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VT03 flow cell user manual

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1. so po ooo so e eooo o wo ooo S C e e C i The addition of chloride to the mobile phase has a few restrictions For example the ISAAC is not recommended at a high working potential gt 1 2 V vs Ag AgCl in 2 mmol l KCI because CI is oxidised and contributes to the background current In ion chromatography the addition of Cl may lead to undesired chromatographic changes In case of a silver working electrode the addition of CI to the mobile phase will cause formation of an AgCI coating on the working electrode leading to inactivation At high pH or high modifier concentrations the ISAAC is less suitable and a HyREF is recommended 14 VTO3 flow cell user manual ed 8 Ag AgCl KCI sat d KCI crystals cotton wool Fig 7 Schematic representation of the Ag AgCl reference electrode Salt bridge Ag AgCl reference electrode The reference electrode of the Ag AgCl type with salt bridge consists of a silver rod coated with solid AgCI immersed in a solution of saturated KCI containing KCI crystals Electrical contact with the other electrodes in the flow cell is made through a salt bridge consisting of a wetted cotton wool frit which is electrically conducting and slows down leakage of KCI This REF for the VT 03 flow cell is factory filled with KCI For certain applications another chloride salt is to be preferred In case of perchlorate containing mobile phases sodium chloride is mandatory because potassium perchlora
2. Antec Industrieweg 12 2382 NV Zoeterwoude The Netherlands VT 03 Electrochemical Flow cell User manual 110 0010 Edition 9 2012 Q so M 9001 T 31 71 5813333 F 31 715813334 info myantec com www myantec com certified Copyright 2012 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 INTRO Flexcell 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 Table of contents Table of contents 3 The electrochem
3. 5 working electrode 5 6 working electrode diameter 9 working potential limits 7
4. EC it affects both the signal and the noise For the VT 03 flow cell several glassy carbon WE diameters are available 0 7 2 and 3 mm In a standard LC system the signal and the noise increases linearly with the WE diameter This means that he S N ratio remains more or less the same In case of micro LC an increase of the WE diameter will increase the noise more than the signal Therefore in micro LC a decrease of the WE diameter will result in a better S N ratio Table III Flow cell recommendations Column diameter mm Recommended flow cell 3 and higher 0 7 mm HEC The choice for a flow cell is primarily based on the HPLC column diameter Table Ill This way the best possible detection limit for a standard microbore or capillary column is warranted The recommended combinations are giving the best S N ratios It should be kept in mind that other combinations are possible that still result in acceptable sensitivities for many applications All VT 03 flow cells are individually tested and meet our high standards of quality and detection sensitivity Spacer thickness The thickness of the gasket affects the linear flow velocity in the cell With a thinner spacer the cell volume is decreased Table IV resulting in a higher linear flow velocity The signal increases with thinner spacers while the noise remains more or less constant Fig 4 Several authors have described the relation between the layer thickness i e spacer thick
5. vibrations This is caused by the high compressibility of the trapped air 30 VTO3 flow cell user manual ed 8 Check your REF regularly If you do not see chloride salt crystals or if you see air bubbles your REF needs maintenance Material 1 An over saturated and thoroughly degassed KCI solution 2 A stainless steel rod of about 5 cm length and a diameter of 1 mm e g a 1mm drill 3 Ordinary cotton wool Procedure Use proper eye and skin protection when working with solvents Turn the cell OFF on the controller Stop the HPLC pump Disconnect the cell from the controller Remove the REF from the inlet block Disassemble the REF by unscrewing the black swivel Fig 23 Inspect the Vyton rings for wearing and especially the cotton wool frit replace if required see below NOa PWN gt Fig 23 Exploded view of the reference electrode The arrow indicates the tip of the AgCI coated silver rod See Table VII for description and numbers 8 Remove the remaining KCI from the salt bridge 9 Clean all parts with demi water 10 The Ag AgCl electrode must be cleaned if the silver on the tip CHAPTER4 Maintenance 31 11 Fig 23 arrow has a non metallic appearance by gently grinding it on sanding paper also the AgCl can be gently resurfaced in this way 12 The frit in the salt bridge ensures electrical contact with the buffer If the frit is discoloured or dried out it has to be renewed In that c
6. AUX and REF are connected using the red blue and black cell cable The LC outlet is placed on top to prevent entrapment of bubbles connector Fig 11 Installation of flow cell DECADE II WORK AUX and REF are connected using the red blue and black cell cable The LC outlet is placed on top to prevent entrapment of bubbles CHAPTER 3 Installation 19 A N Use proper eye and skin protection when working with solvents 1 Check the REF visually for air bubbles and saturation with KCI Some KCI crystals should be visible When no crystals are visible or air bubbles are trapped the REF needs maintenance see page 29 To prevent drying out the REF is sealed with a cap on arrival Remove the cap 2 Tighten the black swivel of the REF a small droplet should appear at the cotton wool frit Do not remove this droplet because it ensures proper contact of the REF with the mobile phase 3 Turn on the HPLC pump Place some tissues as some mobile phase may be spilled during this mounting procedure Connect the column outlet to the flow cell inlet using small bore PEEK tubing 0 3 mm ID and one of the fingertights supplied Use only our factory supplied M fingertights in the flow cell others may cause serious damage Let the tubing protrude for ca 1 5 cm from the fingertight and tighten it carefully n 5 1 5 cm Over tightening affects the flow through the tubing turbulence and de
7. ace Finally it is important that the analyte of interest can be oxidised or reduced with favourable I E characteristics This in fact means that a high signal must be obtained at a low working potential For most applications glassy carbon will be the WE material of choice Under certain circumstances other materials are favourable For example for the analysis of iodide a silver WE can be used At the silver WE the following oxidation reaction occurs for iodide Ag l gt Agl e This reaction already takes place at a very low working potential 1 mV which results in an extremely high selectivity This allows the determination of iodide in urine samples to take place almost without any sample pre treatment CHAPTER 1_ The electrochemical flow cell 7 Table Working potential limits and application area for different WE materials WE material parental limits V major application Glassy carbon catecholamines Gold i i carbohydrates Platinum alcohols glycols Silver i f halides cyanide Copper amino acids carbohydrates Another consideration in choosing a WE is the oxidation or reduction of mobile phase constituents or WE material that occurs when the potential exceeds the limits as given in Table At high positive working potentials the water in the mobile phase electrolyses and results in an strong increase of the background current and noise Formation of metal oxides resulting in an increase in backgro
8. ase continue with Maintenance of the cotton wool frit step 1 Otherwise continue with step 7 Table VII REF parts and description Item Description REF cap for storage and shipment Vyton ring large Salt bridge Vyton ring small Ag AgCl electrode fitting Swivel for REF OORUN Maintenance of the cotton wool frit N Use proper eye and skin protection when working with solvents 1 Use a drill of 1 mm 0 039 inch to push out the frit from the outside Fig 24 Be careful not to damage the frit constriction first arrow Fig 24 Pushing the cotton wool frit out 2 Clean the salt bridge thoroughly by tap water and demi water respectively 3 Saturate a small piece of cotton wool in saturated KCI to exclude trapping of air within the wool 4 Plug the salt bridge with the REF cap and fill the salt bridge for 50 32 VTO3 flow cell user manual ed 8 5 Use the drill to pack the wool from above through the KCI solution into the channel of the salt bridge compress it firmly but not too much since electrical conduction is essential 6 Remove the cap 7 Fill the salt bridge completely add some KCI crystals out of a saturated solution to ensure prolonged saturation 8 Place the small Vyton ring over the Ag AgCl electrode and slowly insert it in an angle of 45 into the salt bridge Make sure not to enclose an air bubble 9 Tighten the black swivel such that a small droplet appears at the end
9. by an integrator or recorder voltage potential l E converter clamp V uit spacer AUX WE LC out Fig 2 Schematic representation of an electrochemical cell with a three electrode configuration 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 AUX versus the WE without REF the working potential would continuously change due to polarisation effects at the electrodes resulting in highly unstable working conditions 6 VTO3 flow cell user manual ed 8 If the working potential would be applied only over the REF versus the WE without AUX the working potential would be very well defined However the potential of a REF is only well defined if the current drawn is extremely low pico amperes resulting in a very limited dynamic range A three electrode configuration combines the best of both electrodes The REF stabilises the working potential and the AUX can supply high currents This results in the tremendous dynamic range of a three electrode system Working electrode Electrochemical detection puts high demands on the WE material The WE 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 surf
10. creases the flow cell performance 4 Connect 0 5 mm ID PEEK tubing to the outlet of the flow cell Use only our factory supplied fingertights in the flow cell others may cause serious damage Again see above do not over tighten the fingertight 5 Fill the flow cell by keeping it in an angle of 45 with the REF fitting on top is best done by blocking the outlet with a finger and letting the air escape via the REF fitting Carefully check the thread of the fitting for trapped air bubbles 6 When the REF fitting is completely filled with mobile phase mount the REF while slowly releasing the outlet Make sure not to include an air bubble 7 Position the flow cell in its clamp in the controller with the REF at the lower side and the outlet at the upper side This excludes trapping of air bubbles by the REF 8 Connect the cell cable as illustrated in Fig 10 The maximum detection stability is attained when not only the flow cell but also the HPLC column is incorporated in the controller The controller has an integrated Faraday cage and an accurately thermostatted oven compartment which ensures stable working conditions Installing the flow cell and column 20 VTO3 flow cell user manual ed 8 within such a controlled environment is the minimum requirement for high quality LC EC trace analyses Never switch ON the flow cell when N the cell cable is not correctly connected the cell is only partly or not at all filled with b
11. e drop will destroy column performance and pulse dampener CHAPTER 3 Installation 25 fused silica capillary Teflon sleeve glass mounting plate Fig 19 Mounting of the fused silica connector in the VT 03 micro flow cell The capillary connector is not necessary with a 1 mm or larger ID column In that case use narrow bore PEEK tubing lt 100 um ID and install as described on page 16 ISAAC REF or on page 18 salt bridge REF To prevent any damage to the flow cell the following steps should be carried out Fig 19 1 Connect the FS capillary to the pump by passing the column and switch on the pump to check that the capillary is open Liquid droplets must come out Otherwise replace with new piece of 100 um FS tubing Note that both endings of the supplied FS capillary are cut and polished If the capillary is open first switch off the pump and wait till the pressure is zero Disconnect the capillary from the pump Insert the fused silica capillary into the tightly fitting Teflon sleeve supplied Protrude both through our factory supplied fingertight fitting Use only our factory supplied fingertights in the flow cell others may cause serious damage J 15cm i Mount this combination carefully in the injection block Fig 19 Let the fused silica slightly lt 0 5 mm protrude through the injection hole Clean the factory supplied glass mounting plate from particles 26 VTO3 flow ce
12. electrode ISAAC reference electrode The ISAAC reference electrode is in direct contact with the mobile phase which contains chloride ions The chloride concentration determines the potential therefore each time a fresh mobile phase is prepared it should contain exactly the same concentration of chloride ions The standard electrode potential of the Ag AgCl electrode in 1 0 mol l CI solution for the following half reaction is defined as E AgCl s lt gt Ag s CI E 0 222 V The potential of the REF is dependent from the chloride concentration as described by the following equation RT Bagh Beeps T In CT cell where R is the gas constant 8 314 Jmol K T is the absolute temperature 293 K and F is the Faraday constant 96485 Cmol The potential of the ISAAC at 2 mmol l KCI is 379 mV Table V The potential difference dE between the saturated KCI Ag AgCl reference electrode and the ISAAC is 189 mV If an application is running at 800 mV vs Ag AgCl with sat d KCl the potential setting using the ISAAC should be 611 mV vs Ag AgCl in 2mmol KCl CHAPTER 2_ Reference electrodes 13 0 4 E cell V 0 3 0 2 0 1 6 1 0 2 0 3 0 KCI mol l Fig 6 Dependence of the Ag AgCI REF potential on the chloride concentration Table V Potential of the Ag AgCl reference electrode dE is the potential difference with Epgagc in saturated KCI CI mmol l E Ag AgCl mV _ dE mV
13. g 8 Never switch ON the flow cell when the cell cable is not correctly connected the cell is only partly or not at all filled with buffer electrolyte the outside of the flow cell is wet particularly the part between the auxiliary and working electrode connection because substantial damage to the working electrode or electronics may occur The maximum detection stability is attained when not only the flow cell but also the HPLC column is incorporated in the controller The controller has an integrated Faraday cage and an accurately thermostatted oven compartment 18 VTO3 flow cell user manual ed 8 which ensures stable working conditions Installing the flow cell and column within such a controlled environment is the minimum requirement for high quality LC EC trace analyses VT 03 flow cell with salt bridge REF The flow cell is assembled properly when it arrives The force on the bolts is pre set to 13 Necm a little bit beyond fingertight Familiarise yourself with this force since over tightening of the bolts strongly deteriorates the S N ratio and eventually the cell itself Also be aware that the black marks on both blocks should be in line This ensures the best performance For instructions about assembling the cell refer to page 20 LC in gt i inlet WE block black REF SHIELD P O O yellow Fig 10 Installation of flow cell in Intro or DECADE WORK
14. ical flow cell 4 Introduction 4 Three electrode configuration 5 Working electrode 6 Detection limit 7 Working electrode diameter 9 Spacer thickness 9 Reference electrodes 12 ISAAC reference electrode 12 Salt bridge Ag AgCl reference electrode 14 HyREF reference electrode 14 Installation 16 VT 03 flow cell with HyREF or ISAAC 16 VT 03 flow cell with salt bridge REF 18 VT 03 micro flow cell 21 Assembling the micro flow cell 21 Capillary connections 24 Maintenance 28 HyREF 28 ISAAC 28 Polishing 28 Ag AgCl salt bridge 29 Saturation and air bubbles 29 Material 30 Procedure 30 Maintenance of the cotton wool frit 31 Working electrode 32 Decreased flow cell performance 32 Polishing 33 Index 34 4 VTO3 flow cell user manual ed 8 CHAPTER 1 The electrochemical flow cell Introduction The VT 08 flow cell is available with a glassy carbon platinum gold silver and copper working electrode In combination with the spacer set 25 50 and 120 um a variety of detection volumes down to 5 nl can be attained As a standard the salt bridge Ag AgCl reference electrode is advised For special applications the HyREF reference electrode is available A third reference electrode is the in situ Ag AgCl ISAAC IN w REF Fig 1 The VT 03 electrochemical flow cell The upper part the inlet block is separated from the working electrode block by means of a gasket spacer not shown The VT 03 electrochemical f
15. ined as the analyte concentration that results in a signal that is 3 times the standard deviation of the noise 3 O ioie E Crop signal where sigma noise is 0 2 x peak to peak noise and c4 is the concentration of analyte injected In Fig 3 a typical S N ratio of a VT 03 glassy carbon flow cell with 2 74 mm WE is shown In this example the concentration detection limit for norepinephrine based on three times the sigma noise is 11 pmol l see Table Il for conditions Expressing the performance of a detection system by only the peak height makes no sense A system can easily be changed in a way that a larger peak height is obtained However if the noise increases similarly it has the same effect as switching a recorder to a higher sensitivity peaks appear higher but the S N ratio is the same Expressing the limit of detection in an absolute amount i e in picomoles without mentioning the injection volume makes a good comparison between different systems difficult 20m peak to peak noise ethos 1 5 pA 15 18 24 24 27 30 t min Fig 3 Typical S N ratio for norepinephrine measured with a VT 03 glassy carbon flow cell peak height 80 nA peak to peak noise 1 5 pA The CHAPTER 1 The electrochemical flow cell amount injected is 20 pmol 1 0 umol The concentration detection limit based on three times the sigma noise is 11 pmoll Working electrode diameter The size of the WE is an important factor in LC
16. ing Fig 21 1 A N firmly to avoid snapping out of the capillary Sudden pressure drop will destroy column performance and pulse dampener CHAPTER 3 Installation clearing air bubbles outlet on top pump Ww Fig 21 Filling a VT 03 micro flow cell at 200 400 uL min using a low dead volume connector 1 Pressure must not rise gt 200 bar Read column instructions first The flow cell is then filled at a higher flow rate After filling the column is reconnected Be careful not to include air bubbles i Never switch ON the flow cell when the cell cable is not correctly connected the cell is only partly or not at all filled with buffer the outside of the flow cell is wet particularly the part between the auxiliary and working electrode connection because substantial damage to the working electrode or electronics may occur 27 28 VTO3 flow cell user manual ed 8 CHAPTER 4 Maintenance HyREF ISAAC The HyREF reference electrode is in principle maintenance free If not in use it should be stored dry after disassembling the flow cell The ISAAC reference electrode requires maintenance usually not more than once in 3 months In practice this means that when the flow cell is opened to service the working electrode the reference electrode should be serviced as well A B reference electrode solution EL AM reference electrode surface Fig 22 Servicing the ISAAC reference e
17. issue wetted with ethanol or acetone CHAPTER4 Maintenance 33 3 Polishing the electrode surface Polishing 1 2 D Shake diamond slurry thoroughly before use Rinse the polishing disc with demi water before applying the diamond slurry Apply a small amount a few drops is sufficient of slurry on the wetted polishing disc and polish the electrode with a figure 8 motion for about one minute Apply only gentle pressure Clean the electrode with an ethanol wetted tissue and check the surface visually repeat the procedure if necessary Reassemble the detector cell Clean the polishing disc with demi water Store the polishing disc dust free in its plastic bag 34 VTO3 flow cell user manual ed 8 CHAPTER 5 Index CHAPTER 5 Index 35 Ag AgCl reference electrode parts 29 standard electrode potential 12 auxiliary electrode 5 detection limit 7 flow cell volume 11 HyREF installation 16 reference electrode potential 14 I E converter 5 installation micro flow cell 21 installation flow cell 16 ISAAC installation 16 reference electrode potential 12 maintenance cotton wool frit 29 HyREF 26 ISAAC 26 salt bridge 27 working electrode 30 micro flow cell installation 21 parts Ag AgCl reference electrode 29 polishing 26 31 reference electrode 5 12 salt bridge 14 installation 18 signal to noise ratio 7 spacer thickness 9 three electrode configuration 5 voltage clamp 5 warranty
18. lectrode polishing A and coating B Servicing the reference electrode is done by polishing the reference electrode surface until the shining metal appears Fig 22A Immediately after polishing the electrode is coated by applying a few drops of the reference electrode solution on the reference surface Fig 22B After 20 min the reference solution is flushed away with distilled water If not in use for longer period of time disassemble the flow cell The flow cell including the reference electrode should be cleaned with distilled water dried with a tissue and stored dry Polishing Polishing the reference electrode is done using the factory supplied polishing kit containing diamond slurry and polishing disc 1 Shake diamond slurry thoroughly before use CHAPTER4 Maintenance 29 2 Rinse the polishing disc with demi water before applying the diamond slurry 3 Apply a few drops of slurry on the wetted polishing disc and polish the electrode with a figure 8 motion for about one minute Apply only gentle pressure 4 Clean the electrode with a wetted tissue and check the surface visually repeat the procedure if necessary until the shining metal REF surface appears 5 Clean the polishing disc with demi water 6 Store the polishing disc dust free in its plastic bag Ag AgCl salt bridge Three aspects determine the proper function of an Ag AgCl reference electrode 1 The chloride concentration must be kept at a st
19. ll user manual ed 8 8 Carefully push the block on the glass plate until the silica capillary is flush with the surface 9 Fix the fused silica capillary firmly with the fingertight while keeping a slight pressure of the block on the glass plate 10 Mount the two flow cell blocks by crosswise tightening of the bolts max 13 Nem 1 am A B TT T Fig 20 Different column connectors with micro flow cell Column type A has no ferrule but ends with fused silica 1 Column type B has a ferrule connector 1 On the left A the standard installation with capillary connector 2 This is suitable for fused silica columns On the right B installation with a ferrule and sleeve connected to the column 11 Connect the other end of the capillary connector as shown in Fig 20 Use either a ferrule with sleeve Fig 20B or couple to the capillary column using the connector sleeve Fig 20A 2 If leakage occurs a low dead coupler can be used with fingertights and sleeve 12 Continue installation as described at point 3 on page 17 ISAAC REF or at point 1 skip point 3 on page 19 salt bridge REF depending on the type of reference electrode used NB To speed up the filling of the micro flow cell the cell can be connected directly to the HPLC thus by passing the column When disconnecting the column always slowly release pressure or damage to the column will occur Tighten low dead coupl
20. low cell has been developed for ultra trace analysis in standard microbore and capillary LC EC After extensive testing it was established that the confined wall jet configuration gave the very best results In addition it was found that the electrode materials quality and the finishing of the electrodes in the flow cell are decisive factors for the performance of an EC detector While competitive designs usually deteriorate when in use this flow cell by design improves in performance The flow cell permits unusually short stabilisation times trace analysis within half an hour CHAPTER 1_ The electrochemical flow cell 5 after starting up may be expected We have so much confidence in our flow cell that we warrant the glassy carbon flow cell for a period of 5 years Three electrode configuration In the VT 03 flow cell a three electrode configuration is used Fig 2 The working potential is set between the working electrode WE and the auxiliary electrode AUX The AUX is kept at a precisely defined reference electrode REF potential by means of the so called voltage clamp This is an electronic feed back circuit that compensates for polarisation effects at the electrodes At the WE which is kept at virtual ground the electrochemical reaction takes place i e electrons are transferred at the WE 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
21. minimize the change of scratching the inlet block surface Then turn the inlet block downwards in horizontal position The inlet block will now lay on top of the WE block with the bolds positioned on the mounting holes The black positioning markers on the sides of the blocks should now be aligned in vertical position Fig 17 Inlet block positioned on top of WE electrode note that black positioning markers are aligned in vertical position 4 Hold the complete cell together with the glass mounting plate and turn it up side down again Take of the glass plate and position it underneath the cell again Tighten the bolds in a crosswise pattern with the hex key tightening force 13 Ncm Now the cell is ready for use again 24 VTO3 flow cell user manual ed 8 Fig 18 Fixing the two cell blocks with the hex key tighten the bolds ina crosswise pattern Capillary connections The micro flow cell is supplied with a low dead volume fused silica capillary connection for coupling with capillary columns lt 1 mm ID fused silica columns as a standard N Use proper eye and skin protection when working with solvents If the capillary connection has already been installed properly continue with step 11 If not start with step 1 When disconnecting the column always slowly release pressure or damage to the column will occur Tighten low dead coupling Fig 21 1 A N firmly to avoid snapping out of the capillary Sudden pressur
22. ness in a thin layer flow cell and the measured current S as S k b where b is the spacer thickness and k a constant Also for the VT 03 flow cell the relation between S and b results in a straight line Fig 5 10 VTO3 flow cell user manual ed 8 Fig 4 The signal and noise for 1 0 umol nor epinephrine measured at variable spacer thickness given in um See Table II for other conditions 200 100 0 02 0 04 0 06 0 08 0 10 0 12 b 2 3 Fig 5 Peak height versus spacer thickness to the power 2 3 Decreasing the spacer thickness is limited by an increased pressure drop over the flow cell which eventually will lead to an obstruction of the flow The minimum spacer thickness available is 25 um Applying these small spacers should be done with care Over tightening of the bolts may cause an excessive pressure built up over the flow cell and increase the noise considerably CHAPTER 1_ The electrochemical flow cell 11 Table IV Flow cell volume WE diameter mm 3 00 2 74 2 54 2 00 1 90 1 00 0 75 0 50 spacer um 25 50 120 cell volume ul 0 35 0 29 0 25 0 16 0 14 0 039 0 022 0 010 0 85 0 71 0 61 0 38 0 34 0 094 0 053 0 024 0 18 0 15 0 13 0 08 0 07 0 020 0 011 0 005 12 VTO3 flow cell user manual ed 8 CHAPTER 2 Reference electrodes The VT 03 flow cell is available with an ISAAC in situ Ag AgCl reference electrode a salt bridge Ag AgCl reference electrode and a HyREF reference
23. of the salt bridge but do not over tighten the swivel 10 Flush the complete mounted REF with demi water dry it with a tissue but keep the cotton wool frit soaked 11 Carefully inspect the REF visually for trapped air bubbles otherwise remove them go back to step 7 or if necessary step 1 A When not in use please store the REF with the cotton wool frit immersed in a saturated KCI solution to prevent drying out Working electrode Cleaning of the working electrode block 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 As a rule of thumb only polish if the surface of the working electrode lacks its mirror like finish which cannot be restored by wiping the electrode surface with a tissue wetted with ethanol or acetone Decreased flow cell performance N Use proper eye and skin protection when working with solvents Several actions can be taken at decreased flow cell performance Avoid unnecessary polishing take the next step only if the previous was not successful 1 Electrochemical cleaning of glassy carbon WE In the pulse mode let the potential jump between 1 and 1 V for 10 min Settings t1 1000 ms t2 1000 ms t3 O ms E1 1V E2 1V 2 Wiping the electrode surface with a t
24. rictly fixed level This is best guaranteed by using a saturated chloride salt solution at a constant temperature 2 The salt bridge must allow proper electrical contact with the mobile phase The higher the leakage through the frit the better the conduction This conflicts with the previous point 3 Air bubbles inside or close to the salt bridge will lead to instability of the three electrode configuration Because of their extreme compressibility changes in conductivity and the ionic equilibrium of the REF occur This increases the noise considerably The REF is factory filled with KCI unless specified otherwise Other chloride salts should be used when the mobile phase contains perchlorate use NaCl or a high percentage of organic modifier use LiCl Saturation and air bubbles After prolonged use the salt bridge in the REF will not be saturated any more which usually leads to a poor reproducibility in electrochemical detection The potential of the REF is determined by the chloride concentration see page 13 If the salt bridge is not saturated and the KCI concentration changes 1 the noise in the system will slowly but continuously increase 2 the background current will increase 3 sensitivity for movements and pump noise will increase If an air bubble is trapped in the salt bridge or in the cotton plug that separates the salt bridge and the mobile phase the flow cell becomes extremely sensitive towards flow fluctuations and
25. te precipitates and will clog the cotton wool frit At high modifier percentages the REF must be filled with lithium chloride for similar reasons HyREF reference electrode The HyREF is a hydrogen reference electrode its potential depends on the pH of the mobile phase The HyREF is fully comparable with the standard Ag AgCl REF as to baseline stability and S N ratio The HyREF is more user friendly and in principle this REF is completely free of maintenance Trapping of air bubbles like in the salt bridge Ag AgCl type is impossible because of the absence of a salt bridge Consequently refilling the REF with saturated KCl is not longer required Due to the absence of a salt bridge and its inertness the HyREF is an excellent alternative for the Ag AgCl REF especially in case of high modifier concentrations i e analysis of fat soluble vitamins or high pH analysis of carbohydrates PAD Depending on the pH of the mobile phase the potential setting of the working electrode vs the HyREF may differ significantly compared to Ag AgCl I E curves show a shift of more than 200 mV at pH 3 1 e g catecholamines no shift appears at pH 12 e g PAD of carbohydrates Therefore it is CHAPTER 2_ Reference electrodes 15 advisable first to construct a hydrodynamic or scanning voltammogram when using the HyREF In Table VI the potential of the HyREF is measured against the Ag AgCl in sat d KCI electrode at different pH values Table VI Meas
26. trode requires 2 mmole chloride ions KCI or A N NaCL in the mobile phase Add and equilibrate before installation of the ISAAC See manual electrochemical detector for optimisation of working potential 1 Connect the column outlet to the flow cell inlet using small bore PEEK tubing 0 3 mm ID and one of the fingertights supplied Use only our factory supplied fingertights in the flow cell others may cause serious damage Let the tubing protrude for ca 1 5 cm from the fingertight fitting and tighten it such that the tubing is not or slightly indented by the fitting 1 5 cm 2 Do not over tighten the fingertight Over tightening affects the flow pattern through the tubing turbulence and may strongly decrease the flow cell performance 3 Connect 0 5 mm ID PEEK tubing to the outlet of the flow cell Use only our factory supplied fingertights in the flow cell others may cause serious damage Again see above do not over tighten the fingertight 4 Turn on the HPLC pump Keep some tissues at hand as you probably will spill some mobile phase during this mounting procedure 5 Fill the flow cell by keeping it in an angle of about 45 with the outlet LC out on top to force the air through the outlet 6 Position the flow cell in its clamp in the controller with the REF at the lower side and the outlet at the upper side This excludes trapping of air bubbles 7 Connect the cell cable as illustrated in Fi
27. uffer the outside of the flow cell is wet particularly the part between the auxiliary and working electrode connection because substantial damage to the working electrode or electronics may occur When not in use please store the REF with the cotton wool frit immersed in a saturated KCI solution to prevent drying out Fig 12 Always install flow cell with outlet on top CHAPTER 3 Installation 21 VT 03 micro flow cell The micro flow cell is assembled properly when it arrives The force on the bolts is pre set to 13 Ncm a little bit beyond fingertight Familiarise yourself with this force since over tightening of the bolts strongly deteriorates the S N ratio and eventually the cell itself Also be aware that the black marks on both blocks should be in line This ensures the best performance Assembling the micro flow cell The VT 03 micro flow cell has an 0 7 mm WE and is equipped with a 25 um spacer Older models of the VT 03 micro are equipped with a metal centring ring In that case both blocks WE and inlet block have an edge groove to facilitate the metal ring and are fixed in the metal ring to align the inlet in the centre of the WE ANTEC LEYDEN Fig 13 Disassembled VT 03 micro flow cell with spacer Note new VT 03 micro cells purchased after 2009 are delivered without metal centring ring and have no groove in WE and inlet block To assure proper installation and optimal performance please follo
28. und current is a limiting factor for metal electrodes Glassy carbon and platinum have the highest positive potential limits and are therefore often used in oxidative ECD For negative potentials the use of platinum electrodes is limited by the ease of reducing hydrogen ions to hydrogen gas Detection limit One of the most important parameters used to characterise the performance of a detection system is the signal to noise ratio S N ratio from which the concentration detection limit is derived It enables objective comparison not only between different electrochemical detectors but also between complete analytical methods irrespective what detection system is used Table II LC EC conditions for analysis of norepinephrine column ODS 2 3 um 100 x 4 6 mm flow rate 1 0 ml min mobile phase H3PO 50 mM citric acid 50 mM 20 mg l EDTA 100 mg l octane sulphonic acid OSA pH 3 1 with KOH 5 methanol sample 1 0 umol I norepinephrine 20 yl injection temperature 30 C flow cell VT 03 flow cell with 3 mm GC WE mounted with 50 um spacer E cell 800 mV vs Ag AgCl filled with saturated KCl leel ca 3 nA 8 VTO3 flow cell user manual ed 8 In literature several ways are described to determined the detection limit In principle it does not matter which definition of detection limit is used as long as the definition is precisely described In this manual the concentration detection limit CLop for a certain compound is def
29. ured cell potential HyREF Ag AgCl versus pH Epyrer Ag agci MV 7s eo mse fo So if an Ag AgCl REF is replaced by a HyREF the pH effect must be taken into account Table VI The pH vs voltage relation is described by EnyReF Engiagcl 328 29 9 pH 1 For example a working potential of 800 mV vs Ag AgCl with sat d KCl at pH 3 has to be changed to Enyrer 800 328 29 9 3 561 7 mV vs HyREF 16 VTO3 flow cell user manual ed 8 CHAPTER 3 Installation VT 03 flow cell with HyREF or ISAAC The flow cell is assembled properly when it arrives The force on the bolts is pre set to 13 Nem a little bit beyond fingertight Familiarise yourself with this force since over tightening of the bolts strongly deteriorates the S N ratio and eventually the cell itself Also be aware that the black marks on both blocks should be in line This ensures the best performance For instructions about assembling the cell refer to page 20 yellow Fig 8 Installation of flow cell Intro or DECADE WORK AUX and REF are connected using the red blue and black cell cable LC out should be on top to prevent entrapment of bubbles connector Fig 9 Installation of flow cell DECADE II WORK AUX and REF are connected using the red blue and black cell cable LC out should be on top to prevent entrapment of bubbles CHAPTER 3 Installation 17 The ISAAC reference elec
30. w the instruction below for assembling of a VT 03 micro flow cell It is also strongly advised to assemble normal VT 03 flow cells 2 mm and 3 mm GC using a similar procedure Mounting the cell in another manner may lead to damage of the spacer and flow cell blocks For cells without metal centring ring discard skip the sections in the procedure related to placement of the centring ring 22 VTO3 flow cell user manual ed 8 1 Hold the WE block in horizontal position with the WE facing downwards and insert the 4 bolds in the 4 mounting holes of the WE block Place the glass mounting plate on top of it turn the glass plate with WE block up side down and place it on a flat surface table Fig 14 WE block with inserted bolds on the glass mounting plate 2 Place the metal ring on the WE block Make sure that you hold the metal ring horizontally when pushing it onto the block Subsequently gently place the 25 um spacer on top of the WE block with bolds using a pair of tweezers The spacer should lay flat on the WE surface with the bolds centred in the spacer bold holes Fig 15 Placement of 25 um spacer on the WE block 3 Place the inlet block on top of the WE block as depicted in the next figure When placing the block keep it tilted and gently try to position the two bold holes in the inlet block on top of the bolds without CHAPTER 3 Installation 23 touching the surface of the inlet block with the bold ends this will

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