for HPLC-ECD systems (carbohydrates)
Contents
1. Antec Industrieweg 12 2382 NV Zoeterwoude The Netherlands PQ for HPLC ECD systems carbohydrates 180 0029 Edition 4 2014 ISO 9001 T 31 71 5813333 F 31715813334 info myantec com www myantec com certified Copyright 2015 Antec The Netherlands Contents of this publication may not be reproduced in any form or by any means including electronic storage and retrieval or translation into a foreign language without prior agreement and written consent from the copyright of the owner The information contained in this document is subject to change without notice ROXY ALEXYS DECADE DECADE II DECADE Elite INTRO Flexcell SenCell ISAAC HyREF are trademarks of Antec Whatman word and device and Whatrnan word only are trademarks of Whatman International Ltd SOLVENT IFD and AQUEOUS IFD are trademarks of Arbor Technologies Inc Clarity DataApex are trademarks of DataApex Ltd Mi crosoft and Windows are trademarks of Microsoft Corporation Excel is a registered trademark of the Microsoft Corporation The software and the information provided herein is believed to be reliable Antec shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of software or this manual All use of the software shall be entirely at the user s own risk Chapter 1 Table of contents 1 Table of contents Fable Of contents icc2. 2uA V 300mV 600nA Use the proper unit multiply V output with range if necessary Note in case of using the Clarity software with the Antec device drivers this will give signal directly in current units nA or pA 14 PQ for HPLC ECD systems edition 4 CHAPTER 5 PQ results summary Electronic test results and HPLC test results Specified Measured Result ELECTRONIC TESTS Dummy cell test Current I cell 2 67 0 05 nA N lll acia Noise p p lt 2 0 pA o DAS lit Analog output test Output at 5 nA V 530410 mV NN EA HPLC TESTS Signal Height VT 03 gt 50 nA o AA rea Height Flexcell gt 100 nA _s NA lance Repeatability RSD area lt 30 anan Se E E Linearity Correlation coefficient r gt 0 997 as as In case of using a VT 03 cell check the Flexcell as n a and vice versa fill in passed or failed ROXY noise p p spec lt 3 pA Final result passed failed Verified by customer coooococccccccccnccccncncnccnnn Deviations Y N Comments Chapter 6 PQ certification 15 CHAPTER 6 PQ certification The Performance Qualification PQ tests were carried out in accordance with the PQ procedure and have been carried out to the satisfaction of both parties All tests as described in this document have been successfully completed and all results are within specifications Antec Leyden representative Compa
3. mM NaOH water should be HPLC grade low TOC Flow rate pump 1 1 00 mL min Flow rate pump 2 0 50 mL min Chapter 4 HPLC performance test 13 Processing PQ results The PQ procedure results in 5 chromatograms for calculation of linearity and 8 chromatograms for calculation of repeatability 1 Analyze the chromatograms and create reports containing retention time peak heights and peak areas 2 Based on the data from the repeated chromatograms n 8 calculate the average values and the relative standard deviation as percentage of the average RSD 3 Based on the data from the 5 chromatograms for calculation of linear ity calculate the correlation coefficient between peak area and con centration or injection volume 4 Write down the results in the PQ results table on page 14 If the final result of the HPLC PQ procedure is failed beware that it is not necessarily related to the detector Contact your supplier in case you need support with troubleshooting To avoid clogging of the restrictor tubing flush it with water after use When flushing the system SWITCH OFF THE CELL Recalculation of signal units from mV to nA If the signal from the detector is acquired with an AD convertor the associ ated units are generally mV Use the range setting and the following for mula to convert to current units Signal nA range uA V Signal mV For example at the 2 A V range a signal of 300 mV would be Signal
4. the auto zero and compensation to off 2 Measure the analogue output with cell off 3 Switch on the cell and measure the analogue output 4 Calculate the difference in output voltage measured with Cell on and Cell off Fig 3 5 Enter the results of the Analogue output test in the PQ results table on page 13 mV cell on 500 4007 300 530 mV 2004 1007 cell off cell off tht T T T 0 1 2 3 mir Fig 3 Measuring output with dummy cell off and on at 5 nA V for other settings see Table 1 If passed continue with the HPLC analysis If failed follow maintenance procedures check knowledge base on our website or contact your supplier Chapter 4 HPLC performance test 9 CHAPTER 4 HPLC performance test For a successful performance test it is important that the HPLC EC system has been optimized and is in top condition The system must be installed as described in the user manual Test parameters Parameters to characterize and check the performance of a system are re peatability linearity and responsiveness Configuration For testing the performance of an HPLC system with an Antec electro chemical detector in PAD mode on a gold working electrode under alkaline conditions the column from the HPLC system is replaced by an assembly of restriction and mixer capillary tubing Fig 4 A kit containing the test substance as well as the parts between injector and cell can be ordered unde
5. 17 2 PQ for HPLC ECD systems edition 4 CHAPTER I Introduction This document describes the Performance Qualification PQ for HPLC systems with an Antec electrochemical detector ECD as advised by the manufacturer It is the result from our interpretation of many regulations and laboratory practices In addition feedback from users and representa tives helped us to finalize this procedure A complete PQ for an HPLC system with aROXY DECADE II DECADE or INTRO electrochemical detector consists of an electronic noise test and a detection performance test by analyzing a standard solu tion All qualification checks in this document must be approved or must be marked n a if not applicable Any deviation observed must be docu mented in the non conformance record All relevant documents regarding this operational qualification must be filed together in one location As regulations and customer requirements may change manufacturer re serves the right to introduce changes without prior notice For details on functionality operation and theory references are made to the instrument user manual Supported configurations The PQ procedure in this document is applicable for an HPLC ECD sys tem with an Antec electrochemical detector and Flexcell or VT 03 flow cells Fig 1 with gold Au working electrodes running under alkaline con ditions Note PQ procedure on glassy carbon and gold electrodes under acidic condition
6. a aa a a are dabei aia 1 Chapter di AS 2 Introduction aa en iron eee aaae riaa da 2 Supported configurations oooncccnnncnnnnnnnncccnonccnno rana nr cnc 2 Chapter 2 oi SGLTS ea ae SETE SEK TRE STE SEERE SEE SE SEERE 3 Identifica dao 3 Engng ain a ei ta ei eee 3 Reviewer Customer ais thai bin niin nae 3 INSTRUMENTS tai des a e emt eine te eee lee 4 TestimatenalS uiii dene fetes td ilogico leet 5 Testidevic es Tiara ir lata ria lares as 5 Ghapter noia 6 Electronicitests cda dia 6 Required Sii SES ulin ivi Aiello 7 Dummycolltest ii A aa ai are 7 Analogue output test esera ae ede ein eee 8 A A caves scouts soteuetutere 9 HPLC performance test ccccceceeeeeeeeeeeeeeeceeeeesaeeeeeeeseeeeesaeeseaeeeeeeees 9 Test parameters comicios e hdi 9 Configura he ae tia eee Es eee 9 Required COMSUMADIES ODER tede 10 Test SUBStanCe cui di ri 10 Prepara iii ac 10 PO procedure aa ea aaa kia ai arr reen En 11 HPLC ECD test conditions for PQ ccceesceeeeeeeseeeeeeneeseeeeeteaeeeseeeeneees 12 Processing PQ result Unida dida 13 Recalculation of signal units from MV to NA eeeeeeeeeeenteeeeeeneeeeeenaes 13 Chapter Su AA acters een Re tba 14 PQ results SUMMALY norii aaaea aa aada aaia 14 Chapter Di ici cst See inana aae aaas e Ee ieoi ais 15 POCO e a a aa e lag 15 Chapter ii 16 COM 5 4 1 series cis 16 Chapter BR SEE E va VE Nares 17 Non conformance record W W W u u u dseseeeeeve eee en nonannnn nono nano nn nana nnnnnncnnannnns
7. ctronic tests use a dummy cell and checks the following parame ters electronic noise level background current stability of the electrochemical detector temperature stability resistor and capacitor that both require con stant temperature to meet the noise and stability specifications analogue output The Dialogue software can be used for automated testing and will gener ate a report with results As an alternative the acquired noise data can be evaluated in Excel Details can be found in the appendixes document 180 0028C Data acquisition with exactly the same noise evaluation pro cedure can be used as well Noise definition There are different methods for calculation of noise The specifications in this document are based on the following method The noise level is the average of 30 peak to peak noise measurements over a period of 30 s to tal of 15 min as given in the following formula and visualized in Fig 2 pA 0 200 400 600 800 t s Fig 2 Noise measurement on a baseline trace Chapter 3 Electronic tests 7 Required tools Partno Description 171 9005 Dialogue PQ software with noise_template xIt m and Microsoft Excel 2003 or newer or alternative Data acquisition software 250 0040 Dummy cell x part of detector accessories one per cell 250 0128 Output cable part of detector accessories AD convertor or calibrated voltmeter Dummy cell test Prepa
8. ng to the conditions as indicated in Table 3 1 If necessary first flush all system components with pure water to re move any storage liquid such as organic solvents to avoid precipitation of the mobile phase 2 Clean the flow cell see user manual polish the gold electrode if the gold color is not visible and assemble with a 50 um spacer 3 Prepare the mobile phase and flush the system up to the cell 4 Connect the cell and ensure it is air free and completely filled with mo bile phase before turning on the cell Chapter 4 HPLC performance test 11 Do not turn the cell on when mobile phase is not yet flowing through the cell This can damage the cell 5 Set the conditions as given in Table 3 and wait for the system to stabi lize until a stabile baseline is obtained before starting the PQ HPLC tests 6 Dissolve the Glucose in 3 mL of mobile phase abbreviated as MP prior to use to make a 20 mmol L stock solution Close the vial shake well and dilute this solution 1000x 7 For example dilute in 3 steps mark 3 empty vials clearly as 1 2 3 and add 900 uL mobile phase to each of the 3 vials Add 100 uL stock solution in vial 1 close and shake well With a new clean pipet take 100 uL solution from vial 1 and add to vial 2 close and shake With a new clean pipet take 100 UL solution from vial 2 and add to vial 3 Vial 3 now contains 1 mL of the 20 mol L Glucose standard for PQ The PQ standard sol
9. ny CC a Penmormer goa ias S FALSE REDE ske i erat Customer authorized to sign Company Dept cesar eds Reviewer CustOMer oocccccconcnconcnconnnconnnccnoncnnonconincnnincnnincinincnns Date Signature 16 PQ for HPLC ECD systems edition 4 CHAPTER 7 Comments Chapter 8 Non conformance record 17 CHAPTER 8 Non conformance record Any case of non conformance found during the PQ procedure should be documented and signed for acceptance or corrective action taken Table 4 Non conformance record Ref Non conformance and action taken Signature Sign executing customer engineer 1 Verified by Customer cceeeeeee eee eee Deviations Y N Comments 18 PQ for HPLC ECD systems edition 4
10. r pn 250 3044 PQ for HPLC ECD kit carbohydrates Pump Sampler Pump 3m Red 10m Orange Cell Configuration with second post column addition pump Sampler Pump O KON Cell 3m Red 10m Orange Configuration with single pump Fig 4 For PQ the analytical column is replaced by an assembly of 3 m PEEK tubing with ID of 125 um and 10 m PEEK tubing with ID of 500 um PQ for HPLC ECD systems edition 4 Two hardware configurations are supported Fig 4 HPLC with single pump HPLC with secondary pump for post column addition In case of performing the PQ procedure on an ALEXYS system with Clarity software pre configured method files are available on our website De tailed description of performing a PQ using the Clarity software can be found in the appendixes document 180 0028C also downloadable from our website Required consumables To perform a PQ the following items have to be present on location e Autosampler vials e Sodium hydroxide 50 solution HPLC grade or better e 1 Lultra pure water resistivity gt 18 MOhm cm low TOC Test substance The test is based on the analysis of Glucose under alkaline conditions Prepare the test solution according to the label Table 2 Reordering info for test substance for PQ on gold cell under alka line conditions This item is part of pn 250 3044 PQ for HPLC ECD kit carbohydrates Glucose for PQ 250 1067 Preparations Set up the system accordi
11. rations Before running the test make sure the system has stabilized for more than an hour with a dummy cell installed and ON at the right temperature working potential E and range setting see Setting below in Table I Settings Table I Dummy cell test settings Parameter Sting Cell potential Oven Zero First available filter setting 0 1 s or 0 5 Hz 100 pA Acquisition Data rate lt 10 Hz Output test INTRO DECADE REC output DECADE ll or ROXY Output Procedure 1 For detailed instructions on running a Dummy cell test with Dialogue software or how to use the noise template see the Appendixes docu ment 180 0028C 2 Make sure the system has stabilized for at least one hour before run ning the test PQ for HPLC ECD systems edition 4 3 Measure the noise during 15 minutes Acquisition frequency must be set to less than 10 Hz 4 Read the cell current from the display I cell 5 Enter the results of the Dummy cell test in the PQ results table on page 13 Analogue output test The analogue output of the detector is tested by measuring the difference in output signal from a dummy cell with the working potential switched off zero level and on The measurement is taken from the rear panel Output connector which is either connected to some software through an AD con vertor or alternatively measured with a calibrated voltmeter 1 Use the settings from Table I but set the detector range to 5 nA V and set
12. s is described in document 180_0028 Working electrode material other than Au or GC are not supported with PQ procedures SO Fig 1 Flexcell left and VT 03 cell right Chapter 2 Identification 3 CHAPTER 2 Identification Engineer The undersigned engineer certifies to be trained and qualified to perform PQ on Antec instruments Company Performer Title Signature Reviewer customer The undersigned reviewer customer accepts that the above mentioned en gineer is trained and qualified to perform a PQ on Antec instruments Company Reviewer Customer Title Signature Owner designated authorized person PQ for HPLC ECD systems edition 4 Instruments Device p n s n Pump Autosampler injector Electrochemical detector Flow cell with Au WE Reference electrode Acquisition software amp version s n entering more than one s n is allowed if more than one unit is used Others Verified by customer eeeeeeeeeee ees Deviations Y N Comments Chapter 2 Identification Test materials Glucose solution Mobile phase Test devices Device p n s n Dummy cell Volt meter or AD signal Verified by Customer W u sssseeeeererer rener Deviations Y N Comments 6 PQ for HPLC ECD systems edition 4 CHAPTER 3 Electronic tests The ele
13. ution is 20 pmol L Glucose in mobile phase MP Dissolve and dilute the glucose powder prior to use PQ procedure Test injection Run a single test chromatogram with the 20 umol L Glucose standard Check the chromatogram and optimize the automated integration Linearity test Linearity is checked based on a 5 point calibration The correlation coeffi cient can be based on different injection volumes 5 10 15 20 and 25 uL injections of the test solution or based on 20 uL injections from manual dilutions 20 80 40 60 60 40 80 20 and 100 0 dilutions of test solution with water Repeatability test Repeatability is evaluated based on 8 subsequent analyses of the test so lution 12 PQ for HPLC ECD systems edition 4 HPLC ECD test conditions for PQ Table 3 HPLC EC conditions for PQ using Glucose Temperature 35 C flow cell and column Integrator Data acquisition frequency lt 5 Hz PAD mode E1 E2 E3 150 750 800 mV t1 t2 13 ts 500 130 120 20 ms Other settings Offset 0 Filter Off Indicative parameters Icell is about 0 5 1 5 UA noise is about 3 6 nA Settings for single pump configuration see Fig 4 Mobile phase 100 mM NaOH water should be HPLC grade low TOC and gt 18 MOhm cm Flow rate 1 50 mL min P about 100 bar Settings for two pumps configuration see Fig 4 Mobile phase pump 1 Water HPLC grade low TOC and gt 18 MOhm cm Post column solution 300
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