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1. 1 LCD DISPLAY Used to display messages selected syringe auto mode 2 SYRINGE SELECTOR Selects the syringe for the manual control 5 3 TRIGGER INPUT Input for an external signal to trigger the drive sequence 4 SYNCHRO PULSE OUTPUT TTL Pulse output to trigger the recording system or any electronic device to be synchronized with the instrument 5 MANUAL MOVEMENT Manual control of the syringes 6 MOTOR ON INDICATOR Lit when at least one of the motors is activated 7 START STOP Initiates or stops the programmed sequence in the automatic mode The instrument may also be started and stopped using the keyboard of the PC 8 PROGRAM RESET Resets the MPS 52 instrument does NOT reset syringe values 9 MAIN POWER FUSE 3 A for 220 V or 6A for 115 V 10 AC LINE CONNECTOR 11 MAIN POWER SWITCH 12 MOTOR POWER CONNECTOR Sends the power pulses to the stepping motors 13 LOGIC CONNECTOR Connects the MPS 52 controller to the PC 14 MOTOR FUSES 5A 15 HARD STOP SF BNC CONNECTOR 3 2 AC Power and Connections Before connecting the SFM to the local AC line verify that the setting of the instrument matches the local line voltage Prepare the SFM for operation by connecting the mechanical subsystem to the MPS 52 unit Connect the MPS 52 to the serial port of the microcomputer Finally plug the MPS 52 into the appropriate AC line 3 3 Temperature Regulation The SFM module may be connected to a circulating temperature
2. executed by pushing the button or the start stop button on the front panel of the MPS Figure 2 The button can be used to terminate an experiment prematurely if necessary If the Single button was used to transfer the driving sequence to the MPS only a single shot can be made The LE button must then be pushed to return to the Stopped Flow Program window and the Single button must be pushed again to re transfer the driving sequence to the MPS for a subsequent shot If the Multiple button was used to transfer the driving sequence to the MPS the button can be used to execute shots until the Program Run window shows that 0 shots remain The Exi button is then pushed to return to the Stopped Flow Program window IMPORTANT Before running in automatic mode verify that the syringe valve handles of the syringe used in the driving sequence are set to C and unused syringes set to R Data Acquisition Software Data acquisition is generally made using the Bio Kine software The Bio Kine software may be loaded onto the same microcomputer as the MPS software The Bio Kine software can be started from within the MPS software using the button The button can also be used to switch to the Bio Kine program if it has already been started Data acquisition is triggered by a synchronization pulse from the MPS see section 7 8 All acquisition parameters need to be configured correctly in the Bio Kine software before dat
3. Delay Lines Device Delay Line 1 ul Singe No 0 25 5 ELI Windows Cuvette Delay Line 2 ul Delay Line No 0 31 7 Serial Port Limits Cancel 11 4 Serial Port Configuration The serial port must be configured before the MPS and MPS software can communicate Serial port configuration is made in the Config Serial Port window available under the Config menu Figure 56 Select the serial port used to connect the MPS and the microcomputer in section 3 2 11 70 SFM 3 4 User s Manual ver 1 1 Figure 56 MPS Software Serial Port Configuration Config Serial Port x Serial Port e Windows Device Syringes Cuvette Delay Line Serial Port Limits Connect Device cnc 11 5 System Limits Configuration The system limits are configured in the Config Limits window available under the Config menu Figure 57 The Config Limits window contains several sections for configuration of the hard stop valve lead time acceleration phases mode and overheating protection Each section and options are described below Figure 57 MPS Software System Limits Configuration BUE windows Valve Lead ms o Default Device Acceleration Phases Syringes A Automatic Cuvette Delay Line C Manual Serial Port Limits X Overheating Protection Exit Valve Lead Time This section of the windows allows one to enter the number of milliseconds before the flow stops that the exit va
4. FC type cuvettes have blackened edges to reduce light scattering in fluorescence configuration The FC 15 and FC 20 cuvettes are the best choices for CD experiments in the far UV Their large aperture facilitates low noise recording at these wavelengths TC type cuvettes have been primarily designed for absorbance and transmittance experiments however in the TC xx yyF models both sides of the light path are transparent These models of cuvettes can also be used for fluorescence experiments using dilute samples and excitation with a laser or any other low divergence light source Cuvettes of the TC xx 10 type have a 1x1 mm cross section and cuvettes of the TC xx 15 type have a 1 5x1 5 mn cross section SFM 3 4 User s Manual ver 1 1 Figure 5 SFM Cuvette Specifications CUVETTE OPTICAL SPECIFICATIONS Cuvette Drawing Light path mm a Aperture mm b Main application FC 08 Fluorescence light scattering FC 15 Fluorescence light scattering high absorbance FC 20 CD fluorescence TC 50 10 Absorbance CD fluorescence TC 50 15 Absorbance CD fluorescence TC 100 10T Absorbance CD TC 100 10F Absorbance CD fluorescence TC 100 15T Absorbance CD TC 100 15F Absorbance CD fluorescence Notes 1 All cuvettes are made of Suprasil transparent from 185 to 2500 nm HS Black Quartz Transparent Quartz CUVETTE DEAD VOLUMES AND DEAD TIMES With Berger Ball Mixer With H
5. 8 1 8 2 8 3 8 3 1 8 3 2 8 3 3 8 3 5 9 1 9 2 9 3 9 4 9 4 1 9 4 2 9 5 SFM 3 4 User s Manual ver 1 1 Syringe Configuration 6 32 Cuvette Configuration 6 33 Delay Line Configuration 6 33 Serial Port Configuration 6 34 System Limits Configuration 6 34 7 INSTRUMENT OPERATION 7 36 Manual Syringe Control 7 36 MPS 7 36 7 1 2 SOFTWARE 7 36 Syringe Initialization 7 37 Filling the Syringes 7 38 SFM Cleaning and Storage 7 39 Long term Storage of the SFM 7 40 Creating a Driving Sequence 7 40 Acceleration Phases 7 42 Programmable Synchronization Pulses Triggers 7 44 Saving or Loading Driving Sequences 7 44 Running in Automatic Mode 7 45 Data Acquisition Software 7 45 8 A SHORT STOPPED FLOW PRIMER 8 46 General Principle of Stopped Flow Experiments 8 46 Design and Execution of Stopped Flow Experiments 8 47 General Advice for Stopped Flow Experiments 8 47 ACHIEVEMENT OF FASTEST DEAD TIMES 8 47 WASHING 8 48 CAVITATION 8 48 8 3 4 SIGNAL AMPLITUDE 8 48 FLOW RATE 8 49 9 TEST REACTIONS 9 50 Reduction of 2 6 Dichlorophenolindophenol by Ascorbic Acid 9 50 Evaluation of the Dead Time 9 50 Evaluation of Washing and the Quality of the Stop 9 51 Variable Ratio Mixing 9 52 REDUCTION OF DCIP BY ASCORBIC ACID 9 52 ALCOHOL DEHYDROGENASE ACTIVITY 9 53 Mixing Solutions of Unequal Density and Viscosity 9 55 10 INSTALLATION OF THE QUENCHED FLOW COMPONENTS 10 60 SFM 3
6. If the volume collected is not substantially larger than the SFM flow line volume contamination of samples by old reacted solution 10 65 SFM 3 4 User s Manual ver 1 1 may occur It is recommended to collect sample volumes a minimum of 3 5x flow line volumes section 10 3 In addition it is recommended to wash old solution out of the SFM and tube with buffer between sample collections and perform test experiments to verify the level of sample contamination is minimal 10 4 2 PARTIAL LIQUID COLLECTION This method is the most preferred method used for quenched flow experiments It is similar to the Figure 50 Pipette Syringe method in section 10 4 1 2 in that the sample is Collection collected in a pipette or syringe Figure 50 It differs from the total liquid collection method because only the portion of the liquid exiting the SFM that corresponds to completely new uncontaminated sample is collected The exit valve is programmed to divert contaminated sample to waste so that only uncontaminated sample is recovered Because of this even very small volumes 10 s of ul of sample can be collected and sample economy is high The programming of the exit valve is described in section 12 6 As discussed in the previous section it is recommended that a pipette be used for collection rather than a syringe Undue back pressure from a collection syringe plunger can force liquid to exit through the waste outlet instead of being coll
7. window Figure 24 and Figure 28 The dead time is calculated for the last valid phase according to its flow rate and of the cuvette dead volume Figure 5 The dead time is calculated according to the equation show in Figure 28 The ageing times are calculated for the current phase selected based upon the syringes flow rates delay lines installed and intermixer volumes Figure 8 The ageing times are calculated according to the equations shown in Figure 28 Figure 28 MPS Software Dead Ageing Times z Cuvette Dead Volume Dead Time pend Nine Total Flow Rate 7 8 ms Ageing Time DLI 128ms_ SFM 4 Delay Line 1 Intermixer Volume M1 M2 12 8 ms Ageing Time Total Flow RateS1 S2 DL2 10 6 ms Delay Line 2 Intermixer Volume M1 M2 Ageing Time Total Flow RateS1 S2 SFM 3 Delay Line Intermixer Volume M1 M2 Ageing Time Total Flow RateS1 S2 The MPS software provides the possibility to repeat phases a number of times in virtually any order This is accomplished Figure 29 MPS Software though a macro sequence entered in the Drive Sequence Drive Sequence Macro frame of the Stopped Flow Program window Figure 29 The macro sequence can be edited to run a single phases or many phases in a different order than present in the 1 1 20 program grid The syntax of the macro is described in detail in the MPS Software User s Manual chapter 6 Drive Sequence 7 7 Acceleration Phases With o
8. 13 3 3 13 3 4 SFM 3 4 User s Manual ver 1 1 14 TEST REACTIONS 14 3 Washing Efficiency SYSTEM SPECIFICATIONS 13 88 TEST EXPERIMENTS 13 88 MINIMIZING SAMPLE CONTAMINATION 13 89 MINIMIZING REACTANT CONSUMPTION 13 89 14 91 14 1 Alkaline Hydrolysis of 2 4 Dinitrophenyl Acetate DNPA 14 91 14 2 Calculation of Hydrodynamic Volumes from Kinetic Data 14 93 14 94 14 4 Recovery of Uncontaminated Material in Intermixer Volume 14 95 1 5 SFM 3 4 User s Manual ver 1 1 SECTION I GENERAL INFORMATION SFM 3 4 User s Manual ver 1 1 TABLE OF CONTENTS SECTIONI 1 WARRANTY 2 INTRODUCTION AND SPECIFICATIONS 2 General Description 2 1 1 THE MECHANICAL DESIGN 1 8 2 9 2 9 2 9 2 1 2 INTELLIGENT POWER SUPPLY 2 9 2 1 3 MICROCOMPUTER COMMANDS 2 9 2 2 Modes of Operation 2 2 1 STOPPED FLOW SF MODE commercial reference SFM X S 222 QUENCHED FLOW QF MODE commercial reference SFM X Q 2 3 Specifications 2 Principle of Operation 2 5 Description of the Mechanical Design 2 6 The Delay Lines 3 GENERAL INSTRUCTIONS FOR INSTALLATION 3 1 Operating Features 32 AC Power and Connections 3 3 Temperature Regulation 2 10 2 10 2 10 2 12 2 13 2 13 2 13 3 15 3 15 3 16 3 16 SFM 3 4 User s Manual ver 1 1 1 WARRANTY BIO LOGIC WARRANTS EACH INSTRUMENT IT MANUFACTURES TO BE FREE FROM DEFECTS IN MATERIAL AND WORKMANSHIP UNDER NORMAL USE AND SERVICE FOR THE PERIOD OF ONE YEAR
9. Several stop modes that can be used with the SFM are describe in the following sections 5 4 1 5 4 2 FREE FLOW SYSTEM In this mode the outlet of the observation head is connected to a waste tube and the outlet is continuously open This procedure may to be used in case of pressure sensitive organelles or to avoid any pressure artifact on the cuvette material as for some CD measurements To use this mode the exit tube provided in the standard equipment is attached to the outlet of the observation head Figure 10 This tube has a vent permitting the entry of air Care should be taken to break the liquid column as close as possible to the observation head outlet It is recommended to connect the exit tube to a larger PVC tube to do this and permit further air entry If these precautions are not taken and a long continuous tube is connected to the outlet a long column of liquid will be pushed during the flow At the motor stop the inertia of this Figure 10 Exit Tube Installation Exit Tube gt LU To Waste Observation liquid column will inevitably generate underpressure in the cuvette and lead to artifacts from cavitation HARD STOP SYSTEM In this mode the flow will be immobilized by a combination of two mechanisms first from the stepping motors stop and second by a high speed electrovalve hard stop which closes the output of the SFM cuvette This hard stop is actuated by the programmable p
10. how to design and perform quenched flow experiments using the SFM It is not meant to be an exhaustive reference as there are many variations on the quenched flow experiment too numerous to describe here We invite the user to explore the references listed below to learn more about rapid mixing and the quenched flow technique 13 1 Barman T E and Gutfreund H 1964 in Rapid Mixing and Sampling Techniques in Biochemistry Ed B Chance R H Eisenhardt Q H Gibson and K K Lonberg Holm Eds Academic Press London pp 339 344 Gutfreund H 1969 Methods in Enzymology 16 229 249 Barman T E and Travers F Methods of Biochemical Analysis 1985 Vol 31 1 59 General Principle of Quenched Flow Experiments The simplest quenched flow experiment consists of three stages mix age and quench Complex experiments may involve more stages but for example purposes only a three stage experiment is discussed here Figure 70 shows a schematic of a quenched flow experiment The reaction considered is A B gt C where the reaction can be stopped at any time by the addition of quencher Q Mix In the first stage flow is initiated by two plungers The Figure 70 Quenched Flow Experiment Scheme plungers force the reactants A and B through a mixer where they are mixed and the reaction initiated and starts to Waste Collect produce C DELAY LINE MIXER1 MIXER2 Age In the second stage the Cc plungers push the sampl
11. model HDS Figure 51 that includes an internal siphon like frame and allows blockage of convection created by density or temperature differences Using this mixer quenched flow experiments traces can be performed for time points from the first millisecond to several 100 seconds Out Installation of the HDS mixer is identical to that of a standard Berger Ball mixer Instructions are provided in the Technical Section of this manual 10 67 SFM 3 4 User s Manual ver 1 1 10 5 4 DIRECT EXIT ATTACHMENT Short ageing times can be achieved and sample economy improved in the free flow method of Figure 52 Direct Exit sample collection section 10 4 1 1 by Attachment minimizing the volume from the last mixer to the exit tube This can be accomplished by replacing Delay Line 2 or the exit valve with a direct exit attachment Figure 52 This attachment allows the exit tube to be connected as close as possible to the last mixer Further details and pricing may be obtained from Bio Logic or its nearest representative 10 68 SFM 3 4 User s Manual ver 1 1 11 SOFTWARE CONFIGURATION The SFM is controlled by computer and it is delivered with the MPS software that is common to all Bio Logic rapid kinetics instruments This section briefly describes the configuration the software Pleas note that the procedures and examples have been generalized and configuration choices should be made based
12. 1 PHASE 2 PHASE 3 TOTAL VOLUME 45ms Syringe 1 Syringe 2 25yl 5 ml s 360yl 8 ml s Syringe 3 30pl 6 ml s 270yl 6 ml s 12 80 SFM 3 4 User s Manual ver 1 1 IMPORTANT Because flow rate is reduced during acceleration phase and that the total time does not change the total volume of the accelerated syringe will be ess than the total volume of the original driving sequence Figure 67 The MPS software does NOT update the driving sequence on the screen to reflect an acceleration phase It is therefore recommend that the automatic mode of accelerations phases be used only when the volume delivered is not a critical factor in the experiments Manual Mode In this mode the MPS software will not calculate an acceleration phase It is left to the user to manually design a driving sequence including any necessary acceleration phases For more information about acceleration phases see the MPS Software User s Manual chapter 6 12 9 Programmable Synchronization Pulses Triggers The MPS can be programmed to deliver synchronization pulses triggers These pulses are TTL pulses 0 or 5 Volt and delivered from BNC connectors Synchro out 1 Synchro out I and Synchro out 2 on the front panel of the MPS Figure 2 Both Synchro out 1 and Synchro out 2 are rising triggers 025 V Synchro out I is the simply the inverse of Synchro out 1 and is a falling trigger 50 V The triggers can be
13. 5 21 5 23 5 26 58 5 28 5 28 5 29 5 29 5 29 5 30 5 30 5 31 6 32 i 6 32 E 3B 6 33 6 34 6 34 7 36 7 36 7 36 7 36 Qi 7 38 7 39 7 40 7 40 7 42 7 44 7 44 7 45 SFM 3 4 User s Manual ver 1 1 7 11 Data Acquisition Software 8 ASHORT STOPPED FLOW PRIMER 8 1 8 2 8 3 8 3 1 General Principle of Stopped Flow Experiments 7 45 8 46 8 46 Design and Execution of Stopped Flow Experiments General Advice for Stopped Flow Experiments ACHIEVEMENT OF FASTEST DEAD TIMES 8 3 2 WASHING 8 3 3 CAVITATION 8 3 4 SIGNAL AMPLITUDE 8 3 5 FLOW RATE 9 TEST REACTIONS 9 1 Reduction of 2 6 Dichlorophenolindophenol by Ascorbic Acid 9 2 Evaluation of the Dead Time 9 3 Evaluation of Washing and the Quality of the Stop 9 4 Variable Ratio Mixing 9 4 1 REDUCTION OF DCIP BY ASCORBIC ACID 9 4 2 ALCOHOL DEHYDROGENASE ACTIVITY 9 5 Mixing Solutions of Unequal Density and Viscosity 8 47 8 47 8 47 8 48 8 48 8 48 8 49 9 50 9 50 9 50 9 51 9 52 9 52 9 53 9 55 SFM 3 4 User s Manual ver 1 1 INSTALLATION OF THE OPTICAL SYSTEM The Bio Logic stopped flow module should be used with a Bio Logic Modular Optical System MOS Each MOS has been designed to match our SFM instruments to obtain the highest performance possible for any kinetic system Installation instructions for each MOS can be found in their respective sections of the Bio Logic Modular Optical
14. DL2 28ms SFM 4 Delay Line 1 Intermixer Volume M1 M2 Ageing Time Total Flow RateS1 S2 Delay Line 2 Intermixer Volume M1 M2 Ageing Time Total Flow RateS1 S2 The MPS software provides the possibility to repeat phases a number of times in virtually any order This is accomplished Figure 66 MPS Software though a macro sequence entered in the Drive Sequence Drive Sequence Macro frame of the Quenched Flow Program window Figure 66 The macro sequence can be edited to run a single phases or many phases in a different order than present in the 1 1 20 program grid The syntax of the macro is described in detail in the MPS Software User s Manual chapter 6 Drive Sequence Incubation Period An incubation period between two flow phases can be programmed by simply entering a phase duration for the phase and zero for all the syringe volumes 12 8 Acceleration Phases With ordinary aqueous solutions the SFM motors can drive the syringes up to a flow rate of 6 ml s for a 20 ml syringe without acceleration phase Table 1 It is possible to push solutions at faster flow rates provided an acceleration phase is added to the driving sequence As noted in section 11 5 12 79 SFM 3 4 User s Manual ver 1 1 the MPS Software can be configured for two different acceleration phases modes automatic and manual CAUTION Because a motor could stall even with the use of acceleration phases it is stron
15. FROM DATE OF PURCHASE THIS WARRENTY EXTENDS ONLY TO THE ORIGINAL PURCHASER THIS WARRANTY SHALL NOT APPLY TO FUSES OR ANY PRODUCT OR PARTS WHICH HAVE BEEN SUBJECT TO MISUSE NEGLECT ACCIDENT OR ABNORMAL CONDITIONS OF OPERATION IN THE EVENT OF FAILURE OF A PRODUCT COVERED BY THIS WARRENTY THE PRODUCT MUST BE RETURNED TO AN AUTHORIZED SERVICE FACILITY FOR REPAIR AND CALIBRATION AND TO VALIDATE THE WARRANTY THE WARRANTOR MAY AT THEIR DISCRETION REPLACE THE PRODCUT IN PLACE OF REPAIR WITH REGARD TO ANY INSTRUMENT RETURNED BECAUSE OF DEFECT DURING THE WARRENTY PERIOD ALL REPAIRS OR REPLACEMENTS WILL BE MADE WITHOUT CHARGE IF THE FAULT HAS BEEN CAUSED BY MISUSE NEGLECT ACCIDENT OR ABNORMAL CONSITIONS OF OPERATION REPAIRS WILL BE BILL AT NORMAL COST IN SUCH CASES AN ESTIMATE WILL BE SUBMITTED BEFORE WORK IS STARTED IN CASE ANY FAULT OCCURS NOTIFY BIO LOGIC OR NEAREST SERVICE FACILITY GIVING FULL DETAILS OF THE DIFFICULTY AND INCLUDE THE MODEL NUMBER TYPE NUMBER AND SERIAL NUMBER UPON RECEIPT OF THIS INFORMATION SERVICE OR SHIPPING INSTRUCTIONS WILL BE FORWARDED TO YOU EXCEPTION ARC LAMPS SOLD BY BIO LOGIC ARE ONLY WARRENTIED FOR A PERIOD OF 3 MONTHS FROM DATE OF PURCHASE 1 8 2 SFM 3 4 User s Manual ver 1 1 INTRODUCTION AND SPECIFICATIONS 2 1 General Description Each Bio Logic stopped flow module SFM consists of a mechanical subsystem and a motor power supply MPS There are two SFM conf
16. LEX IE RIS IEC EC MEE CILEQ LOL J ED 4 e l 0804 ades CITET mies TEETE E j 2 0604 ye eere oce ee Sey a ee tas tne ane Se pease o i 1 1 1 1 t a e 4 Bop ne de M S 0407 amp 2 i i 1 1 j 1 02041 0fAb 70194 id aie T te ae ee E ee 0 00 aaa a 0 0 50 0 100 0 150 0 200 0 250 0 300 0 350 0 400 0 Ageing Time ms This experiment was done entirely using the continuous flow ageing method The interrupted flow ageing method could also have been used for the longer time points with the same results 14 2 Calculation of Hydrodynamic Volumes from Kinetic Data As indicated in section 10 3 the volumes supplied in this manual are the mechanical volumes The hydrodynamical volumes may vary slightly around these values and in some instances it may be necessary to determine the hydrodynamic intermixer volumes The results of the DNPA experiment in the previous section can be used to determine the hydrodynamical intermixer volumes A procedure for determining the hydrodynamical volume of Delay Line 2 is provided below 1 Using the data from the DNPA experiment in the previous section calculate the fraction of reaction complete Y for each ageing time according to the equation Y A t A 0 A A 0 where A t is the absorbance at 325nm at ageing time t A 0 is the absorbance at tage 0 and A ce is the absorbance at tage oo 2 Using Y from step 1 a
17. Software Syringe Control Syringes Command x Syringe Control V inp Syringe 2 Syringe 3 26 mi Eug sigse Manual Speed ao X Up And Low Limits Tose 7 2 Syringe Initialization The MPS that controls the SFM follows the movements of the syringes so that the actual residual volumes are displayed at all times in the MPS software Syringes Command Load window Figure 22 When the MPS is turned on and the software started turned on the syringe volume counters show MAEHE and have to be initialized Figure 22 The syringes are initialized by setting the syringes to their uppermost empty position and resetting the syringes in the MPS software The syringes can be selected and moved to their uppermost positions either directly with the MPS section 7 1 1 or through the MPS software section 7 1 2 Once a syringe has reached its uppermost position the syringe motor will oscillate and vibrate as it becomes out of phase with the driving pulses There is no danger to the SFM or syringe motors when this occurs but there is no reason to unnecessarily prolong this treatment either The syringes can be reset individually by pushing the button for each syringe or all at once by pushing the button in the software Syringes Command Load window Figure 22 CAUTION Measurement of residual syringe volume is made by counting the logic pulses from the controller for each syringe If for a
18. Systems User s Manual However the Bio Logic stopped flow module can be adapted to any good quality optical system This is accomplished using fiber optic light links see below or through direct connection of the SFM to the optical system Please contact Bio Logic or it s nearest representative to determine the best method of connecting the SFM to your existing optical system Figure 3 Connection of an SFM to other Optical Systems Light link in absorbance mode Observation Head SFM 3 4 User s Manual ver 1 1 5 INSTALLATION OF THE STOPPED FLOW COMPONENTS 5 1 The Observation Head The stopped flow observation head Figure 4 has four optical windows one window for excitation and three for observation This allows measurements of transmittance single or double wavelength fluorescence emission and light scattering or fluorescence polarization without adding any reflecting or beam splitting elements The two windows at right angles to the incoming light can be equipped with lenses to increase the efficiency of light detection The observation head can be equipped with several types of cuvettes described in Figure 5 The cuvettes can be installed and remove Figure 4 Stopped Flow Observation Head very easily and quickly Removal and replacement of the observation cuvette is shown in Figure 11 There are two general styles of cuvettes FC fluorescence cuvette TC transmittance cuvette
19. a Teflon piston Hamilton type Observation Plastic syringes are too soft and do not give Head good results It is also recommended that a Pd the syringe with be filled with about 1 ml solution and all bubbles eliminated before use PN 5 5 Special Accessories Several accessories are available to expand the functions of the SFM Below are the descriptions of the accessories and their functions Custom accessories can also be designed and we invite you to contact Bio Logic or its nearest representatives to discuss your particular needs 5 5 1 SMALL DRIVE SYRINGE The SFM standard syringes 20 ml have a large driving speed range This allows each syringe to be programmed for different speeds and can be used to make mixing ratios different from 1 1 Ratios as high as 1 20 can be obtained with the standard syringes Beyond ratios of 1 20 the results can be poor due to the extremely slow movement of the syringe motor delivering the sample to be diluted For operation with dilution ratios higher than 1 20 we advise the use of a 5 ml syringe for injecting the solution to be diluted This enables the motor pushing the 5 ml syringe to run at a faster and smoother rate The specifications of the 5 ml syringe are give in Table 1 Syringes of 5 ml can be ordered from Bio Logic or its representatives Syringe disassembly and reassembly is discussed in the Technical Instructions section of this manual We recommend that the user be familia
20. a programmed incubation period before being mixed with the quencher Under these conditions taxe depends on the intermixer volume the total flow rate as the sample enters the intermixer volume the total flow rate as the sample exits the intermixer volume and the incubation period of the sample in the intermixer volume tage tag entry tpause ta exit where tage entry Intermixer volume Flow rate through intermixer during entry t exit Intermixer volume Flow rate through intermixer volume during exit tpause Time sample is transiently stored in the intermixer volume As with the continuous flow method the intermixer volume and flow rates can be modified by introducing different delay lines and modifying syringe flow rates in the MPS software IMPORTANT To obtain uniform ageing of the sample tage entry must always equal t exit This imposes the requirement that the sample must enter and exit the intermixer volume with the same flow rate An example driving sequence using the interrupted flow method is shown in Figure 72 The experiment is performed in Phases 2 4 In phase 2 the reactants are mixed and the intermixer volume filled with sample In Phase 3 the sample is allowed to age for 300 ms In phase 4 the sample is pushed out of 13 85 SFM 3 4 User s Manual ver 1 1 the intermixer volume quenched and collected No delay line is used The intermixer volume for mixers 2 and 3 is then 27 6
21. frame Figure 26 MPS Software Syringes Contents of the Stopped Flow Program Syringes contents window Figure 24 and Figure 26 The text Is entered from the keyboard and the BACKSPACE and DEL keys can be used for corrections IMPORTANT It is strongly recommend that users take advantage of this feature of the MPS software to keep track of the samples loaded into the SFM syringes Each time a program grid cell value is changed information about the current syringe current phase and driving sequence is updated displayed below and to the right of the grid Figure 27 This information indicates 1 Current phase number and total number phases in the driving sequence 2 Volume delivered by the current syringe during the current phase or current phase total volume if an entire phase is selected 3 Flow rate of the current syringe during the current phase or current phase total flow rate if an entire phase is selected 4 Total volume delivered by each syringe during the driving sequence SFM 3 4 User s Manual ver 1 1 Figure 27 MPS Software Driving Sequence Information Syringes Total time ms Volumes pl 100 Syr 2 ul inn Syr 3 pl q0 Syr 4 ul inn Synchro 1 Phase 2 5 Volume 1UU pl How Hate 1 U ml s l An indication of the Dead Time and Ageing Times for a driving sequence are also displayed in the Stopped Flow Program
22. initialized Figure 59 The syringes are initialized by setting the syringes to their uppermost empty position and resetting the syringes in the MPS software The syringes can be selected and moved to their uppermost positions either directly with the MPS section 12 1 1 or through the MPS software section 12 1 2 Once a syringe has reached its uppermost position the syringe motor will oscillate and vibrate as it becomes out of phase with the driving pulses There is no danger to the SFM or syringe motors when this occurs but there is no reason to unnecessarily prolong this treatment either The syringes can be reset individually by pushing the button for each syringe or all at once by pushing the button in the software Syringes Command Load window Figure 59 CAUTION Measurement of residual syringe volume is made by counting the logic pulses from the controller for each syringe If for any reason a syringe is blocked during a run the pulses will not correspond to the true volume delivered and the value displayed may become erroneous e g in the case of incorrect positioning of a valve In this case it is advisable to reinitialize the syringes If by accident a syringe is returned to its uppermost position the syringe volume counter will again show BEBE and the syringe must be reinitialized To avoid such accidents the Up and Low Limits checkbox may be checked When this box is checked the MPS software will not allow the s
23. interrupted flow step of Figure 75 To minimize sample contamination such movement of the sample should only be done if the flow line volume is much large than the volume to be collected 13 89 SFM 3 4 User s Manual ver 1 1 Figure 75 Ageing Methods Comparison Example In this example a double dilution experiment is performed where the sample from the first dilution is aged before for a time Ta before the second dilution quench S1 Protein S2 Label S3 Quencher Delay lines used are noted below For a Ta 100ms both the continuous and interrupted flow ageing methods are evaluated for sample economy Continuous Flow This method uses a 90u1 delay line intermixer volume of 116 7 Time ms 308 Total Volume Used ul S1 Protein 60 60 S2 Label 300 300 S3 Quencher 1800 1800 Using this driving sequence Ta 116 7u1 1 169ml s 100 ms 99 8 ms Interrupted Flow In this method a 17 1 delay line is used intermixer volume of 43 81 Time ms 110 40 30 Total Volume Used ul S1 Protein 20 20 3x less than continuous flow method S2 Label 100 33 140 2 1x less than continuous flow method S3 Quencher 165 9x less than continuous flow method In the first phase the flow between the two mixers is 1 1 ml s ageing the sample a total of 40 ms The second phase incubates the sample for 20 ms The third phase mixes the sample in the intermixer volume with S3 NOTE The sample is pushed out of th
24. is set by pressing W for Waste and C for Collect on the keyboard Selected values entered in the program grid can be cut copied and pasted using the Cut Copy and Paste functions available under the Edit menu To perform a cut copy or paste operation select the area of the grid desired by dragging the mouse with the left mouse button pushed in and then choose the Cut Copy or Paste functions desired under the Edit menu The values will be stored in the Windows clipboard for the Cut and Copy functions Values will be pasted from the Windows clipboard for the Paste function If copy area is bigger than paste area the operation is done only for values that can fit inside paste area CAUTION Blank and non numeric values entered in the program grid are considered as zero values A phase duration of Oms will cause the phase to be skipped in the execution of the drive sequence The contents of the syringes can be J entered in the Syringe Contents frame Figure 63 MPS Software Syringes Contents of the Quenched Flow Program Syringes contents i window Figure 61 and Figure 63 The text is entered from the keyboard and the BACKSPACE and DEL keys can be used for corrections IMPORTANT It is strongly recommend that users take advantage of this feature of the MPS software to keep track of the samples loaded into the SFM syringes Each time a program grid cell value is changed information about t
25. mixer has been installed at that position SFM 3 4 User s Manual ver 1 1 Figure 9 SFM 4 S Flow Line and Delay Line Volumes SFM 4 S FLOW LINE VOLUMES Line Number Flow Line Volume ul 156 Delay Line 1 9 165 7 Delay Line 2 22 188 Cuvette Figure 5 MIXER2 MIXER1 RESERVOIR1 e N e vt 9 5 S z 2 z x x g x o o o o DELAY LINE AND INTERMIXER VOLUMES Delay Line N 1 17 N 2 40 N 3 90 N 4 140 N 5 190 N 6 500 N 7 100 Volume ul Intermixer Volume 25 5 Miss M2ss ul ntermixer Volume 31 7 M2ss M3ss ul ntermixer Volume 27 4 M2ss M3ups pl Notes Intermixer volumes are measured from the mixing point of one mixer to the mixing point of the next mixer BB indicates a Berger Ball mixer has been installed at the position noted HDS indicates a Hiah Densitv mixer has been installed at the position noted SFM 3 4 User s Manual ver 1 1 5 4 Liquid Outlet System During the injection phase the liquid in the cuvette can reach linear velocities greater than 20 meters per second At the flow stop the liquid column has to be immobilized in a fraction of a millisecond Several different stop modes can be used to immobilized the liquid column Depending on the stop mode this can result in overpressure or underpressure conditions that are potential sources of stop artifacts
26. more information about acceleration phases see the MPS Software User s Manual chapter 6 Programmable Synchronization Pulses Triggers The MPS can be programmed to deliver synchronization pulses triggers These pulses are TTL pulses 0 or 5 Volt and delivered from BNC connectors Synchro out 1 Synchro out I and Synchro out 2 on the front panel of the MPS Figure 2 Both Synchro out 1 and Synchro out 2 are rising triggers 05 V Synchro out I is the simply the inverse of Synchro out 1 and is a falling trigger 50 V The triggers can be used to synchronize the SFM and data acquisition system or other instruments If the Bio Kine software is being used for data collection acquisition will start on the falling edge of the synchronizing pulse 1 e at the end of the first active phase with a synchro set to On The triggers can also be used for synchronizing the SFM with other devices The timing of the triggers with respect to the drive sequence is programmed in the last line s of the program grid in the Stopped Flow Program window The duration of the pulse will be equal to the duration of the phase Synchro out 1 or Synchro out 1 and 2 can be used depending on the instrument configuration section 5 4 2 If the hard stop is in Automatic mode only Synchro out 1 is available If the hard stop is 1n Manual mode Synchro out 1 is available and Synchro out 2 is used to control the hard stop If the hard stop is not used None both Sync
27. of 2 6 Dichlorophenolindophenol by Ascorbic Acid A complete description of the reduction of 2 6 dichlorophenolindophenol DCIP by ascorbic acid AA and its use can be found in Tonomura et al Analytical Biochemistry 1978 84 370 383 DCIP has a strong absorbance at 524 nm and reduction by ascorbic acid results in a nearly complete decoloration The second order reduction rate constant is highly dependent on pH and varies from about 10 5 M s at pH 2 0 to 107 M s at pH 8 0 If the concentration of DCIP is sufficiently smaller than AA the reaction can be treated as a pseudo first order reaction whose rate constant will be directly proportional to the AA concentration All these properties make this reaction a very useful tool for stopped flow calibration The fast reaction at acid pH can be used measure the dead time of the SFM instrument The slow reaction at neutral pH slow reaction to check the quality of the stop to evaluate the washing of the observation cell and to test the variable ratio mixing capabilities The following sections describe the use of this reaction for testing the and exploring its capabilities Evaluation of the Dead Time The dead time of the SFM can be measured using both the fast and slow reduction reactions of DCIP An example dead time evaluation is shown in this section As discussed in section 8 3 1 the dead time of a stopped flow experiment depends on many factors besides simply the flow rate and cuvette vol
28. pipette or syringe The partial liquid collection method can be used with either the continuous or interrupted flow ageing method section 13 2 1 Example driving sequences using the partial liquid collection method with continuous flow ageing and interrupted flow ageing are shown in Figure 73 and Figure 74 respectively Partial Liquid Collection with Continuous Flow Ageing PURGE COLLECT time ms Syr 1 nl Syr 4 nl Synchro 1 Phase 3 5 Syringes Total Volumes pl a iD 105 imo 105 iO Waste Collect Waste Waste Off Off Off Off Total Volume 135 pl Total Flow Rate 9 0 ml s Syringes contents Shots Drive Sequence Ageing Time z DLI 85ms 1 1 20 DL2 46ms Multiple Comments Close 13 87 Quenched Flow Program x SFM 3 4 User s Manual ver 1 1 Figure 74 Partial Liquid Collection with Interrupted Flow Ageing PURGE COLLECT v y time ms Syr 1 nl Syr 2 ul Syr 3 ul Syr 4 ul Valve Synchro 1 Phase 1 Phase2 Phase3 Phase4 Phase5 3Y nges Total 50 30 Volumes pl 0 150 90 300 150 90 300 90 I Waste Waste Waste Collect Off Off Off Off Phase 5 5 Volume 60 pl Flow Rate 3 0 ml s Syringes contents Shots Buffer DNPA Drive Sequence Ageing Time DL1 8 5 ms 1 1 20 z EZ DL2 46ms Comments Clo
29. shown in Figure 7 The observation head may be installed using the mixing blocks labeled 0 MIX 0 0 MIX DL DL MIX 0 DL MIX DL or no mixing block The installation of the different mixing blocks is described in Table 3 Table 3 SFM 4 Observation Head Installation MIXING BLOCK COMMENTS 0 MIX 0 Installed with no additional delay lines 0 MIX DL Installed with one delay line between the mixer block and the observation head DL MIX 0 Installed with one delay line between the SFM body and the mixer block DL MIX DL Installed with delay lines on both sides of the mixer block NONE Only a delay line is installed between the SFM body and the observation head and the SFM 4 functions as an SFM 3 Figure 6 Syringe 3 is blocked by the delay line and only syringes 1 2 and 4 are useable In this case syringe 3 does not need to be filled SFM 3 4 User s Manual ver 1 1 Figure 6 SFM 3 Installation of Delay Lines OBSERVATION HEAD SECOND MIXER M2 DELAY LINE FIRST MIXE M1 SFM 3 4 User s Manual ver 1 1 Figure 7 SFM 4 Installation of Mixing Blocks and Delay Lines OBSERVATION HEAD THIRD MIXER M3 Na HEAD SPACERS PEE DELAY LINE TWO 2 e y MIXER BLOCK SECOND MIXER MIXER BLOCK ges hU M2 SECOND MIXER 6 9 7 M2 a ca DELAY LINE ONE E IUe DL1 5 25 SFM 3 4 User s Manual ver 1 1 5 3 Flow Line and Intermixer Volumes Figure
30. upon the equipment purchased and intended experiments The user may refer to the MPS Software User s Manual for more details about the MPS software This sections assumes that the user has already installed and started the MPS software on the host microcomputer For installation instructions see chapters 2 and 3 of the MPS Software User s Manual 11 1 Device Configuration The device to be installed should be configured according to the instrument purchased and mode chosen for use The device appropriate should be chosen in the Config Device window available under the Config menu Figure 53 Further information may be found in chapter 4 1 of the MPS Software User s Manual Figure 53 MPS Software Device Configuration SFM 2 S amp SFM 20 S Controller type SFM 3 S amp SFM 300 S SFM 3 Q amp SFM 300 Q MPS 51 MPS 52 Syringes SFM 4 S amp SFM 400 S Cuvette SFM 470 amp SEM 400 Q MPS 60 ES i QFM 5 Delay Line SFM 5 C Serial Port Limits ok Cancel 11 2 Syringe Configuration Syringe configuration is made in the Config Syringes window available under the Config menu Figure 54 Select the syringe volumes to indicate the nature of the syringes that have been installed in each syringe position of the SFM using the menu in the Config Syringes window Figure 54 Use the Custom button to enter syringe specifications if you have a custom syringe The SFM comes equipped with standard 20 ml syringes and
31. 00 300 3 NaOH 300 300 4 HCI 300 300 Exit Valve Waste Waste Collect Table 9 DNPA Experiment Parameters INTERMIXER CS VOLUME M2 M3 FLOW RATE ml s 50 12 18 100 6 35 150 4 53 200 3 71 300 2 106 450 1 333 159 600 1 212 1000 0 6 353 IMPORTANT This reaction is very sensitive to contamination The experiments must be performed from smallest tage to the largest least to most DNP produced so that contamination of subsequent shots is kept to a minimum Three shots were performed for each tage The first shot was discarded and the second and third shots kept for analysis A tage 0 ms sample was prepared by hand by mixing 300pl of the DNPA solution with 300 ul of water and 300 ul of 2 M HCI A tage e sample was prepared by hand by mixing 3001 of the DNPA solution with 300 ul of 1 M NaOH and 300 ul of 2 M HCI The absorbance of DNP at 325 nm was measured for each ageing time The absorbance was measured for 500u1 of each sample mixed with 500p1 of water in a 1cm path length cuvette The results were plotted against the ageing times as shown in Figure 77 The apparent first order rate constant determined from Figure 77 is 28 s which yields a second order rate constant of 56 M s for a final OH of 0 5 M 14 92 SFM 3 4 User s Manual ver 1 1 Figure 77 DNPA Experiment Results 1 20 Jt infinity Abs 1 001 T x M lc M E
32. 4 User s Manual ver 1 1 10 1 Introduction 10 60 10 2 Installation of the Mixer Blocks and Delay Lines 10 60 10 3 Flow Line and Intermixer Volumes 10 63 10 4 Sample Collection Methods 10 65 10 4 4 TOTAL LIQUID COLLECTION 10 65 10 4 PARTIAL LIQUID COLLECTION 10 66 10 5 Special Accessories 10 66 10 5 1 SMALL DRIVE SYRINGE 10 66 10 5 2 LARGE DRIVE SYRINGE 10 67 10 5 3 HIGH DENSITY MIXER 10 67 10 5 4 DIRECT EXIT ATTACHMENT 10 68 11 SOFTWARE CONFIGURATION 11 69 11 1 Device Configuration 11 69 11 2 Syringe Configuration 11 69 11 3 Delay Line Configuration 11 70 11 4 Serial Port Configuration 11 70 11 5 System Limits Configuration 11 71 12 INSTRUMENT OPERATION 12 73 12 4 Manual Syringe Control 12 73 12 1 1 MPS 12 73 12 1 2 SOFTWARE 12 73 12 2 Syringe Initialization 12 74 12 3 Filling the Syringes 12 75 12 4 SFM Cleaning and Storage 12 76 12 5 Long term Storage of the SFM 12 77 12 6 Creating a Driving Sequence 12 77 12 7 Incubation Period 12 79 12 8 Acceleration Phases 12 79 12 9 Programmable Synchronization Pulses Triggers 12 81 12 10 Saving or Loading Driving Sequences 12 81 12 11 Running in Automatic Mode 12 81 13 A SHORT QUENCHED FLOW PRIMER 13 83 13 1 General Principle of Quenched Flow Experiments 13 83 13 2 Design and Execution of Quenched Flow Experiments 13 84 13 2 1 AGEING METHODS 13 84 13 2 COLLECTION METHODS 13 86 13 3 General Advice for Quenched Flow Experiments 13 88 1 4 13 3 1 13 3 2
33. 8 SFM 3 and Figure 9 SFM 4 below indicate the volumes of SFM flow lines and delay lines The amount of time a sample ages between two mixers is given by Ageing time between two mixers Intermixer volume Flow rate through intermixer volume It should be noted that the volumes give in the table are the mechanical volumes The hydrodynamical volumes may vary slightly around these values For precise measurement of ageing times it is recommended that the intermixer volumes be determined experimentally with known reactions One such experimental procedure for determining intermixer volumes is described in the Quenched Flow section of this manual Figure 8 SFM 3 S Flow Line and Delay Line Volumes SFM 3 S FLOW LINE VOLUMES Line Number Flow Line Volume ul 103 41 155 156 7 Delay Line 22 188 Cuvette Figure 5 o o c A amp oN CUVETTE MIXER1 DELAY LINE RESERVOIR1 RESERVOIR2 N e Lu ul Lu o z x m rn gt gt gt o o o DELAY LINE AND INTERMIXER VOLUMES Delay Line N 1 17 N 2 40 N 3 90 N 4 140 N 5 190 N 6 500 N 7 100 Volume pl Intermixer Volume Miss M2gg ul Intermixer Volume Miss M2ups Hl Notes Intermixer volumes are measured from the mixing point of one mixer to the mixing point of the next mixer BB indicates a Berger Ball mixer has been installed at the position noted HDS indicates a High Density
34. Phase 3 5 Total Volume 300 pl Total Flow Rate 15 0 ml s Syringes contents Shots Drive Sequence Ageing Time z DLI 51ms Single 1 1 20 nm DL2 28m Comments Close 12 77 SFM 3 4 User s Manual ver 1 1 A driving sequence is entered in the program grid in the Quenched Flow Program window Figure 61 Each column of the grid represents a driving sequence phase Each phase contains actions for the SFM to perform A complete driving sequence may contain from 1 to 20 phases Although only 5 phases are shown initially additional phases may be inserted using the Insert Phase command under the Edit menu Figure 62 shows an expanded view of the program grid The Figure 62 MPS Software Program Grid duration of a phase is entered in ms 1 9999 ms phase on the Phase first line of the program grid Tun T uli The volume in ul delivered by syringe 100 each of the syringes during a Volumes Syr 3 ul 100 phase is entered on the line next Syr 4 ul 100 to the appropriate syringe The Collect Position position of the exit valve is set Synchro 1 Off near the bottom of the program grid To enter the phase duration and syringe volumes delivered click on the corresponding cell or use the keyboard arrows keys to navigate between cells The BACKSPACE key can be used for correction and the DEL key to clear a value The position of the exit valve
35. S Delay Line N 1 17 N 2 40 N 3 90 N 4 140 N 5 190 N 6 500 N 7 100 Volume ul Intermixer Volume 25 5 M1gs M2gg pl Intermixer Volume 27 6 M2ss M3gg ul Intermixer Volume 23 3 M2ss M3ups pl Notes Intermixer volumes are measured from the mixing point of one mixer to the mixing point of the next mixer BB indicates a Berger Ball mixer has been installed at the position noted HDS indicates a High Density mixer has been installed at the position noted 10 64 SFM 3 4 User s Manual ver 1 1 10 4 Sample Collection Methods The result of a quenched flow experiment can be recovered by two different methods total liquid collection and partial liquid collection The method of choice will depend on the experiment The two methods are described below 10 4 1 TOTAL LIQUID COLLECTION In this method all the liquid that exits the SFM during a quenched flow experiment is recovered This includes the result of the quenched flow experiment and any old reaction mixture that remained in the SFM before the start of the experiment Two manners exist to recover the total liquid from a quenched flow experiment These are describe in the next two sections 10 4 1 1 10 4 1 2 Free flow method A tube is connected to the waste outlet of the exit valve to recover the liquid exiting C the SFM Figure 49 The 7 AA liquid may be ejected into a test tube or beaker for simple col
36. S Software User s Manual for more details about the MPS software This sections assumes that the user has already installed and started the MPS software on the host microcomputer For installation instructions see chapters 2 and 3 of the MPS Software User s Manual 6 1 Device Configuration The device to be installed should be configured according to the instrument purchased and mode chosen for use The device appropriate should be chosen in the Config Device window available under the Config menu Figure 15 Further information may be found in chapter 4 1 of the MPS Software User s Manual Figure 15 MPS Software Device Configuration Config Device x SFM 2 S amp uSFM 20 5 Controller type SFM 3 S amp SFM 300 S as SFM 3 Q amp SFM 300 Q MPS 51 MPS 52 Auto Syringes SFM 4 S amp SFM 400 S CM 1 Cuvette SFM 4 Q amp SFM 400 Q C MPS 60 Manua Delay Line ie IC C None Serial Port Limits 6 2 Hard Stop Configuration The hard stop is configure under the Config Device window shown in Figure 15 The hard stop mode should be chosen as desired The different modes are described in section 5 4 2 6 3 Syringe Configuration Syringe configuration is made in the Config Syringes window available under the Config menu Figure 16 Select the syringe volumes to indicate the nature of the syringes that have been installed in each syringe position of the SFM using the menu in the Config Syringes window Figur
37. To accomplish this sufficient volume of mixed samples needs to pass through the cuvette during the shot This volume varies with flow rate viscosity and composition of the sample It is strongly recommended that tests be perform and adequate washing conditions found before starting any series of experiments CAVITATION Cavitation occurs when turbulence creates regions of low enough pressure in a liquid that a cavity is formed This cavity fills with the liquid s vapor These cavities collapse incompletely leaving behind small bubbles of vapor which interfere with optical observation methods As the flow rate increases through a mixer so does the likelihood of cavitation The probability of cavitation also increases with increasing viscosity for a given flow rate De gassing of solutions decreases the probability of cavitation by eliminating gas and lowering the total vapor pressure available to fill the cavities SIGNAL AMPLITUDE Signal amplitude is generally proportional to the path length of the cuvette and the concentration of signal generating reagent An increase in signal can then be accomplished by an increase in cuvette path length or an increase in the concentration of reagent However the researcher may be limited by practical concerns such as value of sample viscosity of sample dead times inherent limitation of signal such as inner filter effect and sample precipitation As with achievement of fastest dead times compromises m
38. VN BioLogic jence Instruments SFM 3 4 User s Manual SFM 3 4 User s Manual ver 1 1 TABLE OF CONTENTS 1 WARRANTY 1 8 2 INTRODUCTION AND SPECIFICATIONS 2 9 2 General Description 2 9 2 1 1 THE MECHANICAL DESIGN 2 9 2122 INTELLIGENT POWER SUPPLY 2 9 2 1 3 MICROCOMPUTER COMMANDS 2 9 2 2 Modes of Operation 2 10 2 2 1 STOPPED FLOW SF MODE commercial reference SFM X S 2 10 2 2 2 QUENCHED FLOW QF MODE commercial reference SFM X Q 2 10 2 3 Specifications 2 12 2 4 Principle of Operation 2 13 2 5 Description of the Mechanical Design 2 13 2 6 The Delay Lines 2 13 3 GENERAL INSTRUCTIONS FOR INSTALLATION 3 15 3 1 Operating Features 3 15 3 2 AC Power and Connections 3 16 3 3 Temperature Regulation 3 16 4 INSTALLATION OF THE OPTICAL SYSTEM 4 20 5 INSTALLATION OF THE STOPPED FLOW COMPONENTS 5 21 5 1 The Observation Head 5 21 52 Installation of the Mixer Blocks and Delay Lines 5 23 5 3 Flow Line and Intermixer Volumes 5 26 5 4 Liquid Outlet System 5 28 5 4 1 FREE FLOW SYSTEM 5 28 5 4 2 HARD STOP SYSTEM 5 28 5 4 3 EXIT IN A SYRINGE 5 29 5 5 Special Accessories 5 29 5 5 1 SMALL DRIVE SYRINGE 5 29 2329 2 LARGE DRIVE SYRINGE 5 30 5 5 3 HIGH DENSITY MIXER 5 30 5 5 4 OBSERVATION HEAD WITH SEPARATE COOLING 5 31 6 SOFTWARE CONFIGURATION 6 32 6 1 Device Configuration 6 32 6 2 Hard Stop Configuration 6 32 6 3 6 4 6 5 6 6 6 7 7 1 7 11 7 2 7 3 7 4 7 5 7 6 7 7 7 8 7 9 7 10 7 11
39. a acquisition can be performed Please refer to the Bio Kine for Windows Software Manual for information on how to configure the data acquisition parameters SFM 3 4 User s Manual ver 1 1 8 ASHORT STOPPED FLOW PRIMER This section describes the basics of the stopped flow technique and provides some general advice about how to design and perform stopped flow experiments using the SFM It is not meant to be an exhaustive reference as there are many variations on the stopped flow experiment too numerous to describe here 8 1 General Principle of Stopped Flow Experiments The simplest stopped flow experiment occurs in two stages In the first stage flow is initiated by two plungers The plungers force liquid Figure 33 A Simple Stopped Flow Experiment through a mixer and along a flow path into an observation cuvette The resulting mixture ages as it travels along the flow path and into the cuvette The amount of ageing depends on the flow rate of the mixture and the volumes of the flow path and cuvette In this first stage the mixer flow path and cuvette are initially washed by the constantly refreshed mixture This continues until a steady state condition arises in which the age of the mixture is completely linear with respect to the distance along the flow path Once the steady state condition is reached any particular point in the flow path represents the mixture at particular age Furthermore the age of the mixture in the cuv
40. ariable ratio mixing method the concentration of one reactant ascorbic acid in this case can easily be varied while another reactant DCIP is kept constant The curves in Figure 38 were analyzed using the Bio Kine software to determine the rate constants The rate constants measured show a satisfactory linear relationship as a function of ascorbic acid concentration Figure 39 SFM 3 4 User s Manual ver 1 1 Figure 39 DCIP Variable Ratio Mixing k vs AA 1 2 MA Ps w e 4 6 Ascorbic Acid mM Dilution factors of 1 50 or higher can be obtained with the SFM Figure 40 shows the results of experiments where 1 mM DCIP in S4 was mixed with various volumes of buffer from S1 In this case there is no reaction but only dilution of the DCIP The results indicate a satisfactory linear relationship between the absorbance measured and the final concentration of DCIP calculated according to the diluted effect Figure 40 DCIP Dilution Experiments Absorbance T 1 21 1 11 Dilution Factor 9 4 2 ALCOHOL DEHYDROGENASE ACTIVITY Experimental Conditions Buffer 100 mM Tris Cl 1 mM EDTA 5 g l semicarbazyde Cl and 25 mM ethanol Syringe 1 20 ml Syringe 2 20 ml Syringe 4 20 ml Wavelength Cuvette Detection method Buffer Buffer 1 mg ml alcohol dehydrogenase ADH Buffer 1 mM NAD 340 nm TC 50 10 Absorbance SFM 3 4 User s Manual ver 1 1 Experimen
41. ation of 0 14 ul per micro step p step when standard 20 ml syringes are used With the damping produced by the rotor inertia this results in an almost continuous linear movement of the syringe even at very low flow rates The motors can be activated manually or automatically The manual mode is mainly used to refill or wash the syringes the syringes can be driven independently and their speed adjusted using the microcomputer with a very simple menu see the MPS Software User s Manual for more details The automatic mode is used for actual experiments The motor impulses are counted in the positive direction refilling or negative direction emptying so that the contents of each syringe can be continuously displayed Zero volume corresponds to the uppermost position of the syringe and referencing the zero volume position can be done using the keyboard of the microcomputer The movements of the syringes are completely controlled by the microprocessor which eliminates the need for a stop syringe Thus the stop artifact present in most conventional stopped flow systems is absent in the SFM The observation system can be synchronized with the syringe start or stop by using the trigger pulses available on the front panel of the MPS unit The independent control of each syringe allows a high versatility of the injection sequence It is possible to make an injection of one syringe only unequal filling of the syringes variable ageing ti
42. ay be necessary to achieve successful a stopped flow experiment Table 6 shows some of the most common actions that can be taken to improve signal amplitude and their consequences 8 48 SFM 3 4 User s Manual ver 1 1 Table 6 Common Actions to Improve Signal Amplitude overuse of reagent Increase Cuvette Path Length increased dead time Inadequate washing Inner filter effect fluorescence Increase Signal overuse of reagent Increased viscosity causing cavitation Increase Reagent Concentration Increased viscosity causing stalled motors Increase viscosity causing inadequate washing 8 3 5 FLOW RATE The flow rate of the SFM is limited by the speed with which the stepping motors can push At the nominal flow rate limit of 6ml s 20 ml syringes all syringes in use and using the smallest cuvette sub millisecond dead times may be accomplished However solutions of increased viscosity will lower the obtainable syringe speed Also lower than room temperatures often lower the obtainable syringe speed The limitation to syringe speed may sometimes be defeated by the use of acceleration phases section7 7 which allows each syringe to come to an intermediate speed for a short time before jumping to the higher speed Experimental conditions should always be tested before use on precious samples SFM 3 4 User s Manual ver 1 1 9 TEST REACTIONS 9 1 9 2 Reduction
43. bath for temperature regulation The coolant flows through two internal circuits one around the injection and reservoir syringe ports and the other through the valve block and observation head SFM 3 4 User s Manual ver 1 1 SECTION II STOPPED FLOW MODE SFM X S SFM 3 4 User s Manual ver 1 1 TABLE OF CONTENTS SECTION II 4 INSTALLATION OF THE OPTICAL SYSTEM 5 INSTALLATION OF THE STOPPED FLOW COMPONENTS 5 1 The Observation Head 5 2 Installation of the Mixer Blocks and Delay Lines 5 3 Flow Line and Intermixer Volumes 5 4 Liquid Outlet System 5 4 1 FREE FLOW SYSTEM 5 4 2 HARD STOP SYSTEM 5 4 3 EXIT IN A SYRINGE 5 5 Special Accessories 5 5 1 SMALL DRIVE SYRINGE 5 5 2 LARGE DRIVE SYRINGE 5 5 3 HIGH DENSITY MIXER 5 5 4 OBSERVATION HEAD WITH SEPARATE COOLING 6 SOFTWARE CONFIGURATION 6 1 Device Configuration 6 2 Hard Stop Configuration 6 3 Syringe Configuration 6 4 Cuvette Configuration 6 5 Delay Line Configuration 6 6 Serial Port Configuration 6 7 System Limits Configuration 7 INSTRUMENT OPERATION 7 1 Manual Syringe Control 7 1 1 MPS 7 1 2 SOFTWARE 7 2 Syringe Initialization 7 3 Filling the Syringes 7 4 SFM Cleaning and Storage 7 5 Long term Storage of the SFM 7 6 Creating a Driving Sequence 7 7 Acceleration Phases 7 8 Programmable Synchronization Pulses Triggers 7 9 Saving or Loading Driving Sequences 7 10 Running in Automatic Mode 4 20 5 21
44. bove and the pseudo first order rate constant measured calculate the actual ageing time Ta for each point from Ta 1000x In C 0 In C t k 1000 x In 0 5 In 0 5 x 1 Y k where C 0 is the DNPA concentration at tage 0 0 5 mM for the experiment in the previous section C t is the DNPA concentration at time t and k is in s The units of Ta are ms 14 93 SFM 3 4 User s Manual ver 1 1 3 The hydrodynamical intermixer volume M2 M3 can then be calculated from Volume F Ta where F is the total flow through the intermixer volume in l s Since the hydrodynamical intermixer volume can be calculated for each Ta the mean and standard deviation of the volume can easily be determined 14 3 Washing Efficiency To obtain the best results from quenched flow experiments it is necessary to minimize sample contamination The most common source of sample contamination is inefficient washing of old reacted sample from the flow lines and intermixer volumes before sample collection The simplest method of determining the volume needed to efficiently wash or purge the flow lines and intermixer volumes is to carry out multiple experiments with increasing purge volumes until there are no differences in experimental results In many cases this is impractical due to the cost or availability of one or more experimental components The procedure below uses the DNPA experiment 14 1 to provide an example of how to determine th
45. chieved with minimal change of delay lines In addition unlike pneumatic based systems the flow rates are independent of viscosity and temperature An example driving sequence using the continuous flow method is shown in Figure 71 It should be noted that the experiment is performed only in Phase 2 of the driving sequence This phase encompasses all stages mixing ageing and quenching of the of experiment No delay line is used The intermixer volume for mixers 2 and 3 is then 27 6 ul Figure 48 and the total flow through the intermixer volume is 6 ml s which indicates tage _27 6ul 4 6ms 6 mL s The continuous flow method is generally used to study reactions from 1 to 100ms It is generally the most economic ageing method with respect to sample consumption within this time range 13 84 SFM 3 4 User s Manual ver 1 1 Figure 71 Example Continuous Flow Experiment Quenched Flow Program EXAMPLE QF4 Syringes Total time ms Volumes ul Syr 1 ul Syr 2 ul Syr 3 ul Syr 4 ul A Collect Collect Waste Waste Synchro 1 Off Off Off Off Phase 2 5 Volume 150 pl Flow Rate 3 0 ml s Syringes contents Shots Drive Sequence Ageing Time z DL1 Spine i ME Single 11 20 DL2 46ms Syringe 2 DNPA Multiple Syringe 3 Singe 4 jun T 13 2 1 2 Interrupted flow method In the interrupted flow method the sample 1s transiently stored in the intermixer volume for
46. d rectangle Figure 59 Syringes are emptied or filled using the Rl v and buttons or with the lt Up gt arrow lt PageUp gt lt Down gt arrow and lt PageDown gt keys on the keyboard The button and lt Up gt arrow move a syringe upwards by one elementary movement and the v button and Down arrow move a syringe downwards by one elementary movement The A button and lt PageUp gt arrow move the piston upwards by 10x elementary movements and the z button and lt PageDown gt move the piston downwards by 10x elementary movements The size of the elementary steps and syringe movement speed is controlled in the Manual Speed section of the Syringes Command Load window Figure 59 The ET and gt buttons to change the manual speed The display shows the speed in flow rate based on the syringe installed and moved 12 73 SFM 3 4 User s Manual ver 1 1 Figure 59 MPS Software Syringe Control Syringes Command x Syringe Control V inpl Syringe 2 Syringe 3 26 mi Eug sigse Manual Speed ao X Up And Low Limits Tose 12 2 Syringe Initialization The MPS that controls the SFM follows the movements of the syringes so that the actual residual volumes are displayed at all times in the MPS software Syringes Command Load window Figure 59 When the MPS is turned on and the software started turned on the syringe volume counters show MAEHAN and have to be
47. d to empty and fill the syringes respectively The LCD panel at the top of the controls will display which syringe has been selected and whether it is being filled or emptied motor on o SOFTWARE Syringe control from the MPS software is made through the Syringes Command Load window available under the Syringes Command menu Figure 22 The syringe to be moved is selected by clicking on the corresponding frame or pressing the lt Left gt or lt Right gt arrows keys on keyboard The new selected syringe will be surrounded with a red rectangle Figure 22 Syringes are emptied or filled using the amp 2 v and B buttons or with the lt Up gt arrow lt PageUp gt lt Down gt arrow and lt PageDown gt keys on the keyboard The button and lt Up gt arrow move a syringe upwards by one elementary movement and the v button and Down arrow move a syringe downwards by one elementary movement The button and lt PageUp gt arrow move the piston upwards by 10x elementary movements and the z button and lt PageDown gt move the piston downwards by 10x elementary movements The size of the elementary steps and syringe movement speed is controlled in the Manual Speed section of the Syringes Command Load window Figure 22 The ET and buttons to change the manual speed The display shows the speed in flow rate based on the syringe installed and moved SFM 3 4 User s Manual ver 1 1 Figure 22 MPS
48. e AGE reaction mixture through a delay line to the second mixer QUENCH The sample ages reacts as it travels through the delay line bp A B until it reaches the second mixer where it is quenched Quench As the sample passes through STAGE 1 STAGE 2 STAGES the second mixer it is mixed with the quencher Q which stops the reaction The resulting solution is then collected for analysis of the quantity of C produced during the experiment The age of the sample tage is the total time between the start of the reaction in and the moment it is quenched The age will depend on the total flow rate through the delay line and the intermixer volume as described in section 10 3 It can also depend on the duration of a pause in the flow that allows the sample to age for long times see section 13 2 1 2 A quenched flow study will consist of numerous experiments where tage is varied for each experiment At the end of the study a kinetic trace can be constructed by plotting tage vs the results of each sample analysis 13 83 SFM 3 4 User s Manual ver 1 1 13 2 Design and Execution of Quenched Flow Experiments Experiments are designed using the MPS software to create a driving sequence This is accomplished by filling in elements of program grid as described in section 12 6 The number of phases contained in an experiment driving sequence and the function of each phase will depend on the process being examined the ageing method and the sam
49. e 16 Use the Custom button to enter syringe specifications if you have a custom syringe The SFM comes equipped with standard 20 ml syringes and these are the default syringes installed in the MPS software Changes only need to be made in the software when syringes of different volumes other than standard have been installed in the SFM TWARNING Incorrect syringe configuration will cause volume and flow rate calculations to be incorrect Further information about syringe configuration can be found in chapter 4 2 of the MPS Software User s Manual SFM 3 4 User s Manual ver 1 1 Figure 16 MPS Software Syringe Configuration Syringe 2 Syringe 3 E 4 Hr 20 ml ol en Diam 17 mm iam 17 mm Pitch 4 mm Pitch E mm ol 20 ml Diam 17 mm Pitch 4 mm rw e IE windows gt Device Syringes Cuvette Delay Line Serial Port Limits 6 4 Cuvette Configuration Cuvette configuration is made in the Config Cuvette window available under the Config menu Figure 17 Select the cuvette according to the cuvette you have installed in the SFM The type of cuvette is chosen in the list shown in the Config Cuvette window Figure 17 TWARNING Incorrect cuvette configuration will cause dead time calculations to be incorrect Further information about cuvette configuration can be found in chapter 4 3 of the MPS Software User s Manual Figure 17 MPS Software Cuvet
50. e intermixer volume using ONLY buffer and with a flow rate EQUAL to the flow rate the sample entered the intermixer volume In this fashion the sample is aged the same entering and exiting the intermixer volume Upon exiting the intermixer volume the sample is aged 40 ms The total ageing of the sample is 100 ms In this example the interrupted flow method is the more sample economic ageing method 13 90 SFM 3 4 User s Manual ver 1 1 14 TEST REACTIONS 14 1 Alkaline Hydrolysis of 2 4 Dinitrophenyl Acetate DNPA A complete description of the alkaline hydrolysis of 2 4 dinitrophenyl acetate DNPA can be found in Gutfreund H 1969 Methods in Enzymology 16 229 249 DNPA can be hydrolyzed by OH to 2 4 dinitrophenol DNP At 20 C the Figure 76 DNPA DNP Absorbance Spectra reaction has a second order rate constant DNPA HCI x EET m in water of56M s Conditions can NE aa easily be set to make the concentration of DNP HCI 100MM _ OH sufficiently larger than that of DNPA so that the reaction occurs under pseudo first order conditions with an apparent rate constant Kapp Of 56 s x OH NOTE The OH is the concentration of OH after mixing with DNPA The reaction can be quenched at any time by addition of excess acid and the amount of DNP produced determined by absorbance at 325nm Figure 76 shows the absorbance spectrum of DNPA and DNP under various conditions of pH It can be see that the absorbance spectr
51. e the contaminating activity but this sets a reasonable limit for the dilutions that can be obtained with the SFM 9 5 SFM 3 4 User s Manual ver 1 1 Mixing Solutions of Unequal Density and Viscosity The SFM can be used to mix solution of unequal density and viscosity in addition to simple aqueous solutions This situation is commonly found when the kinetic of protein renaturation refolding is to be measured Figure 43 shows the result of an experiment performed with cytochrome c Experimental Conditions Buffer 100 mM NaCl 20 mM MOPS pH 7 5 Syringe 1 20 ml Buffer Syringe 2 20 ml Buffer Syringe 3 20 ml 50 uM cytochrome c in 5 5 M guanidine HCl 20 mM MOPS pH 7 5 Wavelength 290 nm Cuvette FC 15 Detection method Fluorescence 320 nm cutoff filter Temperature 25 C Cytochrome c denatured in 5 5 M guanidine HCl was Figure 43 Cytochrome c Renaturation mixed with buffer in a 1 10 ratio and the intrinsic fluoresce of cytochrome c was observed The final concentrations of cytochrome c and gaunidine HCl in the cuvette were 5 uM and 550 mM respectively At this final concentration of guanidine HCl cytochrome c shows rapid renaturation as seen in NI Figure 43 300 Time ms T ec o e 2 oO E 2 I3 o o E Q o o o g o 3 LL The curve in Figure 43 was fitted with two exponentials and rate constants of 83 s and 9 s The amplitudes of the exponentials were 38 and 62 of the total t
52. e volume needed to efficiently wash or purge the flow lines and intermixer volumes using inexpensive and readily available materials In this example the needed volume for intermixer volume M2 M3 is determined The procedure can be adapted as needed to various experimental conditions and systems Experimental Conditions Syringe 1 Water Syringe 2 1 mM DNPA 1 v v DMSO 2 mM HCl Syringe 3 1 M NaOH Syringe 4 2M HCl Delay Line 1 VariableO Delay Line 2 Variable Driving Sequence Time ms 20 T 0 30 S1 Water S2 DNPA V 60 3 NaOH V 60 4 HCI V 60 Exit Valve Waste Waste Waste Collect Where V is varied from 0 in small increments until the results indicate complete washing on the intermixer volume T is varied so that the total flow rate in phase 2 is equal to that in phase 4 14 94 SFM 3 4 User s Manual ver 1 1 The results of an experiment performed with no delay lines installed is shown in Figure Figure 78 Washing Efficiency 78 It should be remembered that the purge 1 04 t inifinity volume is the volume flowing into intermixer volume M2 M3 and equal to S2 S3 2 x V It can be clearly seen that the reaction products collected after using a purge volume of zero are completely contaminated by the old reaction mixture that remained in the instrument before sample collection The results in Figure 78 indicate that a minimum purge volume of 25 30ul is necessary to wash
53. ected CAUTION The volume diverted to waste should be a minimum of 3 5x the flow line volumes section 10 3 to ensure only uncontaminated sample is collected Larger volumes may be necessary and it is recommend that test experiments be performed to optimize the volume needed to minimize sample contamination 10 5 Special Accessories Several accessories are available to expand the functions of the SFM Below are the descriptions of the accessories and their functions Custom accessories can also be designed and we invite you to contact Bio Logic or its nearest representatives to discuss your particular needs 10 5 1 SMALL DRIVE SYRINGE The SFM standard syringes 20 ml have a large driving speed range This allows each syringe to be programmed for different speeds and can be used to make mixing ratios different from 1 1 Ratios as high as 1 20 can be obtained with the standard syringes Beyond ratios of 1 20 the results can be poor due to the extremely slow movement of the syringe motor delivering the sample to be diluted For operation with dilution ratios higher than 1 20 we advise the use of a 5 ml syringe for injecting the solution to be diluted This enables the motor pushing the 5 ml syringe to run at a faster and smoother rate The specifications of the 5 ml syringe are give in Table 1 Syringes of 5 ml can be ordered from Bio Logic or its representatives 10 66 SFM 3 4 User s Manual ver 1 1 Syringe disassembly a
54. ed daily cleaning procedure to be done before shutting off the instrument 1 Remove and remaining samples or buffer from the syringes 2 Wash the syringes and flow lines 2 3 times with water This is done by filling each syringe with water to a volume at least equal to the sample volume used for experiments With the syringe valve handles set to C empty the syringes completely Since the liquid will exit via the exit valve it will wash the flow lines and exit valve as well as the syringes 3 Wash the syringes and flow lines one time with 70 10096 ethanol Use the same procedure as in step 2 12 76 SFM 3 4 User s Manual ver 1 1 4 Dry the syringes flow lines and cuvette with a single wash of air Use the same procedure as in step 2 The syringes should be emptied in reverse numerical order so that all liquid is pushed out of the syringes flow lines and exit valve Set all syringe valve handles to R and move all syringes to their lowermost positions The syringe plungers should exit the SFM so that the plunger tips are completely visible If this is done using the MPS software it will be necessary to uncheck the Up and Low Limits checkbox in the software Syringes Command Load window Figure 59 Note You may observe a few drops of liquid that fall from the syringes when the syringe plungers are completely out of the SFM This is normal as a small amount of liquid is always trapped between the plunger tip and the
55. egassed and filtered before filling the SFM The syringes of the SFM can be emptied and filled manually section 7 1 The filling of the syringes follows the steps below and shown in Figure 23 1 Attach a syringe disposable plastic syringes may be used containing sample or buffer to a syringe reservoir port on top of the SFM Figure 23 Panel 1 2 Set the syringe valve handle to R and fill the syringe manually section 7 1 while exerting a slight pressure on the reservoir syringe Figure 23 Panel 2 and Panel 3 The pressure exerted on the reservoir syringe prevents any vacuum from occurring in the reservoir syringe which could result in bubble formation It is suggested that 20ml syringes be filled using manual speed 4 in the MPS software and 5ml syringes be filled using manual speed 3 3 Eliminate any bubbles in the SFM syringe by driving the SFM syringe up and down several times while it is connected to the reservoir syringe Figure 23 Panel 4 4 Turn the syringe valve handle to C Figure 23 Panel 5 5 Empty one or two elementary movements of the syringe section 7 1 to definitively eliminate any bubbles remaining in SFM and cuvette 6 Repeat the above process for the other syringes It is recommend that the syringes be filled in reverse numerical order to best remove bubbles from the SFM and cuvette IMPORTANT ALL SYRINGES MUST BE FILLED EVEN IF THEY WILL NOT BE USED FOR AN EXPERIMENT The valve handles of the unused
56. ered from a quenched flow experiment with the SFM The sections below describe how sample collection is incorporated into a driving sequence 13 2 2 1 Total liquid collection If the total liquid collection method is chosen for sample recovery the exit valve position is set constant throughout the experiment The exit valve should 13 86 13 2 2 2 Figure 73 Quenched Flow Program SFM 3 4 User s Manual ver 1 1 be set to Waste if the free flow method is used and Collect if pipette syringe collection is used In general total liquid collection will be used only with the continuous flow ageing method section 13 2 1 1 and when large volumes gt 1 ml of sample need to be collected and contamination from previously reacted sample can be neglected In such situations all stages mix age and quench of the experiments will occur in a single phase as shown in Figure 71 Partial liquid collection Sample collection in experiments using the partial liquid collection method is divided into two parts purge and collect Purge Reactants are mixed aged and the reaction quenched but the exit valve is set to Waste and the exiting liquid is not recovered The purge serves to evacuate all old reaction mixture from the SFM wash the flow lines free of any contamination and fill the flow lines with new uncontaminated sample Collect The exit valve is set to Collect and new uncontaminated sample is pushed from the SFM into a
57. es an internal siphon like frame and allows blockage of convection created by density or temperature differences Using this mixer stopped flow traces produced by mixing high density solutions with water can now be recorded from the first millisecond to several 100 seconds Installation of the HDS mixer is identical to that of a standard Berger Ball mixer Instructions are provided in the Technical Section of this manual SFM 3 4 User s Manual ver 1 1 5 5 4 OBSERVATION HEAD WITH SEPARATE COOLING The standard observation head may be replaced with an observation head that has separate cooling Figure 14 Figure 14 Observation Head with Separate Cooling The separate cooling feature permits a temperature regulation of the observation head in addition to that of the main body of the instrument This may be used in cases where mixing of the solution produces a temperature change of the solution flowing into the cuvette and extra cooling is needed SFM 3 4 User s Manual ver 1 1 SOFTWARE CONFIGURATION The SFM is controlled by computer and it is delivered with the MPS software that is common to all Bio Logic rapid kinetics instruments This section briefly describes the configuration the software Pleas note that the procedures and examples have been generalized and configuration choices should be made based upon the equipment purchased and intended experiments The user may refer to the MP
58. eservoir syringe prevents any vacuum from occurring in the reservoir syringe which could result in bubble formation It is suggested that 20ml syringes be filled using manual speed 4 in the MPS software and 5ml syringes be filled using manual speed 3 3 Eliminate any bubbles in the SFM syringe by driving the SFM syringe up and down several times while it is connected to the reservoir syringe Figure 60 Panel 4 4 Turn the syringe valve handle to C Figure 60 Panel 5 5 Empty one or two elementary movements of the syringe section 12 1 to definitively eliminate any bubbles remaining in SFM and cuvette 6 Repeat the above process for the other syringes It is recommend that the syringes be filled in reverse numerical order to best remove bubbles from the SFM IMPORTANT ALL SYRINGES MUST BE FILLED EVEN IF THEY WILL NOT BE USED FOR AN EXPERIMENT The valve handles of the unused syringes should be turned to R after the filling process is complete The SFM is now ready for operation 12 75 SFM 3 4 User s Manual ver 1 1 Figure 60 SFM Syringe Filling Procedure Panel 1 Panel 2 Panel 3 12 4 SFM Cleaning and Storage After each day s experiments the SFM should be cleaned A thorough cleaning of the SFM will ensure that it has a long functional life and diminish any chance of sample contamination for the next user of the instrument The procedure below is the recommend
59. ette at the point of observation during the shot is the theoretical dead time the time before which observation of the mixture is impossible CUVETTE The second stage of the experiment begins when the flow is stopped At this point the mixture in the cuvette and elsewhere becomes stationary but continues to age Observation of the mixture in the cuvette after the stop therefore represents a timecourse of the reaction from the dead time onward Figure 33 shows a schematic of a simple stopped flow experiment In the experiment reagents A and B sce isnot STAGE 2 REACTION are pushed into a mixer where they react to form product C Reagent A has a strong absorbance while reagent B and product C do not Therefore as the reaction proceeds the absorbance of a mixture of A and B should decrease as A is diminished Figure 34 shows a cartoon of the experiment over time Note the two stages of the experiment as described above Figure 34 Stopped Flow Experiment Timecourse is e Nn a IMPORTANT In every stopped flow experiment enough liquid must be pushed to wash the flow path and cuvette and achieve a steady state condition If this is not done all sample is SFM 3 4 User s Manual ver 1 1 contaminated and the resulting signal trace does not represent the true timecourse of the reaction 8 2 Design and Execution of Stopped Flow Experiments Experiments are designed using the MPS software to create a drivi
60. gly recommended that driving sequences using acceleration phases be tested on inexpensive solutions before using precious samples Ideally the driving sequences should be tested using solutions as close as possible to those in the real experiment Automatic Mode In this mode the MPS software automatically calculates an acceleration phase for the driving sequence when a syringe flow rate exceeds the nominal maximum flow rate Table 1 and Table 8 The acceleration phase has a duration of Sms and accelerates the syringe to 83 of it s nominal maximum flow rate An automatic acceleration phase example is shown in Figure 30 The MPS software cannot calculate an acceleration phase for phases that have a duration of 5ms or less The MPS program will request than a longer phase duration be used Table 8 Acceleration Phases AUTOMATIC ACCELERATION PHASES Syringe Size Nominal Maximum Flow Rate Acceleration Phase 30 ml 8 ml s Time 5ms Volume 34ul 20 ml 6 ml s Time 5ms Volume 25ul 5 ml 1 32 ml s Time 5ms Volume 5 5pl Figure 67 MPS Software Automatic Accretion Phase Example In the driving sequence below all syringes are 20 ml PHASE 1 PHASE 2 TOTAL VOLUME 50ms Syringe 1 Syringe 2 400ul 8 ml s Syringe 3 300yl 6 ml s Syringe 2 exceeds the nominal flow rate maximum of 6 ml s The MPS software calculates an acceleration phase as show below PHASE
61. he Config Limits window available under the Config menu Figure 20 The Config Limits window contains several sections for configuration of the hard stop valve lead time acceleration phases mode and overheating protection Each section and options are described below SFM 3 4 User s Manual ver 1 1 Figure 20 MPS Software System Limits Configuration ne Windows Valve Lead ms 0 Default Device Acceleration Phases Syinges Eg Automatic Cuvette Delay Line C Manual OK Serial Port Limits X Over heating Protection Hard Stop Valve Lead Time This section of the windows allows one to enter the number of milliseconds before the flow stops that the hard stop starts closing The default value is zero The lead time may be adjusted from 0 5 ms to fine tune the quality of the stop Acceleration Phases Mode This sections of the windows allows selection of the acceleration phases mode Two modes are possible Automatic and manual These modes are described in detail in 7 7 Overheating Protection Checkbox This checkbox allows the selection or deselecting of over heating protection The MPS that drives the SFM was designed to do fast experiments To do this the motors are powered up and ready to go during the entire driving sequence this is also necessary to keep syringes not used from moving backwards under the force of the back pressure generated by the other syringes It requires a lot of energy to do t
62. he current syringe current phase and driving sequence is updated displayed below and to the right of the grid Figure 64 This information indicates 1 Current phase number and total number phases in the driving sequence 2 Volume delivered by the current syringe during the current phase or current phase total volume if an entire phase is selected 3 Flow rate of the current syringe during the current phase or current phase total flow rate if an entire phase is selected 4 Total volume delivered by each syringe during the driving sequence 12 78 SFM 3 4 User s Manual ver 1 1 Figure 64 MPS Software Driving Sequence Information Phase 2 Phase 3 Phase 5 Leder otal timc ma Yolumes pl Syr 1 ul 0 Syr 2 nl 100 200 Syr 3 ul 100 200 Syr 4 pl 100 200 Waste Waste Synchro 1 Phase 3 5 Total Flow Rate 15 0 ml s 12 7 An indication of the Ageing Times for a driving sequence are also displayed in the Quenched Flow Program window Figure 61 and Figure 65 The ageing times are calculated for the current phase selected based upon the syringes flow rates delay lines installed and intermixer volumes Figure 47 and Figure 48 The ageing times are calculated according to the equations shown in Figure 65 Figure 65 MPS Software Ageing Times SFM 3 Delay Line Intermixer Volume M1 M2 Ageing Time Ageing Time Total Flow RateS1 S2 DL1 51ms
63. heir own microprocessor The sequence of impulses to be sent to the stepping motors are stored in the memory of each motor board One main microprocessor board synchronizes all the power supplies and performs the communication with the microcomputer via a serial interface 2 1 3 MICROCOMPUTER COMMANDS The SFM module is controlled by the MPS software program running on a PC or compatible microcomputer under Windows 3 1x or Windows 95 Various menus and windows permit the user to e know the volume of the solution contained in each syringe e perform manual or automatic movement of the syringes e create a sequence of reaction with complete control of time and volume delivered by the syringes e save or recall the sequences e program the synchronization pulse used to trigger the acquisition system 2 9 SFM 3 4 User s Manual ver 1 1 load the data acquisition software Bio Kine More detail on the functions and use of the MPS program can be found in the MPS Software User s Manual 2 2 Modes of Operation The SFM can be used in two main operating modes that are briefly described below More detail on the two modes of operation can be found in other sections of this manual 2 2 1 2 2 2 STOPPED FLOW SF MODE commercial reference SFM X S In this configuration the SFM is a full stopped flow instrument with an optical observation chamber This configuration is described in Figure 1 In this configuration the SFM has uni
64. his and the motor boards inside the MPS generate heat The instrument was not designed specifically for driving sequences lasting more than a few seconds Because of this sequences longer than a few seconds result in the motors getting very hot and can possibly burn them out To avoid burning out the motors the solution is to allow the motor boards to cool before doing a subsequent shot Overheating protection forces the MPS software to wait 2x the entire length of the driving sequence in between shots ensuring that the motor boards cool sufficiently and avoid burnout The box is checked by default and it is strongly recommended that overheating protection always be left enabled SFM 3 4 User s Manual ver 1 1 7 INSTRUMENT OPERATION 7 1 Manual Syringe Control The syringes of the SFM can be controlled either manual or automatically Automatic control of the syringes is strictly used only for experiments The manual control of the syringes is used for initialization filling and emptying the syringes The manual movement of the syringes can either be made directly from the MPS or though the MPS software Both methods are described in the following sections 7 1 1 MPS Syringe control directly from the MPS is made through the use of the buttons on front Figure 21 MPS Syringe Controls panel of the MPS Figure 21 and Figure 2 The and buttons are used to select the syringe to be moved The up and down buttons are use
65. hors used heavy water in the light buffer for it to match the density of the mixture in the cuvette However the best solution is to use the high density HDS mixer developed by Bio Logic This mixer is described in detail in section 5 5 3 SFM 3 4 User s Manual ver 1 1 SECTION III QUENCHED FLOW MODE SFM X Q SFM 3 4 User s Manual ver 1 1 TABLE OF CONTENTS SECTION III 10 INSTALLATION OF THE QUENCHED FLOW COMPONENTS 10 1 Introduction 10 2 Installation of the Mixer Blocks and Delay Lines 10 3 Flow Line and Intermixer Volumes 10 4 Sample Collection Methods 10 4 4 TOTAL LIQUID COLLECTION 10 60 10 60 10 60 10 63 10 65 10 65 10 4 PARTIAL LIQUID COLLECTION 10 66 10 5 Special Accessories 10 5 1 SMALL DRIVE SYRINGE 10 66 10 66 10 5 2 LARGE DRIVE SYRINGE 10 67 10 5 3 HIGH DENSITY MIXER 10 67 10 5 4 DIRECT EXIT ATTACHMENT 10 68 11 SOFTWARE CONFIGURATION 11 1 Device Configuration 11 2 Syringe Configuration 11 3 Delay Line Configuration 11 4 Serial Port Configuration 11 5 System Limits Configuration 12 INSTRUMENT OPERATION 12 1 Manual Syringe Control 12 1 1 MPS 11 69 11 69 11 69 11 70 11 70 11 71 12 73 12 73 12 73 12 1 2 SOFTWARE 12 73 12 2 Syringe Initialization 12 3 Filling the Syringes 12 4 SFM Cleaning and Storage 12 5 Long term Storage of the SFM 12 6 Creating a Driving Sequence 12 7 Incubation Period 12 8 Accelerati
66. hro out 1 and 2 are available Saving or Loading Driving Sequences Driving sequence can be saved as files on the hard drive of the microcomputer In this way a series of driving Figure 31 MPS Software sequences for different experiments can be prepared in Load Save Driving Sequences advance and subsequently loaded as needed Ff SFM 4 S amp SFM 400 5 Files are saved or loaded using the Save or Load ie Edit Syringes Cor functions under the Sequence Files menu Figure 31 New Files are saved using filenames up to 8 characters in length Close Save Save s More information about saving or loading driving sequences can be found in the MPS Software User s Manual chapter 6 Print 1 C MPSSSAMPLES4 SF4 Exit SFM 3 4 User s Manual ver 1 1 7 10 Running in Automatic Mode 7 11 Once a driving sequence has been entered or loaded it is transferred to Figure 32 MPS Software Shots Frame and the MPS by pushing the Single or Program Run Window Multiple button in the Shots frame of Shoe the Stopped Flow Program window Figure 24 and Figure 32 The MPS is now in automatic mode and the Program Run window will be displayed Figure 32 the number of shots possible based the current volumes in the SFM syringes READY It also indicates whether the SFM is running a driving sequence or ready for Number of shots 16 the next shot A driving sequence is Single
67. igh Density HDS Mixer Cuvette Dead Volume ul Dead Time ms Dead Volume ul Dead Time ms FC 08 6 30 4 3 0 FC 15 k 51 3 FC 20 74 1 TC 50 10 36 8 TC 50 15 54 6 TC 100 10T i 45 0 TC 100 10F 45 0 TC 100 15T i 61 5 TC 100 15F 1 Dead volumes measured from mixing point to the center of the observation area 2 Dead times calculated at 10 ml s flow rate Dead time is inversely proportional to flow rate SFM 3 4 User s Manual ver 1 1 5 2 Installation of the Mixer Blocks and Delay Lines In stopped flow mode the syringes of the SFM can be used to perform many types of mixing experiments It is difficult to list all the possibilities here Some are described below 1 Load several reagents and mix them in different shots with the contents of the last syringe 2 Use syringes loaded with reagents and buffer to vary the concentration of one or two reagents and mix the result with the contents of the last syringe 3 Perform sequential mixing and delays between up to 3 reagents before they are mixed with the content of the last syringe The observation head is installed on the SFM body differently depending on how many syringes are present and the type of experiment performed SFM 3 The observation head and delay line are installed as shown in Figure 6 The smallest delay line comes standard and installed with the instrument SFM 4 The observation head and delay lines are installed as
68. igurations SFM 3 The mechanical sub system consists of three machined syringes one valve block with 3x3 way valves with the possibility to include one or two mixers and one ageing loop SFM 4 The mechanical sub system consists of four machined syringes one valve block with 4x 3 way valves with the possibility to include one to three mixers and one to two ageing loops All SFM syringes valves delay lines and cuvettes are enclosed in a water jacket to allow temperature regulation of the reactants containers The syringe plungers of the SFM are driven by stepping motors via ball screws 2 1 1 THE MECHANICAL DESIGN The mechanical part of the SFM module is carefully constructed The parts in contact with the sample and the buffers are all machined out of materials selected for their inert characteristics stainless steel Teflon Kel F VITON EPDM PEEK and quartz Millisecond dead time can be achieved with the SFM due to the combined effects of high performance control of the stepping motors and low dead volumes Ageing lines of various volumes can be used in the SFM The ageing line s of the instrument can be replaced and secured in a few minutes 2 1 2 INTELLIGENT POWER SUPPLY The high performance of the SFM and the high speed of the stepping motors can be achieved only because of the quality of its power supply The MPS unit contains independent constant current power supplies for each syringe all driven independently by t
69. ilities here A few common types are described below 1 Load several reagents mix them and quench the reaction with the contents of the last syringe 2 Use syringes loaded with reagents and buffer to vary the concentration of one or two reagents mix and then quench the resulting mixing with the contents of the last syringe 3 Perform sequential mixing and delays between up to 3 reagents before they are mixed with the content of the last syringe In all experiments the final sample is recovered for analysis standard with a quench exit valve Figure 44 to simplify sample collection The exit valve and delay line s are installed on the SFM body differently depending on how many syringes are present and the type of experiment performed SFM 3 SFM 4 All SFM Q instruments are shipped Figure 44 Exit Valve Collect The exit valve and delay line are installed as shown in Figure 45 The exit valve and delay line s are installed as shown in Figure 46 The exit may be installed using the mixing blocks labeled 0 MIX 0 0 MIX DL DL MIX 0 DL MIX DL or no mixing block The installation of the different mixing blocks is described in Table 7 Table 7 SFM 4 Exit Valve Installation MIXING BLOCK COMMENTS 0 MIX 0 Installed with no additional delay lines 0 MIX DL Installed with one delay line between the mixer block and the observation head DL MIX 0 Installed with one delay line betwee
70. ines is an easy operation which usually takes only a few minutes Delay lines of nominal volumes up to 1000 yl are available Standard equipment of an SFM X S does not include ageing lines SFM X Q and QS versions are delivered with two sets of ageing lines up to 200 ul Ageing lines of 500 ul and 1000 ul can be SFM 3 4 User s Manual ver 1 1 obtained as additional accessories To evaluate the ageing time of a reaction the entire volume between two mixers has to be taken into account This volume includes the delay line plus the dead volumes the volumes on the both sides of the delay line and the mixers The complete description of the volumes are described in section 5 3 SFM 3 4 User s Manual ver 1 1 GENERAL INSTRUCTIONS FOR INSTALLATION This section of the manual contains information on the installation and preliminary operation of all SFM instruments It is recommended that the contents of this section be read and understood before any attempt is made to operate the instrument In case of difficulties please contact Bio Logic or its nearest representative 3 1 Operating Features The general features of the MPS 52 are shown below in Figure 2 and described in Table 2 Figure 2 MPS 52 Panels MPS 52 Microprocessor unit bio logic struments Science Inst SFM 3 4 User s Manual ver 1 1 Table 2 MPS 52 Panel Descriptions NAME FUNCTION
71. lection or for quenching Bs with an external solution If KEN KL U Connect to the latter method is used the ab L Tube tube acts as an additional i delay line whose volume can be adjusted by the user Figure 49 External Tube Collection CAUTION If the volume collected is not substantially larger than the flow line and tube volume substantial contamination of samples by old reacted solution may occur It is recommended to collect sample volumes a minimum of 3 5x flow line tube volumes section 10 3 In addition it is recommended to wash old solution out of the SFM and tube with buffer between sample collections and perform test experiments to verify the level of sample contamination is minimal WARNING The inner diameter of the tube connected to the waste outlet should always be larger than that of the hole in the waste outlet If this is not respected back pressure can build up inside the SFM during a shot and cause the motors to stall Pipette syringe collection A pipette or syringe is connected to the collect outlet of the exit valve to recover all the liquid exiting the SFM Figure 50 This method allows complete collection of a sample and isolates the collected sample from the environment It is recommended that a pipette be used for collection rather than a syringe Undue back pressure from a collection syringe plunger can force liquid to exit through the waste outlet instead of being collected CAUTION
72. low Rate Acceleration Phase 30 ml 8 ml s Time 5ms Volume 34ul 20 ml 6 ml s Time 5ms Volume 25ul 5 ml 1 32 ml s Time 5ms Volume 5 5yl Figure 30 MPS Software Automatic Acceleration Phase Example In the driving sequence below all syringes are 20 ml PHASE 1 PHASE 2 TOTAL VOLUME Syringe 1 50ms Syringe 2 400ul 8 ml s Syringe 3 300yl 6 ml s Syringe 2 exceeds the nominal flow rate maximum of 6 ml s The MPS software calculates an acceleration phase as show below Syringe 1 PHASE 1 PHASE 2 PHASE 3 45ms Syringe 2 25yl 5 ml s 360yl 8 ml s Syringe 3 30ul 6 m s 270yl 6 ml s 7 43 7 8 7 9 SFM 3 4 User s Manual ver 1 1 IMPORTANT Because flow rate is reduced during acceleration phase and that the total time does not change the total volume of the accelerated syringe will be ess than the total volume of the original driving sequence Figure 30 The MPS software does NOT update the driving sequence on the screen to reflect an acceleration phase It is therefore recommend that the automatic mode of accelerations phases be used only when the volume delivered is not a critical factor in the experiments Manual Mode In this mode the MPS software will not calculate an acceleration phase It is left to the user to manually design a driving sequence including any necessary acceleration phases For
73. lve starts to switch from waste to collect mode The default value is zero The lead time may be adjusted from 0 5 ms to fine tune the quality of the transition Acceleration Phases Mode This sections of the windows allows selection of the acceleration phases mode Two modes are possible Automatic and manual These modes are described in detail in section 12 8 Overheating Protection Checkbox This checkbox allows the selection or deselecting of over heating protection The MPS that drives the SFM was designed to do fast experiments To do this the motors are powered up and ready to go during the entire driving sequence this is also necessary to keep syringes not used from moving backwards under the force of the back pressure generated by 11 71 SFM 3 4 User s Manual ver 1 1 the other syringes It requires a lot of energy to do this and the motor boards inside the MPS generate heat The instrument was not designed specifically for driving sequences lasting more than a few seconds Because of this sequences longer than a few seconds result in the motors getting very hot and can possibly burn them out To avoid burning out the motors the solution is to allow the motor boards to cool before doing a subsequent shot Overheating protection forces the MPS software to wait 2x the entire length of the driving sequence in between shots ensuring that the motor boards cool sufficiently and avoid burnout The box is checked by default and it is
74. ly an external flow line can be connected for direct injection of the mixture into a quenching solution Flash quenching with a photoreactive reagent is also a mode that can be easily implemented with the SFM Many other configurations are possible and you are invited to inquire about their feasibility The commercial reference SFM X QS has all the components for the two applications An SFM X S or a SFM X Q can easily be updated to SFM X IQS SFM 3 4 User s Manual ver 1 1 Figure 1 SFM Modes of Operation STOPPED FLOW MODES Panel 1 SFM 3 Panel 2 SFM 4 E p Detection p Detection v Cuvette p QUENCHED FLOW MODES Panel 3 SFM 3 with diverting valve Panel 4 SFM 4 with diverting valve Collect h Mixer ja Valve Valve Delay line V Waste Waste motor motor SFM 3 4 User s Manual ver 1 1 2 3 Specifications The general specifications of each SFM are listed in Table 1 below Table 1 SFM Specifications GENERAL SFM SPECIFICATIONS Number of syringes 3 SFM 3 or 4 SFM 4 6400 steps per motor turn Programmable trigger for data acquisition and synchronization of accessories Filling range of the drive syringes 500ul to syringe limit 30ml syringe 28 pl Minimum injection volume per syringe 20ml syringe 20 yl standard syringe 5mlsyringe 10 pl 30 ml syringe 0 062 8 ml s syringe 10 ml s with accele
75. mes variable concentration variable mixing ratios and other complicated actions with only a few keystrokes The reproducibility and regularity of the linear translation of the syringes and the absence of pressure artifact allow optical recording during the drive sequence This feature greatly facilitates the determination of the initial phase of the reaction being monitored and makes the equipment suitable for very accurate continuous flow experiments Description of the Mechanical Design The observation chamber and the syringes of the SFM are mounted vertically This allows easy purging of bubbles which are evacuated during refilling by a few up and down movements of the drive syringe The syringes valves and observation chamber are very carefully thermoregulated This thermoregulation prevents the occurrence of temperature artifacts on a very wide temperature range and permits rapid kinetic studies even at temperatures below 0 C The Delay Lines The SFM instrument can be used with delay lines permitting various reaction delays to be obtained between the two SFM 3 or three SFM 4 mixers The delay lines are machined into PEEK Kel F or stainless steel spacers depending on the instrument These spacers can be inserted between the mixers to adjust the volume and ageing time of a reaction between the mixers See sections 5 2 and 5 3 for full description of delay line installation and calculation of volumes Replacement of the delay l
76. most of the old reaction from the intermixer volume 50 100 Volume of Purge ul 14 4 Recovery of Uncontaminated Material in Intermixer Volume In the interrupted mode the reaction mixture is transiently stored in the intermixer volume During this incubation period unwanted mixing occurs at both ends of the intermixer volume so that only a fraction of the mixture contained therein can be recovered The experiment described below is intended to give an estimate of the uncontaminated fraction that can be recovered The procedure provided in the experiment can easily be adapted to various incubation times and experimental conditions Experimental Conditions Syringe 1 Water Syringe 2 1 mM DNPA 1 v v DMSO 2 mM HCl Syringe 3 1 M NaOH Syringe 4 2M HCl Delay Line 1 17 ul Delay Line2 190 ul Driving Sequence Time ms S1 Water 7 S2 DNPA 30 3 NaOH 30 4 HCl 30 Exit Valve Collect Where V is varied from 0 in small increments until 2x the intermixer volume T is varied so that the total flow rate in Phase 6 is equal to that in Phase 7 14 95 SFM 3 4 User s Manual ver 1 1 This experiment is designed to test intermixer volume M2 M3 In Phase 2 DNPA and NaOH are pushed through the delay line and then to waste The second phase is used to wash the last mixer with HCl The reaction mixture is then allowed to age for several seconds in the delay line Phase 4 Phase 6 co
77. n the SFM body and the mixer block DL MIX DL Installed with delay lines on both sides of the mixer block NONE Only a delay line is installed between the SFM body and the exit valve and the SFM 4 functions as an SFM 3 Figure 45 Syringe 3 is blocked by the delay line and only syringes 1 2 and 4 are useable In this case syringe 3 does not need to be filled 10 60 SFM 3 4 User s Manual ver 1 1 Figure 45 SFM 3 Installation of Exit Valve and Delay Lines EXIT VALVE SECOND MIXER M2 HEAD SPACEF 10 61 SFM 3 4 User s Manual ver 1 1 Figure 46 SFM 4 Installation of Exit Valve Mixer Blocks and Delay Lines EXIT VALVE B Z THIRD MIXER M3 b HEAD SPACERS DELAY LINE TWO DL2 MIXER BLOCK SECOND MIXER M2 MIXER BLOCK p SECONDMIXER gt M2 D e M DELAY LINE ONE DL1 FIRST MIXER 1 10 62 SFM 3 4 User s Manual ver 1 1 10 3 Flow Line and Intermixer Volumes Figure 47 SFM 3 and Figure 48 SFM 4 below indicate the volumes of SFM flow lines and delay lines The amount of time a sample ages between two mixers is given by Ageing time between two mixers Intermixer volume Flow rate through intermixer volume It should be noted that the volumes give in the table are the mechanical volumes The hydrodynamical volumes may vary slightly around these values For precise measurement of ageing times it is recommended that the intermi
78. nd reassembly is discussed in the Technical Instructions section of this manual We recommend that the user be familiar with this section before attempting syringe disassembly and assembly 10 5 2LARGE DRIVE SYRINGE As described in the previous section the standard 20 ml have a large driving speed range but are not ideal for all circumstances At times one may need a low ageing time or high flow rate from one or two syringes that is beyond the flow limits of the standard syringes with or without acceleration For these situations a large 30 ml syringe may be used The specifications of the 30 ml syringe are given in Table 1 Syringes of 30 ml can be ordered from Bio Logic or its representatives Syringe disassembly and reassembly is discussed in the Technical Instructions section of this manual We recommend that the user be familiar with this section before attempting syringe disassembly and assembly 10 5 3 HIGH DENSITY MIXER Mixing solutions of different densities offers a formidable challenge for Figure 51 HDS Mixer stopped flow instruments In typical protein folding unfolding experiments heavy solutions of urea or guanidine chloride are mixed with pure aqueous buffers The result is an unavoidable convection 10 to 30 seconds after mixing This convection leads to sample contamination for samples stored in delay lines definitively ruining the kinetic experiment The SFM module can be equipped with a specially designed mixer
79. ng sequence This is accomplished by filling in elements of program grid as described in section 7 6 Every experiment driving sequence will contain a phase that triggers the data acquisition and a phase for the actual shot A sample driving sequence is shown in Figure 35 The sample driving sequence shown utilizes only two phases The first phase is used solely to trigger data acquisition at the end of the phase so that the signal during the shot may be observed The second phase defines the shot Figure 35 Example Driving Sequence Stopped Flow Program EXAMPLE SF4 Ea Syringes Total Volumes pl Phase 1 5 Total Volume 0 pl Total Flow Rate 0 0 mls Syringes contents Shots Drive Sequence i Time Single 11 20 2 9 ms Multiple Ageing Time DL1 Oms DL2 Oms Comments Close A single phase may be used to both trigger the data acquisition and define the shot In this instance the data acquisition will start after the shot has finished so the signal during the shot will not be observed It is recommended that data acquisition be triggered before the shot so that the achievement of the steady state condition may be visually verified 8 3 General Advice for Stopped Flow Experiments 8 3 1 ACHIEVEMENT OF FASTEST DEAD TIMES The dead time of a stopped flow experiment is defined as the time before which observation of the mixture is impossible The dead time depends on a number of factors
80. ny reason a syringe is blocked during a run the pulses will not correspond to the true volume delivered and the value displayed may become erroneous e g in the case of incorrect positioning of a valve In this case it is advisable to reinitialize the syringes If by accident a syringe is returned to its uppermost position the syringe volume counter will again show BEBE and the syringe must be reinitialized To avoid such accidents the Up and Low Limits checkbox may be checked When this box is checked the MPS software will not allow the syringes to be driven beyond their upper and lower limits This also avoids accidentally pulling the syringe plunger completely from the syringe and spilling solution onto the SFM 7 3 SFM 3 4 User s Manual ver 1 1 WARNING The Up and Low Limits only applies to control of the syringe from within the MPS software These limits can be bypassed by manual control of the SFM directly from the MPS Further information about the initialization of syringes can be found in the MPS Software User s Manual chapter 5 Filling the Syringes WARNING Utmost care should be exercised during this operation Normal operation of the system requires that no bubbles are present in the injection syringes Should this occur the buffer flow through the observation chamber will not be correctly controlled by the plunger movement and artifacts may be observed For best results it is recommended that all solutions be d
81. o Mixing The possibility to obtain variable mixing ratios by a simple programming of the instrument i e without changing the syringes is one of the major advantages of the SFM instruments The microprocessor control of the stepping motors gives 6400 steps per revolution of the motor and results in a smooth and quasi continuous movement of the syringe over a very large range of flow rates A few example experiments using the SFM to carry out variable ratio mixing are described below 9 4 1 REDUCTION OF DCIP BY ASCORBIC ACID Experimental Conditions Syringe 1 20 ml 20 mM Ascorbic Acid pH 9 Syringe 2 20 ml Buffer Syringe 4 20 ml 100 uM DCIP Wavelength 524 nm Cuvette TC 50 10 Detection method Transmittance Acquisition was started at the end of the stop A series of experiments were performed in which the concentration of ascorbic acid was varied from 0 8 mM to 10 mM This was accomplished by programming the SFM to deliver a constant volume of DCIP S4 and varying volumes of ascorbic acid S1 and buffer S2 The total volume of each shot was kept constant as was the volume of S1 S2 The total flow rate was also kept constant in all experiments Figure 38 shows the results of the experiments and the dilution factor of ascorbic acid is noted next to each curve Figure 38 DCIP Variable Ratio Mixing Experiments TO apes haeret E B w g S S S B E pz 5 E Using the v
82. on Phases 12 9 Programmable Synchronization Pulses Triggers 12 10 Saving or Loading Driving Sequences 12 11 Running in Automatic Mode 13 A SHORT QUENCHED FLOW PRIMER 13 1 General Principle of Quenched Flow Experiments 13 2 Design and Execution of Quenched Flow Experiments 9 58 12 74 12 75 12 76 12 77 12 77 12 79 12 79 12 81 12 81 12 81 13 83 13 83 13 84 SFM 3 4 User s Manual ver 1 1 13 23 40 AGEING METHODS 13 84 13 2 COLLECTION METHODS 13 86 13 3 General Advice for Quenched Flow Experiments 13 3 1 SYSTEM SPECIFICATIONS 13 88 13 88 13 3 2 TEST EXPERIMENTS 13 88 13 3 3 MINIMIZING SAMPLE CONTAMINATION 13 89 13 3 4 MINIMIZING REACTANT CONSUMPTION 13 89 14 TEST REACTIONS 14 1 Alkaline Hydrolysis of 2 4 Dinitrophenyl Acetate DNPA 14 2 Calculation of Hydrodynamic Volumes from Kinetic Data 14 3 Washing Efficiency 14 4 Recovery of Uncontaminated Material in Intermixer Volume 14 91 14 91 14 93 14 94 14 95 SFM 3 4 User s Manual ver 1 1 10 INSTALLATION OF THE QUENCHED FLOW COMPONENTS 10 1 Introduction This section of the manual contains installation instructions for the quenched flow components of the SFM Please read Section I of this manual before proceeding 10 2 Installation of the Mixer Blocks and Delay Lines In quenched flow mode the syringes of the SFM can be used to perform many types of mixing experiments It is difficult to list all the possib
83. only some of which the researcher can control Ideally the dead time depends only on the flow rate of the mixture exiting last mixer and the volume of the between the last mixer and the cuvette Thus as the flow rate is increased the dead time will decrease In addition as the volume between the last mixer and the cuvette volume decreases so does the dead time Nevertheless an effective stopped flow experiment depends on a number of other inter related factors such as adequate signal complete washing of the cuvette prevention of cavitation and prudent use of valuable reagents The relationships between these factors requires careful consideration and experimentation Compromises are often necessary to 8 47 SFM 3 4 User s Manual ver 1 1 achieve successful stopped flow experiments Some of the most common actions that can be take to achieve fastest dead times and their consequences are shown in Table 5 Table 5 Common Actions to Achieve Fastest Dead Times Lower Dead Times Increase Flow Rate stalled motors cavitation overuse of reagent inadequate washing Decrease Cuvette volume loss of signal 8 3 2 8 3 3 8 3 4 WASHING As mentioned in section 8 1 it is necessary to completely wash the flow path between the last mixer and cuvette and the cuvette itself during the shot This ensures that the signal observed after the shot is only of the recently mixed samples
84. ower supply of the SFM No overpressure is developed in the observation cuvette because synchronization of the hard stop with the motor halt The result is elimination of the stop and overpressure artifact giving high quality stopped flow traces with the lowest dead times There are three operation modes of the hard stop that can be chosen in MPS software section 6 2 The modes of operation are Figure 11 Hard Stop Installation To MPS Hard Stop 2 Cuvette Holder SFM 3 4 User s Manual ver 1 1 1 Automatic mode between two shots the hard stop is always closed During a run the hard stop opens at the beginning of the flow and then closes at a designated number of milliseconds before the flow stops 2 Manual mode the hard stop is programmed to open and close by the user 3 None The valve is always open The installation of the hard stop on the observation head is shown in Figure 11 5 4 3 EXIT IN A SYRINGE In this method a syringe is inserted into the SFM observation head outlet Figure 12 Figure 12 Stop Syringe Installation ej z gt The linear momentum of the liquid flowing out of the cuvette will be dissipated in the liquid contained in the syringe This procedure gives clean stop signal but requires that the user empty the stop syringe from time to time Syringe lt 4 IL For the best results it is recommended to use high quality 10 to 20 ml glass syringes with
85. ple collection method The sections below describe the different methods to age and collect the sample with the SFM IMPORTANT The experiment stages introduced in the previous section are simply a conceptual image It is essential to separate the idea of experiment stages from that of driving sequence phases Although a quenched flow experiment may have many stages it is possible that multiple stages occur at the same time or that an entire experiment can be executed in a single driving sequence phase 13 2 1 AGEING METHODS Samples can be aged with the SFM using two different methods the continuous flow method or the interrupted flow method 13 2 1 1 Continuous flow method In the continuous flow method sample flow is continuous from the start of the reaction through sample collection The sample age is dependent only on the intermixer volume and the total flow rate through the intermixer volume In this case tage Intermixer volume Flow rate through intermixer volume The sample age can then be adjusted by changing the intermixer volume or the flow rate through the intermixer volume The intermixer volume is modified by introducing delay lines of different volumes section 10 2 The flow rate through the intermixer volume is modified by changing the flow rate of the syringes in the MPS software section 12 6 The use of stepping motors in the SFM allows a large range of syringe flow rates to be programmed and many tag values a
86. que features for a stopped flow instrument SFM 3 S Two or three solutions can be mixed and injected into the cuvette and a single delay line can be installed Figure 1 panel 1 SFM 4 S Two to four solutions can be mixed and injected into the cuvette and one to two delay lines can be installed Figure 1 panel 2 The speed capability of the SFM instrument 3 or 4 syringes with all its syringes running gives a dead time below 1 ms in the observation cuvette QUENCHED FLOW QF MODE commercial reference SFM X Q In this configuration the SFM functions as a complete quench flow instrument This configuration allows for various modes of operation as described in the Figure 1 SFM 3 Q It can be used as a three syringe quench flow instrument with one delay line two mixers and a diverting valve for waste and collect Figure 1 panel 3 Alternatively an external flow line can be connected for direct injection of the mixture into a quenching solution This mode may be used with or without an additional delay line It can also be used in a simple 3 syringe mode and direct collection of the sample in a pipette or syringe In another mode the mixture can be injected onto a filter at the same time as it is mixed with a flow of washing buffer SFM 4 Q It can be used as a quench flow instrument with 2 to three syringes up to one delay line either single or double mixing and a diverting valve for waste and collect panel 4 Alternative
87. r with this section before attempting syringe disassembly and assembly 5 5 2 5 5 3 SFM 3 4 User s Manual ver 1 1 LARGE DRIVE SYRINGE As described in the previous section the standard 20 ml have a large driving speed range but are not ideal for all circumstances At times one may need a low dead time or high flow rate from one or two syringes that is beyond the limits of the standard syringes with or without acceleration For these situations a large 30 ml syringe may be used The specifications of the 30 ml syringe are given in Table 1 Syringes of 30 ml can be ordered from Bio Logic or its representatives Syringe disassembly and reassembly is discussed in the Technical Instructions section of this manual We recommend that the user be familiar with this section before attempting syringe disassembly and assembly HIGH DENSITY MIXER Mixing solutions of different densities offers a formidable challenge for Figure 13 HDS Mixer stopped flow instruments In typical Out protein folding unfolding experiments heavy solutions of urea or guanidine chloride are mixed with pure aqueous SS buffers just before the cuvette The result is an unavoidable convection reaching the observation cuvette 10 to 30 seconds after mixing This convection creates a massive artifact definitively ruining the kinetics being recorded The SFM module can be equipped with a specially designed mixer model HDS Figure 13 that includ
88. ransition respectively The fit is shown as a dotted line under the experimental curve CAUTION The mixing of solutions of unequal density and viscosity can result in convection artifacts Convection artifacts are due to the slow rise of light buffer from the last mixer and subsequent entry into the observation chamber after mixing The entry of the light buffer is detected by a sudden and reproducible change in absorbance or fluorescence 10 to 100 seconds or more after the mixing The existence of this artifact and the time at which it is observed are dependent on the relative densities and viscosities of the mixture and of the light buffer In the above example with cytochrome c a large dilution ratio was used so that the final mixture has a density not too different from that of the NaCl buffer As a consequence no convection artifact was visible when data acquisition was prolonged for more than 100 seconds On the other hand if a 1 1 mixing was used the high concentration of guanidine in the cuvette 2 75 M would have resulted in the formation of a large gradient of density at the last mixer Under these conditions if no precautions are taken rapid rise of NaCl buffer in the observation cuvette can be observed about 20 s after mixing SFM 3 4 User s Manual ver 1 1 A method to completely eliminate the convection artifact has been proposed by Blond Elguindi et al 1988 in their work referenced at the end of this manual These aut
89. ration Flow fate range 20 ml syringe 0 045 6 ml s syringe 8 ml s with acceleration 5mlsyringe 0 010 1 32 ml s syringe 1 77 ml s with acceleration Minimum flow rate for efficient mixing 1 ml s total flow rate through each mixer Variable ratio range Continuously variable from 1 1 to 1 20 with single dilution 21 100 with double dilution Minimal dead time SF mode 0 98 ms at 16 ml s total flow rate with FC 08 cuvette Minimal ageing time QF mode 1 63 ms at 16mL s total flow rate with minimal volume delay line PEEK stainless steel or Kel F on special order Syringe volume 20ml standard syringes 5 and 30mL syringes are also available 30 ml syringe 0 19 ul Volume per p step 20 ml syringe 0 14 pl 5mlsyringe 0 03 pl Duration of flow adjustable from 1 ms to 9999 ms per phase 300 Watt 110 220 Volt 50 60 Hz Total weight 12 kg 2 4 2 5 2 6 SFM 3 4 User s Manual ver 1 1 Principle of Operation The syringes of the SFM are driven by independent stepping motors The stepping motors are of hybrid technology with 200 steps per revolution and 4 phases each phase being powered by a constant current supply 2 9 A per phase The power supply of each motor is microprocessor controlled A complex impulse sequence enables micro positioning of the motor s rotor with an accuracy equivalent to 1 32 of the mechanical step This gives an effective number of steps of 6400 per revolution or a volume quantific
90. rdinary aqueous solutions the SFM motors can drive the syringes up to a flow rate of 6 ml s for a 20 ml syringe without acceleration phase Table 1 It is possible to push solutions at faster flow rates provided an acceleration phase is added to the driving sequence As noted in section 6 7 the MPS Software can be configured for two different acceleration phases modes automatic and manual SFM 3 4 User s Manual ver 1 1 CAUTION Because a motor could stall even with the use of acceleration phases it is strongly recommended that driving sequences using acceleration phases be tested on inexpensive solutions before using precious samples Ideally the driving sequences should be tested using solutions as close as possible to those in the real experiment Automatic Mode In this mode the MPS software automatically calculates an acceleration phase for the driving sequence when a syringe flow rate exceeds the nominal maximum flow rate Table 1 and Table 4 The acceleration phase has a duration of Sms and accelerates the syringe to 83 of it s nominal maximum flow rate Figure 30 shows and example An automatic acceleration phase example is shown in Figure 30 The MPS software cannot calculate an acceleration phase for phases that have a duration of 5ms or less The MPS program will request than a longer phase duration be used Table 4 Acceleration Phases AUTOMATIC ACCELERATION PHASES Syringe Size Nominal Maximum F
91. recision of quenched flow experiments depend on quality of the sample collect from the SFM Sample contamination can be minimized by optimizing the volume needed to wash all contamination from the SFM flow lines during a given experiment This is best achieved by performing test experiments similar to that described in section 14 3 and adapting them as close as possible to true experimental conditions temperature viscosity etc 13 3 4 MINIMIZING REACTANT CONSUMPTION In some instances it is desired to minimize the consumption of one or two reactants in a quenched flow experiment e g a protein in low supply or radioactively labeled DNA Some suggestions to do so are provided below 13 3 4 1 13 3 4 2 Choosing the best ageing method As mentioned in sections 13 2 1 1 and 13 2 1 2 the continuous flow and interrupted flow ageing methods work best for ageing times of 1 100ms and 100ms to several seconds respectively These ranges are meant to be guidelines and not strict requirements It is worthwhile to explore the application of both ageing methods to the design of an experiment to see which method best economizes the use of reactants An example situation with a double dilution experiment is shown in Figure 75 Moving sample with non precious components When using the partial liquid collection method a non precious component e g buffer may be used to push the sample during the collect step An example of this is shown in
92. riving sequence tells the SFM to automatically perform several functions such as moving the syringes activating the hard stop and triggering data acquisition Driving sequences are created in the MPS software in the Stopped Flow Program window available under the Sequence Files menu Figure 24 Figure 24 MPS Software Stopped Flow Program Window Phaes 7 Total Volumes pl 100 100 100 100 Phase 2 5 Total Volume 400 pl Total Flow Rate 4 0 ml s Syringes contents Shots Drive Sequence E Time Single 1 1 20 7 8 ms Multiple Ageing Time DL1 12 8ms DL2 106ms Comments Close SFM 3 4 User s Manual ver 1 1 A driving sequence is entered in the program grid in the Stopped Flow Program window Figure 24 Each column of the grid represents a driving sequence phase Each phase contains actions for the SFM to perform A complete driving sequence may contain from 1 to 20 phases Although only 5 phases are shown initially additional phases may be inserted using the Insert Phase command under the Edit menu Figure 25 shows an expanded view of the program grid The Figure 25 MPS Software Program Grid duration of a phase is entered in ms 1 9999 ms phase on the first line of the program grid m Phase 2 Phase zi Duration Syr 1 ul 100 The volume in ul delivered by syringe Syr 2 ul 100 noa of
93. rresponds to the purge of the delay line the solution being pushed and evacuated to waste The purge volume is again equal to S2 S3 2 x V After the purge 601 of the reaction mixture is collected and measured The results of this test are shown as a function of purge volume in Figure 79 Figure 79 Recovery of Uncontaminated Material Due to the long ageing time in Phase 4 the solution collected in the last phase 10 should correspond to the full reaction t co Contamination on the leading edge of the liquid column contained in the delay line is observed when the volume of the purge is zero Contamination on the trailing edge is observed for overly large purge volumes when the fresh reactants pushing the liquid column are collected t infinity E c re N RO 2 Q c o o Q lt These results in Figure 79 shows that 120 240 for a delay line of 190ul1 216 9ul Purge Volume ul nominal volume the first 20 to 30ul and the last 30 to 40ul are contaminated and should be discarded 14 96
94. s observed This allows the amplitude of the reaction to be used as A for evaluation of the dead time At the same time smooth kinetics enable an easy detection of any stop artifact Using the results of the analysis of the two reactions Dead Time 1 330 s 1n 0 23 015 1 3 ms This is very close to the theoretical dead time of 1 39ms calculated for a TC 50 10 cuvette and a flow rate of 16 ml s 9 3 Evaluation of Washing and the Quality of the Stop As mentioned in section 8 1 it is necessary to completely wash the flow Figure 37 Washing and Quality of the Stop path from the last mixer to the point of Observation in the cuvette One method of evaluating the volume needed for washing the flow path is presented in Figure 37 The reaction was the slow reduction of DCIP with ascorbic acid at pH 9 The same experimental conditions as in section 9 2 were used except that transmittance was used instead of absorbance Equal volumes of each reactant were mixed The data acquisition was started 100 ms before the shot to allow clear observation of the start of the shot Transmittance The slow reaction also allows the examination of the data around the stop for any artifacts The results indicate that there are no stop artifacts present and that a minimum of 200 pl per reactant is needed to completely wash the flow path for this reaction SFM 3 4 User s Manual ver 1 1 9 4 Variable Rati
95. s the number of shots possible based the current volumes in the SFM syringes It also indicates whether the SFM is running a driving sequence or ready for the next shot A driving sequence is executed by pushing the ES button or the start stop button on the front 12 81 SFM 3 4 User s Manual ver 1 1 panel of the MPS Figure 2 The button can be used to terminate an experiment Figure 69 MPS Software Shots Frame and prematurely if necessary Program Run Window If the Single button was used to transfer the Shots driving sequence to the MPS only a single Single shot can be made The button must Multiple then be pushed to return to the Quenched Flow Program window and the Single button must be pushed again to re transfer the driving sequence to the MPS for a subsequent shot Sere If the Multiple button was used to transfer the driving sequence to the MPS the Number of shots 16 button can be used to execute shots until the Program Run window shows that 0 shots remain The Ex button is then pushed to return to the Quenched Flow Program window IMPORTANT Before running in automatic mode verify that the syringe valve handles of the syringe used in the driving sequence are set to C and unused syringes set to R 12 82 SFM 3 4 User s Manual ver 1 1 13 A SHORT QUENCHED FLOW PRIMER This section describes the basics of the quenched flow technique and provides some general advice about
96. se IMPORTANT The flow rate of the sample during the purge and collect steps must be equivalent If the flow rate during the two steps is different the sample collected will not have a uniform age CAUTION As noted in section 10 4 2 the purge volume should be a minimum of 3 5x the flow line volumes section 10 3 to ensure only uncontaminated sample is collected Larger volumes may be necessary and it is recommend that test experiments be performed to optimize the volume needed to minimize sample contamination An example procedure to determine the purge volume needed is provided in section 14 3 13 3 General Advice for Quenched Flow Experiments 13 3 1 SYSTEM SPECIFICATIONS To achieve successful results from quenched flow experiments and optimal performance of the SFM it is imperative that the specifications of the SFM and its components be respected at all times The specifications are provide in Table 1 It is recommend that each experiment driving sequence be examined carefully for compliance with the SFM specifications before execution 13 3 2 TEST EXPERIMENTS It is strongly recommend that all driving sequences be tested with non precious samples Although such tests may be time consuming they maximize experiment success by ensuring that a majority of miscalculations and mistakes will be found and avoided 13 88 SFM 3 4 User s Manual ver 1 1 13 3 3 MINIMIZING SAMPLE CONTAMINATION The accuracy and p
97. strongly recommended that overheating protection always be left enabled 11 72 SFM 3 4 User s Manual ver 1 1 12 INSTRUMENT OPERATION 12 1 Manual Syringe Control The syringes of the SFM can be controlled either manual or automatically Automatic control of the syringes is strictly used only for experiments The manual control of the syringes is used for initialization filling and emptying the syringes The manual movement of the syringes can either be made directly from the MPS or though the MPS software Both methods are described in the following sections 12 1 1 MPS Syringe control directly from the MPS is made through the use of the buttons on front Figure 58 MPS Syringe Controls panel of the MPS Figure 58 and Figure 2 The and buttons are used to select the syringe to be moved The up and down buttons are used to empty and fill the syringes respectively The LCD panel at the top of the controls will display which syringe has been in selected and whether it is being filled or 2 lt gt vine gt motor on o emptied 12 1 2 SOFTWARE Syringe control from the MPS software is made through the Syringes Command Load window available under the Syringes Command menu Figure 59 The syringe to be moved is selected by clicking on the corresponding frame or pressing the lt Left gt or lt Right gt arrows keys on keyboard The new selected syringe will be surrounded with a re
98. syringe barrel to make a tight seal IMPORTANT Do not forget this step The syringe plunger tips are made of Teflon Pulling the syringe plungers out of the SFM each night allows the tips to expand and make a tight seal during use minimizing any chance of leaks 5 Turn all syringe valve handles to C 6 Turn off the MPS 12 5 Long term Storage of the SFM If the SFM is not to be used for a long period of time more than several weeks it should be cleaned as above in section 12 4 If the SFM is connected to a circulation temperature bath the temperature bath should be disconnected from the SFM and the SFM drained completely of all cooling liquid Afterwards it is recommended that the SFM cooling circuits be flushed with ethanol followed with air The SFM is now ready to be stored 12 6 Creating a Driving Sequence Experiments are performed with the SFM through the use of a driving sequence A driving sequence tells the SFM to automatically perform several functions such as moving the syringes and activating the exit valve Driving sequences are created in the MPS software in the Quenched Flow Program window available under the Sequence Files menu Figure 61 Figure 61 MPS Software Quenched Flow Program Window Quenched Flow Program EXAMPLE QF4 Ea Phaes uae Total time ms Volumes pl Syr 1 ul Syr 2 nl Syr 3 ul 100 100 Syr 4 ul 100 100 Waste CNN Waste Waste Synchro 1 Off Off Off Off
99. syringe valve handles to R and move all syringes to their lowermost positions The syringe plungers should exit the SFM so that the plunger tips are completely visible If this is done using the MPS software it will be necessary to uncheck the Up and Low Limits checkbox in the software Syringes Command Load window Figure 22 Note You may observe a few drops of liquid that fall from the syringes when the syringe plungers are completely out of the SFM This is normal as a small amount of liquid is always trapped between the plunger tip and the syringe barrel to make a tight seal IMPORTANT Do not forget this step The syringe plunger tips are made of Teflon Pulling the syringe plungers out of the SFM allows the tips to expand each night and make a tight seal during use minimizing any chance of leaks 5 Turn all syringe valve handles to C 6 Turn off the MPS 7 5 Long term Storage of the SFM If the SFM is not to be used for a long period of time more than several weeks it should be cleaned as above in section 7 4 If the SFM is connected to a circulation temperature bath the temperature bath should be disconnected from the SFM and the SFM drained completely of all cooling liquid Afterwards is recommended that the SFM cooling circuits be flushed with ethanol followed with air The SFM is now ready to be stored 7 6 Creating a Driving Sequence Experiments are performed with the SFM through the use of a driving sequence A d
100. syringes should be turned to R after the filling process is complete The Stopped Flow Module is now ready for operation SFM 3 4 User s Manual ver 1 1 Figure 23 SFM Syringe Filling Procedure Panel 1 Panel 2 7 4 SFM Cleaning and Storage After each day s experiments the SFM should be cleaned A thorough cleaning of the SFM will ensure that it has a long functional life and diminish any chance of sample contamination for the next user of the instrument The procedure below is the recommended daily cleaning procedure to be done before shutting off the instrument 1 Remove and remaining samples or buffer from the syringes 2 Wash the syringes and flow lines 2 3 times with water This is done by filling each syringe with water to a volume at least equal to the sample volume used for experiments With the syringe valve handles set to C empty the syringes completely Since the liquid will exit via the cuvette it will wash the flow lines and cuvette as well as the syringes 3 Wash the syringes and flow lines one time with 70 10096 ethanol Use the same procedure as in step 2 7 39 SFM 3 4 User s Manual ver 1 1 4 Dry the syringes flow lines and cuvette with a single wash of air Use the same procedure as in step 2 The syringes should be emptied in reverse numerical order so that all liquid is pushed out of the syringes flow lines and cuvette Set all
101. te Configuration Cuvette Selection x ig Windows Device Syringes Luvette Delay Line Serial Port Limits 6 5 Delay Line Configuration Delay line configuration is made in the Config Delay Line window available under the Config menu Figure 18 Select the delay line s according to the delay line s you have installed in the SFM One or two delay lines must be configured depending on the type of device installed under section 6 1 Each delay line is chosen from a pull down menu in the Config Delay Line window Figure 18 IWARNING Incorrect delay line configuration will cause ageing time calculations to be incorrect SFM 3 4 User s Manual ver 1 1 Figure 18 MPS Software Delay Line Configuration Delay Lines Ir windows Delay Line 1 pl Device Eina No 0 25 5 Cuvette Delay Line 2 pl Delay Line No 0 31 7 Serial Port Limits Cancel 6 6 Serial Port Configuration The serial port must be configured before the MPS and MPS software can communicate Serial port configuration is made in the Config Serial Port window available under the Config menu Figure 19 Select the serial port used to connect the MPS and the microcomputer in section 3 2 Figure 19 MPS Software Serial Port Configuration Config Serial Port Ed Serial Port Syringes Cuvette Delay Line Limits Connect Device Cancel 6 7 System Limits Configuration The system limits are configured in t
102. the ese a Mammes Syr 3 ul 100 ase 1s entered on the line next P O EE LN Synchronizatioi to the appropriate syringe The Synchro 1 status of the synchronization trigger 1s noted on the last line of the program grid M a Trigger To enter the phase duration and syringe volumes delivered click on the corresponding cell or use the keyboard arrows keys to navigate between cells The BACKSPACE key can be used for correction and the DEL key to clear a value The synchronization trigger is toggled on or off by pressing O on the keyboard Selected values entered in the program grid can be cut copied and pasted using the Cut Copy and Paste functions available under the Edit menu To perform a cut copy or paste operation select the area of the grid desired by dragging the mouse with the left mouse button pushed in and then choose the Cut Copy or Paste functions desired under the Edit menu The values will be stored in the Windows clipboard for the Cut and Copy functions Values will be pasted from the Windows clipboard for the Paste function If copy area is bigger than paste area the operation is done only for values that can fit inside paste area CAUTION Blank and non numeric values entered in the program grid are considered as zero values A phase duration of 0ms will cause the phase to be skipped in the execution of the drive sequence The contents of the syringes can be J entered in the Syringe Contents
103. these are the default syringes installed in the MPS software Changes only need to be made in the software when syringes of different volumes other than standard have been installed in the SFM WARNING Incorrect syringe configuration will cause volume and flow rate calculations to be incorrect Further information about syringe configuration can be found in chapter 4 2 of the MPS Software User s Manual 11 69 SFM 3 4 User s Manual ver 1 1 Figure 54 MPS Software Syringe Configuration Syringe 2 Syringe 3 E 4 ol 20 ml Diam 17 mm Pitch 4 mm ol 20 ml ol 20 ml Diam 17 mm iam 17 mm Pitch 4 mm Pitch 4 mm IE windows Device Syringes 68 7300 1 400 i 10 ml 7 20 mm 4 mm for SFM 300 amp 400 sdt Cuvette 3 mm 4 mm for pSFM 20 Delsy Line Jr EIC Serial Port Limits 11 3 Delay Line Configuration Delay line configuration is made in the Config Delay Line window available under the Config menu Figure 55 Select the delay line s according to the delay line s you have installed in the SFM One or two delay lines must be configured depending on the type of device installed under section 11 1 Each delay line is chosen from a pull down menu in the Config Delay Line window Figure 54 TWARNING Incorrect delay line configuration will cause ageing time calculations to be incorrect Figure 55 MPS Software Delay Line Configuration
104. ts were performed in a manner similar to the variable mixing ratio mixing experiments of DCIP in the previous section The volume and concentration of NAD S4 were kept constant the concentration of ADH was varying by varying the volumes of buffer S1 and ADH S2 in each experiment The total volume and flow rate of each shot was kept constant The dilution of ADH varied from 1 2 to 1 120 0 5 to 0 083 mg ml final ADH concentration The results of the experiments are shown in Figure 41 Figure 41 ADH Variable Ratio Mixing Experiments Q c S 2 S6 a f lt The initial rate of each reaction in Figure 41 was measured and plotted as a function of the dilution factor in Figure 42 The rates and dilution factors are plotted on a log log scale Figure 42 shows that there is reasonable alignment of the data to a line of a slope of 1 This indicates a linear relationship between the initial rate and the dilution factor Figure 42 ADH Variable Ratio Mixing Rate vs Dilution Initial Rate relative scale Final Dilution The horizontal dashed line in Figure 42 corresponds to the remaining ADH activity after washing the cuvette To obtain this line the cuvette was washed with two shots containing no ADH only S1 and S4 were used follow by a 1 2 dilution of ADH with NAD only S2 S4 The contaminating activity corresponds to a 1 1000 if the initial ADH concentration Further washing could reduc
105. ul Figure 48 and the total flow through the intermixer volume upon sample entry and exit is 6 ml s which indicates tase 27 60 300ms 27 6ul 1009 2 ms 6 mL s 6 mL s entry pause exit Figure 72 Example Interrupted Flow Experiment Quenched Flow Program Syringes Total time ms 50 Volumes pl 0 150 210 150 210 60 Waste Waste i 300 Synchro 1 Off Off Phase 4 5 Volume 0 ul Flow Rate 0 0 ml s Syringes contents Shots Drive Sequence Ageing Time p DL1 i Buffer Single 1 1 20 6 3 ms DL2 40ms DNPA Multiple NaOH Comments Close It is important to note that not all the sample can be recovered from the intermixer volume without contamination This is because unwanted mixing occurs at each end of the intermixer volume by diffusion during the incubation period The fraction of the sample that remains uncontaminated must be determined experimentally and an example procedure is provided in section 14 4 The interrupted flow method allows samples to be aged for several 10 s of ms to several seconds or longer It generally uses more sample than the continuous flow method This is due to the fact that only a portion of uncontaminated sample can be recovered and sometimes necessitates multiple repetitions of the same experiment to achieve sufficient sample volume for analysis 13 2 2 COLLECTION METHODS Section 10 4 described how sample can be recov
106. um of DNP 350 changes with pH but there is a clear Wavelength nm isobestic point at 325nm Q c c a o D 2 These properties make the alkaline hydrolysis of DNPA a useful tool for the testing of a quenched flow instrument The reaction can also be followed by the stopped flow technique omitting the acid quench Experimental Conditions Syringe 1 Water Syringe 2 1 mM DNPA 1 v v DMSO 2 mM HCl Syringe 3 1 M NaOH Syringe 4 2M HCl Delay Line 1 17ul Delay Line2 190g Sample Preparation Make 1 ml of 100 mM DNPA in fresh DMSO 22 6 mg DNPA ml DMSO The solution may turn slightly yellow as the DNPA dissolves As the solution ages the yellow color will intensify For best results it is recommended to use the freshest possible DMSO and prepare new samples each day 1 Prepare a 2 M HCI solution by mixing 8 3 ml concentrated HCl with 50 ml of water 2 Prepare a 1 M NaOH solution by dissolving 2g of NaOH in 50 ml of water 3 Prepare the working DNPA solution by mixing 49 45 ml water 50 ul 2 M HCl 500 ul 100 mM DNPA in DMSO 14 91 SFM 3 4 User s Manual ver 1 1 Driving Sequence Various ageing times for the reaction achieved by varying the intermixer volume M2 M3 and the flow rate through the intermixer volume The general format of the driving sequence is shown below and the delay lines and flow rates used are given in Table 9 Time ms 20 T T S1 Water S2 DNPA 3
107. ume The technique presented here may be adapted to evaluate the dead time under many experimental conditions Experimental Conditions Syringe 1 2 or 3 20 ml 10 mM Ascorbic Acid pH 2 or 9 Syringe 4 20 ml 0 5 mM DCIP Wavelength 524 nm Cuvette TC 50 10 Detection method Absorbance Total Flow Rate ml s 8 pH 9 reaction 16 pH 2 reaction The decoloration of DCIP was followed by measuring the absorbance at 524 nm during the reaction Equal volumes of the reagents were mixed to start the reaction 200 ul of each Data acquisition was made using Bio Kine software and started before the shot The resulting kinetic traces of the reactions at pH 9 0 and pH 2 0 are shown in Figure 36 Figure 36 Reduction of DCIP by Ascorbic Acid pH 2 0 Va Observed absorbance pH 9 0 f change 7 0 15 Total absorbance Change 0 23 10 Time ms SFM 3 4 User s Manual ver 1 1 Analysis of the two traces yields REACTION pH TOTAL FLOW RATE A ABSORBANCE RATE CONSTANT s CE 8 0 23 not determined The dead time can be calculated according to Dead Time 1 k In Ay Aoy Where k is the observed rate constant for the a first order or pseudo first order reaction Ag is the total amplitude of the reaction and A is the observed amplitude of the reaction of interest The t5 of the slow reaction at pH 9 is around 100 ms ensuring that 100 of the reaction i
108. used to synchronize the SFM and other instruments The timing of the triggers with respect to the drive sequence is programmed in the last line s of the program grid in the Quenched Flow Program window Figure 61 The duration of the pulse will be equal to the duration of the phase In quenched flow mode only Synchro out 1 is available for programming 12 10 Saving or Loading Driving Sequences Driving sequence can be saved as files on the hard drive of the microcomputer In this way a series of driving Figure 68 MPS Software sequences for different experiments can be prepared in Load Save Driving Sequences advance and subsequently loaded as needed Ff SFM 4 S amp SFM 400 5 Files are saved or loaded using the Save or Load uda Edit Syringes Cor functions under the Sequence Files menu Figure 68 New Files are saved using filenames up to 8 characters in length Close Save More information about saving or loading driving Gace ike sequences can be found in the MPS Software User s Manual chapter 6 Print 1 C MPS SAMPLES4 SF4 Exit 12 11 Running in Automatic Mode Once a driving sequence has been entered or loaded it is transferred to the MPS by pushing the Single or Multiple button in the Shots frame of the Quenched Flow Program window Figure 61 and Figure 69 The MPS is now in automatic mode and the Program Run window will be displayed Figure 69 The Program Run window show
109. xer volumes be determined experimentally with known reactions One such experimental procedure for determining intermixer volumes is described in the section 14 2 of this manual Figure 47 SFM 3 Q Flow Line and Delay Line Volumes SFM 3 Q FLOW LINE VOLUMES Line Number Flow Line Volume ul 103 41 155 156 7 Delay Line o 1o0 oO Oo ho Collect EXIT VALVE 6 DELAY LINE MIXER1 RESERVOIR1 RESERVOIR2 N e Ww Ww wW o o o z z z i x x gt gt gt o o o DELAY LINE AND INTERMIXER VOLUMES Delay Line N 1 17 N 2 40 N 3 90 N 4 140 N 5 190 N 6 500 N 7 100 Volume ul Intermixer Volume M158 M2gg ul Intermixer Volume Mas M2ups ul Notes Intermixer volumes are measured from the mixing point of one mixer to the mixing point of the next mixer BB indicates a Berger Ball mixer has been installed at the position noted HDS indicates a High Density mixer has been installed at that position 10 63 SFM 3 4 User s Manual ver 1 1 Figure 48 SFM 4 Q Flow Line and Delay Line Volumes SFM 4 Q FLOW LINE VOLUMES Line Number Flow Line Volume ul 156 Delay Line 1 9 165 7 Delay Line 2 18 188 36 55 Collect 14 EXIT VALVE MIXER1 RESERVOIR1 SYRINGE 1 SYRINGE 2 SYRINGE 3 SYRINGE 4 DELAY LINE AND INTERMIXER VOLUME
110. yringes to be driven beyond their upper and lower limits This also avoids accidentally pulling the syringe plunger completely from the syringe and spilling solution onto the SFM 12 74 12 3 SFM 3 4 User s Manual ver 1 1 WARNING The Up and Low Limits only applies to control of the syringe from within the MPS software These limits can be bypassed by manual control of the SFM directly from the MPS Further information about the initialization of syringes can be found in the MPS Software User s Manual chapter 5 Filling the Syringes WARNING Utmost care should be exercised during this operation Normal operation of the system requires that no bubbles are present in the injection syringes Should this occur the buffer flow through the SFM will not be correctly controlled by the plunger movement and artifacts may be observed For best results it is recommended that all solutions be degassed and filtered before filling the SFM The syringes of the SFM can be emptied and filled manually section 12 1 The filling of the syringes follows the steps below and shown in Figure 60 1 Attach a syringe disposable plastic syringes may be used containing sample or buffer to a syringe reservoir port on top of the SFM Figure 60 Panel 1 2 Setthe syringe valve handle to R and fill the syringe manually section 12 1 while exerting a slight pressure on the reservoir syringe Figure 60 Panel 2 and Panel 3 The pressure exerted on the r

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