Titration-Injection microPump. TIP-2k User Manual
Contents
1. OROBOROS INSTRUMENTS high resolution respirometry Oxygraph 2k Manual 1 3 A Mitochondr Physiol Network 12 10 1 28 2010 2007 2010 OROBOROS Oxygraph 2k Series B D DatLab 4 3 Titration Injection microPump TIP 2k User Manual Mario Fasching Lukas Grad Philipp Gradl Erich Gnaiger OROBOROS INSTRUMENTS Corp high resolution respirometry Schopfstr 18 A 6020 Innsbruck Austria T 43 512 566796 E mail erich qnaiger oroboros at http www oroboros at 1 TIPS 2K Maitl Features sssciosnonianennn aaa aAA 2 2 Setting p th TIP 2K snra a a A A 2 2de FIP 2Kk COMPONEN S rrine ni e Ea 2 D2 MIVSU ANAL Ol a E E A A P A E A E A 3 2 3 Manual Operation sssesssssssssrrrrsrrnrsrrrrrrnrrrrnrrnrrrnnsrrrrnrsrrnrnrrnrnrnrrrenrrrrrrnrerre 3 2s S Y cataauneiesdeuuciee mors cece E oe dine diane EEEE sue oak 4 3 Filling and Mounting the Syringe ccccccccccccccccececeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeens 4 3 1 Filling the TIP SYriNGe ccccccceneceeessencceeencceneessccuuuucaveussanecusaureeneesseuuuuaaeeessogs 5 3 2 Mounting a Filled TIP Syringe cicossciasipsccenacasdacetsaiedenardeesdewisenaanes sterdiierenwriens 7 3 3 Inserting the TIP Needle into the Oxygraph 2k Chamber ccccceeeeseeeeeeeeeeeas 7 3 4 Washing the TIP Needle after USC cccccccccsscesseeeereeeesecessennnteesesseguaaanneeesnngs 8 3 5 Prevention of TIP Needle Clogging cece cccceeee eee e eect eeeeeeeeeeeeeeeeeeeeageaeeeee2. A Select VoFow Volume Memaga TPbackwad Tuy pe vae Dan Acton Pause il gt Vol Time Flow l s 36 006 gt 02 Concentration 4 nmol lt 10 2 Start 600 Flow Time Time s 6 17 Test start v Feedback line Delete Insert Program line Insert Replace Delete Move up Move down Line Mode Del s Vol ul Flow ul Feedback Guantity Value gt a 10 5 000 0 17 3000 l 30 000 02 Concentration A nmol ml lt Injection Volume Flow and Time are entered as described above However if a stop criterion is used only the Flow will be maintained absolutely Time and Volume are rather maximal values for each individual injection i e an injection is stopped when either the maximum OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 1 3 A Titration Injection microPump TIP 2k 18 volume has been reached or a stop criterion is achieved These are limits valid for each individual injection During executing one program line many injections may be started and the total volume added may be higher In the feedback mode it is not possible to predict what the total volume will be therefore below the program line table the message feedback control cannot predict final volume is printed Feedback control cannot predict tinal volume a Load setup Save setup Hide details Duration of feedback control Above the table containing feedback conditions are two fields for d
3. total time is not known in feedback control Pump running While an injection is executed similar informations as described above are displayed but the TIP status is highlighted in green a 37 0 02k Panao TIP Line 1 Direct Cyc 1 1 33 10008 en oS Cali e OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 26 TIP Suspended When the progress of a TIP setup is suspended the available information are displayed the time counter continues to display the evolved time followed by the word suspended The status line is highlighted in yellow it STIG GARRET Te tine 1 Direct Viot 1 000 pl 153 s Suspended E oS Calib Pet Tem Special Operations TIP Manual forward the pump is moved forward manually using the front push button of the TIP 2k This status is highlighted with a green background TIP Manual back the pump is moved backward manually using the rear push button of the TIP 2k This status is highlighted with a red background TIP Errors are highlighted in red additionally a red error field is displayed in the status line The error message can be quitted by clicking on this error field BOG SS fi TE a a Se ae Dobos Ok e 1838 0 4 0165 4 1888 37 000 94 8 39 304 0 0323 2 6475 E Datla 4 3 C 0048 f TIP Rear limit appears with a red background when the rear limit is reached upon moving the pump backward TIP Front limit appears
4. 120 00 2 start Pee tsa sooo 020 20000 soo T02 Concentration Elame gt 6000 alaan 300 SIF 900 20 000 0 100 200 00 300 02 Concentration E nmol ml gt 30 00 2 start a 4D 900 100 000 50 000 2 00 120 1 OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 12 Program lines are executed by the TIP strictly sequentially Each program line may lead to several injections by the TIP or none at all After the execution of a program line the TIP is always in stopped mode all injections are finished before starting the next program line Each program line is identified by a number in the column Line The column Mode indicates whether a particular program line uses direct control D or feedback control F The other entries into the table will be explained when discussing the different modes to program the TIP 4 4 Creating and Editing TIP Setups Editing and Creating TIP Setups has to be done in the detailed view of the Control tab press Show details if necessary Editing of a Setup can only be done when no TIP Setup program is running otherwise the editing functions will be locked For editing and creating TIP Setups a DatLab file has to be open either in on line or off line mode Editing and creating program lines is done by using the input mask in the upper part of the Control tab the control buttons situated between the input mask and the program line table and the program line
5. 2k 25 the Info window is saved with the TIP setup and can be viewed after loading a TIP setup 4 11 TIP Status The current status of the TIP is indicated in the O2k status line at the bottom of the screen af 0ST CRE Tere om ose ajia Set Tem TIF Ready TIP Ready appears in the status bar when the TIP 2k is properly connected to the Oxygrph 2k the Oxygraph 2k is switched on and connected to the PC with the RS232 cable and Datlab 4 is connected F7 TIP Test start the TIP performs a pump operation after clicking on Test start without setting an event The background colour is green if the TIP 2k pumps in the TIP forward mode and red if it pumps in the TIP backward mode Setup running When a setup is running but no injection is taking place at the moment the status line will be highlighted in yellow it STIG E TP Line 1 Direct viot 1 000 ml Cyc 27 3 67 100 E oS Calne e Informations being displayed include Tip Line the program line being executed e g TIP Line 1 Mode the modus used in the current program line e g Direct Vtot total volume injected until the end of the last executed injection volume will be updated after the end of each injection e g Vtot 1 000uI Cyc number of cycles executed total number of cycles e g Cyc 2 3 total number of cycles is not known in feedback control Time time of current activity evolved total time of current activity e g 67 100 s
6. Right Solvent Ethanol Substance FCCP FCCP Conc in TIP 1 8668 1 66608 Left Right refers to the side of the syringes and Oxygraph 2k chambers Solvent is the liquid in which the chemical in the TIP syringe is dissolved Substance is the chemical in the TIP syringe to be titrated into the Oxygraph 2k chamber Conc in TIP refers to the stock concentration mM or uM of the chemical dissolved in the TIP syringe The information entered into the Info window is saved with the TIP setup 4 9 TIP Configuration Window The TIP configuration window is accessible by pressing on the TIP configuration tab always Syringe length mm 68 608 visible in the TIP window It displays the TIP syringe Length of step nrm 3125 volume ul TIP syringe length mm and length of single step nm of the step motor This window is for information only nio Syringe volume pl 566 868 Control Chemicals 4 10 The Info Window By clicking on the Info tab the Info window is Control Chemicals Configuration displayed Here a BG Feedback description of the TIP Setup can be entered There is also a field for an usage URL address that may contain a web link This will be used to supply additional information for TIP setups provided by OROBOROS Instruments The information entered into TIP setup for performing an OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP
7. remove all solvents from the needle Therefore before using the needle again remember to rinse the needle with a pure solvent If a needle was washed with water and an ethanolic solution of a water insoluble substance is filled into the syringe without prior rinsing with ethanol the needle will be blocked The same will happen in the reverse case If a TIP syringe is harder to operate than usually start immediately with extensive cleaning procedures Once a needle is totally blocked it is usually impossible with OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 9 exceptions to clean it Suitable solvents for extensive cleaning procedures depend on the substances used Chloroform was successful for some fatty acids An ultrasonic bath may be helpful in cleaning partially blocked syringes 3 5 Prevention of TIP Needle Clogging Reasons for Needle Clogging To prevent washing out effect when the needle is inserted into the chamber the inner diameter of the TIP needle has to be very small This means that the needle is easily blocked by small particles either precipitated from the solution or introduced with the solution While a TIP syringe has to be considered a consumable it is possible to lengthen its lifetime by carefully following the procedures Suggested in this manual Prevention The main way to prevent clogging of needles is to follow rigorously the cleaning procedure outlined a
8. with a red background when the front limit is reached upon moving the pump forward 4 12 TIP Events After Start Events are set automatically in the graph as vertical lines with an Event name at the bottom of the graph No events are set with Test start Click on the event name at the bottom of the graph to view the event text which automatically reports additional information 0 00 0 05 0 10 TIP start POO POU POUS POO TIP end TIP Event names Tip start is an event marking the start of a new TIP setup program OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k POO1 marks the beginning of issu the first actual pumping Time 000203 event the first injection W max 10 characters The programmed volume Text volumes 100 nl Speed 10 nls and the injection speed will be displayed in the event text In direct Bm a control mode and in feedback control mode Delete event Cancel save without using stop conditions the displayed volume will correspond usually to the injected volume unless the injection was stopped manually In feedback mode with using stop condition this initially programmed volume usually will not be reached The correct volume in this case and in the case of a manual termination is displayed in the PStop event Injections are numbered sequentially POO2 POOS during the entire TIP setup program If the TIP 2k is operated in t
9. 014 mechanical limits of the i TIP when the rear or front limits are reached Simultaneous with this event the status bar of m e the TIP 2k changes to a Delete event Cancel save red background with the text TIP Front limit or TIP Rear limit Click on the Error event name to view the text with information on the actually pumped volume A TIP error will automatically stop the running setup program thereby also generating a TIP end event An additional event ERROR without the volume information may be generated depending on software version In addition to the TIP Error event in the graph the green control light on the push button of the TIP 2k blinks fast when TIP Front limit or TIP Rear limit is reached At this status you cannot operate the TIP 2k with Datlab 4 In order to activate the TIP 2k for further use the pump has to be reset manually using the push buttons Then the status TIP Ready is attained the green light stops blinking and is continuously on OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k
10. 30 000 0 03 2160 Direct control predicted final volume 12 000 pl TIP Setup 01 l Load setup Save setup Hide details Cancel Close Some users may want to quickly run a predefined TIP setup program without any modifications For these users as a first step the following quick start information may be helpful Quickly Loading and Starting a TIP Setup Select a TIP Setup in the W pull down menu in the left lower corner and click on Load setup to load the respective control settings The new control settings for the TIP 2k are now displayed Settings of various setups may be viewed at this stage Start the displayed TIP program by clicking on Start OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k Tl 4 2 The Control Window Overview There are two principal viewing modes for the Control Window In the short view only the current TIP setup and control buttons for starting loading and saving a TIP Ea are visible 5 oe ro 200 00 300 02 Concentration nmol ml gt 120 00 2 start 900 50 000 0 250 200 00 300 02 Concentration 4 nmol ml gt 60 00 2 start 90 20 000 0 100 200 00 900 100 000 50 000 2 00 Feedback control cannot predict final volume BG_Feedback Load setup Save setup Show details b Cancel Ciose By pressing Show details an extended view is displayed that allows editing of a TIP setup Delay s A 00 15 00 Feedback co
11. ameter flexible stainless steel needles 270 mm length All programmed volumes and flows will be calculated erroneously if the syringe mounted does not match the specifications in the Datlab TIP Configuration window Voltage supply The power supply of the TIP 2k and electronic control is integrated into the Oxygraph 2k OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 3 2 2 Installation Placement The TIP 2k_ is placed at the top of the Oxygraph 2k It does not need any extra bench space Align the rear ends of the TIP 2k and the Oxygraph 2k This leaves Space for a syringe cooling box which will be available as an accessory Environment The TIP 2k was tested at room temperature and must be protected from temperature fluctuations and condensation of water The instrument must not be exposed to chemicals or vibrations and the syringes Should not be exposed to direct sunlight during operation Cable A 0 5 m cable connects the TIP 2k to the Oxygraph 2k at the rear with a 15 pin female SubD plug When the TIP 2k is connected to the Oxygraph 2k and the mains of the Oxygraph 2k is switched on the green light within the rear button on the side of the TIP 2k lights up indicating proper connection 2 3 Manual Operation Syringe holders blue POM are mounted on each side of the TIP for a syringe with its needle guided into the 3 left and right 7 Oxygrap
12. bove after each usage Additionally it has to be taken care that no particles are suspended in the used solutions For not very well soluble substances filtering of the solution may be necessary Small particles can also be introduced by laboratory activities e g from paper towels or from powdered gloves OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 10 4 Operation of the TIP 2k with DatLab 4 4 1 TIP Control F8 Quick Start and Introduction Press F8 to open the TIP Control window DatLab 4 C DatLab DLDataVieam files prototyp_testing 009 06 02 WX 01 DLD File Experiment Plots Calculate Marks Graph Layout Oxygraph MultiSensor ie TIF Control Fa 1 6 f j as Setups Default settings are given at the start These settings may be edited step by step or by selecting a TIP setup The main TIP window consists of four tabs Control Chemical Configuration and Info They will be covered in detail in the following chapters Control Chemicals Configuration Info Delay s 186 00 03 00 Mode Direct control Feedback control Vol Flow Volume ull ieaoo TIP backward TIP forward VolTime Flow l s 36 006 Flow Time Time s 6 63 Test start Interval s 186 00 03 00 Cycles 12 Duration s 2168 00 36 00 Program line Insert Replace Delete Move up Move down sft elt MA el ae usen fhe fle eg ae oe Int ts lo 180 1 000
13. d from the two supplied by the user Select one of the following three options for defining the volume flow and time duration of active pumping Lontral Chemicals Configuration Info Delay s 186 00 03 00 Mode Direct control Feedback control 10 0088 C TIP backward 30 000 f TIF forward 8 39 Test start Vol Flow Injection volume ul and flow ul s are entered If you change one value and click into another box the corresponding time s for active pumping is calculated and displayed Vol Time Injection volume ul and time s are entered If you change one value and click into another box the corresponding flow l s is calculated and displayed Flow Time Injection flow l s and time s are entered If you change one value and click into another box the corresponding volume ul is calculated and displayed Error messages at zero volume flow If zero 0 is entered in either of the two editable fields of the TIP Control window and you click on Insert or Replace an error window appears Maximum volume and flow are 500 ul and 50 ul s respectively If higher values are entered and you click on Insert or Replace an error window appears TIP forward is the standard operation mode for titrations and injections TIP backward is selected if the pump should operate backwards This is applied for automatically filling the TIP syringes Be careful not to withdraw any sample from the Oxygrap
14. eccepueueeeenaeeeraceeueieatcesencseseecestere 26 instruments oroboros at www oroboros at 1 3 A Titration Injection microPump TIP 2k 2 1 TIP 2k Main Features TIP 2k 2 Channel Titration Injection microPump The _ TIP 2k provides unique advantages for applications which require highest accuracy of titration volumes and automatic control of complex titration injection protocols The TIP 2k was specially developed for the Oxygraph 2k The main modes of operation are automatic multi step injections continuous injections for steady state respirometry both operated form the direct control mode and feedback controlled operation Direct Control Mode Operation mode for delivering a defined volume in a Short pulse A number of cycles N can be defined and the duration of the interval from injection to injection determined repeating the process N times Several such steps can be programmed to run in sequence to give an entire TIP program Feedback Control Mode Operation mode to reach and maintain a certain parameter e g oxygen concentration by automatic TIP injections 2 Setting up the TIP 2k Operation of the TIP 2k is exclusively possible in conjunction with the Oxygraph 2k and Datlab 4 2 1 TIP 2k Components Dimensions D x W x H 270 x 124 x 160 mm without syringes Stainless steel housing 2 mm thick Weight 3 0 kg TIP 2k Syringes Simultaneous operation of two 500 ul Hamilton syringes 0 09 mm inner di
15. eennas 9 4 Operation of the TIP 2k with DatLab 4 sssssss2 s2 10 4 1 TIP Control F8 Quick Start and Introduction ccccccceseee eee eeeseeeeeeeneeaeees 10 4 2 The Control Window Overview 2 cccccccsccesseeeeeeeeeeeccnnaneeeeeettecegnenntteesessaauaes 11 4 3 The Structure of a TIP SetUDieccccnrisannscscetesstacananiecesemnianteneeedeanmanstadieesou 11 4 4 Creating and Editing TIP Setups ssssssssesssnsrsrrrrnrsrenrsrrsrrnrnrenrnrerrrrnrerenrear 12 4 5 Setting the Parameters for a Program Line ssssssssssrsrersrrsrrrrnrnrrrenrerrrrneear 13 The Direct Control MOG sisasctca sec ceceeanavanaantarresdunntacasneesees tieaeinewecose ETEEN 15 The Feedback Control MOdeC cccssccceeeeeesccsseeeeteeeeeeeessanneeeeensecggeenatteesessaauaes 16 4 6 Saving Loading ANd Managing SetUDPS cccccccceeeeeeeee cece eeeeeeeeeeeeeeeeeeeeanaaes 21 4 7 RUNNING a TIP Setup Prograf s ise esccsecconecscetsccastaccssessceosaceesenncassotdsnareentebe 23 48 The Chemicals WINDOW dsiisscnaaetedesassendidedersetaionnnaweleseehiaiuntaneeelsasenmiiaaeweetain 24 Boe TIP COMmOUrAUON WINGOW sasnnceseseciaanseeneseescsaudeuder sane E EA 24 AO TNC IMO VV GOW wacemcoacnntac tune wresannvananneosesssaennestateesswetanasanaees vraveeaneees esos 24 Bell AEP SSCS vous ance Padawan s napa E tates Dee daaawaumee bewelnadatiddaies inthe eau egreaeunadtanasmee eda 25 pee TAP EY CMe enra aE iecuecesageensicaasoscets
16. ere done to the current Setup To apply a new name to the Setup type the new name into the Setup name field on the left side of the safe button and then press Save setup P dew set g Load setup Save setup Hide details OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 22 Load A new setup is selected by activating the drop down menu in the setup name field click on e i Load setup Save setup Hide details temp TIP Setup 01 TIP Setup U2 TPP_direct3_2rml_closed TPP_direct3_ pm closed TPP direct 3 TPP large _range_ start TPF Test After a new setup was selected the Load setup button will be highlighted in yellow Clicking on Load setup will then load the new Setup into the TIP control window Any unsaved changes to the current Setup will be lost The Load setup and Save setup buttons are available from all tabs of the TIP window Manage Setups To delete or rename setups use the Manage Setups function This is accessible from the main menu under TIP E DatLab 4 C DatLab DLData IOC5OEF 2009 04 19 EF 04 DLD File Experiment Plots Calculate Marks Graph Layout Oxygraph Multisensor Ppl 250 TIP Control F Manage Setups A list with all currently available setups will appear These may be renamed or deleted Import OROBOROS Setups Additional TIP setups supplied by OROBOROS instruments in a template archive can be imported by choosing Import Templat
17. es from the File Menu see the manual O2k C DatLab4 Guide E DatLab 4 C DatLab DLDataViOCSOEF Z00 JER Experiment Plots Calculate Marks Graph L Open Cr Close Ctrl F4 Save Chrl 5 Save Os File Search Ctrl F Delete Export DatLab templates Exit Transferring Setups Setup files name TCS are stored in the directory DatLab APPDATA TIPCTRL Any name TCS file manually copied into this directory will be recognized as a TIP setup file This may be used e g to manually transfer a TIP setup written on one computer to another computer OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 23 4 7 Running a TIP Setup Program Start By clicking on Start the TIP setup program is started as displayed in the TIP Control window and in the status line ee suspend otop immediatly etup show details This does not necessitate that an actual injection is started at the same time The program line being executed will be highlighted in blue in the program line table When an injection is started this is displayed in the status line additionally a green light on the button on the TIP unit blinks Stop immediately At any time the entire TIP program may be Suspend Resume Close terminated by pressing Stop immediately If an injection is running this injection will be stopped The total volume injected is given in a TIP event see below If the TIP se
18. etermining how long the program line should be executed i e how long the feedback control should be maintained 1 Feedback control Stop and next program line after HBA seconds or O cycles 0 for unlimited To maintain feedback control for x seconds only fill in the first field The second field does not set an absolute time limit but stops the program line after y injections have been executed The latter option will probably be useful only in special applications Setting a parameter to zero removes any limit therefore setting both parameters to zero will maintain feedback control indefinitely Feedback conditions The hearth of feedback control is setting conditions that will start or terminate an injection This is done in the feedback conditions table The following paragraphs describe the fields used for defining feedback control How these parameters may be used to actually implement feedback control is described in Implementing Feedback Control below Select Quantity ele Value Data Action Pause s 02 Concentration F r irL 10 1 Start F 02 Concentration F nmol gt 20 1 Stop 0 4 Feedback line Delete Insert OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 19 Quantity gt lt Value DataN Action Pause s There always will be at least one feedback condition line visible more may be generated by pressing Inse
19. ge glass cylinders of both syringes with the screws of the syringe holders 4 Insert the tips of the needles into the titration injection solution 5 Shortly press the rear push button of the TIP 2k to change the status from TIP Front limit to TIP Ready The green light at the centre of the TIP 2k rear button stops blinking and is on 6 In DatLab open the TIP Control window F8 and select TIP backward OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 7 7 Set the Volume ul to be filled in the syringes It is advisable to fill some more volume than required for the titration injection experiments Select the flow to a low value to minimize gassing out from the solution 8 Click on Test start to fill the syringe Provide time for pressure equilibration within the syringe For instance if 100 ul is filled at a flow of 50 ul s about 60 to 90 s are required until the pressure difference is relaxed by the slow flow of solution 9 Remove the syringes from the syringe holders and expel the trapped air bubble as described above 3 2 Mounting a Filled TIP Syringe 1 Move the pump manually to or towards the rear limit 2 Loosen the screws on the syringe holders and insert the syringe with the volume markings facing upward 3 Fix the plunger head with the screw of the rear part of the syringe holder fastening it finger tight 4 Before fixing the f
20. h 2k chamber by using the backward mode This may clog the TIP needles A warning is displayed before starting a TIP setup that includes a backward step OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 25 Test start is used to activate a single operation of the TIP 2k according to the volume flow time settings without delay The Test start mode does not interfere with the programmed TIP Setup i e it neither starts the first cycle nor reduces the total volume of the programmed titration injection setup After clicking on Test start the button changes to Test stop enabling to stop the pump before the injection is completed It is advisable to use Test start before inserting the needles into the Oxygraph 2k chamber No events are set by DatLab when using Test start Mode Selection Some parameters differ between the modes for Direct Control and Feedback Control Appropriate input masks are selected by pressing the Direct Control or Feedback control buttons shown below Lontral Chemicals Configuration Info Delay 3 1860 00 03 00 Mode Direct control a2 Feedback control a The Direct Control Mode In the direct control mode a defined volume is injected into the chamber at a defined speed This process can be repeated as desired in defined intervals While one program line is executed volume flow and interval stay constant To achieve changes in these parameters e g i
21. h 2k es chamber The front part of the OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 4 holders is static for fixation of the glass cylinder of the syringe while the rear part is moved with the step motor of the TIP for fixation of the plunger Push buttons are placed on the right side of the TIP 2k Push the front one for forwards movement and the rear one for backwards movement of the plunger holder For stopping the manual movement simply release the push button Pushing both buttons simultaneously also stops the pump At manual operation of the TIP 2k the step motor moves at maximum speed Front and rear limit When the front or rear limit of the pump is reached the motor stops automatically This status is indicated by the blinking green light on the rear push button After a short push on the TIP button in the other direction the TIP is in the status ready the green light is on without blinking 2 4 Safety Potential Hazards When not inserted into the oxygraph chamber the needles of the TIP syringes may due to their length and flexibility inflict serous injuries to the eyes Dangerous chemicals may be splashed out of the syringes by unintentionally operating the TIP If a blocked syringe is used in the TIP the syringe may fracture under the applied pressure and serious injury especially to the eyes may occur by glass fragments Safety Rules Alway
22. he TIP backward mode the events set by the TIP 2k are the same as in the pumping TIP forward mode but the volume indicated in the event text has a negative sign PStop is an event set at the end of an injection if the injection was stopped manually by the user the injection was stopped by a feedback control stop condition the entire programmed volume was injected and injection time was 30 s This event indicates the time when the actual pumping has switched off In the event text the actually injected volume is displayed Summary Injected Volumes When a PStop event is displayed the corresponding event text will always contain the correct actually injected volume When no PStop event is displayed the POQx event will contain the correct actually injected volume There will be no PStop event for injections that were carried out until the entire programmed volume was injected and the lasted less or equal to 30 seconds TIP end appears at the end of the TIP setup program after the last program line has been executed or the setup program was stopped by the user In the event text the final volume injected during the entire setup program is displayed OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 28 TIP Error appears when the KIGEL programmed injection is Time 00 25 53 Sto ed due to Name EM max 10 characters g P i IAr Text TIP stopped at front limit Volume 423
23. in How the condition should be defined in respect to the limit gt or lt depends on the nature of the process in the chamber and of the agent used in the syringes to counteract this process If e g the analytical parameter used for feedback control is oxygen concentration the concentration will usually decrease due to respiratory activity This can be counteracted by filling the TIP syringes either with oxygen saturated media or with hydrogen peroxide when catalase is present in the medium The start condition will therefore define a lower limit for oxygen concentration and the operator lt will be used So the line can be expressed as If c O2 lt lower limit then start injection Using this method there is no programmed upper limit to oxygen concentration but a de facto upper limit will be reached by setting the injection time and volume to appropriate values Method B Start and Stop Actions are used One feedback condition line is constructed defining the first limit and a start Action A second feedback condition line is constructed defining a second limit and a stop Action Quantity gt Value Datan Action OU Concentration F fn 7 10 1 Start z OY Concentration F nmol gt 20 1 Stop 0 OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 21 The injection Flow is set to a rather low value allowing on line monitoring of the analytical parameter dur
24. ince some solution may be spilled while attempting to remove any trapped air bubbles 3 Allow for a slow pressure equilibration until most of the gas space is filled by the solution A small gas volume fills the space between the plunger and the solution 4 Hold the syringe vertically with the needle pointed up and gently tap at the side of the syringe at the point where the air bubble is trapped The bubble moves up the syringe to the needle outlet Position the bubble at the centre of the glass syringe directly at the opening to the needle by gently tapping at the side of the syringe 5 Slightly press the plunger to squeeze out the bubble whereby some solution may be lost from the syringe If the bubble moves to the side while pressing the plunger stop pushing and gently tap the syringe tip until the bubble is located centrally and continue Avoid any undesired contact with the titration solution and prevent the long needle from moving uncontrolled which may be dangerous for the eyes 6 After the bubble has been displaced from the glass part of the syringe eject further about 10 20 ul to flush the entire needle with solution Filling a syringe with the TIP 2k 1 Before mounting the syringe make sure that it is clean and rinsed with the solvent in which the substance to be titrated injected is dissolved 2 Push the plunger fully into the syringe 3 Move the pump to the front limit Fix the plunger heads and syrin
25. ing the injection Injection Volume or Time have to be set to some maximal value but usually will have no consequences in this method VolFlow Volume ull 5 808 VolkTime Fowls 6 010 Flow Time Time s 546 88 When the condition defined in the line containing the Start Action is fulfilled an injection will start This injection will be stopped when the second limit is reached as defined in the line containing the Stop Action The pump will then be inactive until the condition for the Start Action is fulfilled again Which of the two limits has to be placed in the start and stop lines depends again on the nature of the process in the chamber and the content of the TIP syringes In the example given for method A oxygen concentration being controlled respiratory activity in the chamber and e g peroxide in the syringes the line containing a Start Action will set the lower limit for oxygen concentration while the line containing the Stop Action will define the higher limit for oxygen concentration Thus oxygen concentration will be held between an upper and lower limit This may be formulated as If c O2 lt lower limit then start injection If c O2 gt upper limit then stop injection 4 6 Saving Loading and Managing Setups Save After modifying the TIP Control settings the new configuration may be saved To indicate this possibility the button Save setup will be highlighted in yellow after changes w
26. is faster when enough solution is available for completely filling the syringe If injection solution is limiting however it is possible to fill the syringe with a small volume e g 50 ul using suction force Back filling the TIP syringe requires gt 500 ul solution Remove the plunger from the syringe Fix the syringe in vertical position on a holder Using a standard injection syringe with a long needle fill the TIP syringe from bottom to top without inclusion of gas bubbles at the bottom or on the glass wall The plunger is inserted while a small amount of the solution is first extruded on the top Once a tight fitting of the plunger is obtained the solution is extruded through the needle The small diameter and length of the needle cause a substantial resistance against ejection of the solution The plunger should be inserted to the maximum volume mark of the syringe Place the end of the needle securely into a recipient vessel to avoid any hazards to persons or instruments by jetting the solution out of the needle Manually Filling the TIP syringe through the needle is possible with lt lt 500 ul solution 1 Push the plunger completely into the TIP syringe 2 Immerse the tip of the needle into the titration solution and pull the plunger slowly back to the desired volume OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 6 mark It is advisable to suck in some extra volume s
27. ncreasing volumes in later injection several direct control program lines are run in sequence one for each set of parameters Input Mask In the Direct Control mode only the upper left side of the control window is used for the input mask Control Chemicals Configuration Info Delay 3 186 00 03 00 Mode Direct control Feedback control Wol Flow Wolurme pl 10 008 TIF backward f TIF forward VobTime Flow ul s 30 6008 Flawelane Tmall paa Ee Interval z 188 00 03 00 Cycles 2 Duration a 368 00 06 00 Discard changes OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 16 Interval s is the total time from the start of a pumping cycle to the start of the consecutive cycle It is the sum of the titration injection pumping Time s and a pause between the end of the previous and the beginning of the following pumping state interval Cpumping tpause In applications the Interval is much longer than the pumping Time since the system has to attain a new steady state after the disturbance of the titration Cycles is the number of times the injection is repeated with a pumping phase followed by a pause Duration is calculated by DatLab from Duration Interval x Cycles It is the total duration of the program line excluding the delay After the entered values have been copied to the program lines table by Replace or Insert a direct contr
28. ndition is met for DataN points When an injection is running the program will continually check the feedback conditions table for a line containing a Stop If the condition in such a Stop line is met for DataN data point the injection will be terminated Method A uses only Start actions Method B uses Start and Stop actions is used to set a minimal pause between injections No injection will be started for this time after an injection was finished even if the start condition is met This feature is only meaningful for method A OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 20 Implementing Feedback Control Method A Only Start Actions are used A feedback condition line is constructed defining only one upper or lower limit for the analytical parameter Quantity p lt Value Datan Action Pause s 02 Concentration F nmol lt 10 2 Start E Injection Volume Flow and Time are set to achieve rather short pulses VWol Flow Volurne pl 5 888 VolTime Flow pl s 38 808 Flow Time Time s 0 17 After the injection the condition is checked again When the limit is still not reached the condition is still fulfilled after an optional delay of Pause seconds a second injection identical to the first is performed This is repeated until the limit is reached the condition is no longer fulfilled Then the pump is inactive until the condition is fulfilled aga
29. ntrol Mode Dre coriol al a a Stop and next program line after 386 seconds or 8 cycles 0 for unlimited a Select VolFiow Volume ul Mzamaaa e ye beckwad f Touaniy fpe Vae Daten Action Pause fal Yol Time Flow uls 6 108 E 02 Concentration A nmol gt 2 Start 02 Concentration 4 nmol lt 1 Sto Flow Time Time s 266 66 Test start J 2 Feedback line Delete Insert Program line Insert Replace Delete Move up tee F 1200 a at 0 a 200 00 al m Concentration N gt T 00 a start 2F 900 50 000 0 250 200 00 300 02 Concentration 4 nmol ml gt 60 00 2 start 3 of 20 000 0 100 0 0 02 i gt 2 4D a00 100 000 50 000 2 00 Feedback control cannot predict final volume BG_Feedback Load setup Save setup Hide details 1 Cancel Close By pressing Hide details the view is changed to the short modus again For the following discussion the extended view will be used 4 3 The Structure of a TIP Setup A TIP setup is a predefined set of instructions to be executed by the TIP plus informations about the used chemicals The entire actual setup is displayed in a table program lines table in the lower part of the Control window Each setup consists of at least one but possible many program lines _ Line Mode Del s Vol ul Flow ul Times _ Duration s Cycles Feedback Guantity gt lt Value _ DataN Action Int s TF 1200 100 000 0 500 200 00 300 02 Concentration E nmol ml gt
30. ol program line is described in the program line table by the parameters Del ay Vol ume Flow Time Duration and Cycles All other fields are not used in the direct control mode Int s 20 180 5 000 30 000 0 17 540 3 In the picture above two program lines have been used to increase the volume per injection from 1 ul for the first two injections to 5 ul for the subsequent three injections The expected final volume after all program lines are executed is displayed below the program line table Direct control predicted final volume 225 000 pl TPP_direct4_2pSml_clased_10n gt Load setup Save setup Hide details The Feedback Control mode In the feedback control mode the injection flow and depending on usage also time and volume for a single injection is set by the user The total amount of volume injected into the chamber during the execution of the entire program line is however adapted by DatLab to maintain a certain level of a measured analytical parameter e g oxygen concentration and or to hold OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 17 this parameter between predefined limits Such a feedback control can be implemented in two ways Method A Short injection pulses are programmed Each injection is completely executed once started without regard to the measured analytical parameter DatLab continues to start these injections as long as the star
31. program line table either press Replace to overwrite the selected program line or Insert to insert a new program line before the selected program line with the values form the input mask Only after pressing Replace or Insert the values in the program line table will change Selecting a different program line without pressing Replace or Insert will discard all changes The buttons Replace Insert and Discard changes will be highlighted in yellow when changes have been made in the input mask that have not yet been written to the program lines table The control buttons move up and move down can be used to change the position of a program line in the program line table and hence the sequence of the execution of program lines 4 5 Setting the Parameters for a Program Line This chapter describes the different parameters used in the program lines and how to use the input mask to set these parameters Common Features Some features are common in both direct control mode and feedback control mode Delay s is the time s Delay 3 186 00 03 00 before starting the first instruction of Interval 3 186 00 03 00 any program line prog Cycles 12 OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 14 Injection Volume Flow and Time For each program line two of the tree parameters injection volume injection flow and injection time have to be set The third parameter is calculate
32. ront syringe holder repeat steps 2 3 with the second syringe on the other side of the TIP 5 Use two fingers of the left hand to hold both syringes while pressing the front button on the TIP 2k to move the syringes forwards until the protruding edges of the glass cylinder touch the front syringe holders Release the push button as soon as both syringes have reached this final position 6 Fix the syringes with the screws of the front syringe holders fastening them finger tight Move the syringe manually forward if excess solution should be ejected Place the end of the needle securely into a recipient vessel to avoid any hazards to persons or instruments by jetting the solution out of the needle 3 3 Inserting the TIP Needle into the Oxygraph 2k Chamber Before inserting the needle into the Oxygraph 2k chamber rinse the surface carefully In DatLab open the TIP Control window F8 set a low flow and click on Test start for details see below No events are set when using Test start instead of Start in the F8 window Stop the pump after a small droplet of the solution appears at the tip of the needle Remove the extruded drop preferentially with a plastic tip not OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 8 to withdraw any solution from the capillary The latter might occur when using a paper towel Place the spacer of the needle leaving a length of 55 mm to the end Thi
33. rt or removed by pressing Delete Some fields are selection fields a drop down button will appear when clicking on the field By clicking on the button the drop down menu is activated Select a A i en 02 Concentration F r 7 T 02 Concentration F r8 u mnl OU Pressure kPa F kPa O2 Pressure roimHg F mmHg gt 02 Slope uncorr F prol s ml 02 Flus per Y F pmol s mil Block Temp C Fel Barom Pressure kFa Peltier Power 4 is a selection field Select the analytical parameter that you want to control from the drop down menu is a selection field It determines whether the condition is met when the measured analytical parameter is higher or lower than Value Select gt or lt from the drop down menu is the numerical value for the chosen analytical parameter you want to use as condition The unit is given in the field Quantity Enter a numerical value here The fields Quantity lt gt and Value together define a condition is the number of data points the condition has to be met before Action is executed Entering higher numbers gt 1 here allows to stabilize the system preventing injections being triggered by non significant fluctuations and allowing the system to equilibrate after an injection is a selection field Select Start or Stop from the drop down menu Start will start an injection with the parameters entered in the left side of the input mask when the co
34. s spaces the needle end just above the stirrer in a closed 2 ml Oxygraph 2k chamber Be careful not to touch the stirrer bar with the tip of the needle when inserting the needle through the titanium capillary of the stopper into the chamber of the Oxygraph 2k Alternatively e g for working with a different chamber volume adjust the position of the Spacer interactively Put the spacer in a short distance to the end of the needle insert the needle into the stopper and push the needle further down to the desired depth while visually controlling the position through the window For the next usage the spacer will already be in the correct position 3 4 Washing the TIP Needle after Use Immediately after use the following steps should be done 1 Remove the needles from the chamber 2 Extrude some volume from the needle e g by pressing the manual forward button Place the end of the needle securely into a recipient vessel to avoid any hazards to persons or instruments by jetting the solution out of the needle 3 Rinse the outside of the needle with water to remove any media This must be done before rinsing the interior of the syringe with any non aqueous solvents 4 Rinse the needles several times with an appropriate solvent i e one in which the substances used are soluble Usually this will be the same solvent you used during TIP operation 5 Empty the syringe and store in a safe place It will usually not be possible to
35. s table in the lower part of the window Control Chemicals Configuration Info Delay s 186 8 00 03 00 Mode Direct control Feedback control Vol Flow Volume pl 16 6068 TIP backward TIP forward VoltTime Flow ul s 30 000 FlowsTime Time s 0 33 __leststart Interval s 186 00 03 00 Cycles 2 Duration s 366 00 06 00 Discard changes input mask Program line inset Replace Delete Move up Move down control buttons a ee ee Int gt 180 1 000 30 000 0 03 tnes tabl T o 30 000 017 40 program lines tapie 20 000 0 33 i 1 Select a program line to edit by clicking on it The selected program line in the program line table is highlighted in blue The current settings for this program line will be displayed in the input mask and can be edited If you do not want to change the marked line but want to add a program line to the setup click the control button Insert and a copy of the selected program line will be inserted in the program line table OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 13 2 Set the parameters for the program line using the input mask A detailed description for the different modes is given below The button Discard changes will appear in the input mask The values in the input mask are reverted to those in the program line by pressing this button 3 To transfer the new values from the input mask to the
36. s wear safety goggles Never operate the TIP with a blocked syringe When operating the TIP without the needles being inserted into the chamber Place the end of the needle securely into a recipient vessel to avoid any hazards to persons or instruments by jetting the solution out of the needle 3 Filling and Mounting the Syringe Dust and any remains in the syringe cause clogging of the needle as well as wearing of the plunger When using a blocked needle the pump may be damaged OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k owing to the generation of high pressure Therefore carefully read and observe the instructions for washing the needles and prevention of needle clogging TIP Any inclusion of gas bubbles in the syringe must be avoided Solutions must not be subjected to an increase in temperature during or after filling Otherwise gassing out will give rise to bubbles which cause erroneous titration volumes or undesirable hysteresis effects Do not over bend the stainless steel needles since this causes irreversible damage 3 1 Filling the TIP Syringe Before filling the syringe make sure that it is clean and rinsed with the solvent in which the substance to be titrated injected is dissolved Filling the TIP syringe can be done by back filling after removal of the plunger or through the needle while pulling the plunger backwards The back filling of the TIP syringe
37. t condition is met Method B Injections are started when the start condition is met If Comparison Input mask a stop condition is met while the injection is still running the injection is stopped even if the predefined volume has not been added yet Therefore only the flow speed is strictly maintained at the value set by the user but not time and volume In method A only one condition the start condition is used and the validity of the condition is checked only between injections but not during injections In method B two conditions are used start and stop and while the validity of the start condition is also considered only between injections the validity of the stop condition is checked during injections By continuously monitoring and reacting to the analytical parameter the target can be reached more precisely less overshoot and faster no breaks between single injections than with method A On the other hand the pauses between injections following Method A allow the system to reach equilibrium before the decision for or against a further injection The input mask for Feedback Control utilises additionally the upper right part of the control window for feedback control specific parameters Control Chemicals Configuration Info Delay s 10 00 00 10 Feedback control Stop and next program line after 3668 seconds or 8 cycles 0 for unlimited Mode Direct control Feedback control P prog ycles
38. tup is started again afterwards it will be started at the first program line Therefore if the program should be continued at a certain point after a stop instruction the setup has to be changed accordingly will halt the progress of the current program line if no injection is taking place at the moment The time counter in the status line will continue to show elapsed time for the current program line but no injection will be started A running injection will NOT be stopped by Suspend will continue a setup halted with Suspend If the scheduled time for an injection was reached during the time the TIP suspended this injection will start immediately after pressing Resume will close the control window When pressed while a TIP set up is executed the setup will not be stopped but will run in the background By selecting TIP Control from the TIP menu again F8 the TIP window will be opened again displaying the progress of the TIP setup and giving access to the control buttons Stop immediately and Suspend Therefore the TIP control window can be closed after starting the setup and other DatLab features can be used with the setup running in the background OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k 1 3 A Titration Injection microPump TIP 2k 24 4 8 The Chemicals Window The Chemicals window is selected by pressing on the Chemicals tab always visible in the TIP window Control Chemicals Configuration Info Lett
Download Pdf Manuals
Related Search