817 Bioscan. Bioscan. Instructions for Use CH-9101 Herisau/Switzerland Internet

Transcription

1 Bioscan CH-9101 Herisau/Switzerland Internet Instructions for Use

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3 CH-9101 Herisau/Switzerland Internet Instructions for Use / up

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5 Contents Contents 1 Introduction Instrument description Parts and controls Information about the Instructions for Use Organization Notation and pictograms Safety notes Electrical safety General precautionary rules Installation Setting up the instruments Packaging Check Location Arrangement of the instruments Mains connection Setting the mains voltage Fuses Mains cable and mains connection On/Off switching Capillary connections Connection of the Bottle Rack (option) Connection of the pulsation absorber Connection of the injection valve Mounting the column Connection of the flow cell Connecting the waste tubing Rinsing the tubing Rinsing the column System for sugar analysis MIC Electrical connections Settings in «IC Net 2.1» Connecting a sample changer System for the analysis of anions and sugars MIC Electrical connections Capillary connections Configuration in «IC Net 2.1» Preparing the for the analysis System switch-on Start pump Set operating mode I

6 Contents 3 Operation Handling the Operation using «IC Net 2.1» icon window Basic principles Introduction Measuring conditions Pulsed amperometric detection Optimization of the PAD parameters Optimization of the measuring potential Notes Maintenance Faults Practical notes on ion chromatography Separating columns High-pressure pump Eluents Connections Maintenance and servicing General information Shutdown Cleaning the working electrode Changing separating columns Faults and malfunctions Malfunctions and their rectification Instrument test with the dummy cell Validation / GLP Appendix Technical data Standard equipment Warranty and conformity Warranty EU declaration of conformity Certificate of conformity and system validation Index II

7 Contents List of illustrations Fig. 1: Front of the... 3 Fig. 2: Rear of the... 4 Fig. 3: Interior of the (with permanently attached accessories).. 6 Fig. 4: Setting the mains voltage Fig. 5: Connections at the injection valve of the Valve Unit Fig. 6: Connections at flowcell Fig. 7: Connecting up a simple modular IC system with the MIC Fig. 8: Connecting up a simple MIC 8 system including a sample changer Fig. 9: Connections with a as second detector with a modular IC system MIC Fig. 10: Connections of injection valves A and B in the IC Separation Center ( ) Fig. 11: Potentials applied during pulsed amperometric detection (PAD) Fig. 12: Example of a hydrodynamic voltammogram of a substance (A) with additional presentation of the measured values for the pure eluent (B) Fig. 13: Example of a scan of a substance (A) with additional presentation of the scan of the pure eluent (B) III

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9 1.1 Instrument description 1 Introduction 1.1 Instrument description The Bioscan is a PC-controlled measuring instrument for the sensitive analysis of carbohydrates by ion chromatography using pulsed amperometric detection. The compact housing of the contains several IC system components: Detector flow-through cell with three-electrode arrangement for amperometric detection in the Pulse, DC and Scan modes. Column compartment the perfect insulation of the housing creates not only stable thermal conditions for the separating columns, but also shields the system from electromagnetic interference; in addition to the column it also contains the detection cell, pulsation absorber and preheating capillary. Oven amperometric determinations require extremely stable thermal conditions. The built-in column compartment oven ensures that all important components can be set exactly to a temperature from 10 C above room temperature to 60 C with a stability of 0.1 C. All components that come into contact with the eluent and the sample, except the electrodes, are metal-free. Signal converter the contains its own analog/ digital converter for the detector signal. This means that complete control of the instrument is possible from a PC via an RS232 interface. Together with the 709 IC Pump and the 812 Valve Unit, the forms a complete IC system for carbohydrate analysis. Operation is via a PC connected to the RS232 interface and uses the included «IC Net 2.1» control and evaluation program. This software fulfills all the demands which are placed today on a modern integration software: 1-point or multi-point calibration, internal or external standard, selectable algorithms for non-linear calibration, numerous Integration modes with selectable parameters and integration events, various peak recognition methods, peak editor, free scaling, superimposition of several chromatograms, post-treatment of chromatograms, high-performance GLP-conform report generator with output interfaces for monitor, printer and external databases. 1

10 1 Introduction 2

11 1.2 Parts and controls 1.2 Parts and controls Bioscan Fig. 1: Front of the 1 Door to interior 3 Feed through for tubing e.g. connection of injection valve and preheating capillary or connection to waste 2 Feed through for tubing e.g. connection of 709 IC Pump and pulsation absorber 3

12 1 Introduction Fig. 2: Rear of the 4

13 1.2 Parts and controls 4 Mounting screw for fastening the rear panels 5 / 6 5 Detachable rear panel access to top part of the inner compartment 6 Detachable rear panel access to bottom part of the inner compartment 7 Analog signal (REC.) AD/DA-converted signal (not used with IC Net; see chapter 6.1 for details) 8 Analog signal (Int.) Original signal (not used with IC Net; see chapter 6.1 for details) 9 Manual valve (not used with IC Net) 10 Model plate with technical data serial number 12 Lower connections for remote lines (not used with IC Net) 13 Outlet for spilled liquid for discharge of spilled liquid from the inner compartment 14 RS232 interface connection of the PC; see chapter 6.1 for details 15 Upper connections for remote lines only Aux 1, Aux 2 as well as 1 and 2 are used with IC Net 16 Fuse holder changing the fuses, see section Mains connection plug mains connection, see section Mains switch to switch instrument on and off: I = ON 0 = OFF 11 Electric valve (not used with IC Net) 5

14 1 Introduction Metrosep Carb Fig. 3: Interior of the (with permanently attached accessories) 6

15 1.2 Parts and controls 19 Electrode cable (red) connection for working electrode 20 Electrode cable (blue) connection for auxiliary electrode 21 Column connection capillary PEEK capillary 27 Connection for elektrode cable connection '1' 28 Connection capillary connection 709 IC Pump pulsation absorber 29 Connection capillary connection pulsation absorber injection valve 22 Measuring cell holder 30 Pulsation absorber Au - Flow cell Electrode cable (black) connection for reference electrode 25 IC column e.g. Metrosep Carb 1 ( ) for carbohydrate analysis 31 Preheating capillary , length: 3 m 32 Oven 33 Oven heating fan 26 Column holder 34 Connection capillary connection flow cell waste 7

16 1 Introduction 1.3 Information about the Instructions for Use Please read through these Instructions for Use carefully before you put the into operation. The Instructions for Use contain information and warnings to which the user must pay attention in order to assure safe operation of the instruments Organization These Instructions for Use for the provide a comprehensive overview of the startup procedure, operation, fault rectification and technical specifications of these instruments. The Instructions for Use are organized as follows: Section 1 Section 2 Section 3 Section 4 Section 5 Section 6 Introduction General description of instruments, parts and controls and safety notes Installation Setting up, mains connection, attachment of accessories, connection to IC system Operation Detailed description of the operation Basics Information about the pulsed amperometric detection Notes Maintenance Faults Notes on ion chromatography, maintenance, fault rectification, diagnostic tests, validation Appendix Technical data, standard equipment, options, warranty, declarations of conformity, index To find the required information on the instrument, you will find it an advantage to use either the Table of contents or the Index at the back. As a supplement to the Instructions for Use, the Metrohm Monograph "Ion chromatography" is also supplied. This provides an introduction to the theoretical fundamentals and general information on separating columns and sample pretreatment. The Metrohm Monograph "Practical Ion Chromatography" is a practical textbook, which provides an illustrative introduction to the basic principles of ion chromatography and also describes 22 experiments covering the whole world of ion chromatography. IC Application Notes for different applications of Metrohm IC systems are also supplied. 8

17 1.3 Information about the Instructions for Use Notation and pictograms The following notations and pictograms (symbols) are used in these Instructions for Use: <PARAM> Key 'Range' Parameter or entry value 35 Part or control of Part or control of 709 Hazard This symbol draws attention to a possible danger to life or of injury if the associated directions are not followed correctly. Warning This symbol draws attention to possible damage to instruments or instrument parts if the associated directions are not followed correctly. Caution This symbol marks important information. First read the associated directions before you continue. Comment This symbol marks additional information and tips. 9

18 1 Introduction 1.4 Safety notes Electrical safety While electrical safety in the handling of the is assured in the context of the specifications EN / IEC , the following points should be noted: Mains connection Setting of the mains voltage, checking the mains fuse and the mains connection must be effected in accordance with the instructions in section 2.4. Opening the If the is connected to the power supply, the instrument must not be opened (apart from the front door) nor must parts be removed from it, otherwise there is a danger of coming into contact with components which are live. Hence, always disconnect the instrument from all voltage sources before you open it and ensure that the mains cable is disconnected from mains connection 17! Protection against static charges Electronic components are sensitive to static charging and can be destroyed by discharges. Before you touch any of the components inside the, you should earth yourself and any tools you are using by touching an earthed object (e.g. housing of the instrument or a radiator) to eliminate any static charges which exist General precautionary rules Handling of solvents Check all lines of the IC system periodically for possible leaks. Follow the relevant instructions regarding the handling of flammable and/or toxic solvents and their disposal. Never block drain opening for spilled liquids On the bottom of the interior directly below the front door there is a drain opening 1. Spilled liquids can flow directly to the outlet 13 on the back. Due to safety consideration, take care that these openings may never be blocked. 10

19 2.1 Setting up the instrument 2 Installation The is an electrochemical detector and can be included in a modular IC system in two different ways: 1) The as the only detector in an IC system. Apart from the 709 IC Pump, you also require an 812 Valve Unit with one injection valve. In this MIC-8 modular IC system the is intended for the analysis of sugars only. 2) The is included in an IC system as the second detector. An example of this is given in Section 2.5 where an IC system (MIC-9) is described for the simultaneous determination of sugars and anions with chemical suppression. These modular IC system configurations (MIC-1, MIC-2, etc.) are possible instrument combinations that have been properly tested. Under you will find further examples; these are continually updated. Following the general information about the installation of the instruments, Sections 2.4 and 2.5 describe the two configurations mentioned above including their control by the «IC Net 2.1» software. 2.1 Setting up the instrument Packaging The is supplied together with the separately packed accessories in special packaging containing shock-absorbing foam linings designed to provide excellent protection. The actual instruments are packed in an evacuated polyethylene bag to prevent the ingress of dust. Please store all this special packaging as only it can assure transport of the instruments free from damage Check After receipt, immediately check whether the shipment is complete and has arrived without damage (compare with delivery note and list of accessories in section 6.2). In the case of transport damage, see instructions in section "Warranty". 11

20 2 Installation Location Position the instruments in the laboratory at a location convenient for operation, free from vibrations and protected against a corrosive atmosphere and contamination by chemicals. The same applies to all other components of the IC system. To avoid disturbing temperature influences on the insulated column compartment, the entire system including pump and eluent reservoir must be protected against direct sunlight Arrangement of the instruments If the is used as a single detector (see chapter 2.4), the 709 IC Pump, 812 Valve Unit and are best stacked on top of one another in this order. In a two-channel system (see chapter 2.5), the optimum arrangement (1, 2 or 3 towers) depends on the laboratory space available. In general, the IC pumps should be set up at the very bottom and the IC detectors at the very top. 2.2 Mains connection Follow the instructions below for connecting to the power supply. If the instrument is operated with a mains voltage set wrongly and/or wrong mains fuse, there is a danger of fire! Setting the mains voltage Before switching on the for the first time, check that the mains voltage set on the instrument (see Fig. 4) matches the local mains voltage. If this is not the case, you must reset the mains voltage on the instrument as follows: 1 Disconnect mains cable Disconnect mains cable from mains connection plug Remove fuse holder Using a screwdriver, loosen fuse holder 16 below the mains connection plug and take out completely. 3 Check and change fuse if necessary Carefully take the fuses installed for the desired mains voltage out of fuse holder and check their specifications: V 3.15 A (slow-blow) Metrohm No. U V 1.6 A (slow-blow) Metrohm No. U

21 2.2 Mains connection 4 Insert fuse Change fuses if necessary and reinsert in fuse holder. 5 Install fuse holder Depending on the necessary mains voltage, insert fuse holder in the so that the corresponding mains voltage range can be read normally and the adjacent white arrow points to the white bar imprinted below the fuse holder (see Fig. 4) V V Mains switch Mains connection plug V V 16 Fuse holder V V Fig. 4: Setting the mains voltage Fuses Two fuses 3.15 A/slow-blow for V or 1.6 A/slow-blow for V are installed in fuse holder 16 of the as standard. Ensure that the instrument is never put into operation with fuses of a different type as this could cause a fire! For checking or changing fuses, proceed as described in section

22 2 Installation Mains cable and mains connection Mains cable The instrument is supplied with one of three mains cables with plug SEV 12 (Switzerland, ) with plug CEE(7), VII (Germany, ) with plug NEMA 5-15 (USA, ) which are three-cored and fitted with a plug with an earthing pin. If a different plug has to be fitted, the yellow/green lead (IEC standard) must be connected to protective earth (protection class 1). Any break in the earthing inside or outside the instrument can make it a hazard! Mains connection Plug the mains cable into mains connection plug 17 of the (see Fig. 2) On/off switching of the instruments The is switched on and off using mains switch 18. Do not switch the IC components on before all cable connections have been established. 14

23 2.3 Capillary connections 2.3 Capillary connections The capillary connections described below are necessary for operating the in a simple IC system for carbohydrate analysis (MIC- 8). The electronic connections and control by IC Net 2.1 are described in Section 2.4. Section 2.5 contains the description of a combination of this IC system with an IC system for the determination of anions with chemical suppression (MIC-9). The necessary changes to the capillary connections are described there Connection of the Bottle rack (option) The optionally available Bottle rack for supply vessels can be placed on top of the. The tubing connections to the 709 IC Pump are described in the corresponding leaflet Connecting the pulsation absorber To protect the column material from any pressure shocks which the injection may cause we recommend the insertion of the MF Pulsation absorber between the high-pressure pump and the injection valve. This is done as follows (see Fig. 3): 1 Connect the pulsation absorber Place pulsation absorber 30 on the floor in the interior of the. 2 Connection to pump Connect the PEEK Filter unit described in Section of the Instructions for Use to connection 23 of the 709 Pump by using PEEK capillary 22. Lead PEEK capillary 28 or 43 from connection piece 47 of the filter unit outwards through opening 2 of the Bioscan and attach it to one of the 52 connections on the top side of the pulsation absorber. 3 Connection to injection valve Connect PEEK capillary 29 to the second connection 30 of the pulsation absorber, lead it out through 2 of the Bioscan and connection it to the injection valve on the 812 Valve Unit (connection 17). The pulsation absorber is filled with isopropanol and must be rinsed with eluent before a separating column is connected (see Section 2.3.7). The Pulsation absorber can be operated in both directions. 15

24 2 Installation Connection of the injection valve Connect all capillaries to the injection valve according to the following description: Fig. 5: Connections at the injection valve of valve unit 42 Connection for syringe tubing PTFE tubing with coupling ( ) and syringe ( , without needle) 43 Connection for aspirating tubing PTFE tubing for aspirating sample 44 Connection for sample loop 20 µl PEEK sample loop ( ) 45 Eluent outlet Connection to preheating capillary 31 (PEEK, ) 46 Eluent inlet PEEK capillary connection 29 to pulsation absorber 47 Connection for sample loop 20 µl PEEK sample loop ( ) Mounting the column The IC separating column (e.g. Metrosep Carb 1, ) is first mounted in the as follows (see Fig. 3): 1 Connect column to the preheating capillary Remove the caps from column 25. Screw eluent outlet of preheating capillary 31 on to the inlet end of the column (observe flow direction). 2 Connect column to flow-through measuring cell Screw outlet end of column 25 on to eluent inlet 41 of the flow-through cell using the KELF pressure screw. 3 Fix column Fix the column in column holder

25 2.3 Capillary connections Connection of the flowcell The Flowcell ( ) is a part of the standard equipment of the. It is already mounted and ready to use Fig. 6: Connections at flowcell 35 Check marks For correct orientation of the top and bottom part of the flow-cell 39 Auxiliary electrode connection for blue electrode cable Mounting screws (4x) 40 Reference electrode connection for black electrode cable Outlet for eluent Connection of PEEK capillary by KELF pressure screw. PEEK pressure screws may damage the threads of the cell. 41 Inlet for eluent Connection of PEEK capillary by KELF pressure screw. PEEK pressure screws may damage the threads of the cell. 38 Working electrode connection for red electrode cable 19 1 Before starting up check all screws 36 Use the Allen key provided to loosen all four of the 36 screws by one rotation. Then slightly tighten the screws again, e.g. by holding the Allen key between the thumb and index finger only. Finally tighten all four screws by a quarter rotation. Always tighten opposing screws, i.e. tighten one screw and then the opposite screw. Avoid overtightening the screws as this could damage the Distance piece! 17

26 2 Installation 2 Connect the electrode cable The included Cable for connecting the measuring cell is attached to connections 38, 39 and 40 of the measuring cell using the three connectors 19, 20 and 24 (see Fig. 6). The other end of the cable is inserted into connection '1' of the 817 with connector 27 (see Fig 3). 3 Connect the column Screw column connection capillary 21 of the column on to eluent inlet 41 of the flow-through measuring cell using the Pressure screw. 4 Connect the waste container In order to ensure an adequate backpressure, screw a PEEK capillary (approx. 1 m) on to eluent outlet 37 of the measuring cell. Lead the end through opening 3 into a sufficiently large waste container and attach it there. 5 Mount the measuring cell Attach the flow-through measuring cell to the holder Bioscan which is provided for it (see Fig. 3). Rotate the holder and the cell so that the cell outlet is located as high as possible. In this way it is easier for any air bubbles that may occur to escape from the cell. Never switch on the flow-through cell when this is (a) not being rinsed through with a conducting eluent at the time, (b) not completely connected up, or (c) when its outside is moist so that so a short circuit could occur between the connections of working electrode 38 and auxiliary electrode 39! Connecting the waste tubing The has an outlet 13 on its rear panel for discharged liquids to which the waste tubing can be attached. Proceed as follows: 1 Connect the waste tubing Attach matching tubing (e.g. Silicone tubing, optional) into outlet 13 (see Fig. 2). 2 Lead the waste tubing to a drain Place the other end of the waste tubing in a drain and attach it there. 18

27 2.3 Capillary connections Rinsing the tubing The pulsation absorber is supplied permanently mounted and is filled with isopropanol. In order to remove this and any air that may be present in the capillaries and the measuring cell all the eluent-carrying tubing or capillary connections must first be installed; the column and the flow cell must not be connected during the beginning of the process. Proceed as follows: 1 Deconnect preheating capillary from column Deconnect the eluent outlet of preheating capillary 31 from the column and fix it at a waste bottle. 2 Set injection valve to Inject Press the <INJECT> key on the 812 Valve unit, which should not yet be connected to the. The green LED in the key lights up to show that the injection valve is in the Inject position. 3 Vent 709 IC Pump before start up Proceed according to Section 2.7 of the Instructions for Use of the 709 IC Pump. 4 Rinse with dist. H 2 O Immerse the aspiration capillary of the 709 IC Pump in distilled or deionized water. Switch on the 709 IC Pump, rinse the IC system for approx. 10 min. and switch off the 709 IC Pump. 5 Rinse with eluent Immerse the aspiration capillary of the 709 IC Pump in the eluent to be used (e.g. 100 mm NaOH). Switch on the 709 IC Pump, rinse the IC system for approx. 10 min. and switch off the 709 IC Pump. 6 Connect flowcell and rinse with eluent Connect the preheating capillary 31 to flowcell using column connection capillary 21 and Coupling. Switch on the 709 IC Pump, rinse the IC system for approx. 10 min. with eluent and switch off the 709 IC Pump. 7 Remove the coupling Remove the Coupling between column connection capillary 21 and preheating capillary 31. Remount the column between column connection capillary 21 and preheating capillary 31 as show in Fig Rinsing the column In order to rinse or precondition the column the connection capillary 21 to flowcell must be unscrewed from the column. Proceed as described in point 3 and 4 in section

28 2 Installation 2.4 System for sugar analysis MIC Electrical connections The MIC-8 system with manual sample injection is described below. Of course, you can also install a Metrohm sample changer. This is described in Section 2.5 or in the Instructions for Use of the sample changer. In order to connect a sample changer and control it completely you need a further free COM port on your PC. However, we recommend the use of the 762 IC Interface, to which you can then also connect the 709 IC Pump to be controlled by IC Net 2.1. The electrical connections of the MIC-8 system, which consists of the, the 812 Valve unit and the 709 IC Pump, are made according to Figure 7: 817 AUX 1 AUX PC Ground Fill Inject 709 Fig. 7: Connecting up a simple modular IC system with the 817 Bioscan MIC 8 20

29 2.4 System for sugar analysis MIC Settings in «IC Net 2.1» 1 System As in this configuration only the is to be directly controlled by IC Net then only this instrument is to be installed as the detector with the help of the system assistant New system wizard. 2 Method You should first select the standard method supplied for use with your particular column (e.g. for Metrosep Carb 1: CARB1_250.mtw). You can save any alterations under a different file name. 3 Data source Select: Cell( 817 BioScan) as the signal source. 4 Save system Then save the system (e.g. 817_MIC-8.smt). The following system window should now appear: 5 Connect system and workplace Under SYSTEM / Control / Connect to workplace connect the new system with the workplace. At the same time the symbol bar. will appear in the symbol 6 Check measuring cell If the is switched on without the flow-through cell being constantly rinsed by a conducting eluent (e.g. 0.1 M NaOH) then the measuring cell must not be switched on! You should now check this in the instrument window of the 817 Bioscan (accessed with a double-click with the mouse on instrument symbol) under Manual / Cell. 7 Check start mode Make sure that in this system the is standing under Start with determination under Setup / Start mode.. 21

30 2 Installation Connecting a sample changer If the MIC-8 system described above is to be operated with a Metrohm sample changer then the following connections and settings must be made: 817 AUX 1 AUX CELL OFF CELL ON + 24 V GROUND PC 766/788/ Ground Fill Inject Fig. 8: Connecting a simple MIC 8 system including a sample changer The IC Sample Processor 766, the IC Filtration Sample Processor 788 or the Compact Autosampler 813 can be used as the sample changer. In this combination of the MIC-8 system the is operated as the "Master". The start impulse for the instrument methods to the connected sample changer is transferred via the remote connection ( , optional). You can then select any instrument method at the sample changer that is described as being for control with the PC as master. Information about this is given in the corresponding Instructions for Use. If a second COM port on the PC or a 762 IC Interface is available for an RS232 connection then the 766 IC Sample Processor or the 788 IC Filtration Sample Processor can be connected up via this connection. In this way you can generate your own time programs in IC Net 2.1 for these sample changers. Details are given in the Instructions for Use for the particular instrument. 22

31 2.4 System for sugar analysis MIC-8 Creating the time program In order to control the 766 IC Sample Processor and the 788 IC Filtration Processor the following program is created in the Bioscan: In this way shortly after the injection valve has been switched to 'Fill' at the 812 Valve unit, the first remote line 'AUX 1' (see Fig. 6) will be switched briefly to 'ON'. This signal will be scanned at the start of the suitable sample changer programs. Terminate the program entry by activating the program with Enable and store it in the system with Save. The control of the 813 Compact Autosampler is carried out similarly. The only thing to be done here is to set the remote lines to the required times as described above for each of the three working steps: autosampler start, pump start and pump stop in order for a brief impulse to be transferred. In order for the connection Autosampler to function properly the program must first be started at the sample changer and then the "Sample queue" in IC Net

32 2 Installation 2.5 System for the analysis of anions and sugars MIC Electrical connections The combination of an anion system with chemical suppression with the IC system for sugar analysis is described as an example of a complete IC system for the simultaneous determination of anions and sugars. As ideally the two sample loops for the different separation pathways should be connected by as short a piece of capillary as possible for sample injection, a 733 IC Separation Center with two injectors ( ) is used. This means that the 812 Valve Unit is no longer required for sample injection for the Bioscan. The MSM Suppressor Module, which is frequently located in the 733 IC Separation Center, is then replaced by the 753 IC Suppressor Module which is used instead of the 752 IC Pump Unit. The electrical connection of the system, consisting for example of two 709 IC Pumps,, 732 IC Detector, 733 IC Separation Center (2 injectors), 753 IC Suppressor Module, 762 IC Interface and 766 IC Sample Processor is made according to the Figure 9: B A 762 PC MSM Fig. 9: Connections with a Bioscan 817 as second detector with a modular IC system MIC 9 24

33 2.5 System for the analysis of anions and sugars MIC Capillary connections This section describes the capillary connections required for the combination of the IC system for sugar analysis (MIC-8) with an anion system with chemical suppression (e.g. MIC-2). It is assumed that the capillary connections for the anion system are already known. Arrange the necessary components as shown in Fig. 9. Please also observe the recommendations about the arrangement of the IC modules given in Section The capillary connections for the correspond to a large extent to the connections described in Section 2.3. Only the connection of the injection valve to the 812 Valve Unit is not necessary. Instead the second injection valve B of the 733 IC Separation Center is connected for injecting the sample for the sugar determination (see below). For the components for determination of the anions the typical arrangement of the capillaries for such an anion system is assumed to be present. The necessary alterations concern the injection valve A in the 733 IC Separation Center, which must be connected for injection valve B so that the two sample loops can be filled simultaneously. In addition the MSM Suppressor Module connections must be transferred to the 753 IC Suppressor Module if the 752 IC Pump Unit has previously been used with the MSM Module contained in the 733 IC Separation Center. The necessary modifications are described below. Injections valves A and B are designated as follows: Injection valve A: sample injection for anion determination Injection valve B: sample injection for sugar determination All the connections for the two injection valves are shown in Figure 10 on the following page. 1 Connection injection valve A injection valve B For simultaneously filling the two sample loops as short as possible piece of PEEK capillary is attached to sample outlet 48 of injection valve A and sample inlet 55 of injection valve B. 2 Connection pulsation absorber injection valve B Connect PEEK capillary 29 to pulsation absorber 52 in the Bioscan, lead it out through opening 2 or 3 of the Bioscan, and in through opening 27 or 28 of the 733 IC Separation Center and connect it to injection valve B (eluent inlet 58). 3 Connection injection valve B preheating capillary Connect PEEK capillary at injection valve B (eluent outlet 57), lead it out through opening 27 or 28 of the 733 IC Separation Center and in through opening 2 or 3 of the Bioscan and use a PEEK coupling to connect it to preheating capillary

34 2 Installation A B Fig. 10: Connections of injection valves A and B in the IC Separation Center ( ) Injection Valve A - Anions - 48 Sample outlet PEEK capillary connection to injection valve B 49 Sample inlet PEEK capillary connection to sample processor (18 in Instructions for Use for 766 IC Sample Processor) 50 Connection for sample loop PEEK sample loop for anion analysis 51 Eluent outlet PEEK capillary connection to anion separation column 52 Eluent inlet PEEK capillary connection, e.g. to pulsation absorber for anion eluent 53 Connection for sample loop PEEK sample loop for anion analysis Injection Valve B - Carbohydrates - 54 Sample outlet PTFE tubing connection to waste 55 Sample inlet PEEK capillary connection to injection valve A 56 Connection for sample loop 20 µl PEEK sample loop ( ) 57 Eluent outlet Connection of preheating capillary 31 (PEEK, ) 58 Eluent inlet PEEL capillary connection 29 to pulsation absorber 59 Connection for sample loop 20 µl PEEK sample loop ( ) 26

35 2.5 System for the analysis of anions and sugars MIC Configuration in IC Net 2.1 The complete IC system is installed in IC Net using the New system wizard: IC Interface IC Detector IC Pump 27

36 2 Installation IC Pump IC Sample Processor IC Separation Center Suppressor Module 28

37 2.5 System for the analysis of anions and sugars MIC-9 9 Second data recorder 10 Anion method and data source 11 System overview 12 Final settings In the system window of the 753 Suppressor Module the option Start pump with hardware should be activated under Manual. 29

38 2 Installation 13 Bioscan method and data source The Bioscan method and corresponding data source must be selected in the Setup menu which can be accessed by a righthand mouse click on the second data recorder symbol. 14 Connect system with workplace Under SYSTEM / Control / Connect to workplace connect the new system with the workplace. At the same time the symbol appears in the symbol bar. 15 Check measuring cell If the is switched on without the flow-through cell being constantly rinsed by a conducting eluent (e.g. 0.1 M NaOH) then the measuring cell must not be switched on! You should now check this in the instrument window of the 817 Bioscan (accessed with a double-click with the mouse on instrument symbol) under Manual / Cell. 16 Check connections You can now check the electronic connections to all modules. This is done by double-clicking with the left-hand mouse key on each instrument symbol. If the connection is functioning properly then the current status of the instrument parameters will be shown. Remarks The event-line for the suppressor of the 753 Suppressor Module is only set up as a "dummy connection" (Step 8). The real command for switching the suppressor further is made by the 766 Sample Changer via a remote line. For the installation described above the option System face / Stacked recorder icons must be activated in IC Net 2.1 under Options / Global preferences. This is why only a symbol for the data recorder is visible in the system overview. With a right-hand mouse click and the offered Setup option the setting possibilities described for both recorders man (Step 10 and Step 13) can be accessed. 30

39 2.5 System for the analysis of anions and sugars MIC-9 Creating the time program 1. Input of the program for the 766 IC Sample Processor (Master): 001 Ctrl INIT 002 Move sample 003 Lift work 004 Ctrl ZERO Ctrl FILL A Ctrl STEP MSM Ctrl FILL B/Step Pump 120 s 009 Ctrl INJECT A Ctrl INJECT B 1 Initialize remote interface Move needle to sample position Move lift with needle to working height Trigger Autozero on 732 IC Detector Switch injection valve A on 733 to "Fill" Switch on 753 Suppressor-Module Switch injection valve B on 733 to "Fill" Fill sample loops A + B with sample for 120 s Switch injection valve A on 733 to "Inject" Switch injection valve B on 733 to "Inject" 1. Terminate the program entry by activating the program with Enable and store it in the system with Save. 2. Finally check that in the IC system the 766 Sample Processor is entered under Start with inject under Setup / Start mode. Remarks If only one of the detection pathways is to be used in this IC system then the modules which are not required can be removed from the system (remove recorder and instrument symbols with the right-hand mouse key and Unlink) and save the system under a new name. However, please note that in this instrument configuration the recording of the Bioscan data nevertheless begins only when the injection valve A on the 733 IC Separation Center is switched from Fill to Inject, as there is only a corresponding connection to the 762 IC Interface from this valve (see Fig. 9). This means that even if only sugar analysis is to be carried out, the 733 IC Separation Center and the 732 IC Detector must still form part of the system described above. The program described above for the 766 IC Sample Processor must then remain unchanged. 31

40 2 Installation 2.6 Preparing the for the analysis System switch-on This section describes the necessary steps for preparing the to carry out an analysis. A more detailed description of the operation of the using IC Net 2.1 can be found in Section 3 and in the Instructions for Use of the Metrodata IC Net 2.1 software. 1 Mains on Start up the individual instruments and the IC Net program in the following sequence: 1. Computer 2. IC instruments 3. IC Net Check measuring cell For safety reasons check the status of the measuring cell in the instrument window of the Bioscan (accessed by a double-click with the mouse on the instrument symbol) under Manual / Cell. If the is switched on without the flow-through measuring cell being constantly rinsed through a conducting eluent (e.g. 0.1 M NaOH) or correctly connected up then the measuring cell must not be switched on! 3 Check the external control of the 709 IC Pump Depending on the control of the 709 IC Pump (MIC-8: manual; combination with 762 IC Interface: external) the setting of the external control of the IC Pump should be checked with key 8 <EXT.> Start pump 1 Column/ Eluent With the Metrosep Carb 1 ( ) column use 0.1 M NaOH (quality) as the eluent for sugar analysis. This should be made up and degassed freshly every day; it should at least be degassed before use each day. 2 Flow rates / Pressure The Metrosep Carb 1 column should not be subjected to large variations in pressure. For this reason each time that the eluent is used it should be pumped for a period of 10 minutes both before and after use at 0.3 ml/min. For normal operation a flow rate of1.0 ml/min is used. Set a maximum pressure P max of 15 MPa. At 1 ml/min and with 0.1 M NaOH this should establish a pressure of approx MPa with the Metrosep Carb 1. 32

41 2.6 Preparing the for the analysis Set operating mode 1 Manual control of the 817Bioscan In the instrument window of the Bioscan (double-click with the mouse on the instrument symbol) select Mode: Pulse for pulse amperometric detection. The cell must remain deactivated for as long no eluent flows through the flow-through measuring cell. The oven is set to e.g. 32 C. In general this temperature should be at least 10 C higher than the ambient room temperature. 33

42 2 Installation 2 Parameters for the pulse mode Set these method parameters for the pulse mode. Please note that this menu can only be accessed when the option Pulse has been selected for Mode under Manual (see above). Finally transfer the current parameters to Bioscan with <Set> and use System / Save to save them in the system file (*.smt). The IC system with the is now ready for use. 34

43 3.1 Handling the 3 Operation 3.1 Handling the General Amperometric detection takes place with a flowing current and therefore with a chemical conversion of the analyte. The course of a chemical reaction depends directly on various physical parameters, among other things. In order to obtain optimum measuring conditions (e.g. stable baseline or reproducible signals) it is necessary to take the following points into consideration. Further information can be found in Section 4.2. Constant temperature The allows the interior of the instrument to be kept at a constant temperature of up to max. 60 C. The lower limit is the ambient temperature plus 10 C. Under IC Net you should activate the appropriate oven-operating mode in the 817 method parameters and define the temperature (see Section 3.2.2) Constant ph The characteristic current/potential curves of all electrochemically reactive substances depend very strongly on the ph of the ph eluent used. This means that you should prepare the eluent very carefully and check its ph at regular intervals. Pulsation-free eluent flow Changes to the eluent flow should be avoided as far as is possible. The Pulsation Absorber is available for this purpose; it is mounted between the 709 IC Pump and the injection valve as described in Section Never make dry measurements The flow-through measuring cell must not be switched on if it is not being rinsed constantly by the eluent or if the measuring cell is not connected up properly! 35

44 3 Operation 3.2 Operation using «IC Net 2.1» icon The 817 icon is available in the system window if an has been installed with the New system wizard or by using the Setup/New devices/install new device option of the SYSTEM window (see section 2.4.2). If the system is connected and the 817 icon is clicked with the right-hand mouse button then the following menu appears: Open Unlink Opens the window for parameter settings (this window can also be opened by double-clicking the icon). Deletes the 817 icon from the system. 36

45 3.2 Operation using «IC Net 2.1» window 817 icon / Open The window for parameter settings is opened by selecting this menu option with the right-hand mouse button or by double-clicking the 817 icon in the SYSTEM window. It consists of the four tabs Manual, Method parameters, Program, and Links. Manual The Manual tab of the window is only available for a connected system. Mode DC Pulse Scan Selection of detector mode: In the DC mode a constant potential is applied; the analytes are oxidized or reduced in accordance with their electrochemical properties. The Pulse mode works with three different potentials that are applied cyclically. This frees the electrode surface from any adhering reaction products during each sweep and an activated surface is produced for the next measurement. The Scan mode allows potential/current curves to be recorded. 37

46 3 Operation Cell <On> <Off> Manual control of amperometric detector cell: Switches on detector cell. Switches off detector cell. Attention Switch on the detector cell only if a conducting eluent is being pumped through it continuously! Oven <On> <Off> Temperature Manual control of column oven (heater): Switches on oven. Switches off oven. Temperature of column oven. Range: C (> room temp C) Control Manual setting of 4 remote lines. <Set> Sets remote lines to selected states. Aux1, Aux2, Cont1, Cont2 Setting of 4 remote lines. Selection: 0, 1, * Valve <Fill> <Inject> Analog output <Zero> Actual Mode Cell Oven Control Manual switching of the injection valve, e.g. at the 812 Valve Unit: Switches injection valve to "Fill" position (control line Cont1 is set to 0). Switches injection valve to "Inject" position (control line Cont1 is set to 1). Manual setting of the analog output signal to zero. For RS232 data the full-scale range (see next page) is shifted until the signal is located in the center of this range again (s. also Chapter 6.1). Triggers the autozero function. Display of actual values. Display of current detector mode. Display of current cell state. Display of current oven state. Display of current control line settings. 38

47 3.2 Operation using «IC Net 2.1» Method parameters DC mode The DC mode subtab of the Method parameters tab on the 817 Bioscan window is used to set the parameters for the DC mode. This window is only available if Mode has been set to DC on the Manual tab. Method data Cell potential Electric potential applied to the amperometric detector cell in V Range: V. Oven Switches column oven on/off and sets operating temperature. Range: C (> room temp C) Range Selection of measuring range: pa pa. na na. µa µa. Full scale The full-scale range (operating range) sets the desired sensitivity for the analog output. The possible values of the full-scale range depend on the preset measuring Range. For RS232 data (IC Net 2.1), always the maximum settings should be used. 39

48 3 Operation Filter Offset Electronic damping of the analog output (REC) and RS232 signal. Selection: 0.1, 0.2, 0.5, 1, 2, 5 s Offset of the zero point. Selection: % (in steps of 10 %) Actual Cell potential Display of current cell potential in V Oven Display of current oven state. Range Display of current measuring range. Full scale Display of current full-scale range. Filter Display of current damping constant. Offset Display of current offset. <Set> Sends current parameters immediately to the. Parameters are not stored in the system file (*.smt) as long as the file is not saved. 40

49 3.2 Operation using «IC Net 2.1» Pulse mode The Pulse mode subtab of the Method parameters tab on the window is used to set the parameters for the pulse mode. This window is only available if Mode has been set to Pulse on the Manual tab. Method data Cell Definition of three different working potentials that are applied to the amperometric detector cell cyclically. The set potentials are graphically displayed if the Method option is enabled above the graphic window. E1, E2, E3 Potentials. Range: V. t1, t2, t3 Time duration to apply the potentials. Range: s Sample time Measuring time at the end of working potential E1. Selection: 20, 40, 60, 80, 100 ms Oven Switches column oven on/off and sets operating temperature. Range: C (> room temp C) Range Selection of measuring range: pa pa. na na. µa µa. In the Pulse mode, always the µa range should be selected. 41

50 3 Operation Full scale Offset The full-scale range (operating range) sets the desired sensitivity for the analog output. The possible values of the full-scale range depend on the preset measuring Range. For RS232 data (IC Net 2.1), always the maximum settings should be used. Offset of the zero point. Selection: % (in steps of 10 %) Actual E1, E2, E3 Display of current potentials in V. t1, t2, t3 Display of current time durations for potentials in s. Sample time Display of current sample time in ms. Oven Range Full scale Offset Display of current oven state. Display of current measuring range. Display of current full-scale range. Display of current offset. Cycle potentials Graphical display of cycle potentials. Method Actual <Set> Display of potentials set in the Method data frame. Display of current potentials. Sends current parameters immediately to the. Parameters are not stored in the system file (*.smt) as long as the file is not saved. 42

51 3.2 Operation using «IC Net 2.1» Scan mode Parameters The Scan mode subtab of the Method parameters tab on the window is used for parameter setting and curve display for the scan mode. This window is only available if Mode has been set to Scan on the Manual tab. Method data Cell potential Definition of start and end potential for a user-defined potential sweep that is applied to the amperometric detector cell. E1 Start potential for sweep. Range: V. E2 End potential for sweep. Range: V. Scan cycle Definition of scan cycle: full double scan E1... E2... E1 half single scan E1... E2 Selection: full, half Scan rate Definition of scan rate in mv/s. Selection: 1, 2, 5, 10, 20, 50 mv/s Oven Switches column oven on/off and sets operating temperature. Range: C (> room temp C) Range Selection of measuring range: pa pa. na na. µa µa. 43