v1 19 Oct 04 14 58 53 a XQ_3_UV Linearity_NAN ULT_FMS_SW_CAP v1 19 Oct 04 14 58 58 ia XQ_4_Autosampler Reproducibiity_NAN ULT_FMS_SW_NAN v1 19 Oct 04 14 59 02 i XQ_5_Autosampler Linearity_NAN OQ PQ DGU_FMS_SW pan 19 Oct 04 16 24 52 23 KB ff XQ_6_ Gradient Formation Test Pump1_NAN OQ PQ ULT_FMS_SW pan 19 Oct 04 12 15 20 17 KB fa XQ_6_Gradient Formation Test Pump2_CAP OQ PQ U
46. lude an UV detector or for the UltiMate Dual Gradient system a standalone UV detector is required and all UV Detector related tests must be performed on this unit as described in this manual OQ PQ Operating Instructions M1393RO Page 21 of 60 Process 3 4 Evaluating the Tests The templates provided with CD ROM IQOQPO on UltiMate Plus systems and thus all copies made from it for OO and PQ are linked to the corresponding report Do not change this report except for items that are described in Section 4 14 In the report many references link the separate data sheets When lines or columns are inserted or deleted the references are lost and thus the calculations will be wrong To ensure that the data are 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 Select Batch Report on the File menu and start printing by clicking OK For the system 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 Troubleshooting Th
47. mmands 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 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 M1393RO Page 55 of 60 CHROMELEON Listings 0 000 7 000 7 010 30 000 Page 56 of 60 D Equate UV Autozero pump Flow B C D Wait Sampler Inject UV_VIS_1 AcqOn Pump_pressure AcqOn Column_pressure AcqOn pump Flow B C D B B UV_VIS_1 AcqOff Pump_pressure AcqOff Column_pressure AcqOff pump Flow B C D End M1393RO pD 0 400 100 0 0 UV Ready and Pump Ready and Pump_FlowSensor Ready and Sampler Ready 0 400 100 100 OQ PQ Operating Instructions CHROMELEON Listings 6 8 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 625 CalibrateCRP When BeforeFirstSample ParkPercentage D
48. mn 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 um 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 Page 20 of 60 M1393RO OQ PQ Operating Instructions Process 3 3 Performing the Checks The OQ PQ tests that need to be performed depend on the UltiMate system configuration For a complete system e g UltiMate FAMOS and Switchos all test procedures 4 2 to 4 13 must be performed For different system configurations refer to TABLE 3 6 TABLE 3 6 Tests to be performed for the different System Configurations Test Description Duration System Configuration No approx T P R D e Sallas az MEW aa 2 g o Cee 2pc SE S oO D 22e 2Q 23 22 Ss gt 6 gt Do DED SEO gt UltiMate and FAMOS tests 4 2 Lamp Intensity of the UV 10 min e Detector 4 3 Wavelength Check 10 min e e 4 4 Flow Cell Check 10 min e e 4 5 UltiMate Fluid Path Test 15 min e e 4 6 Baseline Noise and Drift 4h e T
49. n the middle of the step 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 in the example in FIGURE 4 15 starts at 8 minutes and ends at 9 minutes Check that the test result is correct and save the report 4 14 2 Completing the Cover Page and Printing Some of the instrument parameters required 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 b c d e f g h Open the report PQ_OQ LCP rdf Choose the Specifications sheet Select the Instruments section and enter the missing information e g S N of the FAMOS flow cell thermometer etc Select the Additional Information section and enter the customer information e g name operator position etc Choose the Other Tests sheet and enter the result of the manual tests e g Section 4 2 4 3 4 4 etc Check the result of the Gradient Accuracy Test as described in Section 4 14 1 Save the report Print the report using the Batch Report option form the File menu Page 42 of 60 M1393RO OQ PQ Operating Instructions mw Troubleshooting 5 1 Overview This section provides troubleshooting information which is related to the OQ PQ procedures More information
50. nformation 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 Auto Zero Scar FIGURE 3 4 Installing the Temperature Probe re 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 OQ PQ Operating Instructions M1393RO Page 19 of 60 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 A2 0 25 ug ml Caffeine CAP system only A3 0 5 ug ml Caffeine CAP system only A4 1 0 ug ml Caffeine A5 2 5 ug ml Caffeine A6 5 0 ug ml Caffeine A7 10 0 ug ml Caffeine A8 20 0 ug ml Caffeine B1 40 0 ug ml Caffeine NAN system only B2 60 0 ug 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 Microcolu
51. npressure value column_pressure Type Analog Ultimate pump settings Pump A Equate Pump B Equate Pump C Equate OQ PQ Operating Instructions M1393RO 100 water 0 1 FA Mob phase A 0 8 acetone C Page 49 of 60 CHROMELEON Listings 0 000 8 00 Page 50 of 60 Pump D Equate Pump Flow B C D UV Autozero Wait Inject UV_VIS_1 AcqOn pump_pressure AcqOn column_pressure AcqOn UV_VIS_1 AcqOff pump_pressure AcqOff column_pressure AcqOff End M1393RO D 0 4 0 0 0 Sampler Ready and UV Ready and Pump Ready and pump_flowsensor ready OQ PQ Operating Instructions CHROMELEON Listings 6 5 Autosampler Reproducibility NAN Configuration Sequence XQ 4 Autosampler Reproducibility NAN Wait Sampler Ready Program file for injection reproducibility test System must be setup for Nano LC experiments For this test time constant is 0 5 instead of 2 0 Program version 08 10 2004 Settings for restrictor 320cm 15um ID Diameter 75um Length 15cm StationaryPhase C18 3um_100A CRP 625 CalibrateCRP When BeforeFirstSample ParkPercentage Disabled Pump limits settings MasterPressure LowerLimit 0 0 MasterPressure UpperLimit 400 0 Pump columnPressure LowerLimit 0 0 Pump columnPressure UpperLimit 200 0 TrapColumnPressure LowerLimit 0 0 TrapColumnPressure UpperLimit 200 0 Settings for UV detector Data_Collection Rate 2 UV_V
52. nt modules a cytochrome C separation should be performed according to the LC Packings factory qualification protocol as described in the user s manual e For Ultimate Dual Gradient system configurations the gradient accuracy test Section 4 10 must be performed on both pumps e Channel C amp D should only be tested on customer request e If the customer ordered a system with two configurations e g a NAN configuration and a CAP upgrade kit the OQ PO should be performed for the system configuration that the customer will be using on a routine basis e f required test any other configuration according to the factory qualification protocol cytochrome C digest separation e 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
53. on 12 5 000 Valve_A Position 10 1 10 000 Valve _A Position 12 15 000 Trap _Column_Pressure AcqOff Loading Pump Flow 0 030 End Page 58 of 60 M1393RO OQ PQ Operating Instructions CHROMELEON Listings 6 9 Stop Flow Program Wait Sampler Ready Program file to stop the flow and lamp at the end of experiments Disable the stop sample when further experiments have to be carried out System must be setup for Nano LC experiments Program version 08 10 2004 Settings for restrictor 320cm 15um ID Diameter 75um Length 15cm StationaryPhase C18 3um_100A CalibrateCRP When BeforeFirstSample CRP 625 ParkPercentage Disabled Pump limits settings MasterPressure LowerLimit 0 0 MasterPressure UpperLimit 400 0 ColumnPressure LowerLimit 0 0 ColumnPressure 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 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 Sampler Temperature UpperLimit 30 00 Virtual channel settings pump_pressure Formula
54. ort 10 Verify that the valve position corresponds to the LED indicator Page 40 of 60 M1393RO OQ PQ Operating Instructions 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 Due to this the calculated result of the Gradient Accuracy test Section 4 10 may be wrong and the system may not pass the test In such a case the delay times should be checked and adjusted if necessary This applies for the 5 50 95 and 100 steps or if the result 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 report layout Open the Pump Gradient sheet and go to line 137 d Make sure to modify the correct columns only FIGURE 4 14 e D E F G for capillary CAP configurations e J K L M for nano NAN configurations 132 133 Calculation of Gradientaccuracy and rep
55. p file on the CD ROM IQOQPO 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 Chromeleon Guillaume Laptop IQOQPQ_UltiMate template OQPQ v1 Browser Jee UB fe edt vew Workspace Quaificaton Batch Toos window Hep ax Osa SS shaw ogeg vi a OGU_FMS_SW_NAN CAP v1 a XQ_1_Wavelength Check_NAN a XQ_2 UV Nowe and Drift Oven Test_NAN V Lnearity_NAN Q hosampler Reproducbiity_NAN a XQ_S_Autosampier Lnearty_NAN a XQ_6_Gradient Formation Test Pump1_NAN E XQ_6_Gradient Formation Test Pump2_CAP Ea XQ_7_Switchos Pump Test_NAN ULT_FMS_CAP v1 a XQ_1_Wavelength Check_CaP T XQ_2_UV Nose and Dr
56. roducibility 134 135 136 137 138 Name 140 141 142 143 Equ dration 144 Nan low pressure gradiendi 145 Nan low pressure gradient 146 Nan low pressure gradien 147 slop 148 149 Observed Values for Pump in capillary mode Observed Values for pump in nano mode Note The first part is dedicated to cap LC and the other to Nano LC FIGURE 4 14 Section of the Report Table to be modified e Double click on each column to open the Properties Report Column box FIGURE 4 15 OQ PQ Operating Instructions M1393RO Page 41 of 60 Test Procedures Properties Report Column Categories Variables General Channel Name a OK Sequence Raw Data File Path Name Sample Number of Peaks c Audit Trail _ Select Peak all Preconditions Count Peaks If Chromatogram Sum Peak Results If Detection Parameters Start Time relative to Inject Time Peak Results End Time relative to Inject Time Peak Calibration Signal Value Peak Table Signal Dimension Peak Purity and Identification y Signal Noise m Explain Variable Quantification Mathad Qamnle Rata E Formula chm sig_velue average 8 9 Parameter Header Signal Step 5 Dimension chm sig_dim Format 9 000 Peak Channel Se Selected Channel C Fix A Fixed Channel s aim FIGURE 4 15 The Properties Report Column Box f g h Check that the segment that is used for calculation is i
57. s 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 Page 2 of 60 M1393RO OQ PQ Operating Instructions DANGER WARNING CAUTION 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 action that if not done correctly or adhered to could lead to personal injury or loss of life 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 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 sta
58. s presented in FIGURE 4 9 140 OE External NMS 1 Ai J T 7 T 100 150 20 0 250 030 0 350 400 FIGURE 4 9 Example of a Calibration Curve Nano LC system 99 96 Correlation Coefficient 4 9 1 Performing the Test a 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 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 uL min 4 0 uL min CRP 625 50 Injection loop 1 0 uL 1 0 uL Injection type Partial Loop Fill 0 2 uL Partial Loop Fill 0 5 uL To evaluate the result of the Linearity of the Injection Test a Use the quantization file caffeine qnt b Check the integration and correct if necessary c Calculate the correlation coefficient d Verify that the correlation coefficient meet the acceptance criteria TABLE 2 3 Page 34 of 60 M1393RO OQ PO Operating Instructions 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 Di
59. sembly and degas the solvent The four channels should be well flushed using the Purge function of the pump Connect a 30 um I D tubing P N 160182 to port 2 of the Switchos valve A Connect the Switchos output to the port 1 of the Switchos valve A using a 130 um I D tubing P N 160180 Connect port 10 to waste using the same type of tubing e f Start the Switchos pump from the panel g After equilibration start the sequence XQ_7 Switchos Pump Test_NAN or CAP respectively h i Verify that the parameters meet the specifications presented in TABLE 2 3 and fill in the appropriate field in the Other Tests page of the report The result for the pressure stability test is on the Switchos page of the report gt Measure the flow rate with a syringe and chronometer watch for 5 min OQ PQ Operating Instructions M1393RO Page 39 of 60 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 Put the Switchos in LOCAL mode Switch valve A and valve B from position 1 2 to position 10 1 using the manual control button on the front panel a b gt c d e Connect a syringe adapter P N 160259 to port 1 of valve A Flush with a 250 ul syringe water through port 1 into p
60. 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 Page 8 of 60 M1393RO OQ PQ Operating Instructions Requirements for a Successful 00 P0 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 Standard Inert Flow Sensor Capillary H
61. st Flow rate too low e Solvent filter e Check replace 4 5 clogged Baseline Noise and Drift Drift too high e Old lamp e Replace Test of the UV Detector 4 6 Oven accuracy 4 6 Linearity of the UV Detector 4 7 Temperature readout not within the specifications Signal is out of range Temperature probe not properly installed Acetone concentration too high Install properly e g use a 4mm screw Dilute with mobile phase A Reproducibility of the Injection Volume 4 8 Linearity of the injection 4 9 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 Air in the syringe Air at the bottom of the sample vial Purge syringe Remove air Gradient Accuracy 4 10 Considerable drift of the 95 to 100 B steps No too little formic acid in mobile phase A and B Check replace solvents Result not within specification Wrong delay time Hardware Check modify Check filter valves etc Switchos Fluid Path Check 4 11 Flow rate too low See above 4 5 See above 4 5 Switchos Flow Rate and Pressure Stability Test 4 12 Pulsation too high Check valve not working properly Clean rep
62. te 0 4 ul min 4 0 ul min Note Page 36 of 60 a The acetonitrile is used to perform a wash step during the FAMOS test procedure 4 8 M1393RO OQ PQ Operating Instructions Test Procedures 4 10 1 Performing the Test a Setup the UltiMate system and the FAMOS Microautosampler as discussed in Sections 3 2 2 A and 3 2 2 B and TABLE 4 6 Degas the solvents properly b The four channels should be well flushed using the Purge function of the Micropump c Start the flow delivery of channel 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 IES 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 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 OQ PQ Operating Instructions M1393RO Page 37 of 60 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 spe
63. tement 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 OQ PQ Operating Instructions M1393RO Page 3 of 60 The operator should follow all safety precautions warnings etc provided with the instruments in addition please note the items presented below 1 10 11 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 If a leak occurs turn off power to the instrument and remedy the situation immediately If the mobile phase includes volatile or flammable solvents avoid open flames and sparks Many organic solvents and buffers are toxic Make certain that you know the toxicological properties of all mobile phases that you are using 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 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
64. tion 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 um O D to the flow cell inlet 280 wm O D Use P N 161405 for the 360 um tubing and P N 161498 for the 280 um 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 E Caution Older revisions of the UltiMate system may be equipped with the E older type of the C and D solenoid valves P N160051 Due to their D limited resistance against strong organic solvents do not expose these lg valves to acetonitrile for a longer period than required to perform the wash A i cycle of test 4 8 e g not longer than 4 h Page 18 of 60 M1393RO OQ PO Operating Instructions Process 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 I
65. ty test System must be setup for Nano LC experiments For this test time constant is 0 5 instead of 2 0 Program version 08 10 2004 Settings for restrictor 320cm 15um ID Diameter 75um Length 15cm StationaryPhase C18 3um_100A CRP 625 CalibrateCRP When BeforeFirstSample ParkPercentage Disabled Pump limits settings MasterPressure LowerLimit 0 0 MasterPressure UpperLimit 400 0 Pump columnPressure LowerLimit 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 FlushVolume 5 0 WashVolume 50 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 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 M1393RO Pag
66. urrent 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 e f the system includes an oven run the warm up and oven test pgm FIGURE 3 3 e f the system does not include an oven run the warm up pgm FIGURE 3 3 WE default qnt 05 May 04 14 48 3 f Drift and Noise_CAP pgm ULT_FMS 08 Oct 04 11 41 58 8 warm up and oven test pgm ULT_FMS 19 Oct 04 15 26 10 warm up pgm ULT_FMS 18 Oct 04 10 05 15 Po inj Vol inj Date Time 1 system warm up Blank Al 1 00 warm up and ov default Single 2 Drift and noise test Blank A2 1 00 Drift and Noise_ default Single 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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