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SX20 Hardware User Guide

1. 80 Updated January 2006 Applied 2 5 4 Asymmetric Variable Ratio Mixing The SX20 is fitted with 2 5mL Kloehn manufactured drive syringes as standard This provides a 1 1 stopped flow mixing ratio Other mixing ratios may be achieved by fitting drive syringes of different volumes to the sample handling unit For single mixing stopped flow measurements the following ratios may be achieved using one standard 2 5mL Kloehn syringe and one of the Hamilton manufactured Salt Line syringes Syringe Configuration 2 5mL 2 5mL 2 5mL ImL 2 5mL 500uL 2 5mL 250uL 2 5mL 100uL 2 5 4 1 Setting the Drive Pressure for Asymmetric Mixing Experiments Caution To prevent damage to the flow circuit from excessive pressures produced when using drive syringes smaller than the standard 2 5mL Kloehn syringe the pneumatic drive pressure must be reduced via the pressure regulator fitted to the rear of the sample handling unit The drive pressure is set at 4bar for standard 1 1 mixing experiments For 1 5 1 10 and 1 25 mixing ratios the drive pressure must be reduced to 2 bar using the regulator connected to the gas inlet on the rear of the sample handling unit Left Hand Drive Ram Pressure Regulator Updated January 2006 Applied _ 2 5 4 2 Setting the Drive Volume for Asymmetric Mixing Experiments The total
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3. A liedPhotophysic J STEP 2 Flow Circuit Configuration The flow circuit should be set up according to the following instructions and referring to the diagram below oo Optical Cell Ea i Stop Valve Actuator wer AN wet Li sran _ Waterbath Adapter oe 115ul Ageing Loop se ease 4 Way Coupler Valve C to Cell Tube Straight Connector PK19 Stop Syringe SD PK18 Flush Ram Pre mix Ram 74 Updated January 2006 Applied _ Drain the thermostat solution from the water bath by removing the bleed screw from the top of the cell block Remove the front cover of the water bath Remove the aging loop from the straight through connector that connects to the flush line Remove the plugs from the pre mixer and the end of the extension tube Connect the extension tube to the straight through connector on the end of the flush line and the aging loop to the pre mixer Pressure test the flow line to make sure that it is not leaking Replace the front of the water bath and the bleed screw STEP 3 Adjusting the Pre Mix Drive Volume Screw the fine adjuster on the pre mix drive ram fully upwards so that the ram will have no upward travel this is the zero drive volume position Screw the fine adjuster five turns to increase the drive volume This equates to a pre mix drive volume of approximately 220uL It is essential that the pre mix drive volume should be 220uL 10uL Use of the Profi
4. The 5uL rapid kinetics cell allows collection of stopped flow data with a dead time of 0 45ms The cell is mounted in an interchangeable cell cartridge supplied in a protective storage case and is supplied with a I1mL stop syringe 2 2 10 QFA 1 Quench Flow Adaptor Option The quench flow accessory utilises the sequential mixing capability of the SHU The adaptor is mounted in the standard interchangeable cell cartridge and is fitted with a basic sample collection loop 2 2 11 AN 1 Anaerobic Accessory The anaerobic accessory improves the ease with which anaerobic experiments can be performed without the need for a glove box environment It consists of the following components 1 Pack of ten 3 way valves for loading anaerobic samples into the SHU 2 Nitrogen purging manifold that connects to the sample handling unit 9 Updated January 2006 Applied _ 2 3 SX20 Instrument Set up This section describes the basic set up procedure and configuration of the SX20 stopped flow reaction analyser More detailed information relating to the set up and connections of each component of the system is provided later in this chapter 2 3 1 Site Requirements The SX20 should be set up in a clean laboratory environment on a sturdy work surface The laboratory temperature should be maintained within the range 15 35 C The minimum space requirement for the SX20 length x width x height is as follows SX20 system 2 0m x 0 7m x 0 5m SX20 system f
5. Systems not equipped with the sequential mixing option are only configured for single mixing experiments No additional preparation is required a Optical Cell A Stop Valve Actuator cae P me ae ae ic acl i Waterbath Adapter aa yo ar Valve F to Cell Waste Tube E Valve C to Cell Tube gt DE Straight Connector l PK1 9 Stop Syringe c IA Co cre ae aes iy rF Loe f Drive Ram 70 Updated January 2006 Applied _ STEP 3 Adjusting the Drive Volume Screw the knurled volume adjuster on the Auto Stop fully clockwise i e front edge moves from left to right then screw it three and a half turns anti clockwise counter clockwise This equates to a total flow volume of approximately 140 uL STEP 4 Flushing the Flow Circuit Fit two reservoir syringes filled with distilled water to the C and F luer fittings It is recommended that 5 or 10mL plastic disposable syringes be used Turn the C and F drive valves to the Load side position Ensure that the drive ram is pushed fully down Fill the C and F drive syringes by pushing down the plungers of the reservoir syringes Expel any air bubbles by flushing backwards and forwards between the drive syringes and the reservoir syringes several times Turn the F drive valve to the Drive forward position and manually push the water from the drive syringe thro
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7. The light guide is normally connected to the short pathlength port lower right hand side of the cell housing Electronics Unit The electronics unit should be positioned to the left of the sample handling unit Workstation Monitor and Printer The workstation is normally positioned to the left of the electronics unit The monitor is positioned above the electronics unit 14 Updated January 2006 Applied _ 2 3 3 SX20 Electrical Set up This section describes the basic electrical set up of the SX20 stopped flow reaction analyser It systematically lists the connections that must be made between the various components of the system Mains Electricity Supply The lamp power supply unit and electronics unit feature a universal mains power supply and do not need to be set for the mains electricity voltage of your particular country These units are provided with mains cables terminated in IEC plugs These plugs should be connected to the distribution board provided The distribution board should be fitted with a plug suitable for the mains sockets fitted in your laboratory This means that only one socket is required to power the entire system The workstation PC monitor and printer are each supplied with a mains cable for connection to the distribution board The diagram overleaf shows the mains supply connections Earth Ground Connections The electronic circuitry used in the stopped flow spectrometer is very sensitive and m
8. 5 5 4 Procedure for Bench Top Anaerobic Stopped Flow Studies ccccceeees 85 4 Updated January 2006 Applied _ 2 0 Introduction This is a reference guide to the hardware of the SX20 stopped flow reaction analyser It describes the set up operation and routine maintenance of the hardware of the instrument A trouble shooting guide is also included to help the user fault find commonly encountered problems 2 1 SX20 Stopped Flow Reaction Analyser This section describes the experimental capabilities of the standard configuration SX20 stopped flow reaction analyser and the components that comprise the system 2 1 1 Description The SX20 stopped flow system in its standard configuration without any of the optional accessories described later in this section allows the user to carry out single mixing stopped flow measurements The user can select between detection of absorbance kinetics at a single wavelength or fluorescence kinetics using an optical filter to isolate the fluorescence signal 2 1 2 Components The standard configuration SX20 stopped flow system consists of the following major hardware components 1 150W Xenon lamp power supply and igniter unit 2 150W Xenon lamp housing lamp fitted 3 Monochromator Mono 1 4 Optical rail for mounting of the lamp housing and monochromator 5 Fibre optic light guide 6 Sample handling unit fitted with 20uL cell cartridge 7 Absorbance detector 8 Fluoresc
9. Operation Please refer to the Hewlett Packard user manual supplied with the computer for full details on operation of the computer The operation of the SX20 via the workstation is described fully in the software chapters of the User Manual 67 Updated January 2006 AppliedP _ 2 4 9 Photo Diode Array Detector Option PDA 1 2 4 9 1 Description The SX20 may be equipped with an optional photodiode array detector for collection of time resolved absorbance spectra from a single stopped flow drive There are two versions of the 256 element photodiode array accessory The visible region PDA has an operational spectral range of 300 1100nm with a wavelength resolution of 3 3nm The UV region PDA has an operational spectral range of 190 735nm with a wavelength resolution of 2 2nm Each PDA is capable of an integration speed of 1 4ms per scan 2 4 9 2 Basic Set Up and Electrical Connections The cream coloured photodiode array unit is normally positioned on the rear of the sample handling unit The PDA is connected to the cell housing via a fibre optic light guide The light guide is attached to the cell housing and the PDA via two finger screws The light guide has a rectangular bundle of fibres of dimensions 7mm x Imm at the SHU end and a circular bundle of fibres of 3 5mm diameter at the PDA end The PDA unit is connected to the PDA module in the Electronics Unit via a black cable fitted with 26 pin D connectors The yellow diode lab
10. and adsorbed into the wall material of the sample flow circuit The accessory comprises a purging manifold and a set of 3 way valves 2 5 5 1 Purging the Drive Syringes The most significant source of oxygen ingress is between the plunger and barrel of the drive syringe Although the syringes are gas tight the tips of the plungers are made of Teflon which does not act as a barrier to oxygen In order to stop oxygen from passing between the barrel and plunger the end of the syringe barrel needs to be maintained in an inert environment This is achieved using the anaerobic purging manifold shown attached to the sample handling unit in the picture below 83 Updated January 2006 Applied _ To fit the accessory push it over the drive syringe plungers and screw the accessory to the underside of the sample handling unit water bath so that the ends of the drive syringe plungers are enclosed Connect an inert gas supply nitrogen or argon to the male fitting on the side of the manifold and gently purge so that the ends of the drive syringes are in an inert environment The previous diagram shows a cut away view of the assembled accessory 2 5 5 2 Flow Circuit Preparation Another significant source of oxygen contamination is through the flow circuit The SX20 sample handling unit is fitted with PEEK tubing and PEEK drive valves as standard This minimises the danger of oxygen contamination from the tubing and the valves The thermostatic liqu
11. drive volume should be increased as the ratio increases in order to obtain good reproducible results The table below lists some recommended drive volumes that may be used as a guide during experimental set up Preparing the SHU for Asymmetric Mixing 1 2 3 4 5 6 7 Check that the drive pressure has been set to 2bar Drain the water bath housing and remove the front plate Unscrew the F drive syringe from the drive valve retaining plate by gripping the metal tip Withdraw the syringe down through the rubber water bath seal Repeat the reverse of the removal procedure to fit the Hamilton syringe It will be necessary to fit a rubber sleeve to the glass barrel of smaller diameter syringes to prevent water bath leaks Tighten the syringe into the drive valve retaining plate carefully to provide a leak free fit Test the flow circuit for leaks using the procedure described previously and tighten the drive syringe further if necessary Replace the front plate of the water bath housing Adjust the drive volume on the Auto Stop according to the values listed in the table above 82 Updated January 2006 AppliedPhotophysics 2 5 5 Anaerobic Operation using option AN 1 The AN 1 anaerobic accessory equips the SX20 with a high performance bench top anaerobic capability In such an environment operating under anaerobic conditions requires stringent care with respect to eliminating oxygen dissolved in the thermostat medium
12. dual channel fluorescence detection or fluorescence polarisation options the second fluorescence detector must be connected to Channel 1 of the Electronics Unit PMT Module This channel is normally used for absorbance measurements and the fluorescence detector is best connected by disconnecting the detector cable from the absorbance detector and connecting to the second fluorescence detector For dual channel fluorescence detection the second fluorescence detector should be mounted on the upper right port of the cell block according to the picture below using the twist lock fittings supplied The male fitting is connected to the cell block using 4 countersunk screws ensuring that the light sealing O ring is fitted The female fitting is connected to the end of the fluorescence detector using 3 screws 46 Updated January 2006 AppliedPhotophysics 2 4 4 Monochromator 2 4 4 1 Description The high precision automated monochromator contains a 250nm_ holographic diffraction grating that provides a useful operating wavelength range of 190 to beyond 850nm limited by PMT detector range The monochromator is accurately pre aligned and calibrated using a He Ne laser and mercury emission lines The wavelength of monochromatic light is selected by rotation of the grating using a stepper motor housed within the monochromator unit This motor is controlled via Control Module 1 in the Electronics Unit This allows the monochromator wavelength to
13. flow from the optical cell to the stop syringe and from the stop syringe to the waste collection vessel lstop syringe that is emptied and filled during the stopped flow process The sequential double mixing flow circuit features two additional drive valves and drive syringes plus extra flow tubing This configuration will be described fully later in this Chapter 24 Updated January 2006 AppliedPhotophysics Stop Valve Actuator Left Valve to Cell Tube oe is a o ak Water Bath Adapter Waste Tube TPA ates Right Valve to Cell Tube Stop Syringe WZ Drive Ram Optical Cell Updated January 2006 Applied _ 2 4 1 4 Drive Syringes Two 2 5mL drive syringes are fitted to the standard single mixing SHU The drive syringe is screwed into the base plate of the drive valve and is filled with the reagent solution that is mixed during the stopped flow drive The plastic piston is constructed of PEEK to protect against corrosion The tip of the piston is Teflon The SHU water bath is fitted with rubber grommets to provide a leak free seal between the drive syringe and the water bath housing In addition to the standard Kloehn manufactured 2 5mL syringe use of Hamilton syringes of different volumes may be used to achieve asymmetric mixing ratios This is described in detail later in this Chapter However it is essential that if asymmetric mixing ratios are attempted the drive pressure mu
14. of stray light Fluorescence measurements are normally carried out using the 2mm pathlength of the standard 20uL cell or 5mm pathlength of the SuL cell These are accessed by fitting the excitation light guide to the lower right port on the cell block Remember to blank off any unused ports 44 Updated January 2006 Applied _ Caution Before removing the detector from the cell block it is essential that any high voltage applied to the detector is switched off via the software to prevent damage to the PMT For SX20 systems fitted with a single detection channel there will be one cable connected to the PMT Module of the Electronics Unit When switching between absorbance and fluorescence detection the cable must be connected to the appropriate detector For SX20 systems fitted with dual detection channels there will permanently be two detector cables connected to the PMT Module of the Electronics Unit The absorbance detector is connected to Channel 1 and the fluorescence detector is connected to Channel 2 2 4 3 3 PMT Characteristics The response characteristics of the Hamamatsu R6095 PMT are displayed in the chart below AW EE ees im im QUANTUM OyT Y O EFFICIENCY KA C _ TIVITY mA GY 5 QUANTUM EFFICIENCY 9 SENE CATHODE RADIANT ay ka 200 40 600 B00 WAVELENGTH nm 45 Updated January 2006 AppliedPhotophysics 2 4 3 4 Dual Channel Fluorescence Set Up For SX20 systems fitted with the
15. source uses a 150W gap shortened xenon bulb and is ignited and powered by the lamp power supply unit Three different lamps may be fitted to the housing to accommodate experimental requirements A manually operated shutter is located on the exit port of the lamp housing to control the passage of light to the monochromator The shutter is open when the shutter is pushed to the rear The shutter is closed when pulled to the front Lamp Shutter 50 Updated January 2006 Applied gt gt 2 4 5 2 Basic Set Up and Electrical Connections The 150W lamp housing is mounted on the right hand side of the optical rail A magnet assembly is sometimes attached to the rear of the housing to provide greater stability of the xenon arc lamp The lamp housing is fitted with two input terminals black negative and red positive that are connected to the lamp power supply unit The earth post should be connected to the earth post on the optical rail using the braided cable supplied Lamp Stability Magnet ve Terminal ve Terminal a Earth Point 2 4 5 3 Lamp Stability Magnet The SX20 lamp housing is sometimes fitted with an adjustable magnet assembly that stabilises the xenon arc The magnet is attached to the rear of the lamp housing in the orientation that provides the greatest stability 51 Updated January 2006 Applied _ 2 4 5 4 Lamp Types There are three types of 150W arc lamp approved for use in the SX20 lamp housing The choi
16. switched off by moving the rocker switch to the off O position 2 4 7 3 Maintenance The Electronics Unit contains no user serviceable components and should not require any maintenance In case of a damaged fuse the 1A mains fuse holder is located above the mains inlet socket 2 4 7 4 Electrical Characteristics The electrical characteristics of the system are as follows Supply voltage 85 264V Supply frequency 47 63Hz Power rating SOVA Fuse IA 65 Updated January 2006 AppliedP hotophysics 2 4 8 Workstation 2 4 8 1 Description The SX20 features a Hewlett Packard PC fitted with a fibre optic interface card that is linked to the Electronics Unit of the SX20 and provides computer control of the stopped flow data acquisition via the SX Control Software The PC is connected to a flat screen monitor ink jet printer and network socket A programmable water circulator may also be connected to provide temperature control of the stopped flow unit 2 4 8 2 Basic Set Up and Electrical Connections The set up of the computer is fully described in the Hewlett Packard PC user manual supplied with the computer Please refer to this manual for detailed instructions for setting up the computer The interface card is connected to the Electronics Unit via a fibre optic cable Fibre Optic PC Interface Card Fibre Optic Cable To Electronics Unit 66 Updated January 2006 al I E z WhArTnnnveire O Ok re We oe j J 2 4 8 3
17. the exception of those that connect to the optical cell face These tubes are attached to the cell using a pressure plate which enables both inlet tubes and the outlet tube to be tightened or loosened simultaneously The arrangement of the pressure plate and flow lines is displayed in the picture below For the purpose of clarity the drive valves have been removed Securing Screw Pressure Plate Waste Flow Tube Reagents Out Valve C Flow Tube Reagent In Valve F Flow Tube Reagent In 29 Updated January 2006 Applied _ The single mixing SHU flow circuit comprises the following tubes Part Number Left Hand Valve to Cell Right Hand Valve to Cell Cell to Stop Valve The sequential double mixing SHU flow circuit comprises the following tubes Part Number Valve A to Pre Mixer Valve B to Pre Mixer Pre Mixer Pre Mixer to Connector Connector Pre Mixer to Cell Valve F to Connector Valve C to Cell Cell to Stop Valve 30 Updated January 2006 Applied PEEK is an inert thermoplastic material that is compatible with a wide range of organic solvents acids and bases making it an ideal choice for the stopped flow circuit PEEK also offers high anaerobic performance for oxygen sensitive stopped flow experiments provided that the flow circuit has been prepared carefully The compatibility of PEEK with various reagents encountered in stopped flow measurements is outlined in the table below 1 1 1 Trich
18. the lower left hand port A plastic blanking plug should be placed over the unused port Sample Handling Unit Pneumatic Connections The stopped flow operation of the sample handling unit is driven by a compressed gas supply A nitrogen cylinder is recommended for this purpose and must be regulated to 8bar 125psi A length of flexible tube 6mm internal diameter is supplied for connecting the cylinder to the sample handling unit The cylinder pressure regulator should be fitted with a push on fitting to accommodate the flexible tube which is clamped onto the fitting with the finger tightened hose clamp supplied with the system 12 Updated January 2006 AppliedP hot The main pneumatic inlets labelled 1 amp 2 are located on the rear of the sample handling unit Port is internally connected to the auto stop and on sample handling units equipped for sequential mixing the right hand drive ram Port 2 is connected to the left hand drive ram An h shaped pneumatic tubing arrangement allows both of these ports to be connected to the flexible tube of the compressed gas supply see picture The arrangement also incorporates an in line pressure regulator that is used to adjust the drive pressure of the left hand drive ram This connector may additionally be used for supplying nitrogen to the lamp housing when using ozone producing lamps or when using the anaerobic purging manifold Detailed instructions for operating with ozone
19. 24 2 4 1 3 Stopped FlOW Circuit eeeneeesseesssssssereeerereeessssssssrnesesrreeeeessssssssnsseserereeeesssss 24 2 ANA DIVES VES CS asin sve desresvecanvedvcesdeshanntu aE a RAEES 26 BAM Diye VARY CS e a a ects 26 Os UO OEIC onr eens tactendhend secant a EEEE EEE ENE 21 BA T OEN IV a E E E E E E E ET 28 24 LS Plow DIN iero EEEE AE EEEE 29 2 4 1 9 Interchangeable Cell Cartridge System ccsssssseesseeeeeeeeeeeeeeeeeaeaeeeeeeeees 32 Dada CMG reen E E E NE 34 2 4 1 11 Absorbance Pathlength Selection ccccccccssssssseesseeeeeceeeeeeeeeeeeaaeaeeeeeees 35 2 4 1 12 The Auto Stop Mechanism cc cccccccccccecceeeceeeeeeeecsesseeeeeeeeeeeeeeeeauaaaeeesnees 36 2 4 1 13 Pneumatic Drive and Drive Rams cccccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 39 2 4 1 14 Temperature amp OMIIO sx ssicsanensnecieacataosananasonavsansasdessnesnenetwasnesanacancsateesninnsbequavutis 40 24 2 Absorbance Delet tOn craes sonnn unnn EEEE NE RENNE EEEN 42 2 Updated January 2006 Applied gt 24 21 Deso piOn noan r aaa a eiia a ii aari 42 2 4 2 2 Basic Set Up and Electrical ConnectionSs eseesssssseoeeoeeeeeessssssssssssseererreesssssss 42 24 2 3 PMT Characteristi S sicsreceascsnatecsicccnatooscaaqdeapiananseahantunnssebedasaheseaeneuamettadeansurneewaeaes 43 2 AS Fluorescence DELCCLOL irisito ania aian eiia i id eian ia aiiis 44 2A Deser pO eninin Nar a aa a a a aai 44 2 4 3 2 Basic Set Up and Electrical ConnectionSs
20. 4 4 6 Photodiode Array Operation When the SX20 is used with the optional photodiode array accessory PDA 1 the monochromator is automatically set to the zero order position by the software This allows white light from the lamp source to pass directly through the monochromator to the optical cell The user must adjust the entrance and exit slit widths to maximise the signal reaching the photodiode array unit A detailed description of photodiode array operation is provided later in this chapter of the user manual 2 4 4 7 Absorbance Measurements in the near IR gt 650nm It is essential that absorbance measurements collected at wavelengths greater than 650nm use an appropriate filter to eliminate second order stray light from detection Second order stray light is a phenomenon common to all monochromators fitted with diffraction gratings In addition to monochromatic light of the required wavelength a small proportion of light with half this wavelength is also generated All SX20 systems are supplied with a 645nm cut off filter that should be positioned between the optical cell and the absorbance detector when measuring absorbance in the near IR region gt 650nm 2 4 4 8 Maintenance The monochromator should not require any maintenance from the user Under no circumstances should the user attempt to recalibrate the monochromator 49 Updated January 2006 Applied ho gt 2 4 5 150W Lamp Housing 2 4 5 1 Description The arc lamp
21. Applied gt SX20 Hardware User Guide This section provides a complete guide to the hardware of the SX20 stopped flow system It includes a technical description of the hardware instrument set up hardware related aspects of operation and a basic maintenance guide Applied Photophysics Limited 203 205 Kingston Road Leatherhead KT22 7PB United Kingdom Tel 44 0 1372 386537 USA 1 800 543 4130 Fax 44 0 1372 386477 Email techsup photophysics com URL www photophysics com 1 Updated January 2006 Applied _ Contents PATATE CE E Os aica csicesacteasteeaaet aside saeeuseisauecsnesectaseueausestesonensevaanecsuesaeoaseeaatsestecarendeesanecseesaronse 5 D Mig NMC SCT ID OM eseese EOE O ES EE EIES SERET 5 22 COMO TC FILS seirena era ERT E OE REES EAEE OENES SERET 5 2 2 SX20 Optional Accessories eesessesssssssseeeterreesssssssssssserererrrresssssssssseseeeeeereressssss 7 2 2 1 SQ 1 Sequential Double Mixing Option eeesssssssssssseeeeeereessssssssssssseeereeeessssssss 7 2 2 2 SEM 1 Emission Scanning Option cccccccccccccccccceceeeseessseeeeeeeececeeeeeseeaeaaaeeeeees 7 2 2 3 AM 1 Extended Absorbance Option ccccccccceccccccaeeeeeseeeeeeeeecceeeeeeeeeeeaaaaaeeeeees 7 2 2 4 PDA 1 Photodiode Array Option ccccccccceeeccececceeeeeseeseseeeecceeeeeeeeeeeauaaaeeeseess 8 2 2 5 UV 1 Deuterium Light Source Option cc ccccccccssssssessseeeeeceeeeeeeeeeeeaaaaeeeeeee
22. be set via the SX20 control software Entrance Slit Control Exit Port Exit Slit Control Sn l dn Screws The monochromator entrance and exit slits are manually adjustable using the dials on the front of the monochromator The dials are calibrated in millimetres and are mechanically linked to a pair of slit blades on the entrance and exit A 1mm monochromator slit opening is equivalent to a wavelength bandwidth of 4 65nm The slits provide a bandwidth range from below 0 5nm up to 37 nm 47 Updated January 2006 Applied _ 2 4 4 2 Basic Set Up and Electrical Connections The monochromator is located on the optical rail to the left of the lamp housing It is essential that the lamp housing and monochromator are correctly coupled together The two units must be pushed together so that the exit port of the lamp locates firmly with the entrance port of the monochromator To prevent vibration both units should be locked in position on the optical rail using the four plastic locking screws on each unit The monochromator control cable is connected to Port 1 of Control Module 1 in the Electronics Unit For systems with two monochromators Mono 1 must be connected to the Drive 1 port and Mono 2 must be connected to the Drive 2 port 2 4 4 3 Selecting a Suitable Monochromator Slit Width Band Pass The monochromator slit width setting is an important variable in the set up of a stopped flow experiment Typically these values lie in the ra
23. ce of lamp will depend on individual experimental requirements Systems are normally supplied fitted with a 150W ozone free xenon lamp unless requested otherwise This lamp has excellent long term stability and a spectral range of 250 1000nm For studies in the far UV region a I50W ozone producing xenon lamp is recommended with a spectral range of 200 1000nm Operating with an ozone producing lamp requires the lamp housing to be purged with an inert gas such as nitrogen The purging procedure is described later in this section For fluorescence based measurements a 150W mercury xenon lamp is available that offers significantly higher output at particular wavelengths The lamp emission profile relative to the standard 150W source is shown overleaf and is accompanied by a table listing the most significant mercury emission lines Fluorescence excitation at these wavelengths provides an improved signal to noise level in fluorescence data compared to a standard 150W xenon lamp Additional lamps can be supplied mounted on an extra lamp housing unit back plate and storage case This allows for more rapid lamp changeovers and eliminates the need for lamp alignment following the swap The table below lists the three lamps approved for use with the SX20 system Osram 150W Xe O free XBO 150W CR OFR Osram XBO 150W 4 Hamamatsu Sensitive L2482 fluorescence SF general SF 150W Xe O producing Far UV region SF 150W Hg Xe 52 Updat
24. cking on Empty in the Pro Data control software Repeat the above two steps until the drive syringe is empty Flush from the other three drive syringes in the same manner as described above Fill all four drive syringes with water and turn all of the drive valves to the Drive position Enable the sequential mixing mode in the Pro Data control software With a delay time of 1s carry out a stopped flow drive by clicking the Drive button in the window shown below The drive profiles will be displayed automatically after the drive and should be used to check the accuracy of the drive and 2 volumes SHU Setup Cell Sequential Solutions Syringes Waste timers General Enable Delay time s 05 76 Updated January 2006 AppliedP _ STEP 6 Checking the Drive Volume Using the Profiles Function Carry out several drives and check that the drive 1 volume is 220uL 10uL and the drive 2 volume is 180uL 10uL from the Drive Profiles data shown in the window below The drive volumes and measured delay time should be consistent from run to run Desi ime C001 TET i AgnaTine C0501 Tomy T aoura AH Fe ul Dielom 710 Ml ul De m 159 732 ul Fina elocie 14279 Volurmevul imes STEP 7 Loading the Reagents Ensure the all four drive valves are set to the Load position Replace the reservoir syringes with ones containing the samples The F flush reservoir syringe should be filled with a neutral buffer solutio
25. control knob 90 between the Load and Drive positions The picture below shows the orientation of the valves in both positions tor LP heteee myn i hn F P y A i i Valves in Drive Position Valves in Load Position 2 4 1 6 Stop Syringe A 2 5mL stop syringe is fitted to the SHU Its purpose is to act as a small reservoir of waste solution from which a volume equal to the drive volume may be displaced prior to the stopped flow drive At the time of the drive the stop syringe is refilled and the action of the plunger striking the trigger indicates that the drive is completed and activates the acquisition of data The stop syringe is screwed into the lower threaded port of the stop valve and supported in this position by the stop syringe clamp The plastic piston is constructed of PEEK to protect against corrosion The tip of the piston is Teflon 27 Updated January 2006 AppliedP hot In addition to the standard Kloehn manufactured 2 5mL stop syringe use of other stop syringes may be used for specific experiments For rapid kinetic measurements rate constants in excess of 500s a smaller volume 1mL Hamilton manufactured stop syringe is recommended for improved data fitting This is described in detail later in this Chapter 2 4 1 7 Stop Valve The stop valve controls the flow of solution from the flow circuit into the stop syringe and from the stop syringe to the waste vessel The stop valve may b
26. d flow drive for 10s with the drive pressure held 3 During the 10s drive closely observe the tips of the drive syringe pistons 4 Ifthe pistons do not creep upwards the flow circuit is leak free The flow circuit is full when the stop syringe contacts the auto stop trigger initiating data acquisition At this moment the drive syringes should not be able to push more solution from the drive syringes and the pistons should not creep upwards If the flow circuit leaks the pressure held on the drive syringes over 10s will push the solution from the drive syringes out of the flow circuit at the location of the leak In this case the pistons will appear to creep upwards during the 10s period A more detailed description of identifying leaks and their repair is given in the maintenance section of this chapter 33 Updated January 2006 Applied 5 1 10 Optical Cells All optical cells available for use on the SX20 are construction from quartz and feature an integral flow circuit and T mixer located in the quartz The dimensions of the optical windows of the cell provide the user with different optical pathlengths and dead times The diagram below shows the internal design of the 20uL optical cell 2mm Pathlength Observation Window Reagent Inlet Port Waste Outlet Port Fluorescence Observation Window 10mm Pathlength Observation Window Reagent Inlet Port T Mixer Cell Pathlengths and Dead Times The table belo
27. displays the number of hours that the lamp has been running Adjacent to the timer is a recessed button to reset the timer when the lamp is replaced The lamp is switched off by moving the rocker switch to the off O position 2 4 6 4 Use of Xenon and Mercury Xenon Lamps The lamp PSU is designed to supply a running current appropriate for a 150W Xenon lamp and a 150W Mercury Xenon Lamp A switch on the rear panel labelled 8 5A and 7 5A provides a simple way to control the running current for these lamps When using a xenon lamp ensure the switch is set to the 8 5A position When using a mercury xenon lamp set the switch to the 7 5A position An incorrect setting will result in increased noise visible on data traces 59 Updated January 2006 AppliedF physics O 2 4 6 5 Maintenance The lamp PSU contains no user serviceable components and should not require any maintenance In case of a damaged fuse the 3 15A mains fuse holder is located above the mains inlet socket 2 4 6 6 Electrical Characteristics The characteristics of the lamp power supply and igniter unit are as follows Supply voltage 85 264V Supply frequency 47 63Hz Power rating 250VA Fuse 315A 60 Updated January 2006 AppliedPhotophysics O _ 2 4 7 Electronics Unit 2 4 7 1 Description The Electronics Unit provides automated control of the sample handling unit and monochromator wavelength setting plus signal acquisition from the photomultiplier tube det
28. e control knob anti clockwise 180 to the front This is the Drive position Stop Syringe Brake Mechanism The brake mechanism see Auto Stop picture is provided to improve the results obtained when performing sequential double mixing experiments It is used to provide friction on the stop syringe plunger and hence prevent over run after the first drive This would result in cavitation in the flow line and inconsistent results The friction is increased by tightening the mechanism decreased by loosening it It must be loose for single mixing stopped flow so that it does not impair the dead time of the instrument but tightened up as necessary when performing sequential mix experiments Note that the sequential mixing brake assembly is only fitted to SHUs equipped with the sequential double mixing option SQ 1 38 Updated January 2006 AppliedPhotophysics 2 4 1 13 Pneumatic Drive and Drive Rams The SX20 Sample Handling Unit features a pneumatic drive system The drive ram acts as the interface between the pneumatic drive system and the flow circuit To satisfy the differing needs of the single mixing stopped flow and sequential double mixing stopped flow techniques three drive rams are supplied on the full sequential flow machine On the single mixing instrument only one drive ram is supplied The picture below shows the three drive rams compatible with the SX20 Single Mix Drive Ram Flush and Pre Mix Drive Rams Sequen
29. e drive ram There are three steps to this process Firstly the stop valve is turned to the Empty position Secondly the return cylinder is raised to displace a fixed volume of solution from the stop syringe Finally the return cylinder returns to its initial position and the stop valve is switched to the Drive position 2 The drive ram fires pushing the reagents from the two drive syringes through the flow tubes into the optical cell The optical cell contains a T mixer that accounts for thorough mixing of the two reagents prior to the observation window 3 The flow of the mixed solution is stopped at the moment the stop syringe plunger contacts the copper trigger on the Auto Stop mechanism I e once the selected drive volume has been mixed 4 A signal is monitored as set up in the software 2 4 1 3 Stopped Flow Circuit A simplified view of the single mix flow circuit is portrayed in the diagram overleaf The flow circuit consists of the following key components 2 drive valves for loading the reagents into the drive syringes 2 drive syringes for storing the reagents prior to mixing 2 flow tubes for transferring the reagents from the drive syringes to the optical cell 1 optical cell with an integral T mixer where the reagents are mixed and an observation chamber with two different optical pathlengths 1 waste flow tube for transferring the mixed reagents from the optical cell to the Auto Stop unit 1 stop valve that controls the
30. e fluorescence detector is connected to Channel 2 via a black cable fitted with a 10 pin D connector PDA Module For systems equipped with the photodiode array option this module controls the PDA unit which is mounted on the back of the sample handling unit The PDA unit is connected to the module via a black cable fitted with a 26 pin D connector Workstation Monitor and Printer Refer to the user manuals supplied with these units for detailed instructions relating to electrical connections The workstation houses a PCI card that is connected to the communications module of the Electronics Unit via a fibre optic communication cable 19 Updated January 2006 AppliedP hotophysics 2 3 4 SX20 Optional Accessories Basic Set up This section describes the set up of the optional accessories for the SX20 system The basic instrument configuration does not alter significantly for any of the optional accessories with the exception of options SEM 1 and AM 1 which feature a second monochromator Instrument set up for other optional accessories will be described fully in the relevant operating instructions section 2 3 4 1 SEM 1 Scanning Emission Monochromator Option This option features a two tiered optical rail and second monochromator The instrument layout for this option is shown in the picture below Sample Handling Unit Emission Light Guide Emission Mono 2 Extended Optical Rail Power Supply Unit Excitation Light Guide Excitati
31. e turned manually using a control knob or automatically via the software controlled Auto Stop mechanism The Drive position is used to control the flow from the flow circuit to the stop syringe The Empty position is used to control the flow from the stop syringe to the waste vessel The Drive and Empty positions are shown in the diagrams below Stop Valve in Drive Position Stop Valve in Empty Position The body of the valve is constructed of PEEK with a plastic spindle The 180 rotation of the spindle provides control of the flow direction The spindle is retained in the valve body by a stainless steel plate A stainless steel adaptor is screwed into the entrance port of the stop valve to accommodate the PEEK flow tube connected to the optical cell A polypropylene tube is connected to the exit port to flow to the waste vessel 28 Updated January 2006 AppliedP n oto D ry ys cs 2 4 1 8 Flow Tubing The flow circuit utilises custom made PEEK polyetheretherketone tubing terminated with compression fittings to link the drive valves with the optical cell and the cell with the stop valve The rigidity and chemical inertness of PEEK make it ideally suited for this purpose The internal diameter of all the flow tubing before the cell entrance is 1 56mm The waste tube connected between the cell and stop valve is 3 12mm All the flow tubes are connected to the various valves and connectors in the flow circuit using threaded fittings with
32. each stopped flow drive Circulator Unit Connections A circulator unit is connected to the SHU water bath via 8mm tubing The SHU is fitted with quick fit Legris type fittings to allow the circulator to be connected and disconnected rapidly without the need to drain the water bath The rear port is the outlet and the front port is the inlet For circulator units equipped with an external temperature probe to compensate for temperature loss between the circulator and the external water bath a probe port is provided on the right side of the Software Controlled Circulator Units Certain circulator units may be connected to the serial port of the workstation PC via a RS232 cable to provide temperature control in the SX20 control software The following circulator units may be linked to the workstation PC Neslab RTE 200 Fisher Scientific 3016 For further information on circulator unit compatibility please contact the Technical Support Team at Applied Photophysics 40 Updated January 2006 Applied ysic _ Recommended Thermostatic Fluids Applied Photophysics recommend the addition of an anti bacterial agent and antifreeze to distilled water as a suitable thermostatic fluid Draining the Water Bath The water bath can be drained according to the following procedure 1 Switch off the circulator unit 2 Loosen the bleed screw on top of the cell block using a 2 5mm hexagonal wrench 3 Allow the water bath to drain 4 Tighte
33. ectors and data processing The modular design of the SX20 electronics means only required features need be installed and any faults that develop are localised and can be easily repaired by substitution of the appropriate module All modules have a built in self test capability which communicates any operational problem to the user 61 Updated January 2006 AppliedP hot The rear view of the SX20 Electronics Unit is shown in the picture below F E 3 The full functionality and connections of the Electronics Unit modules are described in order from left to right Mains Inlet The Electronics Unit mains electricity supply inlet is connected to the mains distribution board The universal mains 12V Power Supply Module This provides 12V power to all electronics modules of the SX20 except the Communications Module A red LED indicates that the module is operational Two sockets are provided for monitoring the output level of the module 5V Power Supply Module This provides 5V power to all electronics modules of the SX20 A red LED indicates that the module is operational Two sockets are provided for monitoring the output level of the module Communications Module This is the interface of the electronics and the fibre optic link to the computer it also provides several extra inputs for analogue temperature probes and a general purpose digital I O The module is connected to the PCI card housed in the workstat
34. ed January 2006 AppliedP hot ysics The output profiles for the xenon lamp red and mercury xenon lamp blue are displayed in the diagram below Voltage v 150 200 250 300 350 400 450 500 550 600 650 700 VVavelength nm The principle emission lines of the mercury xenon lamp are listed in the table below Wavelength Wavelength 53 Updated January 2006 AppliedP hot 2 4 5 5 Purging the Lamp Housing Ozone Producing Lamp Only Caution Ozone inhalation is harmful to health This procedure applies only to lamp housings fitted with an ozone producing xenon lamp Ozone is produced by the reaction of oxygen in the atmosphere with UV light Inhalation of ozone is harmful to health and will rapidly degrade seals and optical surfaces in the lamp housing and monochromator Purging the lamp housing with an inert gas such as nitrogen will ensure there is no build up of ozone in the lamp housing A fitting is provided on the right hand side of the lamp housing to connect a purging line from the main stopped flow drive supply Connection of the Lamp Housing Purging Accessory If nitrogen is being used as the main drive gas supply then the elbow on the h shaped tubing arrangement used to connect the gas supply to the back of the sample handling unit can be replaced with an additional flow regulator assembly The tubing on the outlet of the flow regulator should be connected to the purge inlet on the right hand side of the lamp ho
35. elled 256 will be illuminated when a PDA is attached 68 Updated January 2006 AppliedPhotophysics 2 5 SX20 Experimental Operation This section provides detailed information relating to experimental operation of the SX20 stopped flow reaction analyser hardware 2 5 1 Single Mixing Stopped Flow Operation The following procedure describes the use of the syringes and the various valves during sample loading sample flow circuit flushing and stopped flow mixing This is the basic experimental template for stopped flow operation Variations of this template will be described in a later section of this chapter STEP 1 Sample Handling Unit Preparation Sequential Mixing Systems Only For systems equipped with the sequential mixing option SQ 1 the sample handling unit should be set up as shown in the diagram below with the single mix drive ram connected to the left hand ram platform The single mixing experiment always utilises this drive ram and the reagents are always loaded in the F left and C right drive syringes The black plastic dust cover should be fitted to the right hand Pre Mix ram platform 69 Updated January 2006 AppliedPhotophysi STEP 2 Flow Circuit Configuration Sequential Mixing Systems Only For systems equipped with the sequential mixing option SQ 1 the flow circuit should be set up as shown in the diagram below with the flow tubing connecting directly between drive valve F and the optical cell
36. ence Measurements Optical filters are used in fluorescence measurements to eliminate the scattered excitation light from detection by the fluorescence PMT The fluorescence signal is measured at a 90 angle to the excitation light and the filter is mounted between the optical cell and detector The filter 25mm diameter is mounted in the circular filter holder and retained in position using the split ring The filter holder shown below is designed for use with cut off and band pass filters Filter Holder 78 Updated January 2006 AppliedPhotophysics Cut Off Filters Cut off filters eliminate light of shorter wavelength than its cut off value Applied Photophysics stock the following range of Schott manufactured filters suitable for use with the SX20 360 550 The transmittance profiles for these filters are displayed below AMA fii M A E RN TAL TING z EA n o wo ESE ZA 7K 1200 e n NE Wavelength nm Figure 10 Glass thickness 3 mm all other types 79 Updated January 2006 AppliedPhotophysics O Band Pass Filters Band pass filters transmit a band of light of a certain wavelength Band pass filters are most suited to applications where more than one fluorophor is present in the reaction mixture Applied Photophysics offer a 435 465 and 505nm filters for use with the SX20 The transmittance profiles for these filters are displayed in the diagram below
37. ence detector 9 Electronics Unit fitted with single detection channel 10 Computer fitted with fibre optic interface card 11 Monitor 12 Printer 5 Updated January 2006 Applied _ These components will be described in further detail later in this section In addition to the major components the SX20 system is supplied with the following ancillary components 1 Mains electricity distribution board and mains cables 2 Set of cables for electrical connection of the SX20 major components 3 Pneumatic tubing and connectors for connecting the system to an 8bar gas supply 4 In line pressure regulator for controlling drive pressure 5 Storage case including toolkit and consumable spares kit 6 User manual and back up software 6 Updated January 2006 Applied _ 2 2 SX20 Optional Accessories This section describes the optional accessories that are available for the SX20 system These options are available for new systems or as upgrades to existing systems as the research interests of the user evolve 2 2 1 SQ 1 Sequential Double Mixing Option The sequential double mixing option equips the sample handling unit with four drive Syringes and permits the mixing of two reagents A B with a first mixing drive and after a user defined delay period mixes the aged solution with a third reagent C This option consists of the following components 1 A sequential mixing sample handling unit 2 2 sequential mixing drive
38. es 8 2 2 6 DD 1 Dual Channel Detection Option 2 0 0 ccccssssssseeeeeeeeceeeeeeeeeeeaaeaeeeeeess 8 2 2 7 DF 1 Dual Channel Fluorescence Detection Option cccccccceeeeeeeeeeeeeeeeeseees 9 2 2 8 FP 1 Fluorescence Polarisation Option ccccccccccccssssssssssseseeeeccceeeeeeeeeeeaaeeeeeneees 9 2 2 9 RC 1 5uL Rapid Kinetics Cell Option cc ccccssssssseseeeeeeeeeeeeeeeeeeaeeaeeeeeeees 9 2 2 10 QFA 1 Quench Flow Adaptor Option eeesssssssssssssseeeeereessssssssssssseeeeeresssssssss 9 2 2 11 AN 1 Anaerobic ACCOSSOTY ccssssssssssseeececccecceeessacaeeessseeeeeeeecceeeesesseaeageeesssses 9 2 D gt X20 UMS MCE Se UP ecrire nirre a E N 10 Fy Se Regurements rirmi a ante cddnedn E 10 DD SA2 Hardware Sep erospen irena a E Na 1 Vir ie ESP OU 3 als gers elsif Omen mete mene eee e EE A Serre Renter mrs 15 2 3 4 SX20 Optional Accessories Basic Set up cccccccccccsssssssssseeeeeeceeeeeeeeeeseaeeeeeeeees 20 2 3 4 1 SEM 1 Scanning Emission Monochromator Option ccccceceeeeeeeeeeeeeeeeeees 20 2 3 4 2 AM 1 Extended Absorbance Option cccccccccssssssssssssseeeeececceeeesenseeeeeseseees 21 2A SA adware 01 1 021018 Rene eee eer ence nem acer meee eer tener rer Te mee ree 22 2 4 1 Sample Handling Unit sssicieceiiriin innsinn inienn aani aiaei iai 22 2A MN nrod Oian Ea EN TEE E i 22 2 4 1 2 Description of a Stopped FIOW Drive esesesessssssssssseeeeersssssssssssssseeereeeesssssss
39. housing 2 4 5 8 Lamp Stability Test The stability of a lamp is a performance measure that will determine the quality of the data that can collected on the SX20 Lamp stability can be measured according to the following procedure 1 The sample handling unit should be configured in the absorbance mode with a 2mm optical pathlength 2 Set the monochromator to 350nm with entrance and exit slits set to 0 5mm 3 Click the Reference button to apply a high voltage to the detector 4 Acquire data over a 10s period The maximum peak to peak noise on the signal during the acquisition period should not exceed 0 001AU for a new system As systems and lamps age the stability level decreases It is recommended that the lamp alignment should be checked if the lamp stability has deteriorated significantly If the stability exceeds 0 003AU the lamp should be considered for replacement 57 Updated January 2006 AppliedPhotophysics _ 2 4 6 Lamp Power Supply Unit 2 4 6 1 Description The lamp power supply unit PSU is a purpose designed unit for igniting and running the 150W xenon arc lamp The unit contains a safe start igniter that is harmless to surrounding electronic equipment The igniter delivers a high voltage pulse to strike the arc lamp and once ignited supplies a stable running voltage to the lamp 2 4 6 2 Basic Set Up and Electrical Connections The 150W xenon lamp power supply unit is generally located to the right of the optical
40. id in the circulator should be purged of oxygen by gently bubbling nitrogen through it at 25 C 1 C 1g of sodium dithionite should be added to the thermostat liquid The circulator must be left running for the duration of anaerobic operation A solution of 600ng ml Glucose Oxidase and 10mM glucose in 100ml of sodium acetate pH5 should be flushed through the internal flow lines of the sample handling unit After 1 hour deoxygenated buffer should be used to flush out the Glucose Oxidase solution The sample handling unit is now ready for anaerobic use 2 5 5 3 Sample Introduction Care Another source of oxygen contamination is at the point where the samples are introduced into the drive syringes For basic anaerobic work the use of gas tight luer tip reservoir syringes 1s recommended Prepare the anaerobic samples as usual draw them into gas tight syringes bring them quickly to the sample handling unit as you are about to fit them to the luer connections expel a small amount of the sample to prevent an air bubble from being trapped between the reservoir syringe and the fitting Advanced anaerobic work requires the use of three way valves to introduce the samples into the system These are supplied as part of the AN 1 accessory and may be used according to the following procedure 84 Updated January 2006 Applied _ 2 5 5 4 Procedure for Bench Top Anaerobic Stopped Flow Studies 1 2 3 4 5 6 7 Flush the drive s
41. ion PC via a fibre optic communication cable The Temp Inputs port is currently redundant on the SX20 62 Updated January 2006 Applied _ The function of the red Tx and Rx LEDs are identical to those on the front panel of the Electronics Unit These LEDs flash to indicate that information is being transmitted and received The green Status LED reports a fault on the module electronics This is constantly illuminated if the module passes the self test routine and flashes if the test is failed Control Module 1 This provides stepper motor control used to drive the monochromators For systems with one monochromator the monochromator must be connected to the Drive 1 port For SX20 systems fitted with the SEM 1 or AM 1 options i e two monochromators Mono 1 must be connected to the Drive 1 port and Mono 2 must be connected to the Drive 2 port The green Status LED below each port indicates the operational status of the respective Drive The LED is constantly illuminated if the Drive passes the self test routine and flashes if the test is failed KSHU Module This provides control of the sample handling unit pneumatics It also manages inputs from the drive ram transducers and temperature probe The sample handling unit is connected via a black cable fitted with a 44 pin D connector The Lamp Cont port is currently redundant on the SX20 system The green Status LED indicates the operational status of the module electronics This i
42. itted with option AM 1 2 5m x 0 7m x 0 5m The SX20 requires a stable mains electricity supply of 85 264V The sample handling unit is pneumatically driven and requires a compressed gas supply preferably nitrogen capable of operating at 8bar 120psi The supply should be fitted with a push on tube adaptor to accept the flexible hose 6mm internal diameter that connects to the SHU inlets For connection to a water circulator unit the SHU is provided with an inlet and outlet fitting compatible with a flexible hose of 8mm internal diameter 10 Updated January 2006 AppliedPhotophysics 2 3 2 SX20 Hardware Set up The picture below shows the standard arrangement of the major hardware components of the system PC Workstation Sample Handling Unit Monochromator Lamp Housing Electronics Unit Light Guide Optical Rail Power Supply Unit The major components of the SX20 spectrophotometer should be assembled according to the picture above with the lamp housing and monochromator positioned on the optical rail to the right of the sample handling unit Note The sample handling unit is shown with the sequential mixing SQ 1 option fitted SX20 systems equipped with the SEM 1 and AM 1 options are supplied with two monochromators and an extended optical rail The set up of these systems should be assembled according to the instructions provided later in this Chapter The following descriptions are provided to assist the user with the set
43. ive valve The direction of flow is manually selected by the user via the drive valve control knob The SHU features a pneumatic drive system that drives the reagents from the drive syringes through two micro volume flow tubes into an optical flow through cell The optical cell contains an integral T mixer which under pressure causes highly efficient mixing of the two reagents This reaction mixture is stopped in the observation chamber of the optical cell Each pneumatic drive displaces the current reaction mixture from the observation chamber to the stop syringe via a waste flow tube and subsequently to a waste vessel The volume of each drive is accurately controlled via the Auto Stop mechanism This mechanism is also responsible for triggering the data acquisition procedure The cell housing employs a removable cartridge design in which the optical cell is mounted This provides the capability for rapidly changing the optical cell according to experimental requirements The cell housing features five observation ports that provide user friendly flexibility in measurement of absorbance and fluorescence based signals 23 Updated January 2006 Applied _ 2 4 1 2 Description of a Stopped Flow Drive The following sequence of events occurs for each stopped flow drive 1 The stop syringe is emptied of a set volume of solution by the auto stop mechanism This volume equates to the drive volume that will be mixed later in the sequence by th
44. les function in the Pro Data control software allows accurate measurement of the drive volume and will be described in STEP 6 STEP 4 Adjusting the Total Drive Volume Screw the volume adjuster on the Auto Stop fully clockwise i e front edge moves from left to right until the pneumatic connection starts to rotate then screw it eight and a half turns anti clockwise counter clockwise This equates to a total drive volume of approximately 400uL and will result in a drive 2 volume of 180uL 10uL assuming a pre mix drive 1 volume of 220uUL 10uL Use of the Profiles function in the Pro Data control software allows accurate measurement of the drive volume and will be described in STEP 6 75 Updated January 2006 Applied _ STEP 5 Flushing the Flow Circuit Fit four reservoir syringes filled with distilled water or another suitable solvent to the A B C and F luer fittings It is recommended that 5mL or 10mL plastic disposable syringes are used Turn the A B C and F drive valves to the Load position Ensure that the drive rams are pushed fully down Fill the drive syringes by pushing down the plungers of the reservoir syringes Expel any air bubbles by flushing backwards and forwards between the drive syringes and the reservoir syringes several times Turn the F drive valve to the Drive position and manually push the water from the drive syringe through the flow circuit and into the stop syringe Empty the stop syringe by cli
45. loroethane Urea 1 2 Dichloroethane Ammonium Chloride Carbon Tetrachloride Calcium Salts Chloroform Copper Salts Dichloromethane Iron II Chloride Dichlorobenzene Iron III Salts Ethylene Dichloride Manganese Salts Trichloroethylene Magnesium Salts Aliphatic Esters Nickel Salts Butyl Acetate Potassium Salts Ethyl Acetate Silver Nitrate Diethylether Sodium Salts Dioxane Tin I Chloride Petroleum Ether Sulphites Tetrahydrofuran Hydrogen Peroxide Benzene Bleach Toluene Soap Solution Xylene Sodium Hydroxide Hexane Nitric Acid Conc Phenol dilute Sulphuric Acid Con Phenol conc Hydrochloric Acid Dimethylsulphoxide Diphenylsulphone Acetonitrile SFWWASFr rr rPrrrrrr rrr rTP raS rWmrrrrrrrrrrrrrwUPrryr e A No attack no or little adsorption B Slight attack C Severe attack 31 Updated January 2006 AppliedP hot 2 4 1 9 Interchangeable Cell Cartridge System The SX20 sample handling unit features a novel cell cartridge system that facilitates cartridge mounted optical cells to be interchanged with a minimum of work The diagram below shows a cell cartridge removed from the cell block Sealing O Ring Observation Windows Sealing O Ring Captive Fixing Screws Procedure for changing the optical cell The cell currently fitted in the cell block of the sample handling unit should be removed according to the following procedure 1 2 3 4 5 Remove the light guide fluorescence detect
46. lt on the module electronics This is constantly illuminated if the module passes the self test routine and flashes if the test is failed Two vacant slots in the electronics rack are available for future instrument expansion 2 4 7 2 Operation Refer to the picture below for aspects relevant to operation of the Electronics Unit ELECTRONICS UNIT SX CAUTION gt TO PREVENT ELECTRIC SHOCK DISCONNECT AC LINE BEFORE A REMOVING COVERS REFER TO USER MANUAL WANING THIS APRARATLES MUST Di The mains electricity supply to the Electronics Unit is controlled by the black rocker switch on the front panel of the unit The power is switched on by moving the switch to the on I position The green LED labelled Status indicates that power is present This LED should be constantly illuminated 64 Updated January 2006 Applied a When the Electronics Unit is first switched on the instrument will automatically check its zero positions the monochromator motor will be audible and perform an electronics self test procedure If the self test procedure fails the green Status LED will flash indicating an electronics error In the event of a self test failure restart the Electronics Unit to remedy the fault If the fault persists please contact the Technical Support Team at Applied Photophysics The two red LEDs labelled Tx and Rx indicate that the Electronics Unit is transmitting and receiving information respectively The Electronics Unit is
47. m It is fitted with one mains supply input The modules are fitted with a range of sockets and must be connected as follows F E 3 12V and 5V Supply Modules These modules are not connected to other components on the SX20 Communications Module This is connected to the PCI card housed in the workstation PC via a fibre optic communication cable The Temp Inputs port is currently redundant on the SX20 Control Module 1 This provides control of the monochromators For systems with one monochromator the monochromator must be connected to the Drive 1 port For SX20 systems fitted with the SEM 1 or AM 1 options i e two monochromators Mono 1 must be connected to the Drive 1 port and Mono 2 must be connected to the Drive 2 port KSHU Module This provides control of the stopped flow sample handling unit The sample handling unit is connected via a black cable fitted with a 44 pin D connector The Lamp Cont port is currently redundant on the SX20 system PMT Module This provides control of the photomultiplier tubes There are two versions of the PMT module The single channel PMT module provides a single channel for fluorescence or absorbance measurement The detector is connected via a black cable fitted with a 10 pin D connector In the case of the dual channel PMT 18 Updated January 2006 Applied gt module the absorbance detector is connected to Channel 1 via a black cable fitted with a 10 pin D connector Th
48. n Fill the drive syringes by pushing down the pistons of the reservoir syringes this ensures that there is no cavitation inside the drive syringes and therefore no bubbles Ensure that there are no air bubbles in the drive syringes Set the four drive valves to the Drive position STEP 8 Data Acquisition The experimental procedures for the various modes of data acquisition are described in detail in the Operation Manual chapter of this user manual The first trace acquired with new reagents will prime the flow lines and should be discarded The drive syringes must be refilled after each acquisition 77 Updated January 2006 AppliedP hot _ 2 5 3 Optical Filters The SX20 is designed to operate with a variety of optical filters according to experimental requirements 2 5 3 1 Absorbance Measurements in the near IR gt 650nm It is essential that absorbance measurements collected at wavelengths greater than 650nm use an appropriate filter to eliminate second order stray light from detection Second order stray light is a phenomenon common to all monochromators fitted with diffraction gratings In addition to monochromatic light of the required wavelength a small proportion of light with half this wavelength is also generated All SX20 systems are supplied with a 645nm cut off filter that should be positioned between the optical cell and the absorbance detector when measuring absorbance in the near IR region 2 5 3 2 Fluoresc
49. n the bleed screw on top of the cell block 41 Updated January 2006 AppliedPhotophysics 2 4 2 Absorbance Detector 2 4 2 1 Description The SX20 is fitted with a side window 9 stage photomultiplier tube for absorbance detection measurements The standard detector features a Hamamatsu R928 PMT capable of operation in the wavelength range 185 900nm 2 4 2 2 Basic Set Up and Electrical Connections The detector is mounted to one of the two upper ports on the cell block using two mounting screws The detector position must always be opposite that of the light guide fitted to one of the lower ports and this configuration determines which optical pathlength of the cell is selected for the measurement See the section describing the cell block and optical cells for further information on available pathlengths Absorbance measurements normally employ the 10mm pathlength of the standard 20uL cell or 5mm pathlength of the 5uL cell These require the detector to be mounted to the upper right port and the light guide to the lower left port of the cell block Remember to blank off any unused ports 42 Updated January 2006 Applied _ Caution Before switching the detector between ports it is essential that any high voltage applied to the detector is switched off via the software to prevent damage to the PMT For SX20 systems fitted with a single detection channel there will be one cable connected to the PMT Module of the Electronics U
50. ndling Unit 2 4 1 1 Introduction The Sample Handling Unit SHU is the heart of the SX20 stopped flow reaction analyser where reagents are loaded mixed and monitored for a signal change The picture below shows an SHU equipped with the sequential double mixing SQ 1 option Principal components of the system are labelled for reference purposes and are described in detail in this section Reservoir Syringes Optical Cell Housing 3 eae AN _ Drive Valve Controls J Auto Stop Assembly Top Water Bath Housing Stop Valve Stop Syringe Drive Syringes Sequential Mix Drive Ram Auto Stop Assembly Bottom Single Mix Drive Ram 22 Updated January 2006 Applied _ The picture overleaf shows the SHU fitted with the sequential double mixing option This option equips the SHU with 4 drive syringes and 2 pneumatic drive rams The standard single mix system is equipped with 2 drive syringes and 1 pneumatic drive ram The operation of each SHU is identical for single mixing stopped flow experiments The flow circuit comprises 2 drive syringes 2 drive valves 3 flow tubes optical cell stop valve and stop syringe With the exception of the last two components the flow circuit is enclosed by a water bath to provide temperature control of the reagents and their reaction Reagents are loaded into separate drive syringes from reservoir syringes The direction of flow into and out of the drive syringe is controlled by a dr
51. nge 0 25 to 2mm A suitable value will depend on numerous factors the most important factors being the type of signal being measured and the stability of the reagents to photochemical degradation photo bleaching The entrance and exit slit widths are normally set to identical values A suitable setting for absorbance experiments is 0 5mm Fluorescence experiments tend to be more sensitive and require higher light throughput using wider slit width settings 1mm and 2mm settings are commonly used It should be noted that samples which are susceptible to photochemical reaction should be investigated with a narrower slit setting to reduce the possibility of photo bleaching 2 4 4 4 Band Pass Calculation The wavelength bandpass is calculated by multiplying the slit width readings in mm by a factor of 4 65 Bandpass below 2 nm are most accurately set using feeler gauges The final bandpass adjustment should always be set by moving the dial in a clockwise direction This procedure will give maximum accuracy and reproducibility 48 Updated January 2006 Applied _ 2 4 4 5 Dual Monochromator Operation Systems equipped with two monochromators are labelled Mono 1 and Mono 2 Mono I is always connected to the lamp housing on the lower rail Mono 2 may either be positioned on the upper rail in the emission configuration or on the lower rail to the left of Mono I in the UV absorbance configuration according to the experimental requirement 2
52. nit When switching between absorbance and fluorescence detection the cable must be connected to the appropriate detector For SX20 systems fitted with dual detection channels there will permanently be two detector cables connected to the PMT Module of the Electronics Unit The absorbance detector is connected to Channel 1 and the fluorescence detector is connected to Channel 2 2 4 2 3 PMT Characteristics The response characteristics of the Hamamatsu R928 PMT are displayed in the diagram below TPMES0001 ER CATHODE RADIANT SENSITIVITY aes d QUANTUM ErriciEency n y D ee a A CATHODE RADIANT SENSITIVITY mA QUANTUM EFFICIENCY D 100 200 300 400 500 600 700 800 800 1000 WAVELENGTH nm 43 Updated January 2006 AppliedPhotophysics 2 4 3 Fluorescence Detector 2 4 3 1 Description The SX20 is fitted with an end window 1 1 stage photomultiplier tube for emission detection measurements The standard detector features a Hamamatsu R6095 PMT capable of operation in the wavelength range 300 650nm In addition to the standard PMT Applied Photophysics offer alternative PMTs for applications requiring different detection characteristics 2 4 3 2 Basic Set Up and Electrical Connections The detector is mounted to the fluorescence port at the rear of the cell block using 3 finger screws When fitting the detector ensure that the detector light seal and the seals in the cell block are present to prevent detection
53. on Mono 1 Lamp Housing The general assembly of the components is similar to the basic system However the following points should be noted The lamp is always connected to monochromator and never to monochromator 2 Monochromator 2 is connected to the Drive 2 port of Control Module 1 in the Electronics Unit The excitation light guide has a rectangular bundle of fibres of dimensions 5mm x 1mm at the SHU end The emission light guide has a circular bundle of fibres of 3 5mm diameter at the SHU end 20 Updated January 2006 AppliedPhotophysics 2 3 4 2 AM 1 Extended Absorbance Option This option features an extended optical rail and second monochromator The instrument layout for this option is shown in the picture below Sample Handling Unit Monochromator 2 Monochromator 1 Lamp Housing Power Supply Unit Light Guide Opto Coupler Extended Optical Rail The general assembly of the components is similar to the basic system However the following points should be noted The lamp is always connected to monochromator and never to monochromator 2 Monochromator 2 is connected to the Drive 2 port of Control Module 1 in the Electronics Unit The monochromators are connected using the mono to mono opto coupler 21 Updated January 2006 AppliedPhotophysics 2 4 SX20 Hardware Operation This section provides detailed information relating to the hardware of the SX20 stopped flow reaction analyser 2 4 1 Sample Ha
54. or and fluorescence cut off filter holder from the cell block Drain the contents of the thermostat bath housing Loosen the pressure plate that seals the three flow tubes against the face of the cell cartridge No more than one turn is sufficient Undo the four fixing screws that secure the cell cartridge to the cell block Carefully withdraw the cell cartridge to the rear The refitting procedure is the reverse of the removal 1 2 3 4 5 Ensure that the O ring seals are present Insert the cell cartridge into the cell block so that the bevelled corner of the cartridge is aligned with that on top of the cell block Secure the cartridge in position with the four captive fixing screws Tighten the tubing pressure plate screw to ensure a good seal between the three flow tubes and the cell Replace the light guide and detectors 32 Updated January 2006 Applied _ 6 Flush the flow circuit with distilled water until all bubbles have been expelled 7 Reattach the thermostat bath cover Whenever any component of the flow circuit has been altered upon reassembly the user is recommended to check the flow circuit for leak free operation Checking the flow circuit for leaks Leaks in the flow circuit will result in collection of unreliable data and loss of sample The flow circuit may be checked for leaks using the following procedure 1 Fill the flow circuit with water or solvent 2 In the software set up a stoppe
55. p Valve Stop Syringe The emptying of this syringe controls the drive volume A number of syringe Sto options are available Stop Syringe Stop Syringe Clamp This provides support for Clamp the stop syringe Brake Assembly Brake Assembly This is required only for sequential double mixing experiments ah Stopped Flow Trigger This completes a circuit Rotten Cylinder once contacted by the piston of the stop syringe to start the data acquisition Return Cylinder This upward movement of the cylinder empties the stop syringe It also contains the adjuster for setting the drive volume The Return Cylinder is pneumatically driven by compressed gas 36 Updated January 2006 AppliedPhotophysics Setting the Drive Volume The volume of a stopped flow drive is controlled using the adjuster screw on the return cylinder This adjuster sets the vertical limit of travel for the return cylinder The zero drive volume is set by turning the screw clockwise to the limit of its travel The volume is increased by releasing the screw a set number of turns Using the standard 2 5mL stop syringe one full turn of the adjuster screw equates to a total drive volume of approximately 40uL The exact drive volume required for a stopped flow experiment will depend on a number of factors including the type of measurement the acquisition time and physical properties of the reagents Recommended drive volumes are provided in the relevant sec
56. pplied 2491 NES C11 Oia aed cccrtncdensaaauncentediee snayaatideaeasoanes nacdsonhece sus reted dana saasauueneenesoasedenneteans 68 2 4 9 2 Basic Set Up and Electrical Connections cccccccssssseeseseeeeeeeeeeeeeeeeeeaeeeeeeees 68 2 5 SX20 Experimental Operation ccsesseseeeeccceceeeceeaaesesseseeeeeeceeeeeeeeseeeaaaaesseees 69 2 5 1 Single Mixing Stopped Flow Operation ccccccccsssssssesssseeeeeeeceeeeeeeeaeaaeeeeenees 69 2 5 2 Sequential Mixing Stopped Flow Operation cccccsssssssesseeeeeeeeeeeeeeeeeeeaeeeeeeees 73 Ze OOP Cal PCRS orca yasiciiesnotue see E A A E E A E E T 78 2 5 3 1 Absorbance Measurements in the near IR gt 650nm ec eeccceeeceeeeceeee 78 2 9 3 2 Fluorescence Measure Menis sesternice e e 78 2 5 4 Asymmetric Variable Ratio Mixing seeeseesssssssseoeerrressssssssssssseerereressssssssssssee 81 2 5 4 1 Setting the Drive Pressure for Asymmetric Mixing Experiments 068 81 2 5 4 2 Setting the Drive Volume for Asymmetric Mixing Experiments 00 82 2 5 5 Anaerobic Operation using option AN 1 cc ceessssssseeeeececeeeeeeeeeaesseesseseeeeeeeess 83 2 5 5 1 Purging the Drive Syringes cccccccccccccececceneeesseseeeeeccceeeeeesesaaeasesessseeeseeeess 83 25 5 2 Flow Circuit Pre par aiOm secciones oiie a a aaia 84 2 5 5 3 Sample Introduction Care cccccccccccccceccccceaaeesssseeeeeeccceeeeeeessausasacssseseeeeeeees 84 2
57. producing lamps are provided in the xenon lamp operation section of the manual 13 Updated January 2006 Applied _ Stopped Flow Circuit The flow circuit of the sample handling unit requires connection of the waste flow tube to the outlet of the stop valve Place the free end of the waste tube into a suitable receptacle Note To prevent damage to the optical cell during shipping the flow circuit is filled with an antifreeze solution The flow circuit should be flushed thoroughly with distilled water prior to use Water Circulator Connections Two tubing connections on the lower right of the sample handling unit water bath permit connection of a circulating thermostatic water bath to the stopped flow sample handling unit The front port is the inlet and the rear port is the outlet It is important that the inlet and outlet are connected correctly otherwise the sample handling unit water bath will not fill correctly It is recommended that the circulating thermostatic water bath is positioned below the sample handling unit to aid the drainage procedure for the water bath Absorbance Detector The absorbance photomultiplier tube detector is connected to one of the two upper ports of the cell housing and opposite the port used for the light input A plastic blanking plug should be placed over each of the unused ports Fluorescence Detector The fluorescence photomultiplier tube detector is connected to the rear port on the cell housing
58. rail Suitable clearance to the rear and left hand side should be allowed for the ventilation fan to operate efficiently The PSU has one mains inlet socket and two sockets red and black for connecting the cables to the lamp housing The red cable is the positive The black cable is the negative and also carries the 13000V ignition pulse to start the lamp hence the heavy insulation The earth post should be connected to the earth post on the optical rail using the braided cable supplied The Lamp Cont socket on the rear of the PSU is currently redundant 58 Updated January 2006 AppliedP hot ra T j Wiel gt So ae joie VSS mE 2 LAMP CURRENT BSA g TSA ITSA T REMOVING COVERS BEDEIN AC iN REFER TO USERS MANUAL fete WARNING THIS APPARATUS MUST BE gE EARTHED ZofbJos md CE Made in United Kingdom CAUTION TO PREVENT ELECTRIC SHOCK DISCONNECT AC LINE BEFORE 2 4 6 3 Operation The mains electricity supply to the power supply unit is controlled by the black rocker switch on the front panel of the PSU The power is switched on by moving the switch to the on I position The red LED indicates that power is present To ignite the lamp press the red Start button on the front panel of the PSU If ignition is successful the digital timer display will be activated Xenon arc lamps typically require 30 minutes to warm up and stabilise The digital timer
59. rams 3 Stop syringe brake mechanism 2 2 2 SEM 1 Emission Scanning Option This emission scanning option extends the fluorescence detection capability of the SX20 An emission monochromator located between the optical cell and the fluorescence detector allows collection of fluorescence kinetic traces at a single wavelength and collection of steady state and time resolved emission spectra It consists of the following components 1 Emission monochromator Mono 2 2 Second optical rail and four supports 3 Emission fibre optic light guide 2 2 3 AM 1 Extended Absorbance Option The extended absorbance option provides improved stray light performance in the far UV enabling accurate absorbance measurements up to 2AU below 250nm It consists of the following components 7 Updated January 2006 Applied _ 1 Second monochromator Mono 2 2 Extended optical rail 3 Mono 1 to Mono 2 optical coupler 2 2 4 PDA 1 Photodiode Array Option The Photodiode Array PDA option allows collection of time resolved absorbance spectra from a single stopped flow drive There are two different versions available A UV region PDA operates in the wavelength region 180 750nm A visible region PDA operates in the wavelength range 350 1100nm The option consists of the following components 1 Photodiode array unit UV or visible version 2 PDA fibre optic light guide xenon light source version 3 Electronics Unit PDA module 2 2 5 UV 1 Deute
60. ringe Completely fill both of the drive syringes and ensure that there is no gap between the drive ram and the drive syringe plungers Set both drive valves to the Drive forward position STEP 6 Data Acquisition The experimental procedures for the various modes of data acquisition are described in detail in the SX20 Operation chapter of this User Manual Twenty five drives can be made in quick succession before the drive syringes will need to be refilled 72 Updated January 2006 AppliedP noto D ry yS ICS 2 5 2 Sequential Mixing Stopped Flow Operation The sequential mixing stopped flow experiment allows investigation of the reactivity of reaction intermediate species The following procedure describes the use of the syringes and the various valves during sample loading sample flow circuit flushing and sequential stopped flow mixing The sequential mixing experiment employs a double pneumatic drive The first Pre Mix drive mixes the contents of the two right hand drive syringes A and B in an aging loop After a user defined aging period from 0 010s to 1000s the second Flush drive mixes the contents of the aging loop with a third reagent C STEP 1 Sample Handling Unit Preparation The sample handling unit should be set up as shown in the diagram below with the flush drive ram connected to the left hand ram platform and the pre mix drive ram connected to the right hand ram platform 73 Updated January 2006 i
61. rium Light Source Option The deuterium light source option allows the collection of PDA data using the UV version PDA accessory down to 200nm The option consists of the following components 1 Deuterium lamp power supply unit 2 Deuterium lamp housing lamp fitted 3 PDA fibre optic light guide deuterium light source version 2 2 6 DD 1 Dual Channel Detection Option The dual channel detection accessory allows fluorescence and absorbance data to be collected simultaneously using the absorbance and fluorescence PMT detectors It consists of the following components 1 Dual channel PMT module fitted in the Electronics Unit 2 Detector cable for connecting the detector to the PMT module 8 Updated January 2006 Applied _ 2 2 DF 1 Dual Channel Fluorescence Detection Option The dual channel fluorescence detection accessory allows fluorescence data to be collected simultaneously using two fluorescence PMT detectors It requires the DD 1 dual channel option and consists of the following additional components 1 Additional fluorescence detector 2 Fluorescence detector mounting fittings and filter holder 2 2 8 FP 1 Fluorescence Polarisation Option The fluorescence polarisation accessory allows collection of polarisation and anisotropy signals It consists of the following components 1 Additional fluorescence detector 2 T format excitation and emission polarizer accessory 2 2 9 RC 1 5uL Rapid Kinetics Cell Option
62. s constantly illuminated if the module passes the self test routine and flashes if the test is failed The red TRG LED indicates triggering of the Auto Stop of the sample handling unit PMT Module This controls the photomultiplier tube detector high voltage and signal measurement There are single and double channel versions that allow for connection and measurement on one and two channels respectively For systems with one data acquisition channel the detector cable is connected to the Channel port For systems fitted with two data acquisition channels the absorbance detector is connected to Channel 1 of the module The fluorescence detector is connected to Channel 2 63 Updated January 2006 AppliedP The green Status LED below each port indicates the operational status of the Channel The LED is constantly illuminated if the Channel passes the self test routine and flashes if the test is failed PDA Module only fitted on systems equipped with the optional photodiode array detector This controls all communication with the externally mounted PDA accessory The PDA unit is connected to the module via a black cable fitted with a 26 pin D connector The three yellow LEDs labelled 1024 512 and 256 indicate the type of diode array that is connected to the module The diode labelled 256 should be illuminated when a PDA is attached as the currently supplied units are 256 element diode arrays The green Status LED reports a fau
63. st be reduced using the external pressure regulator fitted to the pneumatic inlet on the rear of the SHU The normal temperature operating range of the drive syringes determines the operating range of the SX20 to 5 50 C Above 50 C the adhesive used to bond the syringe will deteriorate Below 5 C the syringe piston tip does not seal with the glass barrel A special low temperature syringe is available for work below 10 C 2 4 1 5 Drive Valves Two PEEK drive valves are fitted to the standard single mixing sample handling unit There are left hand and right hand versions of the drive valve The drive valve is fixed in position using a stainless steel base plate and two securing screws SHUs fitted with the sequential double mixing option will be fitted with four drive valves at The picture to the right shows a right hand drive valve with its respective base plate and screws Note that the two O rings fitted to seal the valve are not shown th 26 Updated January 2006 AppliedPhotophysics The drive valves control the flow of solution between the drive syringe and either the reservoir syringe or the flow circuit The Load position is used to control the flow of the drive syringe to and from the reservoir syringes via a female leur fitting The Drive position is used to control the flow from the drive syringe towards the optical cell via a PEEK micro volume flow tube Each drive valve is controlled manually by turning the
64. t screws 4 The sample handling unit should be configured in the absorbance mode Set the monochromator to a suitable wavelength for aligning the lamp Applied Photophysics recommend a value of 350nm with monochromator slits set to 0 5mm 5 Click the Reference button in the baseline panel of the control software to set the absorbance baseline 6 Adjust the horizontal alignment setting by turning the hexagonal wrench until the signal reaches an optimum level i e minimum absorbance 7 Adjust the vertical alignment setting by turning the hexagonal wrench until the signal reaches an optimum level i e minimum absorbance 8 Repeat the horizontal and vertical alignment steps until the optimum signal is reached 9 Retighten the lamp alignment mechanism locking screw and refit the magnet assembly to the rear of the lamp housing 10 Replace the two plastic plugs covering the alignment screws 56 Updated January 2006 Applied _ 2 4 5 7 Lamp Lifetime The 150W arc lamps are generally rated for 1000 operational hours The life time of a lamp often exceeds 1000 hours depending on the type of work it is used for Symptoms of an aging lamp include difficulty to ignite poor lamp stability and reduced output particularly in the UV region When the lamp starts to exhibit these symptoms it should be replaced Note that lamps operated in excess of 1000 hours are more susceptible to break or explode causing damage to the interior of the lamp
65. the 13000V ignition pulse to start the lamp hence the heavy insulation The red cable is the positive 150W Xenon Arc Lamp Housing One loose braided cable provides the earth link between the lamp housing and the optical rail The lamp housing is fitted with two input terminals black negative and red positive that are connected to the lamp power supply unit Monochromators For systems with one monochromator the grey monochromator cable fitted with a 9 pin D connector is connected to the Drive 1 port on the control module of the electronics unit For SX20 systems fitted with the SEM 1 or AM 1 option i e two monochromators Mono 1 must be connected to the Drive 1 port and Mono 2 must be connected to the Drive 2 port on the control module of the Electronics Unit Sample Handling Unit The sample handling unit is connected to the KSHU module in the Electronics Unit via a black cable fitted with a 44 pin D connector Absorbance Detector The absorbance detector is connected to channel of the PMT module on the electronics rack via a black cable fitted with a 10 pin D connector Fluorescence Detector The end window fluorescence detector is connected to channel 2 of the PMT module on the electronics rack via a black cable fitted with a 10 pin D connector 17 Updated January 2006 AppliedP hot Electronics Unit The electronics unit contains a number of plug in modules for controlling the various operations of the stopped flow syste
66. tial mixing experiments require great care in setting up the volumes for the first and second mixing drives To assist the user in setting up the correct drive volumes an electronic transducer is fitted in each drive ram mounting platform Movement of the transducer is reported in the software and may be used to calculate the drive volume The limited travel of the transducer necessitates the use of special sequential mix drive rams with a similar travel limit In addition the pre mix drive ram contains a self stop mechanism that allows the volume of the pre mix drive to be set via this mechanism The flush ram is not fitted with this mechanism and the corresponding drive volume is set via the auto stop mechanism Further information on the use of the transducer profile information to set up sequential mixing experiments is provided later in this chapter 39 Updated January 2006 Applied _ 2 4 1 14 Temperature Control The sample handling unit is fitted with a water bath to provide temperature control of stopped flow experiments A circulator unit may be connected to pump thermostatic fluid into the water bath housing surrounding the drive syringes drive valves and flow tubing The thermostatic fluid also fills the cell block ensuring temperature control of the entire flow circuit A thermocouple located in the PEEK housing reports an accurate temperature measurement inside the water bath to the software This temperature is recorded with
67. tions of this chapter that discuss Software Control of the Auto Stop Mechanism The stop syringe is emptied by clicking on the Empty button in the Pro Data software Control Panel This action completes the empty cycle of the Auto Stop mechanism This function is useful for priming the flow circuit during the experimental set up routine to maximise sample economy The software provides control of three solenoid valves that open and close to apply high pressure to the actuator and return cylinder via three plastic tubes labelled A B and C A rotates the stop valve to the Empty position B raises the return cylinder to empty the stop syringe by a set volume and C rotates the stop valve back to the Drive position The timing of these operations is critical and is controlled via the Waste Timings function in the Pro Data software Control Panel Under normal circumstances the user will not need to alter these timings 37 Updated January 2006 Applied _ Manual Control of the Auto Stop Mechanism The Auto Stop mechanism may be manually operated to empty the stop syringe This is particularly useful for cleaning the flow circuit before and after measurements To empty the stop syringe the following procedure should be used 1 Turn the control knob clockwise 180 to the rear This is the Empty position 2 Grip the lower part of the Stop Syringe plunger and push upward expelling the contents of the syringe to the waste vessel 3 Return th
68. ts to the respective ports on the cell block The absorbance pathlength also applies to using the optional photodiode array detector Further information on use of the PDA is provided later in this chapter Fluorescence Excitation Pathlength Selection The fluorescence detector mounting port E is at the rear of the cell block and the emission PMT detector should be mounted using the rubber light seal finger screws and nylon washers provided When measuring fluorescence the excitation light guide should be connected to the D port to give the best illumination and lowest inner filter effect 35 Updated January 2006 Applied hotophysics 2 4 1 12 The Auto Stop Mechanism The Auto Stop mechanism is mounted on the front left hand side of the Sample Handling Unit It has two main functions 1 To control the volume of each stopped flow drive 2 To trigger data acquisition at the moment the flow is stopped The Auto Stop mechanism shown below comprises a number of important components Auto Stop Actuator This provides automated stop valve control via the software It switches between the Drive and Empty positions driven by compressed gas 8bar The plastic control knob De provides an option to control the stop valve Control Knob manually Auto Stop Actuator Stop Valve This controls the flow of reagents into the stop syringe Drive position and out of the stop syringe to the waste vessel Empty p osition Sto
69. ugh the flow circuit and into the stop syringe Empty the stop syringe by clicking on Empty in the Pro Data control software Repeat the above two steps until the drive syringe is empty Turn the F drive valve to the Load side position and the C drive valve to the Drive forward position and manually push the water from the drive syringe through the flow circuit and into the stop syringe Empty the stop syringe by clicking on Empty in the Pro Data control software Repeat the above two steps until the drive syringe is empty If there are bubbles in the stop syringe that cannot be expelled by manual pushing the syringe must be removed and the bubbles removed To collect quality data it is essential that air bubbles are not present in the flow circuit 71 Updated January 2006 Applied _ STEP 5 Loading the Reagents Ensure that all the control valves are set to the Load side position Replace the C and F reservoir syringes with ones containing the samples Where applicable the solution of greatest density must be loaded in the F drive syringe If both samples are of the same density then the most precious sample should be loaded in the C syringe as it has the smaller priming volume Fill the drive syringes by pushing down the pistons of the reservoir syringes this ensures that there is no cavitation inside the drive syringes and therefore no bubbles Prime the sample handling unit by manually pushing 200uL from each drive sy
70. up of the major components of the SX20 Lamp Power Supply Unit The 150W xenon lamp power supply unit is generally located to the right of the optical rail Suitable clearance to the rear and left hand side should be allowed for ventilation of this unit 11 Updated January 2006 Applied _ Xenon Arc Lamp Housing The 150W xenon lamp housing is mounted on the right hand side of the optical rail A magnet assembly is sometimes attached to the rear of the housing This provides magnetic stabilisation of the lamp arc Monochromator The monochromator is located on the left hand side of the optical rail to the right of the 150W xenon arc lamp housing It is essential that the lamp housing and monochromator are correctly coupled together The two units must be pushed together so that the exit port of the lamp locates firmly with the entrance port of the monochromator Both units should be locked in position on the optical rail using the four plastic screws on each unit Fibre Optic Light Guide The fibre optic light guide transmits light exiting the monochromator to the optical cell of the stopped flow sample handling unit The large coupler of the light guide is pushed into the exit slit assembly of the monochromator and secured using 2x M3 finger screws The other end is connected to one of the two lower ports of the optical cell block The short pathlength is accessed from the lower right hand port and the long pathlength is accessed from
71. using as shown in the picture below 54 Updated January 2006 AppliedPhotophysics Purging Procedure Before igniting the lamp the flow regulator should be opened to allow a minimal steady flow of nitrogen through the lamp housing This flow should be continued for at least 15 minutes prior to igniting the lamp This will ensure that all the air in the lamp housing is replaced with nitrogen The nitrogen flow must be maintained when the lamp is running 55 Updated January 2006 Applied _ 2 4 5 6 Lamp Alignment Procedure The lamp alignment should be checked frequently to ensure optimum performance of the SX20 system Any misalignment will result in excess noise due to both a lower light level and more importantly increased vibrational noise The alignment procedure involves adjusting the horizontal and vertical position of the lamp in the lamp housing to optimize the signal monitored in the live display window of the software Caution To prevent risk of electrical shock and damage to the instrument the alignment procedure must only be performed using the insulated 4mm hexagonal wrench supplied with the SX20 system 1 Remove the magnet assembly from the rear of the lamp housing Note the orientation of the magnet if fitted 2 Loosen the lamp alignment mechanism locking screw on the rear of the lamp housing Generally no more than one quarter turn should be necessary 3 Remove the two plastic plugs covering the alignmen
72. ust be correctly grounded The lamp power supply unit the lamp housing and the mains distribution board all have earth posts that must be connected to the earth post on the optical rail via the braided earth straps provided with the system The system is earthed through the mains supply earth It is vital that the laboratory mains electrical supply where the system will be used is correctly earthed The diagram overleaf shows the basic configuration of the earth connections Note that the other components of the system are separately earthed 15 Updated January 2006 AppliedPhotophysics Monitor Sample Handling PN Monochromator zs 150W Lamp Housing Lamp Supply to Monitor to Computer to Printer Earth Strap _ Laboratory Mains Supply SX20 mains supply and earth connections Fluorescence PMT MAINS COMMS CONTROL SHU PMT Monochromator PDA Optional 150W Lamp Housing Lamp Supply Ye Computer ee Absorbance PMT SX20 electrical connections 16 Updated January 2006 Applied _ Lamp Power Supply Unit One loose braided cable provides the earth link between the lamp power supply and the optical rail In addition to the mains inlet socket there are sockets for two output cables from the safe start igniter of the lamp power supply These outputs are connected to the inlet sockets on the lamp housing unit using the loose black and red cables The black cable is the negative and also carries
73. w lists the pathlengths and approximate stopped flow dead times of the optical cells available from Applied Photophysics Cell Volume _Pathlengths 20uL 10mm 2mm Standard SF lms SuL 5mm Imm Rapid SF 500us All SX20 systems are supplied with the standard 20uL optical cell This cell is the most sensitive for emission based measurements and has a dead time of approximately I ms The SuL cell provides extra pathlength options and a shorter dead time 500us however the fluorescence sensitivity is lower than the standard cell owing to the smaller fluorescence window in the SuL cell Note that the SuL cell must be operated with the ImL stop syringe provided with the cell Rapid kinetic measurement will be described in detail later in this chapter of the User Manual 34 Updated January 2006 AppliedP hot 2 4 1 11 Absorbance Pathlength Selection The optical cells feature a long and short pathlength 10mm and 2mm for the standard 20uL cell The pathlength is selected according to the positioning of the light guide and absorbance detector on the cell block The diagram shows the rear of the cell block and the configuration of the light guide and detector to select each pathlength Cell Cartridge Screws Cell Cartridge Ports The long pathlength is between ports C and A The short pathlength is between ports D and B The optical pathlength is selected by moving the absorbance PMT detector A or B ports and light guide C or D por
74. yringes and flow circuit of the SHU with anaerobic buffer After flushing a little anaerobic buffer should be left in the drive syringes Prepare all reagents in a glove box and load these into gas tight luer lock syringes with a 3 way stop valve on the end of the syringe ensure this valve is closed The syringe and 3 way valve should be transferred to the stopped flow instrument Connect an empty disposable syringe on to the unused port on the 3 way valve With the 3 way valve in the same position i e closed turn the drive valve to the Load position and push up on the drive syringe to expel the anaerobic buffer from the drive syringe into the empty disposable syringe on the side of the 3 way valve This effectively scrubs the drive valve luer fitting and part of the 3 way valve making these components anaerobic Open the 3 way valve by turning it 90 degrees and introduce a small amount of the anaerobic sample into the drive syringe Turn the 3 way valve back to the closed position and then push this small volume of anaerobic sample out through the 3 way valve to the disposable syringe containing anaerobic buffer This fills the valve syringe and luer fitting with sample Open the 3 way valve and load the sample into the drive syringes The system is now ready for the stopped flow experiment 85 Updated January 2006 AppliedPhotophysics O 86 Updated January 2006

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