SynthesisCell™ user manual
Contents
1. Antec Industrieweg 12 2382 NV Zoeterwoude The Netherlands TM SynthesisCell User manual 206 0010 Edition 1 2014 ISO 9001 T 31 71 5813333 F 31715813334 info myantec com www myantec com Copyright 2014 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 Il INTRO Flexcell ReactorCell SenCell VT 03 SynthesisCell SynthesisCell 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 Microsoft 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 INTRODUCTION Table of contents 3 Intended use For researc2. Oxidation of heteroatom N S S methyltiopurine lidocaine phenothiazines 2 7 under basic conditions i d R C NH R gt R C N R ZA R R 4 Acetaminophen amodiaquine amsacrine mitoxantrone 3 8 o So OH gt A NSR NS Dehydrogenation Table 2 Examples of solvents used for electrochemical oxidation Compound Mobile phase Electrode Acetaminophen 20mM ammonium acetate in Glassy Carbon Magic 25 methanol Diamond Amodiaquine 1 20mM ammonium formate pH Glassy Carbon Magic 7 4 adjusted with ammonium Diamond hydroxide in 50 acetonitrile Amodiaquine 2 50 methanol Glassy Carbon and 50 10 mM aqueous lrinotecan 20mM ammonium formate with Glassy Carbon Magic 0 1 formic acid pH 3 3 in Diamond 50 acetonitrile on both different oxidation profiles were obtained Angiotensin 0 1 formic acid in 50 Magic Diamond acetonitrile Adenosine 20mM ammonium formate Magic Diamond OH 7 3 adjusted with ammonium hydroxide in 50 acetonitrile Toremifene 20mM ammonium formate pH Glassy Carbon 7 4 adjusted with ammonium hydroxide in 50 methanol LYL LWL 90 10 1 v v v Glassy Carbon water acetonitrile formic acid oe References 1 A Baumann et al J Chromatogr A 121 6 3192 3198 2009 2 T Johansson L Weidolf and U Jurva Rapid Commun Mass Spectrom 21 2323 2331 2007 S M van Leeuwen et al Anal Bioanal Chem 382 742 750 2005 W Lohmann and U Kar
3. 19 Storage 21 Specifications SynthesisCell 22 Accessories SynthesisCell 23 Ordering Information SynthesisCell 23 CHAPTER 1 The SynthesisCell Introduction Congratulations on your purchase of the SynthesisCell The SynthesisCell is a bulk electrochemical cell designed for small scale electrosynthesis of metabolites reactive intermediates and other oxidation and reduction products in milligram quantities The SynthesisCell is available with different working electrodes WE Tubular Reticulated Glassy Carbon RGC Flat Smooth Magic Diamond MD and Tubular Mesh Platinum Pt Moreover there are two types of reference electrodes the Pd H2 HyREF and a salt bridge Ag AgCl electrode SB REF A coiled platinum wire is used as an auxiliary electrode AUX For successful and efficient conversion the parameters as the potential composition of the solution Supporting electrolyte organic solvent concentration pH need to be optimized depending on the type of compounds or desired reaction In general the samples can be oxidized in the solutions containing supporting electrolyte at concentrations of 10mM or higher The higher concentration of supporting electrolyte ca 100mM can improve conversion In case MS is used for analysis the solvent also affects the ESI response ionization suppression In that case the solvent must be compatible with ESI MS and to fulfill this requirement ammonium acetate ammonium formate formic a
4. Fig 1 pH Fig 1 Potential difference between HyREF and Ag AgCl REF versus pH So if an Ag AgCl REF is compared to a HyREF the pH effect on HyREF compared to Ag AgCl must be taken into account The pH voltage relation is described by Exyrer Eagagci 328 29 9 pH 1 Example If an Ag AgCl REF is exchanged for a HyREF a working potential of 800 mV vs Ag AgCl at pH 3 has to be changed to Enyrer 800 328 29 9 3 560 mV vs HyREF As a rule of thumb the working potential should be chosen such that a similar background signal I cell is measured with the HyREF as in using the Ag AgCl REF Working electrodes Electrochemical conversion puts high demands on the working electrode material The working electrode should be made of a electro chemically inert material Furthermore to avoid an irregular flow profile over the electrode it should have a very well defined surface In the table the working potential limits are listed of the WE materials available for SynthesisCell The values presented in the table are 12 SynthesisCell user manual only the estimation and will depend on mobile phase composition pH supporting electrolyte and the analyzed compound itself At high positive working potentials the water in the mobile phase electrolyses and results in decrease in metabolites formation In case of electrolysis of the water mobile phase the cell current Icell readout will display the mes
5. needs maintenance the cell has to be disassembled Before disassembling the flow cell read General precautions 1 Switch off the cell and disconnect the cell cable 2 Remove the AUX and the HyREF 3 To remove the WE Gently push the WE downwards using the white Teflon contacts than pull it up to remove the retaining rings Gently rest the WE on the bottom of the cell Remove the vessel cap and remove WE from the vessel N Use proper eye and skin protection when working with solvents 17 Maintenance Magic Diamond electrode The Magic Diamond working electrode consists of an ultra thin crystalline Diamond layer deposited on a carrier metal Therefore such electrode cannot be polished to restore the electrode surface in case of contamination An effective method to restore the performance is by electrochemical reactivation of the electrode surface under acidic conditions Activation of the electrodes using pulse mode This activation procedure is suitable for both MD and GC electrodes 1 Refill the bottle with clean solvent containing the supporting electrolyte e g the same solvent used for oxidation of the sample 2 Go to the Options pull down menu and choose Detection mode Fig 17 ROXY Dialogue File Edit Options Help Data f Devices Timed Events Connect Not connected m Pulse Oven Detection Mode Pulse settings Step T Settings e1 200 v 1000 ms Temp f FW update wizard E2 2 00 y 211
6. the diamond electrode was seen within days of operation delamination blister formation of the MD layer 21 Storage If the cell is not in use switch off the cell wash and store with water If not in use for longer time several days weeks wash all parts with water and store clean and dry Before disconnecting the cell from the detector turn off the cell first 22 SynthesisCell user manual CHAPTER 4 Specifications SynthesisCell Cell type Cell volume Working electrodes WE Reference electrode RE Auxiliary electrode AUX Port plug Electric connections Three electrode synthesis cell consisting of working electrode WE reference electrode RE and auxiliary electrode AUX Up to 80 mL of sample solution in glass reaction vessel Optional Water jacketed reaction vessel for cooling exothermic reactions Tubular Reticulated Glassy Carbon RGC Optional Flat Smooth Magic Diamond MD Tubular Mesh Platinum PT Pd H2 reference electrode HyREF Optional Ag AgCl reference electrode Coiled platinum wire in glass isolation tube Access port for sample collection dispensing of reagents or venting of cell Cell cables for use with ROXY Potentiostat CHAPTER 5 23 Accessories SynthesisCell The Antec SynthesisCell p n 206 4300 is shipped together with a number of parts listed below in shipkit Use these part numbers when re ordering parts Part no 206 0037 20
7. 000 ms No sense Users Hake E3 0 00 y 310 ms Cell PQ Noise Test ts 20 ms Cell of OQ System A One Off cell a Start Run Stop Run Clear All Shift Del Sequence F2 D2D System Suitability Fig 2 Pull down menu Options gt Detection Mode 3 Select Pulse mode Fig 18 and click OK F Select mode cell 1 Mode Fig 3 Select mode window 4 Set the parameters in the cell control window Fig 19 18 SynthesisCell user manual Parameter Value E1 2V E2 2V E3 OV t1 1000 ms t2 1000 ms t3 0 ms Ts 20 ms It is recommended to set the Output Range to 200 uA 5 Set Run time value to 5 min and turn on the cell 6 Go to Options pull down menu and click Start Run Fig 20 Program will ask to save the data The excel file will be created And start the acquisition ROXY Dialogue File Edit Options Help Data Celi Devices Events Connect Not connected Pulse Oven Acquisition Output Pulse settings Step T g JSk Run time 500 min Range 200uA FS E1 2 00 Von 1000 ms Delay 0 00 min E2 200 v 100 ms Sr ly Filter off Hz Marker E3 0 00 Yy t3 o ms Cell 1 ts 20 ms Cel B Onc Of Temp C LM35 No sensor selected ROXY Dialogue File Edit Help Devices Timed Events Connect Not connected r Pulse settings m Pulse Oven Detection Mode Step T Temp J Settin
8. 6 0300 206 0306 206 0322 206 0314 206 0304 206 0310 206 0900 206 0340 Description Complete SynthesisCell consisting of 80 mL reaction vessel with Tefloncap WE Tubular Reticulated Glassy Carbon RE HyREF and AUX electrodes stir bar electrode cables etc all parts included for immediate use with ROXY Potentiostat Optional Water jacketed reaction vessel Flat Smooth Magic Diamond MD working electrode Tubular Mesh Pt PT working electrode Ag AgCl reference electrode Spare Parts Tubular Reticulated Glassy Carbon RGC working electrode Auxiliary Pt electrode in glass tube Glass reaction vessel 80 mL Glass coated magnet stir bar length 8mm
9. and monitoring and control instruments Nevertheless we have taken steps to eliminate all restricted substances from our products Antec Leyden is an ISO 9001 2008 certified company 4 SynthesisCell user manual ZN Symbols The following pictograms are used in this user manual Warning caution sign It calls attention to a procedure or practice which if not adhered to could result in severe injury or damage to parts or all of the equipment Do not proceed beyond a warning sign until the indicated conditions are fully understood and met The attention sign signals relevant information Read this information as it might be helpful The note sign signals additional information It provides advice or a suggestion that may support you in using the equipment INTRODUCTION Table of contents 5 Safety practices Perform periodic leak checks on LC tubing and cell connections Do not allow flammable and or toxic solvents to accumulate Follow a regulated approved waste disposal program Never dispose of such products through the municipal sewage system LC equipment should be used by trained laboratory personnel only Use proper eye and skin protection when working with solvents Additional safety requirements or protection may be necessary depending on the chemicals used in combination with this equipment Make sure that you understand the hazards associated with the chemicals used and take appropriate measure
10. ation procedure event table The syringe pump commands are ignored as no pump is connected In case that no significant improvement of the cell performance is seen A For MD electrode use the electrochemically reactivation of the electrode surface under acidic conditions B For RGC electrode replace the electrode for a new one Activation of the electrodes using scan mode in acidic conditions 1 Disassemble the SynthesisCell as described on page 12 Keep the MD electrode disc fixed inside the working electrode assembly 2 Wipe the electrode surface with a tissue wetted with methanol or acetone and subsequently with demi water to clean the electrode surface Wear gloves Under all circumstances try to avoid direct contact of the electrode surface with fingers The skin contains fatty substances which will foul the electrode 20 SynthesisCell user manual 3 Assemble the SynthesisCell with MD electrode as described in the previous section 4 Prepare a solvent of 0 5 M Nitric acid HNOs fill and install the synthesisCell f Make sure that all parts that are not acid resistant are not connected in the system during this step 5 Set the ROXY in SCAN mode with the following settings E1 3 00 Volt E2 3 Volt Scan rate 50 mV s scan cycle continuous and range 200 uA V 6 Start scanning under acidic conditions for 1 hours 7 After scanning switch off the cell and the flow rate replace the solvent by HPLC grade wa
11. cid or acetic acid can be used It is not recommended to use acetic acid and its derivatives with Magic Diamond electrode The information how to acquire the MS Voltammogram and use the Scan or DC mode for metabolite synthesis are described in the ROXY Potentiostat User manual 210 7010 and Dialogue for ROXY user guide 210 7017 Table 1 Typical enzymatic CYP reactions that can be done electrochemically Compound indicated with asterisk is drawn as an example Table is adapted from Lohmann et al 2010 The electrochemical oxidation reactions are among others S oxidation N dealkylation hydroxylation and dehydrogenation 8 SynthesisCell user manual Enzyme catalyzed Electrochemically simulated Reference Allylic and aliphatic hydroxylation Tetrazepam requires high potential O OH R CH2 CH3 gt R CH CH3 Benzylic hydroxylation Metoprolol only in low yields 2 R R O Desalkylation of amines Amodiaquine lidocaine clozapine 3 4 C Z N CH2 R gt NH R CHO om NH phe Desalkylation of ethers Metoprolol dibutylsulphide low yield only under acidic conditions 2 5 R O CH2 R gt R OH R CHO ae Hydroxylation of aromatics Metoprolol mephenytoin diclofenac especially for aromatics 2 6 with electron donating groups gt a P N R R H OH Cl Epoxide formation Benzo a pyrene Not mimicked electrochemically f 4 LN TT a N ALN f C C gt C C gt C C or C C gy a TD a i i R i R R
12. ctrode without reference electrode the working potential would continuously change due to polarisation effects at the electrodes resulting in highly unstable working conditions If the working potential would be applied only over the reference electrode versus the working electrode without auxiliary electrode the working potential would be very well defined However the potential of a reference electrode is only well defined if the current drawn is extremely low pico amperes which woud make this configuration useless for our purpose A three electrode configuration combines the best of both configurations The reference electrode stabilises the working potential and the auxiliary electrode can supply high currents This results in the tremendous dynamic range of a three electrode system with sufficient yield in electrosynthesis HyREF reference electrode The SynthesisCell is standard equipped with a HyREF Pd Hz2 reference electrode The HyREF can be used under harsh conditions and is free of maintenance under normal use An important characteristic of the HyREF is the pH dependence of the reference potential It is important to realise that if the pH of the solvent is changed also the optimum conversion potential changes In such case it is advisable to re determine the optimum potential by creating a voltammogram 11 HyREF versus Ag AgCl Reference electrode The reference potential of an Ag AgCl and HyREF are different
13. gs FW update wizard E1 2 00 y E2 2 00 y E3 0 00 y ti 1000 ms t2 1000 ms t3 0 ms Users Marker PQ Noise Test OQ System A One Off I cell E Start Run ts 20 ms Stop Run Ctrl Q Clear All Shift Del Sequence F2 D2D System Suitability Fig 5 Start the activation procedure 19 After the activation procedure turn OFF the cell and leave the flow for additional 1 2min The activation procedure is available also as a program Activation _pulse_rev01 evt and can be executed automatically via events window Fig 22 The method can be found in My Documents and in the subfolders Dialogue Templates The detailed background information about the supplied events files and relevant Dialogue settings are provided in the Dialogue for ROXY User guide 210 7017 r ROXY Dialogue File Edit Tools Options Help Data Cell 1 Devices l Events e Events Activation_pulse_rev01 evt Connect_ Not connected time min parameter i Sort 0 00 Syr Flow Rate 20 ulmin Syr Pump Set the Detection mode to Add row 0 00 Syr Pump Start infuse Syr Pump Pulse mode first 0 05 Pulse E1 3 t1 3 ts 2 00 2 00 0 00 10C 00001 0 05 Cell on off on 00001 5 00 Cell on off off 00001 6 00 Syr Pump Stop Syr Pump status Output 6 10 End events Repeats 0 M Start stop with run C Users Agnieszka Kraj D ocuments Dialogue 10M ADC 2 Max 250 n Fig 6 Activ
14. h purposes only While clinical applications may be shown this instrument is not tested by the manufacturer to comply with the In Vitro Diagnostics Directive WEEE directive Antec Leyden is a Business to Business producer of analytical analysis equipment which fall under WEEE Annex IA categories 8 and 9 includes medical devices and monitoring and control instruments All equipment of Antec Leyden which are subjected to the WEEE directive shipped after August 13 2005 are labelled with the crossed out wheelie bin The symbol on the product indicates that the product must not be disposed as unsorted municipality waste Collection amp recycling information business to business Antec Leyden offers the possibility for disposal and recycling of their instrument at an appropriate recycling facility if requested there may be costs involved with this service Please contact Antec Leyden for more information about this service and to register the return and disposal of end of life instruments info myantec com To assure hygienic amp personal safety all instrument must be returned with a signed decontamination form which is available on the website Shipping address for end of life products Antec Leyden Industrieweg 12 2382NV Zoeterwoude The Netherlands ROHS directive Our instruments are currently exempt from the RoHS directive because they fall under WEEE Annex IA categories 8 and 9 which includes medical devices
15. isCell with RGC WE and HyREF 1 WE contact 2 AUX contact 3 HyREF contact 4 Additional port for gas purging or sampling 5 SynthesisCell cap 6 RGC WE 7 Glass reaction vessel This hardware should be used by trained laboratory personnel only Use proper eye and skin protection when working with solvents Additional safety requirements or protection may be necessary depending on the chemicals used in combination with this equipment Make sure that you understand the hazards associated with the chemicals used and take appropriate measures with regards to safety and protection The SynthesisCell is delivered pre assembled however some protectors for safe transportation must be removed before use 14 SynthesisCell user manual Never switch ON the flow cell when the cell cable is not correctly connected the cell is not filled with buffer electrolyte because substantial damage to the working electrode or electronics may occur Preparations 1 Take the SynthesisCell from the shipping box and remove the protecting foam parts 2 Prepare a solution containing the substance s and a supporting electrolyte ammonium formate formic acid or acetic acid 0 1 1 in water 3 Download and install the latest version of Dialogue software http www myantec com support documents and downloads download software Picture 2 A RGC WE fixed in the vessel cap B WE fixed in the glass reaction vessel C AUX in the f
16. pending on the substance s and the applied potential the cell current I Cell can go up to 2 20 mA Monitor the formation of product by taking aliquots from the vial through the sampling port A 0 1 mmol L 1 5 mg MOPEG solution is for 90 converted in about 40 min under optimized conditions When finished switch off the cell and collect the solution in another vial When mixing of electrode solutions is not allowed first take out the AUX without mixing 16 SynthesisCell user manual CHAPTER 3 Maintenance Working electrode maintenance Activation of the working electrode is necessary if the electrode surface has been electrochemically changed This may be due to fouling by oxidation reduction reaction products Excessively high currents also may change the electrode surface This is noticed by a strongly decreased sensitivity after prolonged use Decreased cell performance Several actions can be taken at decreased flow cell performance Take the next step only if the previous was not successful 1 Rinse the electrodes with clean solvent water with ca 50 organic solvent Clean wipe the REF and AUX with a wetted tissue with solvent 2 Electrochemical activation of the GC and MD electrodes using pulse mode see separate section of this manual 3 The re activation procedure for Diamond electrodes using scan mode see separate section of this manual Disassembly of the SynthesisCell If the working electrode
17. ritted glass tube D AUX assembled in the vessel cap Assembling the SynthesisCell 4 Fill the glass reaction vessel with a solution containing electrolyte s and substance s The maximum allowed volume is 80mL Put the stirrer in the vessel 5 Place the vessel cap on the WE and fix the WE position height with the retaining rings as indicated in the picture 2A 6 Place the WE in the vessel Picture 2B Adjust the position of WE if needed by careful pushing or pulling the white Teflon contacts The RGC Operation 11 12 13 14 15 15 WE is fragile and must be handled with care Keep the wires connecting the WE with the Teflon tubes dry Place the AUX electrode in the fritted glass tube Picture 2C and mount it in the vessel cap Picture 2D The AUX tube should be filled with mobile phase which takes a minute The fritted glass tube keeps the AUX separated from the WE reaction products on both electrodes are not mixed An additional tube without frit is available for cases where the mixing of products is allowed Place the HyREF in the vessel cap Picture 2D Place the SynthesisCell on the magnetic stirrer and adjust the rotation Connect the cell cable as shown in the picture 4 Red WE blue AUX and black REF Set the measurement conditions using Dialogue The user manual 210 7017 Dialogue for ROXY is available on our website The reaction is started as soon as the SynthesisCell is started De
18. s with regards to safety and protection Use of this product outside the scope of this guide may present a hazard and can lead to personal injury Spare parts and service availability Manufacturer provides operational spare parts of the instrument and current accessories for a period of five years after shipment of the final production run of the instrument Spare parts will be available after this five years period on an as available basis Manufacturer provides a variety of services to support her customers after warranty expiration Repair service can be provided on a time and material basis Contact your local supplier for servicing Technical support and training can be provided by qualified chemists on both contractual and as needed basis 6 SynthesisCell user manual Table of contents Safety practices 5 Spare parts and service availability 5 Table of contents 6 The SynthesisCell 7 Introduction 7 References 9 Three electrode configuration 10 HyREF reference electrode 10 HyREF versus Ag AgCl Reference electrode 1 1 Example 11 Working electrodes 11 Installation 13 Unpacking 13 Preparations 14 Assembling the SynthesisCell 14 Operation 15 Maintenance 16 Working electrode maintenance 16 Decreased cell performance 16 Disassembly of the SynthesisCell 16 Maintenance Magic Diamond electrode 17 Activation of the electrodes using pulse mode 17 Activation of the electrodes using scan mode in acidic conditions
19. sage overload and auxiliary potential Eaux will have the extreme value 9 9V Furthermore in case of EC MS the electrospray signal will become very unstable because of gas formed in the cell and loss of the signal can be observed When such a phenomenon is observed it is recommended to adjust the potential to a lower value Table 3 Working potential limits for electrodes WE used with SynthesisCell WE material potential limits vs HyREF V Glassy carbon 2 5V 1 5V Magic diamond 3 5V 2 5V The MD electrode consisting of an ultra thin film of doped diamond material deposited on a carrier The special properties of doped diamond electrodes such as a wide potential window their inertness and excellent response stability makes them well suited for electrochemical conversion of a wide variety electro analytical application The MD electrode is perfect choice if the oxidation of the analyte requires high potential in aqueous electrolytes 13 CHAPTER 2 Installation Unpacking Inspect the transport box for possible damage as it arrives Immediately inform the transport company in case of damage otherwise she may not accept any responsibility Contact your supplier in case of damage or if not all marked items on the checklist are included Prior to shipment your SynthesisCell has been thoroughly inspected and tested to meet the highest possible demands The results of all tests are included Picture 1 Synthes
20. st Anal Chem 79 6831 6839 2007 K G Madsen et al Chem Res Toxicol 21 1107 1119 2008 S M van Leeuwen H Hayen and U Karst Anal Bioanal Chem 378 917 925 2004 B Blankert et al Electroanalysis 17 1501 1510 2005 8 W Lohmann and U Karst Anal Bioanal Chem 386 1701 1708 2006 oF i e w 10 SynthesisCell user manual Three electrode configuration In the SynthesisCell a three electrode configuration is used Error Reference source not found The working potential is set between the working electrode and the auxiliary electrode AUX The auxiliary electrode is kept at a precisely defined reference electrode potential by means of the so called voltage clamp This is an electronic feedback circuit that compensates for polarisation effects at the electrodes At the working electrode which is kept at virtual ground the electrochemical reaction takes place i e electrons are transferred at the working electrode This results in an electrical current to the I E converter which is a special type of operational amplifier The output voltage can be measured by an integrator or recorder Essentially for the oxidation or reduction reaction it would be sufficient to use only two electrodes However the three electrode configuration has several advantages over a two electrode configuration If the working potential would be applied only over an auxiliary electrode versus the working ele
21. ter and flush wash the cell to remove the acidic solution 8 You can start up the synthesis again In the case there is no significant sensitivity improvement observed repeat step 1 to 10 and increase the total scan time step 7 One can also try to perform the activation procedure under the mobile phase conditions of the application Furthermore in literature anodic treatments are reported for example the application of a static potential of 3 Volt vs Ag AgCl for 5 10 minutes to restore the electrode response Such procedure could be executed as an alternative if the above mentioned procedure does not lead to satisfactory results Electrochemical reactivation procedure for MD electrode is part of the ROXY firmware and available via display panel Diag Activate The scan settings and the duration are as described in points 6 and 7 and only push button action is needed to execute it See ROXY Potentiostat user manual 210 7010 Replace the Magic Diamond electrode disc when no improvement is seen after repeated reactivation conditioning attempts Chemical compatibility The MD electrode exhibits an excellent inertness and can be used with a large variety of mobile phase and chemicals However it has been observed that the MD electrode operational lifetime is strongly reduced when exposed to fluorinated acids such as tri fluoroacetic acid Even at relatively low concentrations 2 in aqueous solution significant damage of
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