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SFM-300/400 User`s Manual Version 2.7

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1. Figure 13 HDS Mixer 25 SFM 300 400 User s Manual ver 2 7 3 7 3 Observation head with separate cooling The standard observation head may be replaced with an observation head that has separate cooling see Figure 14 The separate cooling feature regulates temperature in the observation head as well as the main body of the instrument This may be used in cases where mixing the solution produces a temperature change of the solution flowing into the cuvette requiring extra cooling Figure 14 Observation Head with Separate Cooling 3 7 4 MICROCUVETTE ACCESSORY The standard observation head may be replaced with the microcuvette accessory see Figure 15 The microcuvette accessory consists of one micromixer combined with a special 08 cuvette The uFC 08 is a modified FC 08 cuvette with a dead volume varying from 1 to ul The dead volume of the cuvette is adjusted by the user by setting the special adapter provided with the accessory see Figure 15 Minimum dead time obtained with this accessory is 0 25 ms Figure 15 microcuvette accessory 26 SFM 300 400 User s Manual ver 2 7 3 7 5 LOW TEMPERATURE ACCESSORY Because standard stopped flow cannot be operated below 0 C a special accessory was designed to perform kinetics at temperatures as low as 90 C A description of the low temperature accessory is given in Figure 16 The low temperature ac
2. Cap Figure 76 Mixer Removal Examination 1 Examine the mixer for any material blocking the holes If found the mixer should be cleaned before returning it to the SFM we recommend soaking the mixer 2 Hellmanex Il cleaning solution for 15 min or sonication 2 Measure the height of the mixer The mixer Berger Ball or HDS should measure 5 1 mm in height Figure 77 If the mixer is smaller than this it has been damaged and should be replaced Figure 77 Mixer Height Replacement Insert the mixer into the observation head in the orientation shown in Figure 78 k 2 Insert the o ring on top of the mixer 3 Reattach the observation head to the SFM body 4 Reinsert the cuvette and cuvette holder 5 Screw on the observation head nut Berger Ball mixer HDS mixer UP UP c mf c lt n E DOWN DOWN Figure 78 Mixer Orientation 8 3 Lubrication The drive screws should be lubricated periodically once per year with mineral oil Access to the drives screws is obtained by removing the cover in front of stepping motors 8 4 Thermostated Bath To avoid any corrosion we recommend using a circulating oil bath 82 SFM 300 400 User s Manual ver 2 7 9 INSTALLATION OF THE QUENCHED FLOW COMPONENTS 9 1 Installation of the Mixer Blocks and Delay Lines In quenched flow mode the syringes of the SFM can be used to perform many t
3. Titrator Mode Freeze Quench Click to select syringe 1 1 9 ml 7 8 mm 7 4 mm for SFM 300 400 3 6 ml 12 mm 4 mm for SFM 300 amp 400 Driving Sequence 6 8 ml 17 mm 4 mm for SFM 300 amp 400 10 mi 20 mm 4 mm for SFM 300 amp 400 std Classic mode Stopped flow Advanced mode Cancel _ Custom Figure 88 stopped flow configuration The device to be installed should be configured according to the instrument purchased and mode chosen for use Check Classic mode the advanced mode is not available in quenched flow configuration Syringe configuration is made in the same window The active syringe is displayed in yellow select the nature of the syringes that have been installed in each syringe position of the SFM by clicking on the correct one 91 SFM 300 400 User s Manual ver 2 7 The SFM comes equipped with standard 10 ml syringes which are the default syringes installed in the software Changes only need to be made in the software when syringes of different volumes other than standard have been installed in the SFM Use the button to enter syringe specifications if you have a custom syringe In this condition the window shown in Figure 89 is displayed it is then necessary to enter volume piston diameter and screw pitch of the custom syringe to add it to the standard ones Custom Syringe 5 0 ml 15 00 mm 4 08 m
4. Ascorbic Acid mM Figure 71 DCIP Variable Ratio Mixing k vs AA 76 SFM 300 400 User s Manual ver 2 7 Dilution factors of 1 50 or higher can be obtained with the SFM Figure 72 shows the results of experiments where 1 mM DCIP in S3 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 Absorbance 0 050 ea T T T T T 1 81 1 41 1 21 1 11 1 5 1 3 1 2 Figure 72 Dilution Experiments 77 SFM 300 400 User s Manual ver 2 7 7 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 10 ml Buffer Syringe 2 10 ml Buffer 1 mg ml alcohol dehydrogenase ADH Syringe 3 10 ml Buffer 1 mM NAD Wavelength 340 nm Cuvette TC 50 10 Detection method Absorbance Experiments 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 S3 were kept constant The concentration of ADH varied 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
5. 11 2 Device installation using MPS 60 or MPS 70 with Bio kine version 4 47 and higher 4 49 Once Bio Kine is loaded choose Install device installation in the main menu Figure 17 device installation The stopped flow communication is established from this window by checking the stopped flow device box and choosing the corresponding Serial port for the MPS 60 or USB port for the MPS 70 Accept the parameters using the OK button 90 SFM 300 400 User s Manual ver 2 7 Device Installation Acquisition device Stopped flow device MOS 450 4 810 serial acquisition Use stopped flow 05 250 J 810 analog acquisition Communication mode 05 200 v Extemal device C Serialpot COM 9 1 gt TIDAS diode 4 USB r Acquisition parameters Accessories Serial Port COM lt Peltier temperature controller mT jump PCI mT jump USB Box Acquisition Board No board detected OK Cancel Figure 87 device installation 11 3 Stopped flow Configuration Once the stopped flow device its serial port are selected in the device configuration menu refer to section 11 1 choose the Install stopped flow configuration menu see Figure 88 Stopped Flow configuration Device Syringes Syringe 1 18 Diam 20mm 0 Syringe 2 Diam 20mm 0 E Syringe 3 Diam 20mm 0 SFM 400 S Syringe 4 10 Diam 20mm
6. 51 1 Total volume shot 51 63 wl 15 ml s aj 562 gt 52 126 31 771 00 mL s EM EE ul salf 63 ul 1 6 4 54 188 uL 47 Defaut r Start of data acquisition Sequence oe Estimated dead time 2 8 ms Ready At 10 ms before the stop Configuration Content of syringes Initial concentration Final concentration Synge Synge 2 oe Ur swing 3 M 1 Synge 4 A Load SaveAs Comments Print SFM Options Close Figure 23 driving sequence in advanced mode First operation should be to check the configuration of the stopped flow by clicking on the SFM Options button a x Cuvette 34 Hard Stop Acceleration Phases p Delay Line 1 pl 2 nis Automatic 7 Manual None Manual Delay Line 2 pl 100 10 si Valve Lead TC 100 15 2 ms r Ejection delay Line nl v HDS Mixer Hard Stop closed between shots X Overheating Protection Default OK Cancel Figure 24 SFM options in advanced mode e Select the cuvette and mixer according to the cuvette and mixer installed in the SFM refer to the SFM user s manual for more details WARNING Incorrect cuvette and mixer configuration will cause dead time calculations to be incorrect e Valve Lead This section of the window al
7. 71 1 pl B 118 5 pl E 47 4 yl Ready Close Figure 39 Global sequence window The volumes in the structure of the sequence can be changed manually click on the desired box and enter a new injection volume A color code is used to warn the user about the volumes selected orange colour the volumes used are too small to insure an efficient washing of the cuvette or the delay line Green colour the volumes used are adequate to run the kinetic 49 SFM 300 400 User s Manual ver 2 7 The ageing times window is used in order to define an ageing time Figure 39 The ageing time is equal to the waiting phase from phase 2 minimum ageing time time needed for the solution A B to fill the delay line under a continuous flow When changing the ageing time the waiting phase is automatically calculated It is possible to run several shots using different ageing times for each shot To do so click on the button t from the Edit table This will add a second step in the ageing time window Figure 40 To remove a step click on the button erase all the ageing time sequence click When several steps are created and case the user wants to invert two lines the arrow or amp from the edit table can be used At the bottom of the Global sequence window a function allows to repeat each step For example in case three steps are created as in Figure 40 and a repeat number of 2
8. Ratio steps auto Concentration steps Auto Variation Concentration in Mixer 1 Concentration A uM Step number 5 fep vue un Start of data acquisition Final value uM 4 Atstop Pre trigger Load Print Save As SFM Options Edit Sequence Global sequence Start next acquisition step at the end of measurement C ate 5 sec manual continuation Autovariation concentration steps mode Jl j a2 B 3 g c yg wo g d 2g 2m g o g 51 s 25 g o g 6 2 g s g cw a f ce os o d o Pons 59s Repeat number 1 Total volume Syringe 5032 4 yl 250 yl 5282 4 yl Ready P Close SQ a Is Figure 57 Global sequence in concentration steps mode Ratio step manual mode By selecting Ratios steps Manual in the Mode window you have an access to the window menu then Edit Sequence by clicking on the button the following sequence is edited or the latest sequence is automatically loaded as a default one 62 SFM 300 400 User s Manual ver 2 7 F Concentration dependance studies Concentration dependance studies Content of syringes Initial concentration Mixer 2 conditions Syringe 1 w Pe Ratio Diluant 532 21 Concentration Amax rd mM Syringe 2 Ratio 3 5 C
9. Concentration step Auto mode The variations of the concentration of reactant A in mixer M1 have to be fixed by typing a value in concentration A i e 1 in the example choosing the steps numbers and the value of T Edit Sequence the step By clicking on the button 54 the following sequence is edited SFM 300 400 User s Manual ver 2 7 Concentration dependance studies Concentration dependance studies Content of syringes Initial concentration Mixer 2 conditions Syringe 1 Water Ratio 4 Diluant 3 Concentration Amax 250 uM Syringe 2 Acid Ascorbic 50 0 uM Ratio B 3 ConcentrationB 650 uM Syringe 3 DCIP 130 uM gt Total flow rate 100 mis Estimated deadtime 16 ms Variation Concentration in Mixer 1 Concentration A aM Step number 5 uM Start of data acquisition Final value uM eoo 4 Atstop Pre trigger Mode Start nest acquisition step Ratio steps Auto Concentration steps Auto i at the end of measurement C afte 5 sec manual continuation Load Print SaveAs SFM Options Edit Sequence Global sequence Autovariation concentration steps mode I J1 2s H o2 H 6 j co B so j vj Repeat number 1 Total volume Syringe 5032 4 ul 250 yl 5282 4 yl Ready P Close Figure 47 Global sequence in
10. Ratio steps Auto Concentration steps Auto Variation Ratio in Mixer 1 Start next acquisition step Ratio A l Ratio Diluant 4 at the end of measurement Step number 5 after 5 sec mare Step value of manual continuation Start of data acquisition Final value bs Atstop Pre trigger SFM ptions _ Edit Sequence Autovariation ratio steps mode jp U jJ hm S U E J jJ U d Load Print Global sequence 111111 W Repeat number 1 Total volume Syringe J Di pg ow Bg od Ready P Close 1 lt Figure 42 driving sequence for concentration dependence studies In the sequence the user has to indicate the content of the syringes and the initial concentrations of reactant A as Ao and reactant B thus Bo 52 SFM 300 400 User s Manual ver 2 7 Fl Concentration dependance studies r Concentration dependance studies Content of syringes Initial concentration Mixer 2 conditions Syringe 1 Water Ratio Diluan 3 Concentration 250 Syringe 2 50 uM Ratio B 3 ConcentaionB 650 pM Syringe 3 13d uM Total flowrate 100 mi s Estimated deadtime 16 Mode Ratio steps Auto Concentration steps Aito Vari
11. 30 30 35 Collect Synchro 1 Phase 4 5 Total Volume 180 yl Total Flow Rate 9 0 ml s Syringes contents Shots Drive Sequence Ageing Times 1 1 20 DL1 7 3 ms Single DL2 46 ms Multiple EDL a Load Save Comments Print SFM Options Close Figure 107 Partial Liquid Collection with Interrupted Flow Ageing 110 SFM 300 400 User s Manual ver 2 7 The flow rate of the sample during the purge and collect steps must be Equal If the flow rate is different during the two steps the sample collected will not have the same age As noted in section 0 the purge volume should be a minimum of 3 5x the flow line volumes section 9 2 to ensure only uncontaminated sample is collected Larger volumes may be necessary it is recommended 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 5 General Advice for Quenched Flow Experiments 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 provided in Table 1 SFM specifications It is recommended that each experiment s driving sequence be carefully examined for compliance with the SFM specifications before execution It is
12. If none is selected the hard stop is always open Delay lines 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 3 4 Each delay line is chosen from a pull down menu WARNING An incorrect delay line configuration will cause ageing time calculations to be incorrect 5 7 2 Design of the sequence A driving sequence is entered in the program grid shown in Figure 29 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 _inset Phase button or removed using the Remove Phasel button Figure 29 shows an expanded view of the program grid The duration of a phase is entered in ms 1 60000 ms phase on the first line of the program grid The volume ul delivered by each of the syringes during a phase is entered on the line next to the appropriate syringe The status of the synchronization trigger is noted on the last line 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 synchronization trigger is to
13. Syr 2 pl m Syr Syr 4 pl Valve Collect Waste Waste Waste Synchro 1 Off Off Phase 2 5 Volume pl Flow Rate 0 0 ml s Syringes contents Shots Drive Sequence Ageing Times Syringe 1 Single 1 1 20 DL1 21 5 ms DL2 29 2 ms EDL Load Save Comments Print SFM ptions Close Figure 101 example of continuous flow experiment 13 2 2 Pulsed flow method The pulsed flow method consists of making a few pulses to fill the delay line in order to get a turbulence flow in the delay line followed by an incubation time and then mixing with the quencher Under these conditions tage depends on the number of pulses to fill the delay line flow rate in the delay line and incubation time nx volume of the pulse di ot Flow DL With n number of pulse Flow DL flow rate in the delay line t inc incubation time With this method we only need to enter a different incubation time to reach different ageing time The others parameters remain unchanged An example of a driving sequence using the pulsed method is shown in Figure 102 For this example the 17 Mix 190 delay lines are installed 8 pulses of 27 ul are needed to fill the delay line num 190 216 6ul and 1ml s is the flow rate of the pulse in the delay line To reach the point 286ms the incubation time of 10ms is entered 105 SFM 300 400 User s Manual ver 2 7 FA Qu
14. any electronic device to be synchronized with the instrument _5 MANUAL MOVEMENT Manual control of the syringes 6 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 7 PROGRAM RESET Resets the MPS 60 instrument does NOT reset syringe values 8 MAIN POWER FUSE A for 220 V or 6 A for 115 V 9 LINE CONNECTOR 10 MAIN POWER SWITCH MOTOR POWER CONNECTOR gSends the power pulses to the stepping motors 12 SERIAL CONNECTOR Connects the MPS 60 controller to the 13 REMOTE CONNECTOR For optional remote control 14 HARD STOP SF BNC Connects to Hard Stop Valve 2 2 AC Power Connections of 5 60 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 60 unit Connect the MPS 60 to the serial port of the microcomputer Finally plug the MPS 60 into the appropriate AC line 2 3 Operating Features MPS 70 The connection of the MPS 70 with the PC is done through a USB connector by plugging the USB cable on the rear panel of the MPS and installing the driver from Biokine version gt 4 45 e g 4 49 software from the folder driver MPS USB 441 The selection of the syringes individually or simultaneously can be done manually by pressi
15. s Manual ver 2 7 5 INSTRUMENT OPERATION IN STOPPED FLOW MODE 5 1 Manual Syringe Control The syringes of the SFM can be controlled either manually 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 Manual movement of the syringes can be made either directly from the MPS or though Bio Kine Both methods are described in the following sections 5 1 1 MPS 60 Syringe control directly from the MPS is made through the use of the buttons on front panel of the MPS 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 corresponding light on the front panel will indicate which syringe has been selected Syringe control directly from the MPS is made through the use of the buttons on the front panel of the MPS Figure 2 The selected buttons are used to select the syringe to be moved The left button selects syringes from number 1 to 3 SFM 300 or 4 SFM 400 with increasing numbers The right button selects syringes from number 3 or 4 to 1 with decreasing numbers The up and down buttons are used to empty and fill the syringes respectively The corresponding light at the front panel will indicate which syringe has been selected By pushing the left and right select buttons at the same time
16. solution A and solution B to start the reaction Figure 41 The following window is obtained Infos by clicking on the button Omo of the main mixing sequence window al SIT TG mdiae ON o tic concentration dependence studie SI M 300 CONSE To DG nok te RTE The aim of this mode is to follow the concentration dependency of in the reaction B keeping concentration constant It is done using 3 syringes and 2 mixers according to the following diagram Cuvette Detection sy Delay line Kinetics getting slower as 2 i concentration decreases t 51 Diluant t t 53 Loading of solutions The first mixer 1 is used to change concentration of 4 thus diluting solution is loaded in Syringe 1 and is loaded in syringe 2 The second mixer 2 is used to start the A B reaction so is loaded in syringe 3 The shortest delay line 17 must be installed between M1 and M2 Design of the sequence Users indicates the initial concentration 0 and BO and mixing ratio to apply between amp diluant and B at each step in mixer M2 The same total flow rate is used for all steps so dead time remains constant The user builds the sequence by varying the ratio Diluant 4 in mixer 1 or by changing directly the final concentration of Once all parameters are set Biokine proposes volumes per syringe defined according to mechan
17. 1 10 ml Diam 20 mm Syringe 2 10 ml Diam 20 mm Syringe 3 10 Diam 20 mm SFM 300 Q Syringe 4 10 ml Diam 20 mm SFM 400 Q Titrator Mode Freeze Quench Click to select syringe 1 1 9 ml Z 8 mm 4 mm for SFM 300 400 m 3 6 ml 12 mm 4 mm for SFM 300 amp 400 Driving Sequence 6 8 ml 17 mm 4 mm for SFM 300 amp 400 C Ces 10 ml 2 20 mm 4 mm for SFM 300 amp 400 std assic mode Stopped flow Advanced mode Figure 18 stopped flow configuration The device to be installed should be configured according to the instrument purchased and the mode chosen for use must be selected in the driving sequence The titrator and freeze quench modes are only available in a specific configuration please refer to their respective manual for details Syringe configuration is made in the same window The active syringe is displayed in yellow select the nature of the syringes that have been installed in each syringe position of the SFM by clicking on the right one 29 SFM 300 400 User s Manual ver 2 7 The SFM comes equipped with standard 10 ml syringes and these are the default syringes installed in the software Changes only need to be made in the software when syringes of different volumes other than standard have been installed in the SFM Use the amp 1 button to enter syringe specifications if you have a custom syringe In this condition the window shown in Fig
18. 2 to 1 120 0 5 to 0 083 mg ml final ADH concentration The results of the experiments are shown in Figure 73 x c S 2 S a 2 lt Figure 73 ADH Variable Ratio Mixing Experiments The initial rate of each reaction in Figure 73 was measured and plotted as a function of the dilution factor in Figure 74 The rates and dilution factors are plotted on a log log scale Figure 74 shows that there is reasonable alignment of the data to a line with a slope of 1 This indicates a linear relationship between the initial rate and the dilution factor 78 SFM 300 400 User s Manual ver 2 7 Initial Rate relative scale l 0 001 0 01 1 Final Dilution Figure 74 ADH Variable Ratio Mixing Rate vs Dilution The horizontal dashed line in Figure 74 corresponds to the remaining ADH activity after washing the cuvette To obtain this line the cuvette was washed with two shots containing ADH only 51 and 53 were used follow by a 1 2 dilution of ADH with NAD only S2 53 The contaminating activity corresponds to a 1 1000 of the initial ADH concentration Further washing could reduce the contaminating activity but this sets a reasonable limit for the dilutions that can be obtained with the SFM 7 5 Mixing Solutions of Unequal Density and Viscosity The SFM can be used to mix solutions of unequal density and viscosity in addition to simple aqueous solutions This
19. Concentration dependance studies Content of syringes Initial concentration Mixer 3 conditions Syringe 2 Diluant D Ratio Diluant 3 Concentration 250 uM Syringe 3 Ascorbic Acid 5 M gt Ratio B 3 Concentration B 50 pM Syringe 4 DCIP 130 uM Total flowrate 100 Estimated deadlime 16 Mode Variation Ratio in Mixer 2 Start next acquisition step Ratio steps Auto z Ratio 1 Ratio Diluant 20 at the end of measurement Step number after 5 sec Concentration steps auto f manual continuation Step value 01 a Start of data acquisition Final valu inal value 1 4 Atstop 7 Pre trigger Load Print Saves SFM Options hntos Edit Sequence J Global sequence Is ip lo Lee Figure 51 driving sequence In the sequence the user has to indicate the content of the syringes and the initial concentrations of reactant A as Ao and reactant B thus Bo Figure 52 i Concentration dependance studies Concentration dependance studies 1 Content of syringes Initial concentration Mixer 3 conditions Syringe 2 Diluant Ratio Diluant 3 Concentration Amax 250 pM Syringe 3 Ascorbic Acid 50 uM Ratio 3 Concentration B 650 uM Syringe 4 DCIP 130 uM Total flowrate Estimated deadtime 16 ms Mode Variation Ratio in Mixe
20. To accomplish this a sufficient volume of the 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 performed and adequate washing conditions found before starting any series of experiments 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 6 2 3 Signal amplitude Signal amplitude is generally proportional to the path length of the cuvette and the concentration of the 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 the reagent However the researcher may be limited by practical concerns such as value of sample viscosity of sample dead times inherent limitation of the signal such as inner filter effect and sample precipitation As with the achievement of the fastest de
21. User s Manual ver 2 7 Edit Sequence sequence is edited by clicking on the button Concentration dependance studies as following E x Concentration dependance studies Content of syringes Initial concentration Mixer 2 conditions Syringe 1 Water Ratio 3 1 250 Syringe 2 Acid Ascorbic 500 M RatioB 3 Concentration8 650 pM Syringe 3 DCIP 130 uM Total flow rate 100 ml s Estimated dead time 16 ms Mode Variation Ratio in Mixer 1 Start next acquisition step Ratio steps Auto 9 Ratio 1 Ratio Diluant 20 atthe end of measurement Step number C afte 5 sec Concentration steps z s C manual continuation Step value 01 Start of data acquisition Final val guess Je Atstop Pre trigger JU 2 jH o E 3 Bj 5 Bb ow 28 s W us B G gp sg B s go c go ow s B o g o B vw gy co Bg 5 v gp o g o pg us goo g co Bg wo Ip s go g G gf ou go wo yg vw gp w Repeat number 1 Total volume Syringe 450 yl 300 yl 750 pl I amp SL E Step Ratio Dil Ratio Ratio B Concent A Volume Dil Volume Volume Load Print Save As SFM Options ntos _ZEdit Sequence Global sequence Autovariation ratio steps mode Ready P Close Figure 46 Global sequence in ratio steps mode
22. a minimum of 3 5x flow line volumes section 9 2 In addition it is recommended to wash the old solution out of the SFM and tube with a buffer between sample collections and perform test experiments to verify the level of sample contamination is minimal 9 3 2 Partial liquid collection This method is the most preferred method used for quenched flow experiments It is similar to the previous method in section 9 3 1 2 in that the sample is collected in a pipette or syringe Figure 85 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 the contaminated sample to waste so that only uncontaminated sample is recovered Because of this even very small volumes 10 s of ul of a sample can be collected and the sample economy is high The programming of the exit valve is described in detail 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 collected IMPORTANT The volume diverted to waste should be a minimum of 3 5x the flow line volumes section 9 2 to ensure only uncontaminated sample is collected Larger volumes may be necessary and it is recommended that test experiments be performe
23. all the syringes are selected 5 1 3 Software Syringe control from Bio Kine software is made through the button in the stopped flow status area see Figure 21 The MPS is then initialized and communication established between Bio Kine software and the MPS unit The message MPS on line is displayed in a green window in the stopped flow status area The syringe to be moved is selected by clicking on the corresponding frame or pressing the Left or Right arrows keys on keyboard The new selected syringe will be surrounded by a red rectangle Figure 21 Syringes are emptied or filled using the and buttons or with the lt Up gt and lt Down gt arrows The E button moves a syringe upwards by one elementary movement and the button moves a syringe downwards by one elementary movement The button moves the piston upwards by 10x elementary movements and the Z button moves 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 window Figure 21 Press the 81 and P gt buttons to change the manual speed The display shows the speed in flow rate based on the syringe installed and moved 32 SFM 300 400 User s Manual ver 2 7 syringes command Syringe Control V in pl Syringe 2 Syringe 3 B E 1 2
24. amplitude 68 Flow rate 69 7 TEST REACTIONS IN STOPPED FLOW MODE 70 Reduction of 2 6 Dichlorophenolindophenol by Ascorbic Acid 70 Evaluation of the Dead Time 70 Evaluation of Washing and the Quality of the Stop 74 Variable Ratio Mixing 75 7 4 SFM 300 400 User s Manual ver 2 7 75 7 44 Reduction of DCIP by ascorbic acid 74 2 Alcohol dehydrogenase activity 78 7 5 Mixing Solutions of Unequal Density and Viscosity 8 Technical section 79 81 81 Solvent compatibility 8 2 Mixer Removal Examination and Replacement 8 3 Lubrication 8 4 Thermostated Bath 9 INSTALLATION OF THE QUENCHED FLOW COMPONENTS 91 Installation of the Mixer Blocks and Delay Lines 9 2 Flow Line and Intermixer Volumes 93 Sample Collection Methods 9 3 1 Total liquid collection 81 81 82 82 83 83 86 88 88 9 3 2 Partial liquid collection 89 11 SOFTWARE CONFIGURATION IN QUENCHED FLOW MODE 11 1 Device installation using PMS 60 and Bio kine version up to 4 45 90 90 11 2 Device installation using MPS 60 or MPS 70 with Bio kine version 4 47 and higher 4 49 90 11 3 Stopped flow Configuration 11 4 Stopped flow status area 12 INSTRUMENT OPERATION IN QUENCHED FLOW MODE 12 1 Manual Syringe Control 12 1 1 MPS 91 92 94 94 94 12 1 2 Software 94 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 q
25. and 2 Delay line must be chosen carefully so enough volume can be incubated in phase 2 for second mixing The choice will be done depending on mixing ratio and cuvette chosen Bio logic usually advise using 120 or 220 yl delay line to M2 cover ageing time range from 10 ms to several minutes for shorter ageing time we would recommend doing experiments in continuous flow mode Ageing time M1 Delay line Structure of the sequence The mixing sequence is devided into 3 phases first mixing and washing of the delay line Phase 2 Incubation time to age mixture in ageing line Phase 3 second mixing 124 52 53 Design of the sequence The user must select the mixing ratio 51 52 to apply in phase 1 and the mixing ratio in the second mixer phase 3 In phase 3 the delay line is emptied using 51 and or 52 It is the user s choice usually it would be the less valuable sample to save expensive material Biokine proposes to the user a driving sequence which takes into account the volume of the cell the type of mixer and the size of the delay line Depending on flow rate selected both the theoretical dead time in the second mixer and the minimum ageing time are displayed Theoretical dead time Volume of the cell Flow rate in phase 3 Minimum ageing time Volume of delay line 7 Flow rate in phase 1 Automation Once the sequence is set the user just needs to enter the ageing times he wants to reach Biokin
26. concentration steps mode Ratio step manual mode By selecting Ratios steps Manual in the Mode window you have an access to the window menu then jEdit Sequence by clicking on the button the following sequence is edited or the latest sequence is automatically loaded as a default one 55 SFM 300 400 User s Manual ver 2 7 Fl Concentration dependance studies Concentration dependance studies Content of syringes Initial concentration Mixer 2 conditions Syringe 1 E 3 ha Ratio Diluant 3 Concentration Amas 05 mM Syringe 2 I I 35 mM Ratio B D uv Concentration 05 mM Syringe 3 m Total flow rate 100 Estimated dead time 15 ms Variation Ratio in Mixer 1 Start next acquisition step Ratio 4 Ratio Diluant a atthe end of measurement Step number C alte 5 sec Step value Start of data sii Atstop Pre trigger SFM Options ntos Edit Sequence Manual ratio mode Step RatioDi RatioA Concentj amp VolumeDiL VolumeA Volume B 5 manual continuation d Load Print Global sequence Jp Z 5 es eee j ees ees Ja p 55 4 eee AD8 D3B D B5 h 85 U U BE IK kb lire Figure
27. 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 time course of the reaction from the dead time onward CUVETTE A B Figure 61 A Simple Stopped Flow Experiment Figure 61 shows a schematic of a simple stopped flow experiment In the experiment reagents A and B 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 62 shows a cartoon of the experiment over time Note the two stages of the experiment as described above 66 SFM 300 400 User s Manual ver 2 7 STAGE 1 SHOT STAGE 2 REACTION 5 Figure 62 Stopped Flow Experiment Time course 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 samples are contaminated and the resulting signal trace does not represent the true time course of the reaction 6 2 General Advice for Stopped Flow Experiments 6 2 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
28. made from the same window e Load a sequence using the _ toad button eSave a sequence using the S button e Print a sequence using the LP button e Close a sequence using the Cos button Comments a text window is opened by clicking on the Comments button Comments will be saved with the sequence 5 6 3 Programmable synchronization trigger using the MPS 60 Contrary to the classic mode only Synchroout 1 can be used in the advanced mode In this mode the hard stop cannot be controlled manually from Synchro out 2 The use of Synchro out 1 will result in an incorrect trigger signal and lose of signal Synchro out 1 is a falling trigger 5 gt 0 V The acquisition will start at the end of the pushing phase or few milliseconds before according to the configuration chosen by the user in the driving sequence 5 6 4 Programmable synchronization trigger using the MPS 70 Only Trigger can be used in the advanced mode In this mode the hard stop cannot be controlled manually from Synchro out The use of Synchro out will result in an incorrect trigger signal and loss of signal Trigger is a falling trigger 5 0 V The acquisition will start at the end of the pushing phase or few milliseconds before according to the configuration chosen by the user in the driving sequence 39 SFM 300 400 User s Manual ver 2 7 5 7 Creating a Driving Sequence using the classic mode Experiments are performed with the SFM through the us
29. 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 Figure 11 SFM 400 S Flow Line and Delay Line Volumes 23 SFM 300 400 User s Manual ver 2 7 3 6 Liquid Outlet System Hard Stop Valve 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 must be immobilized in a fraction of a millisecond Several different stop modes can be used to immobilize the liquid column The stop mode used can result in overpressure or underpressure conditions that are potential sources of stop artifacts The mode chosen by Bio Logic is presented below the 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 power 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 toMPS x a 2 i to waste tube Hard stop ss Head nut 2 1 74 5 Ouvette holder Cuvette Obse
30. requires that no bubbles are present in the injection syringes If this occurs 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 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 92 1 Attach a syringe disposable plastic syringes may be used containing a sample or buffer to a syringe reservoir port on top of the SFM Figure 92 Panel 1 2 Set the syringe valve handle to R and fill the syringe manually section 12 1 while exerting slight pressure on the reservoir syringe Figure 92 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 10 syringes be filled using manual speed 4 in Bio Kine and 1 9ml syringes be filled using manual speed 2 3 Eliminate any bubbles the SFM syringe by driving the SFM syringe up and down several times while it is connected to the reservoir syringe Figure 92 Panel 4 4 Turn the syringe valve handle to C Figure 92 Panel 5 5 Empty by one or two elementary movements of the syringe section 12 1 to definitively eliminate any bubbles remaining in SFM and cuvette 6 Repeat the abo
31. situation is commonly found when the kinetic of protein refolding renaturation is to be measured Figure 75 shows the result of an experiment performed with cytochrome c Experimental Conditions Buffer 100 mM NaCl 20 mM MOPS pH 7 5 Syringe 1 10 ml Buffer Syringe 2 10 ml Buffer St 3 10 50 uM cytochrome c in 5 5 guanidine HCl 20 mM MOPS pH Wavelength 290 nm Cuvette FC 15 Detection method Fluorescence 320 nm cutoff filter Temperature 25 C Cytochrome c denatured in 5 5 M guanidine HCI was 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 Figure 75 79 SFM 300 400 User s Manual ver 2 7 The curve in Figure 75 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 transition respectively The fit is shown as a dotted line under the experimental curve T d Q N gt S E s o 9 S B ir Figure 75 Cytochrome c Renaturation WARNING 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 t
32. 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 syringe control window IMPORTANT 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 HEE and the syringe must be reinitialized To avoid such accidents the Up and Low Limits checkbox may be checked When this box is checked Bio Kine 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 95 SFM 300 400 User s Manual ver 2 7 WARNING The Up and Low Limits only applies to control of the syringe from within Bio Kine These limits can be bypassed by manual control of the SFM directly from the MPS 12 3 Filling the Syringes WARNING Utmost care should be exercised during this operation Normal operation of the system
33. time according to the equation _ A t AQ A 0 Where A t is the absorbance at 325nm at ageing time t 0 is the absorbance at tage 0 and is the absorbance at tage 114 SFM 300 400 User s Manual ver 2 7 2 Using Y from step 1 above and the pseudo first order rate constant measured calculate the actual ageing time Ta for each point from Lnlc o Lale k Ta 1000x Ln 0 5 Ln 0 5 x 1 y k Ta 1000x Where 0 is the DNPA concentration at tage 0 0 5 mM for the experiment in the previous section and C t is the DNPA concentration at time t and k is in 5 The units of Ta ms 3 The hydrodynamical intermixer volume M2 M3 can then be calculated from EN volume Ta Where F is the total flow through the intermixer volume in ul 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 due to an inefficient washing phase of previous reacted sample coming from the flow lines and intermixer volumes before sample collection The simplest method of determining the volume needed to create an efficient wash or purge phase between the flow lines and intermixer volumes is to carry out multi
34. 0 0 Max Vol ml 5 Piston Diameter mm 15 Screw Pitch mm 4 Modity Add Suppress Figure 89 custom syringe WARNING Incorrect syringe configuration will cause volume and flow rate calculations to be incorrect 11 4 Stopped flow status area A vertical menu bar on the left of the screen is dedicated to the quenched flow device see Figure 90 This menu bar can be hidden or displayed using the srm button in the main menu This menu bar gives access to the syringe control window using the button Mixing refer to section 12 1 and to the classic mode using the poen button refer to section 12 6 The advanced mode is not available in the quenched flow configuration The volumes of the delay lines installed are indicated in SFM option Once the sequence is ready in the driving sequence window the shot control window is displayed in the area as shown in Figure 90 92 SFM 300 400 User s Manual ver 2 7 Stopped flow SFM 20 5 Figure 90 stopped flow menu bar 93 SFM 300 400 User s Manual ver 2 7 12 INSTRUMENT OPERATION IN QUENCHED FLOW MODE 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 d
35. 0 10 Detection method Absorbance Total Flow Rate ml s 8 pH 9 reaction 8 2 reaction The dead time of the experiment is the age of the solution at the observation point In other words it is the time for the mixed solution to go from the centre of the last mixer to the observation point The dead time depends on many factors besides simply the total flow rate and the cuvette volume But because the hydrodynamics phenomenon is difficult to be taken into account for software calculations a slight difference between the estimated dead time given by Bio Kine and the real dead time may be observed In basic pH conditions the reaction is considered as a slow reaction Therefore the amplitude of the signal at the stop can be assimilated to the total amplitude of the reaction In other words the change in absorbance between the mixing point and the observation point is negligible In acidic conditions the reaction is much faster and the change in absorbance between the mixing point and the observation point cannot be neglected The amplitude of the signal at the stop corresponds to the age of the solution So knowing the amplitude of the signal and its rate constant in addition to the total amplitude measured with the slow reaction results it is possible to determine the real dead time of the experiment 70 SFM 300 400 User s Manual ver 2 7 A simplified drawing of the method used for the dead time calculation is given in Fig
36. 0 or MPS 70 3 4 using Bio Kine version 4 47 and higher 28 SFM 300 400 User s Manual ver 2 7 4 3 4 4 5 INS 5 1 25 14 5 1 2 5 1 3 5 2 5 3 5 4 5 5 5 6 5 6 1 5 6 2 5 6 3 5 6 4 5 7 2414 5 72 3 4 3 5 7 4 5 8 5 9 5 9 5 9 2 5 10 6 1 6 2 6 2 1 6 2 2 6 2 3 6 2 4 7 1 7 2 7 3 Stopped flow Configuration 29 Stopped flow status area 31 TRUMENT OPERATION IN STOPPED FLOW MODE 22 Manual Syringe Control 32 MPS 60 32 MPS 70 32 Software 32 Syringe Initialization 33 Filling the Syringes 34 SFM Cleaning and Storage 36 Long term Storage of the SFM 36 Creating a sequence using the advanced mode 36 SFM options 36 Design of stopped flow sequence 38 Programmable synchronization trigger using the MPS 60 39 Programmable synchronization trigger using the MPS 70 39 Creating a Driving Sequence using the classic mode 40 SFM options 40 Design of the sequence 41 Programmable synchronization trigger using the MPS 60 44 Programmable synchronization trigger using the MPS 70 44 Creating a double mixing experiment 45 Creating a Driving Sequence using concentration dependence studies 51 Creating driving sequences using SFM 300 51 Creating driving sequence using SFM 400 57 Running a shot 65 6 ASHORT STOPPED FLOW PRIMER 66 General Principle of Experiments with the SFM 66 General Advice for Stopped Flow Experiments 67 Achievement of fastest dead times 67 Washing 68 Signal
37. 1 gt Phase 1 conditions Phase 3 conditions Start next acquisition step Ratio A 1 Empty Delay line by using Syringe atthe end of measurement RatioB 1 Ratio Delay line 1 Ratio C 1 C after 5 sec Total flow rate 100 Stat of dila acus Estimated deadtime 1 6 ms Minimum ageing time 95 Atstop Pre trigger Load F Save A SFM Options ntos Sequence Global sequence Structure of the sequence Ageing times Phea Pass 5 Age ms EmEI WE Repeatnumber 1 Totalvolme Syringe Onl Bl On cl On Ready P Close Figure 35 Double mixing experiment main window using SFM 300 S gt Double mixing experiments part Figure 36 The content of syringes and the initial concentration has to be entered Thus the final concentration will be calculated the final concentrations change in function of the ratio assessed see below In the Phase 1 conditions window enter the Ratio A and ratio B In the Phase 3 conditions window select the syringe s used in order to empty the delay line Product from the reaction of A B is stored in the delay line and then mixed with the C solution To empty the delay line of the aged solution A or B or A B can be used This procedure is particularly useful to save a precious solution during this flushing phase Asse
38. 1ms V low limite Figure 21 syringe control window 5 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 see Figure 21 When the MPS is turned on and the software started the syringe volume counters show MEE and must be initialized Figure 21 The syringes are initialized by setting the syringes to their uppermost empty position and resetting the syringes in Bio Kine The syringes can be selected and moved to their uppermost positions either directly with the MPS section 0 or through Bio Kine section 0 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 syringe control window IMPORTANTS 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 syri
39. 48 Global sequence in ratio steps manual mode The Global sequence is created through the following table Click on button to create a step then type the ratio in Ratio A that will be used during the step and indicate the volumes All the volumes volume Dil volume A and volume B have to be more than 40ul 1 Click on 3 button to create a second step 2 Click on 4 to clear all the sequence or to remove step 56 SFM 300 400 User s Manual ver 2 7 Global sequence Manual ratio mode arusti r J fF fF fF fF U y y O 353 fF fF fF fF U y 1 11 1 Ff if 3m 5 8 1 8 fF 11 fe Repeat number 1 Total volume Syringe 100 yl 50 pl 150 pl Ready P Close Figure 49 Global sequence window 1 lt The functions of the different buttons the following is used to create or add step is used to remove step K4 is used to remove all the sequence S si is used to move up or down a step is used to validate the sequence Notice that a stored sequence can be automatically load by clicking on load button Load 5 9 2 Creating driving sequence using SFM 400 The aim of this sequence is to observe different kinetic curves by increasing automatically step by step the concentration of one reactant A against B in A B reaction Thus the concentration of B
40. 7 N 2 40 N 3 90 N 4 140 N 5 190 500 N 7 1000 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 Figure 83 SFM 400 Q Flow Line and Delay Line Volumes 87 SFM 300 400 User s Manual ver 2 7 9 3 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 9 3 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 described in the next two sections 9 3 1 1 Free flow method A tube is connected to the waste outlet of the exit valve to recover all the liquid exiting the SFM Figure 84 The liquid may be ejected into a test tube or beaker for simple collection or for quenching with an external solution If the latter method is used the tube acts as an additional delay line whose volume can be adjusted by the user Connect to Tube Figure 84 Exter
41. SFM 300 400 User s Manual Version 2 7 SFM 300 400 User s Manual ver 2 7 TABLE OF CONTENTS 1 INTRODUCTION AND SPECIFICATIONS 6 11 General Description 6 1 1 1 The mechanical designed 6 1 1 2 Intelligent power supply 6 1 1 3 Microcomputer commands 6 1 2 Modes of Operation 7 1 22 Stopped Flow SF mode commercial reference SFM X00 S 7 1 2 2 QUENCHED FLOW QF MODE commercial reference SFM X00 Q 7 1 3 Specifications 9 1 4 Principle of Operation 10 1 5 Description of the Mechanical Design 10 1 6 The Delay Lines 10 2 GENERAL INSTRUCTIONS FOR INSTALLATION 11 21 Operating Features with MPS 60 11 2 2 Power and Connections of MPS 60 12 2 3 Operating Features MPS 70 12 2 4 Power and Connections of MPS 70 15 2 5 Temperature Regulation 15 3 INSTALLATION OF THE STOPPED FLOW COMPONENTS 16 31 The Observation Head 16 3 2 Mixer Installation and Replacement 16 3 3 Cuvette Installation 16 3 4 Installation of the Mixer Blocks and Delay Lines 19 3 5 Flow Line and Intermixer Volumes 22 3 6 Liquid Outlet System Hard Stop Valve 24 3 7 Special Accessories 24 3 7 1 Small drive syringe 24 3 7 2 High density mixer 25 3 7 3 Observation head with separate cooling 26 3 7 4 MICROCUVETTE ACCESSORY 26 3 1 5 LOW TEMPERATURE ACCESSORY 27 4 SOFTWARE CONFIGURATION IN STOPPED FLOW MODE 28 41 Installation SFM 300 400 with MPS 60 using Bio KIne version up to 4 45 28 4 2 Installation SFM 300 400 with MPS 6
42. Times for a driving sequence is also displayed in the driving sequence window and the calculation is made as shown in Figure 97 The ageing times are calculated for the current phase selected based upon the syringes flow rates delay lines installed and intermixer volumes Figure 82 and Figure 83 Intermixer volume 1 2 300 Ageing time Total Flow Rates 81 82 Ageing Time DLI 51ms DL2 28ms Intermixer volume 1 2 5 400 Ageing time DLI Total Flow Rates 81 82 Intermixer volume 2 3 Ageing time DL2 Total Flow Rates 81 82 53 Figure 97 Ageing Times Bio Kine provides the ability to repeat phases a number of times in virtually any order This is accomplished though a macro sequence entered in the Driving Sequence window shown in Figure 98 The macro sequence can be edited to run a single phase or many phases in a different order than present in the program grid 1 1 20 Figure 98 Sequence Macro 101 SFM 300 400 User s Manual ver 2 7 Standard operations can be made from the same window Load a sequence using the _ Load button eSave a sequence using the Se button e Print a sequence using the P putton Close a sequence using the Ces button Comments a text window is opened by clicking on the Comments button Comments are saved with the sequence 12 7 Running a shot Once a driving sequence has been entered or load
43. VENT 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 PRODUCT OR REPAIR WITH REGARD TO ANY INSTRUMENT RETURNED BECAUSE OF A 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 BILLED AT NORMAL COST IN SUCH CASES AN ESTIMATE WILL BE SUBMITTED BEFORE WORK IS STARTED IN CASE ANY FAULT OCCURS NOTIFY BIO LOGIC OR THE 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 SFM 300 400 User s Manual ver 2 7 1 INTRODUCTION AND SPECIFICATIONS 1 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 configurations SFM 300 The mechanical sub system consists of three machined syringes and one valve block with 3x3 way valves with the possibility to include one or two mixers and one ageing loop SFM 400 The mechanical sub system consists of four ma
44. ad times compromises may be necessary to achieve a successful stopped flow experiment Table6 shows some of the most common actions that can be taken to improve signal amplitude and their consequences Table6 Common Actions to Improve Signal Amplitude overuse of reagent Increase Cuvette Path increased dead time Length Inadequate washing Inner filter effect fluorescence overuse of reagent Increased viscosity causing cavitation Increase Reagent Concentration Increased viscosity causing stalled motors Increase viscosity causing inadequate washing 68 SFM 300 400 User s Manual ver 2 7 6 2 4 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 8ml s 10 ml syringes with 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 69 SFM 300 400 User s Manual ver 2 7 7 TEST REACTIONS IN STOPPED FLOW MODE 7 1 Reduction 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 ab
45. add a step BH is used to remove a step K4 is used to remove all the sequence l is used to move up or down a step is used to validate the sequence Notice that the latest sequence is automatically load in this case the manual mode permits the user to change the parameters or by clicking on K4 to remove all the sequence 64 SFM 300 400 User s Manual ver 2 7 5 10 Running a shot Once a driving sequence has been entered or loaded it is transferred to the MPS by pushing the Single or Multiple buttons in classic mode or Ready in advanced mode The MPS is now in automatic mode and the shot control window appears in the stopped flow status as shown in Figure 60 The Shot control window shows 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 button or the start stop button on the front panel of the MPS The button can be used to stop an experiment prematurely if necessary If the Single button was used to transfer the driving sequence to the MPS only a single shot be made The LES button must then be pushed to return to the driving sequence and the Single button must be pushed again to re transfer the driving sequence to the MPS for a subsequent shot If the Multiple buttons was used to transfer the driving sequence to the MPS the lll ton
46. advanced mode starting the acquisition can be done at the stop of the motors or few milliseconds before the stop this time is fixed at 20 ms and cannot be changed Start next acquisition step atthe end of measurement C after 5 sec C manual continuation Start of data acquisition Atstop Pre trigger Figure 54 acquisition parameters Ratio step Auto mode The variations of the ratios between Diluant and reactant A in mixer M2 have to be fixed by typing a value in Ratio A i e 1 in the example choosing the steps numbers and the value of the step Variation Ratio in Mixer 2 Ratio 1 Ratio Diluant 2 Step number 5 Step value n Final value 34 Ratio steps Auto Concentration steps v Figure 55 Ratio steps Mode 60 SFM 300 400 User s Manual ver 2 7 Edit Sequence sequence is edited by clicking on the button as following H Concentration dependance studies Concentration dependance studies Content of syringes Initial concentration Syringe 1 Water Syringe 2 Acid Ascorbic 49 500 fim Syringe 3 DCIP B0 130 uM Mixer 2 conditions Ratio Diluant ea Ratio B En Total flowrate 100 mis Concentration 250 uM Concentration B 65 0 pM Estimated dead time 1 6 ms Ratio steps Concentration steps i Variation Ratio in Mixer 1 Start next acquisition st
47. ation Ratio in Mixer 1 Start next acquisition step Ratio 1 Ratio Diluant 20 atthe end of measurement Step number 5 C after 5 sec Step value 01 Start of data acquisition Final value 14 Atstop Pre trigger Load Print Save SFM Options Edi Sequence Figure 43 Mixing sequence manual continuation Then the conditions in Mixer 2 have to be entering in the following window Mixer 2 conditions Ratio A Diluant 3 Ratio B 3 Total flow rate 10 0 ml s Figure 44 Ratio in mixer 2 These conditions correspond to a 1 to 1 mixing sequence in Mixer 2 with a flow rate of 10 ml s The concentration maximum of A Amax is automatically calculated while the concentration of B reactant is maintained as a constant value There are two ways to increase the concentration of A the first is done by the increase of the ratio in mixer 1 step by step The second is done by increasing the concentration of A step by step Ratio step Auto mode The variations of the ratios between Diluant and reactant A in mixer M1 have to be fixed by typing a value in Ratio A i e 1 in the example choosing the steps numbers and the value of the step Mode Variation Ratio in Mixer 1 Ratio steps Auto Ratio 1 Ratio Diluant 20 Step number Concentration steps uto Step value 01 Final value 1 4 Figure 45 Ratio steps Mode 23 SFM 300 400
48. ation set to none both Synchro out 1 and 2 are available 5 7 4 Programmable synchronization trigger using the MPS 70 The MPS can be programmed to deliver synchronization pulses triggers These pulses are TTL pulses 0 or 5 Volt and delivered from BNC connector Trigger out Synchro in and Synchro out on the rear panel of the MPS see Figure 2 b Trigger and Synchro in out are falling edge triggers 5 0 V Trigger out is used for most of the acquisition devices sold by Bio Logic The triggers can be used to synchronize the SFM and data acquisition system or other instruments If Bio Kine software is being used for data collection acquisition will start on the falling edge of the synchronizing pulse When the Trigger is used then the acquisition will start at the beginning of the first phase with a synchro set to On The triggers can also be used for synchronizing the SFM with other devices 44 SFM 300 400 User s Manual ver 2 7 The timing of the triggers with respect to the drive sequence is programmed in the last line s of the program grid in the driving sequence window refer to Figure 29 The duration of the pulse will be equal to the duration of the phase Synchro out is used to control the hard stop when the hard stop is programmed by the user configuration set to manual If the hard stop is not used configuration set to none both Synchro in out are available 5 8 Creating a double mixing ex
49. c and inorganic liquids Only concentrated acids like sulfuric and nitric can attack it Methylene chloride DMSO and THF has some swelling effect should be used under control Maximum operating temperature 100 Teflon is chemically inert Viton parts these parts are most vulnerable chemically Other materials are available upon request EPDM Nitrile Isolast Please contact our commercial service for enquiries We highly recommend Isolast o ring with an organic solvent Please refer to Isolast http www superseal hu al catalogs busak shamban isocatal pdf for chemical compatibility guide Using a solvent with a non appropriate o ring material will be not considered under warranty by Bio Logic 8 2 Mixer Removal Examination and Replacement Removal and replacement of the mixer in the observation head is described below using Figure 76 Removal 1 2 Unscrew the nut on top of the observation head and remove it 3 4 Remove the observation head from the SFM body via the four screws at the corners of the observation head Remove any observation head caps or collimators Remove the cuvette holder and attached cuvette 5 Insert a flat end pin diam 1 mm or paper clip through the bottom side of the observation head and gently push out the mixer and the o ring 2 2 x 1 6 81 SFM 300 400 User s Manual ver 2 7 _ Observation Head gt Collimator Cuvette
50. can be used to execute shots until the shot window shows that 0 shots remain The nd button is then pushed to return to the driving sequence Shot Control Figure 60 shot control window 65 SFM 300 400 User s Manual ver 2 7 6 A SHORT STOPPED FLOW PRIMER 6 1 General Principle of Experiments with the SFM There are many variations on the stopped flow experiment such as multiple mixing continuous flows and accelerated flow However the simplest stopped flow experiment occurs in two stages In the first stage the flow is initiated by two plungers The plungers force liquid 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 a particular age Furthermore the age of the mixture in the cuvette at the point of observation during the shot is the theoretical dead time the time before which observation of the mixture is impossible The second stage of the
51. cessory consists of an umbilical connector and a mixing compartment e The umbilical connector allows for a fluidic connection between SFM 300 400 and the observation cell This allows for a physical separation of these two parts of the stopped flow It is the case when working with far subzero temperatures the mixing unit and the solution immerged in PEEK tubes can be cooled by the cryothermostat while the rest of instrument can be kept at a more normal emperature The fluid used for regulating the stopped flow temperature also circulates into the umbilical connector keeping the active solutions at a constant temperature until the end of the connector Mixer and observation cell assembly this part of the setup is immerged in the final cryosolvent It contains a flow lines block that serves as a reactant reservoir These flow lines are made of inert material PEEK and are of sufficient volume so as to act as a heat exchanger and allow equilibration of the reactants at the temperature of the cryosolvent before injection in the observation cuvette In the standard configuration the volume of these lines is 200 ul but can be easily adapted The mixer is fitted to the observation cell which can be any the standard stopped flow cell Figure 7 Detection is made through optical fibers which are installed on the cuvette These optical fibers are protected from the cryosolvent by umbilical tubes It is also possible to flush nitrogen or inert gas th
52. ch time a program grid cell value is changed information about the current syringe current phase and driving sequence which is displayed below and to the right of the grid is updated Figure 31 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 42 SFM 300 400 User s Manual ver 2 7 n Stopped Flow Program Phase 1 Phase 2 Phase 3 Phase 4 Phase 5 Total time ms Syr 1 ul Syr 2 ul Syr 3 ul Syr 4 pl Synchro 1 Phase 1 5 Total Volume 400 pl Total Flow Rate 4 0 ml s 1 2 Figure 31 Driving Sequence Information An indication of the Dead Time and Ageing Times is shown in the driving sequence window Figure 32 The dead time is calculated for the last valid phase according to its flow rate and of the cuvette dead volume Figure 7 The dead time is calculated according to the equation show in Figure 32 Dead Time 08 Dead Time Cuvette Dead Volume Total Flow Rate Figure 32 Estimated dead time The ageing times are calculated for the current phase selected based upon the syr
53. chined syringes one valve block with 4x3 way valves and 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 1 1 11 The mechanical desianed 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 Teflon 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 1 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 their own microprocessor The sequences 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 in
54. cribed in the Quenched Flow section of this manual SFM 300 S FLOW LINE VOLUMES Line Number Flow Line Volume ul 69 7 89 88 10 Delay Line 19 108 Cuvette Figure 7 CUVETTE MIXER1 MIXER2 DELAY LINE RESERVOIR1 RESERVOIR2 RESERVOIR3 SYRINGE 1 SYRINGE 2 SYRINGE 3 Figure 10 SFM 300 S Flow Line and Delay Line Volumes DELAY LINE AND INTERMIXER VOLUMES Delay Line 1031 1027 Notes 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 22 SFM 300 400 User s Manual ver 2 7 SFM 400 S FLOW LINE VOLUMES Line Number _ Flow Line Volume ul 69 7 89 88 7 Delay Line 1 13 94 10 Delay Line 2 19 108 Cuvette Figure 7 CUVETTE 13 MIXER2 MIXER1 MIXER3 DELAY LINE 1 DELAY LINE 2 RESERVOIR1 RESERVOIR2 RESERVOIR3 RESERVOIR4 SYRINGE 2 SYRINGE 3 SYRINGE 4 SYRINGE 1 DELAY LINE AND INTERMIXER VOLUMES None N 1 17 N 2 40 N 3 90 N 4 140 N 5 190 N 6 500 7 1000 Delay Line 1003 1023 1031 1027 Notes Intermixer volumes are measured from the mixing point of one mixer to the mixing point of the next
55. d to optimize the volume needed to minimize sample contamination Figure 85 Pipette Syringe Collection 89 SFM 300 400 User s Manual ver 2 7 11 SOFTWARE CONFIGURATION IN QUENCHED FLOW MODE The SFM is controlled by Bio Kine software which is also used to control acquisition parameters This section precisely describes the configuration of the software Please note that the procedures and examples have been generalised and configuration choices should be made based upon the equipment purchased and intended experiments This section assumes that the user has already installed Bio Kine software on the host microcomputer 11 1 Device installation using 5 60 Bio kine version up to 4 45 Once Bio Kine loaded choose Install device installation in the main menu The stopped flow communication is established from this window by checking the stopped flow device box and choosing the corresponding Serial port Accept the parameters using the OK button Device Installation Acquisition device 7 r Stopped flow device 05 450 J 810 serial acquisition 7 Use stopped flow J 810 analog acquisition Stopped flow communication 05 200 External device TIDAS diode array Serial Port coM2 v Acquisition parameters Accessories Serial Port P Peltier temperature controller Acquisition Board PCI 6052E board detected OK Cancel Figure 86 device installation
56. depends on a number of factors only some of which the researcher can control Ideally the dead time depends only on the flow rate of the mixture exiting the last mixer and the volume 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 an adequate signal complete washing of the cuvette and prevention of cavitations and prudent use of valuable reagents The relationships between these factors require careful consideration and experimentation Compromises are often necessary to achieve successful stopped flow experiments Some of the most common actions that can be take to achieve the fastest dead times and their consequences are shown Table5 Table5 Common Actions to Achieve Fastest Dead Times stalled motors cavitations Increase Flow Rate overuse of reagent inadequate washing Decrease Cuvette volume loss of signal 67 SFM 300 400 User s Manual ver 2 7 6 2 2 Washing As mentioned in section 6 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
57. e 1 17 43 0 Delay Line 2 pl n4 140 168 6 r Ejection delay Line pl zi Figure 94 SFM options cuvette not available in quenched flow configuration e HDS mixer not available in standard please contact our commercial service for special application e Valve Lead This section of the window allows one to enter the number of milliseconds before the flow stops that the valve 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 The precision of the setting is 0 1 ms Overheating Protection Not applicable for the MPS 60 The default mode is checked It is a protection against electronic overheating after a long working day 99 SFM 300 400 User s Manual ver 2 7 Hard stop not available is this mode Delay lines 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 3 4 Each delay line is chosen from a pull down menu WARNING An incorrect delay line configuration will cause ageing time calculations to be incorrect 12 6 2 Design of the sequence A driving sequence is entered in the program grid shown in Figure 95 Each column of the grid represents a driving sequence phase Each phase contains actions for the SFM to perform A complete driv
58. e Hard stop 9 10 11 SFM 300 400 User s Manual ver 2 7 Table 3 MPS 70 Panel Descriptions NAME I LCD DISPLAY 2 SYRINGE SELECTOR START STOP MANUAL UP DOWN MOVEMENT SYNCHRO IN 3 SYNCHRO OUT TRIGGER OUT MAIN POWER SWITCH 9 TEMPERATURE PROBE CONNECTOR 14 HARD STOP SF BNC CONNECTOR MOTOR POWER CONNECTOR 2 USB CONNECTOR FUNCTION Used to display messages selected syringe auto mode Selects the syringe for the manual control Initiates or stops the programmed sequence in the automatic mode The instrument may also be started and stopped using the keyboard of the PC Manual up and down movement control of the syringes Input for an external signal to trigger the drive sequence TTL Pulse output for special application TTL Pulse output to trigger the recording system or any electronic device to be synchronized with the instrument Connects to temperature probe Connects to Hard Stop Valve Sends the power pulses to the stepping motors Connects the MPS 70 controller to the PC SFM 300 400 User s Manual ver 2 7 2 4 AC Power and Connections of MPS 70 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 70 unit Connect the MPS 70 to the USB port
59. e SFM body and the observation head and the SFM 400 functions as an SFM 300 Figure 8 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 Table 4 SFM 400 Observation Head Installation SFM 300 400 User s Manual ver 2 7 20 OBSERVATION HEAD SECOND MIXER ERN M2 DELAY LINE Figure 8 SFM 300 Installation of Delay Lines SFM 300 400 User s Manual ver 2 7 OBSERVATION HEAD THIRD MIXER 1 HEAD ei DELAY LINE TWO a DL2 MIXER BLOCK m Dja SECOND MIXER Ec M2 S MIXER BLOCK m DELAY LINE ONE l DL1 Figure 9 SFM 400 Installation of Mixing Blocks and Delay Lines 21 SFM 300 400 User s Manual ver 2 7 3 5 Flow Line and Intermixer Volumes Figure 10 SFM 300 and Figure 11 SFM 400 below indicate the volumes of the 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 given in the table are the mechanical volumes The hydrodynamical volumes may vary slightly around these values For a precise measurement of ageing times it is recommended that the intermixer volumes be determined experimentally with known reactions One such experimental procedure for determining the intermixer volumes is des
60. e 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 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 13 1 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 100 shows a schematic of a quenched flow experiment The reaction considered is gt Where the reaction be stopped at any time by the addition of quencher Q Mix n the first stage flow is initiated by two plungers The plungers force the reactants A and B through a mixer where they are mixed and the reaction initiated and starts to produce Age Inthe second stage the plungers push the sample reaction mixture through delay line to the second mixer The sample ages reacts as it travels through the delay line until it reaches the second mixer where it is quenched Quench As the sample
61. e 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 5 all syringe valve handles to 6 off the MPS 5 5 Long term Storage of the SFM If the SFM will not be used for a long period of time more than several weeks it should be cleaned as explained in section 5 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 for storage 5 6 Creating a sequence using the advanced mode An advanced menu was created to improve the friendliness of the design of the stopped flow sequence and to optimise experimental settings in order to get the best quality results This mode can only be used for a single mixing experiment It is still necessary to use the classic mode for a double mixing experiment or to perform pre washing phase refer to section 5 7 in this case The advanced mode must be selected in the stopped flow configuration see 4 4 Mixing then click on o in the stopped flow status area The window shown in Figure 23 appears 36 SFM 300 400 User s Manual ver 2 7 FA Mixing sequence m Mixing ratio Volume Total flow rate
62. e handle to R and fill the syringe manually section 5 1 while exerting slight pressure on the reservoir syringe Figure 22 Panel 2 and 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 10 syringes be filled using manual speed 4 in Bio Kine and 1 9ml syringes be filled using manual speed 2 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 22 Panel 4 4 Turn the syringe valve handle to C Figure 22 Panel 5 5 Empty one or two elementary movements of the syringe section 5 1 to definitively eliminate any bubbles remaining in SFM and cuvette 6 Repeat the above process for the other syringes It is recommended that the syringes be filled in reverse numerical order to best remove bubbles from the SFM and cuvette 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 Stopped Flow Module is now ready for operation 34 SFM 300 400 User s Manual ver 2 7 Panel 4 Figure 22 SFM Syringe filling procedure 35 SFM 300 400 User s Manual ver 2 7 5 4 SFM Cleaning and Storage After each day s experiments the SFM should be cleaned A th
63. e of a driving sequence A driving sequence tells the SFM to automatically perform several functions such as moving the syringes activating the hard stop and triggering the data acquisition Driving sequences are created in the window shown in Figure 27 The classic mode must be selected in stopped Mixing flow configuration see 4 4 Then click on EM in the stopped flow status area n Stopped Flow Program eli xj Phase 1 Phase 2 Phase 3 Phase 4 Phase 5 Total J Volumes time ms E Insert Phase Syr 1 ul Remove Phase Syr 2 New Syr Syr 4 pl Synchro 1 Synchro 2 1 5 Total Volume 310 pl Total Flow Rate 6 2 ml s Syringes contents Shots Drive Sequence Ageing Times Single 1 1 20 DL1 270ms Dead Time DL2 12 0 ms Multiple 439m EM Load Save Comments Print SFM Options Close Figure 27 driving sequence in classic mode First operation should be to check the configuration of the stopped flow which is done by clicking on the SPMOpions button refer to Figure 27 x Cuvette Hard Stop Acceleration Phases Delay Line 1 i Automatic nt 17 43 1 Manual Manual Delay Line 2 pl Valve Lead n 17 47 4 TC 100715 a 1 ms Ejection delay Line pl v HDS Mixer Hard Stop closed between shots sx X Overheating P
64. e 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 Figure 5 Stopped Flow Observation Head 3 2 Mixer Installation and Replacement Each SFM comes from the factory with mixers installed The mixers are located in the syringe block between the syringe block and the observation head SFM 400 and at the bottom of the observation head below the cuvette as shown in Figure 8 and Figure 9 As in all stopped flow systems the mixer is one of the most delicate pieces of the instrument It is recommended to check the state of the mixer regularly and also when the SFM has been unused for a prolonged period of time Instructions for removal and replacement of the mixers are described in section 8 2 3 3 Cuvette Installation The observation cuvette is one of the most critical parts of all stopped flow instruments Indeed it is extremely important to adapt the cuvette to the parameter being observed For example it would be inappropriate to use the same cuvette for measuring a small absorbance change and for measuring a fluorescence change of a compound having a high absorbance and producing strong inner filter effects The SFM observation head can be equipped with a number of different cuvettes adapted to a variety of situations If our standard cuvettes do not satisfy your specific experimental requireme
65. e synchronization trigger using the MPS 60 The MPS can be programmed to deliver synchronization pulses triggers These pulses are TTL pulses 0 or 5 Volt are delivered from BNC connectors Synchro out 1 Synchro out 1 and Synchro out 2 on the front panel of the MPS see Figure 2 Both Synchro out 1 and Synchro out 2 are rising triggers 055 V Synchro out 1 is simply the inverse of Synchro out 1 and is a falling trigger 5 0 V Synchroout1 is used for most of the acquisition devices sold by Bio Logic The triggers can be used to synchronize the SFM and data acquisition system or other instruments If Bio Kine software is being used for data collection acquisition will start on the falling edge of the synchronizing pulse If Synchro out 1 is used then the acquisition will start at the end of the first active phase with a synchro set to On if Synchro out T is used then the acquisition will start at the beginning of the first 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 driving sequence window refer to Figure 29 The duration of the pulse will be equal to the duration of the phase Synchro out 2 is used to control the hard stop when the hard stop is programmed by the user configuration set to manual If the hard stop is not used configur
66. e will automatically calculate duration of phase 2 to match desired ageing times Once all ageing times are selected select the number of shots you want to accumulate for each ageing step Your sequence is now ready for automation X Figure 38 Help section 48 SFM 300 400 User s Manual ver 2 7 Once all parameters of the double mixing sequence window are defined click on the button _ Edit Sequence This will automatically create a sequence the global sequence window gt Global sequence window part Figure 39 Once the user has clicked on 958 a structure of the sequence is automatically defined taking in account the ratios and total flow rate The structure of the sequence is made with four rows and three columns phases The rows display times and volumes injection for Syringe 1 syringe 2 and syringe 3 The columns display the three phases Phase 1 first mixing S1 S2 Phase 2 waiting phase Solution is stored in the delay line to be aged Phase 3 solution from the delay lined is emptied using S2 and mixed with solution coming from S3 In our example in Figure 39 47 5 uL of aged solution present in the delay line will be mixed with 47 4 uL of solution coming from S3 t Global sequence Structure of the sequence Ageing times Pai Phsez Phases 99 Avet Editable 77744 x o HN ee ai po Bewr Repeat number 1 Total volume Syringe
67. easurement Ratio B 1 Ratio Delay ine 1 RatioC 11 C 5 sec Total flow rate 100 Start of data acquisition Estimated dead time 16 ms Minimum ageingtime 85 Atstop 7 Pre trigger Load Print Save As SFM Options Ontos Edi Sequence Figure 36 editing a double mixing experiment SFM Options Clicking on SFM option opens a new window Figure 37 Select the right cuvette to allow a correct estimation of the dead time Figure 36 Select the correct delay line For practical reasons we advise the user to choose the delay line number 90 uL or number 5 190uL The volume of the delay line has to be high enough to allow a correct mixing during the third phase A B mixed with C Valve lead this time is driving the closure of the valve of the hard stop to fine tune the quality of the stop of the kinetic HDS Mixer In case a HDS mixer is used tick this box to allow a correct calculation of the dead time Overheating Protection this prevents overheating of the electronic in case of intensive daily work Hardstop closed between shots the hardstop closes at the end of the pushing phase and open once the acquisition is completed Ticking this box will left the valve closed This could be particularly useful in order to run a spectrum at the end of a shot for example Use Synchrout 1 trigger used in case an external device is connected to the MPS for synchroni
68. ected after using a purge volume of zero are completely contaminated by the previous reaction mixture that remained in the instrument before sample collection The results in Figure 110 indicate that a minimum purge volume of 25 30ul is necessary to wash most of the contamination that come from previous reaction i e volume in the intermixer in a way to avoid any cross contamination t inifinity Figure 110 Washing Efficiency 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 116 SFM 300 400 User s Manual ver 2 7 Experimental Conditions Syringe 1 Water Syringe 2 1 mM DNPA 1 v v DMSO 2 mM Syringe 3 1 M NaOH Syringe 4 2 M HCI Delay Line 1 170 Delay Line 2 190 ul Driving Sequence Waste Waste Waste Waste Waste Collect V is varied from 0 in small increments until 2 the intermixer volume T is varied so that the total flow rate in Phase 5 is
69. ed it is transferred to the MPS by pushing the Single or Multiple buttons The MPS is now in automatic mode and the shot control window will be displayed in the stopped flow status as shown in Figure 99 The Shot control window shows 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 gt button or the start stop button on the front panel of the MPS The button can be used to stop an experiment prematurely if necessary If the Single button_was used to transfer the driving sequence to the MPS only a single shot be made The nd button must then be pushed to return to the driving sequence 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 Bl 0 can be used to execute shots until the shot window shows that 0 shots remain The End button is then pushed to return to the driving sequence Figure 99 shot control window 102 SFM 300 400 User s Manual ver 2 7 13 A SHORT QUENCHED FLOW PRIMER This section describes the basics of the quenched flow technique and provides some general advice about how to design and perform quenched flow experiments using the SFM It is not meant to be an exhaustive referenc
70. emains constant The user builds the sequence by varying the ratio Diluant A in mixer 2 or by changing directly the final concentration of Once all parameters are set Biokine proposes volumes per syringe defined according to mechanical limitations size of cuvette mixer the user has access to the volumes to customize the sequence S4 B nce the sequence is ready synchronization can be done with the detection part X Figure 50 Automatic concentration dependence studies Loading of solutions Syringe 1 Si is used to stock water Syringe 2 S2 to stock the diluant solution while Syringe 3 S3 and Syringe 4 S4 are filled with reactants respectively A and B The reaction between reactants A and B starts in the third mixer M3 The increase in A concentration can be achieved in two different ways by varying the ratios in Mixer 2 between syringe 2 and syringe 3 i e ratios steps or by increasing the concentration of the reactant A i e concentration steps In both sequences automatic mode and manual mode are available The delay line installed between mixer M2 and Mixer M3 should be the shortest one corresponding to a dead volume of 19 ul Design of the sequence The driving sequence is created in the window shown in Figure 51 This window can be reached from the 2 button in the stopped flow status area 58 SFM 300 400 User s Manual ver 2 7 HB Concentration dependance studies
71. enched Flow Program Phase 2 Phase3 Phase4 5 Volumes time ms 27 10 27 10 Insert Phase Syr 1 ul Remove Phase Syr 2 ul 13 5 13 5 nou Syr 13 5 13 5 Syr 4 13 5 13 5 Valve Waste Waste Collect Collect Waste Synchro 1 Off Off Off Off Off 4 5 Total Volume pl Total Flow Rate 0 0 ml s Syringes contents Shots Drive Sequence Ageing Times z Single 12501 2 10 Syringe 3 Multiple Syringe 4 Load Save As Comments Print SFM Options Close Figure 102 example of pulsed flow experiment Phase 1 and 2 correspond to the washing phases and are repeated 25 times Phases 3 and 4 the collecting phases and they are repeated 10 times 13 2 3 Interrupted flow method In the interrupted flow method the sample is transiently stored in the intermixer volume for a programmed incubation period before being mixed with the quencher Under these conditions tage depends on the intermixer volume the total flow rate as the sample enters and exits the intermixer volume and the incubation period of the sample in the intermixer volume tage thow lpause Intermixer volume t flow flow rate through intermixer volume lpause Time sample is transiently stored in the intermixer volume As with the continuous flow method the intermixer volume and flow rates can be m
72. enched Flow Program E i ri x Total Phase 1 Phase2 Phase3 Phase 4 Phase5 time ms nl Insert Phase Syr 1 ul 0 Remove Phase Syr 2 pl 0 300 Syr 3 nl 0 39 Ss Syr 4 ul 0 30 Valve Waste Collect Waste Waste Synchro 1 Off Off Off Off Phase 4 5 1 Total Volume 240 pl Total Flow Rate 3 0 ml s Syringes contents Shots Drive Sequence Ageing Times singe Wm ou Syringe 2 yum DL2 1084 ms Syringe 2 EDL 4 Load Save Comments Print SFM Options Close Figure 103 example of interrupted flow experiment The interrupted flow method allows samples to be aged for several 100 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 Because the ending and leading edges need to be eliminated delay lines 5 and 4 are generally used for this ageing method 13 3 Double mixing experiment Double mixing experiment can only be achieved with the SFM 400 Q It is generally the combination of interrupted and continuous flow techniques An example of such a sequence is given in Figure 104 In this example volumes of DL1 and DL2 are respective
73. ep Ratio A 1 Ratio Diluant 20 4 at the end of measurement Step number 5 Step value nf Final value 34 7 after 5 sec C manual continuation Start of data acquisition Atstop Pre trigger Load Print Saves SFM Options Edi Sequence Global sequence I lt KID Li ie Repeat number t Autovariation ratio steps mode Total volume Syringe gp 31 gd 3 gp ow Bg ww g ow g c g 3 go su jJ 12 jJ 3 Fos Bg eo pg ww gd s g oo g 3 goo 450 ul J 300 ul 750 ul Ready Close Concentration step Auto mode Figure 56 Global sequence The variations of the concentration of reactant A in mixer M1 have to be fixed by typing a value in concentration A i e 1 in the example choosing the steps numbers and the value of the step By clicking on the button Edit Sequence the following sequence is edited 61 SFM 300 400 User s Manual ver 2 7 HB Concentration dependance studies Concentration dependance studies Content of syringes Initial concentration Mixer 2 conditions Syringe 1 Water Ratio Diluant 3 Concentration 250 uM Syringe 2 Acid Ascorbic A0 50 0 uM Ratio ConcentrationB 650 Syringe 3 89 130 uM Total flowrate 100 mi s Estimated deadtime 16 Mode
74. equal to that in Phase 6 This experiment is designed to test the intermixer volume M2 M3 In Phase 1 DNPA and NaOH are pushed through the delay line and then to waste The second phase is used to wash the last mixer with The reaction mixture is then allowed to age for several seconds in the delay line Phase 3 Phase 5 corresponds to the purge of the delay line the solution being pushed and evacuated to waste The purge volume is again equal to 52 53 2 x V After the purge 60ul of the reaction mixture is collected and measured The results of this test are shown as a function of purge volume in MERGEFORMAT 111 Due to the long ageing time in Phase 3 the solution collected in the last phase should correspond to the full reaction t 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 These results in MERGEFORMAT 111 show that for a delay line of 19041 216 9ul nominal volume the first 20 to 30ul and the last 30 to 40ul are contaminated and should be discarded 117 SFM 300 400 User s Manual ver 2 7 118 325nm Le 1 o a c o 2 lt x 240 Purge Volume Figure 111 MERGEFORMAT 111 Recovery of Uncontaminated Material
75. es free of any contamination and fill the flow lines with a new uncontaminated sample Collect The exit valve is set to Collect and a new uncontaminated sample is pushed from the SFM into a pipette or syringe The partial liquid collection method can be used with either the continuous or interrupted flow ageing method section 13 2 Example driving sequences using the partial liquid collection method with continuous flow ageing and interrupted flow ageing are shown in Figure 106 and Figure 107 respectively 109 SFM 300 400 User s Manual ver 2 7 Purge Collect Fa Quenched Flow Program Total Volumes Insert Phase 0 Remove Phase 150 150 150 Phase 2 Phase 3 Phase 4 Phase 5 115 time ms Syr 1 Syr 2 nl Syr 3 nl Syr 4 nl Valve 45 45 45 Waste Collect Waste Waste Waste Off Off Off New Synchro 1 Phase 1 5 Total Volume 315 pl Total Flow Rate 9 0 ml s Drive Sequence Ageing Times DL2 46ms EDL Syringes contents Shots Syringe 1 Single Syringe 3 Load Save Comments Print SFM Options Figure 81 Partial liquid collection with continuous flow ageing Purge Collect FA Quenched Flow Program Phase 3 Phase 5 Total Volumes time ms nl Insert Phase Syr 1 nl Remove Phase Syr 2 ul yr 2 Hes 0280
76. es the motor pushing the 1 9 ml syringe to run at a faster and smoother rate The specifications of the 1 9 ml syringe are given in Table 1 SFM specifications 1 9 ml syringes 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 3 7 2 High density mixer Mixing solutions of different densities offers a formidable challenge for stopped flow instruments In typical protein folding unfolding experiments heavy solutions of urea or guanidine chloride are mixed with pure aqueous 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 that is guaranteed to ruin the kinetics being recorded The SFM module can be equipped with a specially designed mixer model HDS Figure 13 that includes 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
77. f 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 500ul of each sample mixed with 500ul of water in 1cm path length cuvette The results were plotted against the ageing times as shown in Figure 109 The apparent first order rate constant determined from Figure 109 is 28 s which yields a second order rate constant of 56 M s for a final OH of 0 5 M Eka E c Te N ex i o o lt t 0 Abs 0 194 100 0 150 0 200 0 2500 300 0 3500 400 0 Ageing Figure 109 DNPA Experiment Results 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 9 2 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
78. ggled on or off by pressing O on the keyboard The 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 pressed and then choose the Cut Copy or Paste functions desired in 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 the copy area is bigger than the paste area the operation is done only for values that can fit inside the paste area 41 SFM 300 400 User s Manual ver 2 7 Stopped Flow Program Phase 100 duration time ms Syr 1 ul Syr 2 nl Syr 3 nl Syr 4 nl Synchro 1 Syringes volume BEES ESARET lt Synchronisation trigger Figure 29 Expanded program grid WARNING Blank and non numeric values entered in the program grid are considered as zero values 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 entered in the Syringe Contents frame of the driving sequence window Figure 30 The text is entered from the keyboard and the BACKSPACE and DEL keys can be used for corrections Syringes contents Figure 30 Syringes Contents Ea
79. he 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 a rapid rise of the NaCl buffer in the observation cuvette can be observed about 20 s after mixing Hence the best solution is to use the high density HDS mixer developed by Bio Logic This mixer is described in detail in section 3 7 2 80 SFM 300 400 User s Manual ver 2 7 8 Technical section 8 1 Solvent compatibility Any solution used in the SFM system will be in contact with the following materials e PEEK the core of syringes blocks and piston caps e Teflon head cap or valves for special version e Vitton o rings PEEK has excellent chemical resistance to organi
80. ical limitations size of cuvette mixer the user has access to the volumes to customize the sequence nce the sequence is ready synchronization can be done with the detection part Figure 41 Concentration dependence setup Loading of solutions Syringe 1 S1 is used to stock the diluant solution while Syringe 2 52 and Syringe 5 are filled with reagents respectively A and B The increase of the concentration of A can be achieved in two different ways by varying the ratios between syringe 1 and syringe 2 i e ratios steps or by increasing the concentration of the reagent A i e concentration steps In both sequences automatic mode and manual 51 SFM 300 400 User s Manual ver 2 7 mode are available The delay line installed between mixer M1 and Mixer M2 should be the shortest one corresponding to a dead volume of 19 pl Design of the sequence The driving sequences is created in the window shown in Figure 42 this window can be reached from the zz button in the stopped flow status area fA Concentration dependance studies Concentration dependance studies Content of syringes Initial concentration Mixer 2 conditions Syringe 1 f lt 7 Ratio Diluant 3 Concentration 05 mM Swine gt Ratio J 3 ConcentrationB 05 mM Spine Dp 14 j nM Total flow rate 100 Estimated deadtime 1 6 Mode
81. ing sequence may contain from 1 to 20 phases Although only 5 phases are shown initially additional phases may be inserted using _inset Phase button or removed using the Remove Phasel button Figure 95 shows an expanded view of the program grid The duration of a phase is entered in ms 1 60000 ms phase on the first line of the program grid The volume in ul delivered by each of the syringes during a phase is entered on the line next to the appropriate syringe The position of the exit valve is set near the bottom of the program grid Phase 1 Phase 2 time ms 100 50 duration Syr 1 ul f Syr 2 ul 200 100 TN Syr ul 200 100 Syr 4 ul 200 100 Valve Waste Collect exit valve position Synchro 1 Off Figure 95 Program Grid To enter the phase duration and syringe volumes delivered click on the corresponding cell or use the arrow 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 is set by pressing W for Waste and 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
82. inges flow rates delay lines installed and intermixer volumes The ageing times are calculated according to the equations shown in Figure 33 Ageing Times SrM 300 _ Intermixer Volume 1 2 ime Total Flow RateS1 S2 Intermi 2 me M2 FM 4 lay Line 1 ixer Volu DL2 34 5 ms s 09 psy e Total Flow RateS1 S2 Intermixer Volume 1 2 Penty nee Total Flow RateS1 S2 S3 Figure 33 Ageing Times calculation Bio Kine offers the ability to repeat phases a number of times in virtually any order This is accomplished though a macro sequence entered in the Driving Sequence window Figure 34 The macro sequence can be edited to run a single phase or many phases in a different order than present in the program grid Drive Sequence 1 1 20 Figure 34 MPS Software Drive Sequence Macro 43 SFM 300 400 User s Manual ver 2 7 Once the sequence ready click on the Single or button to activate the shot control window in the stopped flow status area Standard operations can also be made from the same window e Create a new sequence using the New button e Load a sequence using the _ Lead button eSave a sequence using the Se button e Print a sequence using the P button Close a sequence using the bs button e Comments a text window is opened by clicking on the Comments button Comments are saved with the sequence 5 7 3 Programmabl
83. ipped with 1 9 ml syringes Syringe 1 and 2 Water Syringe 3 Dichloroindophenol 1 mM Syringe 4 10 mM ascorbic acid Mixer Standard Berger ball Cuvette uFC 08 dead volume 3 ul Detection MOS 200 in absorbance mode detection at 524 nm Equal volumes of each reactant were mixed The data acquisition was started 40 ms before the shot for clear observation of the start of the shot The fast reaction also allows the examination of the data around the stop for any artefacts The results indicate that there are no stop artefacts present and that a minimum of 40 ul is needed to completely wash the flow path for this reaction Figure 68 kinetics obtained with total volume 80 ul 74 SFM 300 400 User s Manual ver 2 7 120 100 ii J 80 4 60 H 4 40 4 Washing efficiency ye Li ES Ee Pei ee ee ee apaq sasa 2 qasa 20 30 40 50 60 70 80 90 Total volume in uL Figure 69 determination of the minimum volume to push 7 4 Variable Ratio Mixing The ability 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 expe
84. irectly from the MPS or though Bio Kine Both methods are described in the following sections Syringe control directly from the MPS is made through the use of the buttons on front panel of the MPS 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 selected and whether it is being filled or emptied 12 1 2 Software Syringe control from Bio Kine software is made through the button in the stopped flow status area The MPS is then initialized and communication established between Bio Kine software and the MPS unit The message MPS on line is displayed in a green window in the stopped flow status area The syringe to be moved is selected by clicking on the corresponding frame or pressing the Left and Right arrows keys on keyboard The newly selected syringe will be surrounded with a red rectangle Syringes are emptied or filled using the and buttons or with the Up arrow lt PageUp gt lt Down gt arrow and lt PageDown gt keys on the keyboard The E button and lt Up gt arrow moves a syringe upwards by one elementary movement and the button lt Down gt arrow moves a syringe downwards by one elementary movement The Z button and lt PageUp gt arrow moves the piston upwards by 10x elementary moveme
85. is introduced the sequence will be run sothat step number one is done 2 times then step number two is done in two times etc This procedure allows to record each traces and do an average Global sequence Structure of the sequence Ageing times Phase 1 Phase 2 Phase 3 Time ms 38 3 47 2 Step Ageim o 2 arf Bram 7 Po B E Repeat number 3 Total volume Syringe 574 2 ul B 334 2 ul 420 pl Ready gt Close Figure 40 step creation a SAVING LOADING A DOUBLE MIXING EXPERIMENT 3 T Save As TE It is possible to save a sequence by clicking on the file is saved under a DMX file To load an existing sequence click on the Load button 50 SFM 300 400 User s Manual ver 2 7 5 9 Creating a Driving Sequence using concentration dependence studies The aim of this sequence is to observe different kinetic curves by increasing automatically step by step the concentration of one reactant A against B in A B reaction Thus the concentration of B is maintaining constant while the concentration of A is increasing in constant steps automatic mode or in certain steps values manual mode The first mixer M1 is used to change the concentration of a reagent A by mixing it with a diluant while the concentration of reactant B is constant The second mixer is used to mix
86. is maintaining constant while the concentration of A is increasing in constant steps automatic mode or in certain steps values manual mode The second mixer M2 is used to change the concentration of reactant A by mixing reactant A with diluant while the concentration of reactant B is constant The third mixer M3 is used to start the reaction Figure 50 The following window is obtained by clicking on the button GPinfos of the main mixing sequence window 57 SFM 300 400 User s Manual ver 2 7 PS WIDE The aim of this mode is to follow the c endency of i constant It is done using 3 syringes and 2 mixers according to the following diagram the first syringe is not used in this configuration but must be filled with water or buffer to preserve the system from air bubbles Kinetics getting slower as concentration decreases S1 Water S2 Diuant 53 4 Loading of solutions The mixer M2 is used to change concentration of A thus diluting solution is loaded in Syringe 2 and is loaded in syringe 3 The mixer is used to start the A B reaction so B is loaded in syringe 4 The Mix mixing block 2 must be installed between M1 and M3 to minimize sample consumption Design of the sequence Users indicates the initial concentration 40 and BO and mixing ratio to apply between A diluant and B at each step in mixer M3 The same total flow rate is used for all steps so dead time r
87. is now activated on the left panel of the screen Configuration Content of syringes Initial concentration Final concentration Syringe 1 bue 2 E Syringe 2 NEED Syringe 3 Syringe 4 pon Sua 342uM Figure 25 example of driving sequence e The estimated dead time of the reaction is given in ms The dead time is calculated using the cuvette and mixer selected dead volume and the total flow rate Estimated dead time 1 8 ms _ Cuvette Dead Volume Total Flow Rate Figure 26 Estimated dead time 38 SFM 300 400 User s Manual ver 2 7 A color code is used to warn the user about the choice of the parameters selected Colour code message OK depending on syringe total volume may be insufficient for washing the Total youme cuvette lt 4 times the dead volume Total volume too low for correct washing of cuvette OK Syringe volume may be insufficient for washing of Volume per syringe yring mixer Syringe volume too low OK Flow r low for correct mixin 1 ml Total flow rate ow rate too low for correct mixing 1 ml s Flow rate may be difficult to achieve for this cuvette 15ml s OK Flow rate per syringe Flow rate may be too high will be dependant on cuvette and flow by other syringes Flow rate out of range too low or too high Standard operations can be
88. ith 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 exit valve and the SFM 400 functions as an SFM 300 Figure 80 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 83 SFM 300 400 User s Manual ver 2 7 84 EXIT VALVE SECOND MIXER M2 E HEAD SPACE DELAY LINE EL FIRST MIXER M1 Figure 80 SFM 300 Installation of Exit Valve and Delay Lines SFM 300 400 User s Manual ver 2 7 EXIT VALVE THIRD MIXER M3 eg HEAD SPACERS p DELAY LINE TWO a DL2 MIXER BLOCK NEC M2 SECOND MIXER 2 a DELAY LINE ONE DL1 FIRST MIXER M1 Figure 81 SFM 400 Installation of Exit Valve Mixer Blocks and Delay Lines 85 SFM 300 400 User s Manual ver 2 7 9 2 Flow Line and Intermixer Volumes Figure 82 SFM 300 and Figure 83 SFM 400 below indicate the volumes of the 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 hydrodynamic volumes may vary slightly around these values For precise measurement
89. ll the possibilities here Some are described below T Load several reagents and mix them in different shots with the contents of the last syringe 2 Use syringes loaded with reagents and a 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 which type of experiment is being performed SFM 300 observation head and delay line are installed as shown in Figure 8 The smallest delay line comes standard and installed with the instrument SFM 400 The observation head and delay line s are installed as shown in Figure 9 The observation head may be installed using the mixing blocks labeled 0 0 0 MIX DL DL MIX 0 and DL MIX DL or no mixing block The installation of the different mixing blocks is described in Table 4 SFM 400 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 th
90. lows 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 The precision of the setting is 0 1 ms Overheating Protection Not applicable for the recent 5 60 The default mode is checked It is a protection against electronic overheating after a long working day e Hard stop closed between shots in advanced mode the configuration of the hard stop is automatically set the hard stop closes at the end of the pushing phase or a few milliseconds 37 SFM 300 400 User s Manual ver 2 7 before if a lead time is selected and opens at the end of the acquisition If the user needs to keep the hard stop closed at the end of the acquisition to run a spectrum for example then it is necessary to check the corresponding box e Delay lines In the advanced mode delay lines cannot be changed boxes not active because only a single mixing experiment can be performed 5 6 2 Design of stopped flow sequence The window shown in Figure 23 driving sequence in advanced mode is separated into six areas mixing ratio volume total flow rate start of data acquisition shots and configuration These different areas are respectively described below Mixing ratio it is the first parameter set The ratio of the unused syringes must be set to 0 It is possible to enter a decimal va
91. lue for the ratio Volume it is necessary to set the total volume of the reactants pushed into the cuvette using the up or down arrows The volume is proportional to a micro step volume in order to improve the reproducibility of results Once the total volume is selected the volume to be pushed for each syringe is calculated The total volume selected should be big enough to wash the cuvette efficiently between two shots refer to the color code for the limits Total flow rate total flow rate must be selected using the up and down arrows Once the total flow rate is selected the flow rate for each syringe is automatically calculated 1 ml s is considered the minimum value to get efficient mixing There are also limits for a single syringe according to their respective volume refer to colour code e Start of data acquisition Using the stop option only the kinetics will be recorded The acquisition is started when the hard stop closes To make sure the cuvette is well washed and the stationary state is reached it is advised to start the acquisition a few ms before the stop Configuration In this area it is possible to find the volume of the syringe installed and the type of cuvette The content of the syringes can be entered with initial concentration the final concentrations are calculated using the mixing ratio selected Sequence Once the sequence ready click on the _ fe button Tthe shot window
92. ly 216 2 ul and 60 1 ul 107 SFM 300 400 User s Manual ver 2 7 n Quenched Flow Program Insert Phase Remove Phasel Waste Collect Waste Off Off Off Phase 4 5 Volume 40 pl J Flow Rate 0 6667 ml s Drive Sequence Ageing Times 1 20 DLI 1622 Syringes contents Shots Syringe 2 eem DL2 22 5 ms 4 Load Save As Comments Print SFM Options Close Figure 104 double mixing sequence In the first phase samples 1 and 2 are mixed after which the solution is allowed to age in the first delay line phase 2 After this 300 ms incubation the solution is mixed with sample 3 in the second mixer and allowed to age 22 5 ms in the second delay line using continuous mode Finally the solution is quenched by sample 4 The ageing times in DL1 and DL2 for the sequence proposed are 462 2 ms 162 2300 and 22 5 ms respectively It is just necessary to change the incubation time in phase two to vary the first ageing time and to keep the second one constant 13 4 COLLECTION METHODS Section 9 3 described how sample can be recovered from a quenched flow experiment with the SFM The sections below describe how sample collection is incorporated into a driving sequence 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 be set to Waste if the f
93. matic 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 for high versatility of the injection sequence It is possible to make an injection of one syringe only unequal filling of the syringes variable ageing times 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 artifacts allows for 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 1 5 Description of the Mechanical Design The observa
94. n 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 is described in section 3 5 2 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 The SFM 300 400 can be connected to the MPS 60 or MPS 70 Please refer to the appropriate following operating features 2 1 Operating Features with MPS 60 The general features of the MPS 60 are shown below in Figure 2 and described in Table2 MPS 60 Panel Descriptions Figure 2 MPS 60 Panels Front Back SFM 300 400 User s Manual ver 2 7 Table2 MPS 60 Panel Descriptions NAME FUNCTION LCD DISPLAY Used to display messages selected syringe auto EE 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
95. nal Tube Collection if the volume collected is not substantially larger than the flow line and tube volume 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 9 2 In addition it is recommended to wash the old solution out of the SFM and tube with a 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 9 3 1 2 Pipette syringe collection In this method a pipette or syringe is connected to the collect outlet of the exit valve and all the liquid is collected Figure 85 This method allows for the complete collection of the 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 88 SFM 300 400 User s Manual ver 2 7 ERN if the volume collected is not substantially larger than the SFM flow line volume contamination of samples by old reacted solution may occur It is recommended to collect sample volumes
96. ng the buttons 2 on the front panel select buttons pressing one button syringe 1 2 3 or 4 is selected while the corresponding led is lighted on The select left button allows you to switch from syringe 1 to 2 to 3 with increasing numbers The select right button allows you to switch from syringe 3 to 2 to 1 with decreasing numbers 12 SFM 300 400 User s Manual ver 2 7 pressing on the two buttons the same time all syringes 1 2 3 amp 4 are selected while the entire led are lighted on The up down buttons allows you to move the pistons of the syringe up and down The general features of the MPS 70 are shown below in Figure 3 MPS 70 4 Front Panel and Figure 4 MPS 70 4 Rear Panel described in Table 3 MPS 70 Panel Descriptions Figure 3 MPS 70 4 Front Panel Front Panel MPS 704 imer SYRINGES Selected1 2 3 4 5 Start Stop 00000 4 1 Select 2 PowerO O O O O Down 12345 Li 3 4 ce Etherne 10 100 BaseT 5 6 Synchrg in Synchrg o Trigger Out Motors Temperatur
97. nges If by accident a syringe is returned to its uppermost position the syringe volume counter will again show and the syringe must be reinitialized To avoid such accidents the Up and Low Limits checkbox may be checked When this box is checked Bio Kine will not allow the syringes to be driven beyond their upper and lower limits This also avoids 33 SFM 300 400 User s Manual ver 2 7 accidentally pulling the syringe plunger completely from the syringe and spilling solution onto the SFM WARNING The Up and Low Limits only applies to control of the syringe from within Bio Kine These limits can be bypassed by manual control of the SFM directly from the MPS 5 3 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 If this occurs 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 degassed and filtered before filling the SFM The syringes of the SFM can be emptied and filled manually section 5 1 The filling of the syringes follows the steps below which are shown in Figure 22 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 22 Panel 1 2 Set the syringe valv
98. nto the collection device 3 Wash the syringes and flow lines one time with 70 100 ethanol Use the same procedure as in step 2 4 Dry the syringes flow lines and electrovalve 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 the liquid is pushed out of the syringes flow lines and electrovalve 5 Set all syringe valves 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 Bio Kine it will be necessary to uncheck the Up and Low Limits checkbox in the software syringe control window Figure 91 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 6 Turn all syringe valves handles to 7 Turn off the MPS 12 5 Long term Storage of the SFM If the SFM is not 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
99. nts and the button and lt PageDown gt moves 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 window Figure 91 The El and X buttons change the manual speed The display shows the speed in flow rate based on the syringe installed and moved 94 SFM 300 400 User s Manual ver 2 7 syringes command R Syringe Control V in pl Syringe 2 Syringe 3 78 mi 78 mi Reset Reset m d B Manual Speed 21ms H Reset All Figure 91 syringe control window 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 see Figure 91 When the MPS is turned on and the software started turned on the syringe volume counters show HEE and must be initialized Figure 91 The syringes are initialized by setting the syringes to their uppermost empty position and resetting the syringes in Bio Kine The syringes can be selected and moved to their uppermost positions either directly with the MPS section 12 1 1 or through Bio Kine 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
100. nts we invite you to contact us about custom made cuvettes Figure 7 shows the cuvettes presently available and their specifications There are two general styles of cuvettes FC fluorescence cuvette FC type cuvettes have blackened edges to reduce light scattering in fluorescence configuration The FC 15 and FC 20 cuvettes are the best choices 16 SFM 300 400 User s Manual ver 2 7 for CD experiments in the far UV Their large aperture facilitates low noise recording at these wavelengths TC transmittance cuvette 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 5 1 5 mm cross section The two styles of cuvettes have different holders that are used to install them into the SFM observation head The assembly of the cuvette with their respective cuvette holders is shown in Figure 6 TC Cuvettes FC Cuvettes EE Grooves TC 100 xx Cuvette y a s Note sure to align the grooves of the TC Cuvette holder pieces Figure 6 Cuve
101. odified by introducing different delay lines and modifying syringe flow rates in Bio Kine IMPORTANT To obtain uniform ageing of the sample the flow rate of the sample entering the delay line must always equal the flow rate of the sample exiting the delay line An example driving sequence using the interrupted flow method is shown in Figure 103 The experiment is performed in Phases 1 4 In phase 1 the reactants are mixed and the intermixer volume filled with sample In Phase 2 the sample is allowed to age for 400 ms In phase 3 the leading edge of the line is pushed to the waste to get rid of contamination from syringe 4 during phase 2 see section 14 4 for details In phase 4 the sample is pushed out of the intermixer volume quenched and collected Delay line n 5 is installed for the ageing The intermixer volume for mixers 2 and 3 is then 216 9 yl and the total flow through the intermixer volume upon sample entry and exit is 2 ml s which indicates tow tpause tage 108 4 400 508 4 216 9 2 dx 106 SFM 300 400 User s Manual ver 2 7 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 Qu
102. 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 section 14 2 of this manual SFM 300 Q FLOW LINE VOLUMES Line Number Flow Line Volume pl Delay Line 14 108 36 55 Collect IH EXIT VALVE Waste 9 MIXER1 DELAY LINE RESERVOIR1 RESERVOIR2 RESERVOIR3 SYRINGE 1 SYRINGE 2 SYRINGE 3 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 1000 19 35 92 144 192 498 1003 43 60 116 168 216 523 1027 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 Figure 82 SFM 300 Q Flow Line and Delay Line Volumes 86 SFM 300 400 User s Manual ver 2 7 SFM 400 Q FLOW LINE VOLUMES Line Number Flow Line Volume ul 69 7 89 88 T Delay Line 1 13 94 10 Delay Line 2 14 108 36 55 Collect MIXER2 9 LU DELAY LINE 2 MIXER1 DELAY LINE 1 RESERVOIR4 RESERVOIR1 RESERVOIR3 RESERVOIR2 SYRINGE 1 SYRINGE 2 SYRINGE 3 SYRINGE 4 DELAY LINE AND INTERMIXER VOLUMES Delay Line N 1 1
103. of the microcomputer Finally plug the MPS 70 into the appropriate AC line 2 5 Temperature Regulation The syringes valves and observation chamber of the SFM are designed to be temperature regulated Organic oil like Perfluorinated oil may be preferred for temperature regulation to avoid corrosion but the user should check compatibility with stopped flow materials beforehand Careful temperature regulation minimizes any occurrence of temperature artefacts The SFM module may be connected to a circulating temperature bath for temperature regulation The coolant flows through two internal circuits around the injection syringes and through the isolation valve block and observation head With careful temperature regulation temperature artefacts can be avoided over a very wide temperature range between 10 and 80 C For lower temperatures the use of a cryo stopped flow accessory is necessary Rapid kinetics down to 90 C can be achieved Please contact our commercial service for any inquiries SFM 300 400 User s Manual ver 2 7 3 INSTALLATION OF THE STOPPED FLOW COMPONENTS 3 1 The Observation Head The stopped flow observation head Figure 5 is installed on top of the SFM The observation head has four optical windows one window for illumination and three for observation This allows measurements of absorbance transmittance circular dichroism single or double wavelength fluorescence emission and light scattering or fluorescenc
104. oncentration Dm mM Syringe 3 eee F 38 mM Total flow rate 100 Estimated dead ime 1 6 Mode Variation Ratio in Mixer 1 Start next acquisition step Ratio steps Ratio Ratio Diluant atthe end of measurement Step number C after 5 E Concentration steps Auto manual continuation Step value Start of data acquisition Final value Atstop Pre trigger SFM Options Sequence EL Load Print Global sequence Manual ratio mode Step J Ratio Dil Ratio Ratio Concent 4 Volume Dil Volume Volume B IS lab SL e 11 f f Repeat number 1 Total volume Syringe Onl Opl Ready P Close Figure 58 Global sequence in ratio steps manual mode The Global sequence is created through the following table Click on button to create a step then type the ratio in Ratio A that will be used during the step and indicate the volumes All the volumes volume Dil volume A and volume B have to be more than 40ul 3 Click on button to create a second step 4 Click on 4 to clear all the sequence or El to remove a step 5 Repeat number is used to repeat each step 63 SFM 300 400 User s Manual ver 2 7 ET 4 4 gt Figure 59 Global sequence The functions of the different buttons are the following is used to create or
105. orough 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 70 100 ethanol Use the same procedure as in step 2 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 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 Bio Kine it will be necessary to uncheck the Up and Low Limits checkbox in the software syringe control window Figure 21 Note You may observe a few drops of liquid that fall from the syringes when the syringe plungers ar
106. passes through 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 Collect A B_ C EXIT VALVE Waste DELAY LINE MIXER1 MIXER2 QUENCH A B IL A STAGE 1 STAGE 2 STAGE 3 Figure 100 Quenched Flow Experiment Scheme 103 SFM 300 400 User s Manual ver 2 7 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 9 2 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 3 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 2 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 Continuous flow methoq In the continuous flow method the 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 intermixer volume flow rate through intermi
107. periment The double mixing experiment program is used in order to mix three reagents together This means that a SFM 300 or SFM 400 has to be considered In the SFM 300 S all syringes will be used for SFM 400 the three last syringes will be used 52 S3 and 54 and 51 is not used however this syringe has to be loaded with a solvent water or buffer for example A double mixing experiment is divided in three phases 1 A and B are mixed in a first mixer 2 The solution A B goes through a delay line The solution is aged in the delay line waiting phase 3 The solution from the delay line is mixed with a C solution third reageant in the second mixer and the final solution goes through the cuvette for analysis Once the mixing sequence Double mixing mode is selected from ino instal Stopped Flow ixing Configuration menu clicking on the button mixing sequence Figure 35 double mixing experiment be shared two parts Double mixing experiments all parameters such as content syringe concentration phase conditions are defined Global sequence gives information on the volume consumption for each syringe ageing time assessed etc 709 opens a window 45 SFM 300 400 User s Manual ver 2 7 FA Double mixing experiments Double mixing experiments Content of syringes Initial concentration Final concentration Syringe 1 lf J S Syringe 2 lO ey Syringe 3 I
108. ple 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 the 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 Syringe 3 1 M NaOH Syringe 4 2 M HCI Delay Line 1 None Delay Line 2 N 1 1704 115 SFM 300 400 User s Manual ver 2 7 Driving Sequence Waste Waste Collect V is varying by adding small increments from 0 to V wash where the results indicate that a complete washing phase is achieved into the intermixer volume volume between Mixer 2 and mixer 3 T is adapted to maintain a total flow rate in phase 1 equal to the one in phase 3 The results of the experiments performed with no delay lines installed are shown in Figure 110 It should be remembered that the purge 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 coll
109. r 2 Start next acquisition step Ratio steps Ratio j 1 Ratio Diluant 20 1 at the end of measurement Step number 5 after 5 sec Concentration steps Auto f manual continuation Step value 01 Start of data acquisition Final val 0 VA Atstop 7 Pre trigger Load Print Save As SFM Options Grfos Sequence Figure 52 mixing sequence 59 SFM 300 400 User s Manual ver 2 7 Then the conditions in Mixer 3 have to be entering in the following window Mixer 3 conditions Ra o A Diuan 3 Ratio B 3 Total flowrate 100 Figure 53 Ratio in mixer 3 These conditions correspond to a 1 to 1 mixing sequence in Mixer 3 with a flow rate of 10 ml s The concentration maximum of A is automatically calculated while the concentration of B reactant is maintained as a constant value There are two ways to increase the concentration of A the first is done by the increase of the ratio in mixer 2 step by step The second is done by increasing the concentration of A step by step Acquisition parameters The parameters of the acquisition have to be entered in the following window Figure 54 acquisition parameters Start next acquisition step In case of a repetition of the sequence starting the next acquisition step can be done at the end of the measurement or after a defined time Start of data acquisition as in the stopped flow
110. re possible and you are invited to inquire about their feasibility The commercial reference SFM X00 QS has all the components for the two applications SFM X00 S or a SFM X00 Q can easily be updated to a SFM X00 QS SFM 300 400 User s Manual ver 2 7 Figure 1 SFM Modes of Operation STOPPED FLOW MODES Panel 1 SFM 300 gt Detection Delay line motor motor Collect Mixer Valve Delay Tine E B Waste motor motor Panel 2 SFM 400 Light Detection Collect Mixer e Valve Waste Delay Tine 52 motor motor SFM 300 400 User s Manual ver 2 7 1 3 Specifications The general specifications of each SFM are listed in Table 1 SFM specifications below Table 1 SFM specifications GENERAL SFM SPECIFICATIONS 3 SFM 300 or 4 SFM 400 One stepping motor per syringe 6400 steps per motor turn 1103 25 to 1000 ul Programmable trigger for data acquisition and synchronization of accessories 500ul to syringe limit 10ml syringe 28 ul 6 8ml syringe 20 ul standard syringe 1 9ml syringe 10 ul 10 ml syringe 0 062 8 ml s syringe 6 8 ml syringe 0 045 6 ml s syringe 1 9 ml syringe 0 010 1 32 ml s syringe 1 ml s total flow rate through each mixer Continuously variable from 1 1 to 1 20 with single dilution g
111. re was 3 8 ms so very close to the real one m Mixing ratio Volume Total flow rate 1 0 Total volume shot 1 wl oo mls aj 52 1 z 52 126 ul El 800 mL s EB 54 1 54 126 uL 40 mls Default Start of data acquisition Shots At stop Estimated dead time 3 8 ms Multiple Single At 40 before the stop Comaentz Close Configuration Content of syringes Initial concentration Final concentration Syringe 1 2 Syringe 2 Gmeobieaddpd 3 Syringe 3 sscobicacidpH 2 J V _ Syringe 4 oo Ja C j TC 100 10 Figure 64 driving sequence of the reduction of DCIP at pH 9 Ao 0 787 Figure 65 reduction at pH 9 72 SFM 300 400 User s Manual ver 2 7 n Mixing sequence mimm 10m ascorbic acid pH 3 10m ascorbic acid pH 2 m 100 10 Figure 67 reduction at pH 2 73 SFM 300 400 User s Manual ver 2 7 7 3 Evaluation of Washing and the Quality of the Stop As mentioned in section 6 1 it is necessary to completely wash the flow 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 69 The reaction was the fast reduction of DCIP with ascorbic acid at pH 2 Experimental condition Stopped flow SFM 400 equ
112. ree flow method is used and Collect if the pipette syringe collection is used In general total liquid collection will be used only with the continuous flow ageing method section 13 2 1 and when large volumes 1 ml of a sample need to be collected and contamination from previously 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 105 108 SFM 300 400 User s Manual ver 2 7 quenched R inda Total Volumes time ms Insert Phase Syr 1 ul 8 Remove Phase Syr 2 ul 10 mum Syr 3 10 Syr 4 nl J IDD Valve Ce E Waste Waste Waste Synchro 1 0 I Phase 1 5 Total Volume 300 pl Total Flow Rate 6 0 ml s l Syringes contents Shots Drive Sequence Ageing Times Syringe 2 xd DL2 J 54 2 ms 3 ene EL 4 Load Save As Comments Print SFM Options Close Figure 105 driving sequence for a total liquid collection 13 4 2 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 mixtures from the SFM wash the flow lin
113. riments using the SFM to carry out variable ratio mixing are described below 7 4 1 Reduction ef DCIP ascorbic acid Experimental Conditions Syringe 1 10 20mM Ascorbic Acid pH 9 Syringe 2 10 ml Buffer Syringe 3 10 ml 100 uM DCIP Wavelength 524 nm Cuvette TC 50 10 Detection method Transmittance 75 SFM 300 400 User s Manual ver 2 7 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 S3 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 70 shows the results of the experiments with the dilution factor of ascorbic acid noted next to each curve ai b g g g S aj 3 g H q g g Ed Figure 70 DCIP Variable Ratio Mixing Experiments Using the variable 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 70 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 71
114. rotection Default OK Cancel Figure 28 SFM options e Select the cuvette and mixer according to the cuvette and mixer installed in the SFM refer to the SFM user s manual for more details 40 SFM 300 400 User s Manual ver 2 7 WARNING Incorrect cuvette and mixer configurations will cause dead time calculations to be incorrect Valve Lead 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 The precision of the setting is 0 1 ms Overheating Protection Not applicable for the recent MPS 60 The default mode is checked It is a protection against electronic overheating after a long working day Hard stop auto is the default mode In this position the hard stop is closed at the end of the pushing phase or few milliseconds before depending on the lead time chosen and remains closed until the end of the acquisition As soon as the acquisition is finished the hard stop opens In case the user wants to leave the hard stop closed after the acquisition for example to perform a spectrum it is necessary to choose the Hard stop closed between shots option see Figure 28 When using the manual mode the hard stop is programmed to open and close by the user through the synchro out 2
115. rough these tubes for anaerobic work or to avoid condensation phenomena e A temperature probe is in contact with the cuvette to give the precise temperature of the reaction Hard Stop Connection to the umbilic Optical fiber Figure 16 low temperature accessory 27 SFM 300 400 User s Manual ver 2 7 4 SOFTWARE CONFIGURATION IN STOPPED FLOW MODE The SFM is controlled by Bio Kine software which is also used to control acquisition parameters This section precisely describes the configuration of the software Please note that the procedures and examples have been generalized and configuration choices should be made based upon the equipment purchased and intended experiments This section assumes that the user has already installed Bio Kine software on the host microcomputer 4 1 Installation SFM 300 400 with MPS 60 using Bio KIne version up to 4 45 Once Bio Kine loaded choose Install device installation in the main menu The stopped flow communication is established from this window by checking the stopped flow device box and choosing the corresponding serial port Accept the parameters using the OK button Device Installation 4 Acquisition device Stopped flow device MOS 450 J 810 serial acquisition 05 250 J 810 analog acquisition Stopped flow communication TIDAS diode array External device Serial Pot COMI z Acquisi
116. rvation head Figure 12 Hard Stop installation There are three operation modes of the hard stop that can be chosen in Bio Kine The modes of operation are Automatic mode the hard stop is controlled by the software Using this mode the hard stop is closed at the end of the pushing phase and during the acquisition However the hard stop remains opened between shots Manual mode the hard stop is programmed to open and close by the user e None The valve is always open The installation of the hard stop on the observation head is shown in Figure 12 3 7 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 3 7 1 Small drive syringe The SFM standard syringes 10 ml have a large driving speed range Each syringe can be programmed for different speeds and used to make mixing ratios different from 1 1 Ratios as high as 1 20 can be obtained with the standard syringes Ratios beyond 1 20produce poor 24 SFM 300 400 User s Manual ver 2 7 results 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 1 9 ml syringe for injecting the solution to be diluted This enabl
117. sorbance at 524 nm and reduction by ascorbic acid results in a nearly complete discoloration The second order reduction rate constant is highly dependent on pH and varies from about 104 M s at pH 2 0 to 102 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 to measure the dead time of the SFM instrument The slow reaction at basic pH is used to check the quality of the stop evaluate the washing of the observation cell and test the variable ratio mixing capabilities The following sections describe the use of this reaction for testing and exploring its capabilities 7 2 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 6 2 1 the dead time of a stopped flow experiment depends on many factors besides simply the flow rate and cuvette volume The technique presented here may be adapted to evaluate the dead time under many experimental conditions Experimental Conditions Syringe 1 2 or 3 10 ml 20 mM Ascorbic Acid pH 2 or 9 Syringe 4 10 ml 150 uM DCIP Wavelength 524 nm Cuvette TC 10
118. ss the ratio between the solution coming from the delay line A B and C and finally fix a total flow rate Please notice that the flow rate must be assessed to satisfy the turburlent conditions a color coded window orientates the user The green font colour indicates adequate parameter conditions The orange font color indicates a too low total flow rate Start next acquisition step the next acquisition step starts at the end of the measurement or after a delay defined by the user Start of data acquisition starts the acquisition at the stop of the motors or few milliseconds before the stop this time is fixed at 2 ms and cannot be changed Estimated dead time An estimated dead time is calculated in function of the flow rate assessed and the volume of the cuvette chosen from the SFM option Minimum ageing time this corresponds to the time needed to fill the delay line This minimum ageing time is equal to the delay line volume divided by the flow rate coming from syringe 1 and 2 for an SFM 300 A B 46 SFM 300 400 User s Manual ver 2 7 FA Double mixing experiments Double mixing experiments Content of syringes Initial concentration Final concentration Syringe 1 Omm Syringe 2 B 2mM 05 Syringe 3 05mM Phase 1 conditions Phase 3 conditions Start next acquisition step Empty Delay ine by using Syringe A B atthe end of m
119. strongly recommend that all driving sequences be tested with non precious samples Although such tests may be time consuming they maximize the experiment s success by ensuring that a majority of miscalculations and mistakes will be found and avoided The accuracy and precision of quenched flow experiments depend on the quality of the sample collected 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 and adapting them as close as possible to true experimental conditions temperature viscosity etc As mentioned in sections 13 2 1 and 13 2 3 the continuous flow and interrupted flow ageing methods work best for ageing times of 1 200ms and 200ms 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 design an experiment which best economizes the use of reactants 14 TEST REACTIONS IN QUENCHED FLOW MODE 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 dinitro T DNP At 20 C the reaction has a second order rate cons
120. t 1 100 with double dilution 0 8 ms with FC 08 cuvette 0 25ms with the cuvette accessor 2ms with minimal volume delay line PEEK stainless steel or Kel F on special order 10ml standard syringes 6 8 and 1 9 mL syringes are also available 10 ml syringe 0 19 ul 6 8 ml syringe 0 14 ul 1 9 ml syringe 0 03 pl adjustable from 1 ms to 60000 ms per phase 300 Watt 110 220 Volt 50 60 Hz 13 14 kg SFM 300 400 User s Manual ver 2 7 1 4 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 controlled by a microprocessor 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 quantification of 0 14 ul per micro step u step when standard 10 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 Auto
121. tant in water of 56 M s Conditions can easily be set to make the concentration of 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 5 x OH NOTE The OH is the concentration of OH after mixing with DNPA The reaction can be quenched at any time by the addition of excess acid and the amount of DNP produced determined by absorbance at 325nm Figure 108 shows the absorbance spectrum of DNPA and DNP under various conditions of pH It can be see that the absorbance spectrum of DNP changes with pH but there is a clear isobestic point at 325nm 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 111 SFM 300 400 User s Manual ver 2 7 DNPA HCl DNP 2mM DNP 100mM _ lt Figure 108 DNPA DNP Absorbance Spectra Experimental Conditions Syringe 1 Water Syringe 2 1 mM DNPA 1 v v DMSO 2 mM Syringe 3 1 M NaOH Syringe 4 2 Delay Line 1 17ul Delay Line2 190ul 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
122. terface 1 1 3 Microcomputer commands The SFM module is controlled by Bio Kine software starting from version 4 0 for older versions please download an older version of this manual where the use of the MPS software is fully explained It is also advised to read the Bio Kine user s manual to get more information about software functions 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 reactions 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 SFM 300 400 User s Manual ver 2 7 1 2 Modes of Operation The SFM can be used in two main operating modes that are briefly described below More details on the two modes of operation can be found in later sections of this manual 1 2 1 Stopped Flow SF mode commercial reference SEM X00 8 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 unique features for a stopped flow instrument SFM 300 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 400 S Two to four solutions can be mixed and injected into the cu
123. 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 the copy area is bigger than paste area the operation is done only for values that can fit inside paste area IMPORTANT Blank and non numeric values entered in the program grid considered as zero values Phase duration of Oms will cause the phase to be skipped in the execution of the drive sequence Each time a program grid cell s 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 96 This information indicates e Current phase number and the total number phases used in the driving sequence e Volume delivered by the current syringe during the current phase or current phase total volume if an entire phase is selected 100 SFM 300 400 User s Manual ver 2 7 Flow rate of the current syringe during the current phase or current phase total flow rate if an entire phase is selected e Total volume delivered by each syringe during the driving sequence FA Quenched Flow Program time ms Syr 1 ul Syr 2 ul Syr 3 Syr 4 pl Waste Waste Waste Synchro 1 Off Off Phase f Total Volume IB ul Total Flow 15 0 ml s 1 2 3 Figure 96 Driving Sequence Information An indication of the Ageing
124. tion chamber and the syringes of the SFM are mounted vertically This allows for 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 kinetics studies even at temperatures below 0 C 1 6 The Delay Lines The SFM instrument can be used with delay lines permitting various reaction delays to be obtained between the two SFM 300 or three SFM 400 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 3 4 and 3 5 for full description of delay line installation and calculation of volumes Replacement of the delay lines is an easy operation which usually takes only a few minutes Delay lines of nominal volumes up to 1000 ul are available 10 SFM 300 400 User s Manual ver 2 7 Standard equipment of an SFM X00 S does not include ageing lines SFM X00 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 obtained as additional accessories To evaluate the ageing time of a reaction the entire volume between two mixers must be take
125. tion parameters Accessories Serial Port Peltier temperature controller Acquisition Board PCI 6052E board detected OK Cancel 4 2 Installation SFM 300 400 with MPS 60 or MPS 70 3 4 using Bio Kine version 4 47 and higher Once Bio Kine is loaded choose Install device installation in the main menu Figure 17 device installation The stopped flow communication is established from this window by checking the stopped flow device box and choosing the corresponding Serial port for MPS 60 or USB port for MPS 70 Accept the parameters using the OK button 28 SFM 300 400 User s Manual ver 2 7 Device Installation Acquisition device r Stopped flow device 05 450 4 810 serial acquisition Use stopped flow 05 250 4 810 analog acquisition Communication mode MOS 200 M C Serial port COM 4 1 gt TIDAS diode array g USB Acquisition parameters Accessories Serial Port CDM mf Peltier temperature controller mT jump PCI mT jump USB Acquisition Board No board detected OK Cancel Figure 17 device installation 4 3 Stopped flow Configuration Once the stopped flow device its serial port are selected in the device configuration menu refer to section 0 choose the Install stopped flow configuration menu see Figure 18 Stopped Flow configuration Device Syringes Syringe
126. 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 HCI 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 HCI 500 100 mM DNPA DMSO 112 SFM 300 400 User s Manual ver 2 7 Driving Sequence Various ageing times for the reaction are achieved by varying the intermixer volume 2 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 Table8 Waste Collect Table8 DNPA Experiment Parameters INTERMIXER VOLUME M2 M3 FLOW RATE ml s 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 113 SFM 300 400 User s Manual ver 2 7 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 the sample was prepared by hand by mixing 300 of the DNPA solution with 300 ul of water and 300 ul of 2 M HCI A tage the sample was prepared by hand by mixing 300ul of the DNPA solution with 300 ul o
127. tte Assembly The SFM observation head has been designed so that the observation cuvette can be exchanged in a few minutes This is often recognized by our users as one of the many advantages of the SFM systems Removal and replacement of the cuvette is shown in Figure 12 SFM 300 400 User s Manual ver 2 7 CUVETTE OPTICAL SPECIFICATIONS Cuvette Drawing Light path mm a Aperture mm b Main application Fluorescence light scattering Fluorescence light scattering high absorbance CD fluorescence Absorbance CD fluorescence Absorbance CD fluorescence Absorbance CD Absorbance CD fluorescence Absorbance CD Absorbance CD fluorescence WE Black Quarz Transparent Quartz s a CUVETTE DEAD VOLUMES AND DEAD TIMES With Berger Ball Mixer With High Density HDS Mixer Notes 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 Figure 7 SFM Cuvette Specifications 18 SFM 300 400 User s Manual ver 2 7 3 4 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 a
128. uenched flow sequence 12 6 1 SEM options 95 96 98 98 98 98 100 12 6 2 Design of the sequence 12 7 Running a shot 13 ASHORT QUENCHED FLOW PRIMER 13 1 General Principle of Quenched Flow Experiments 13 2 AGEING METHODS 13 2 1 Continuous flow method 102 103 103 104 104 13 2 2 Pulsed flow method 105 SFM 300 400 User s Manual ver 2 7 13 2 3 13 3 13 4 13 4 1 13 4 2 13 5 14 TEST REACTIONS IN QUENCHED FLOW MODE 14 1 14 2 14 3 14 4 Interrupted flow method 106 Double mixing experiment COLLECTION METHODS Total liquid collection 107 108 108 Partial liquid collection 109 General Advice for Quenched Flow Experiments Alkaline Hydrolysis of 2 4 Dinitrophenyl Acetate DNPA Calculation of Hydrodynamic Volumes from Kinetic Data Washing Efficiency Recovery of Uncontaminated Material in Intermixer Volume 111 111 111 114 115 116 SFM 300 400 User s Manual ver 2 7 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 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 USING A NONCOMPATIBLE SOLVENT WITH THE SFM IS NOT COVERED BY THE WARRANTY IN THE E
129. ure 19 is displayed It is then necessary to enter volume piston diameter and screw pitch of the custom syringe to add it to the standard ones Custom Syringe 5 0 ml 15 00 mm 4 00 m number 0 0 Max Vol ml 5 Piston Diameter mm 15 Screw Pitch 4 Modify Add Suppress Figure 19 custom syringe WARNING Incorrect syringe configuration will cause volume and flow rate calculations to be incorrect 30 SFM 300 400 User s Manual ver 2 7 4 4 Stopped flow status area A vertical menu bar on the left of the screen is dedicated to the stopped flow device see Figure 20 This menu bar can be hidden or displayed using the in the main menu This menu bar gives access to the syringe control window using the button refer to Mixing section 0 to the classic mode and to the advanced mode using the pex button refer to section 5 6 At any time information about the configuration of the stopped flow can be found in this bar such as device delay lines installed and cuvette type Once the sequence is ready in the classic or advanced mode the shot control window is displayed in the area as shown in Figure 20 also refer to section 5 6 Stopped flow SFM 20 5 IL Ready 61 shots Autorun End Delay lines Ld jw 100 10 MPS On Line Temp C 77 5 Figure 20 stopped flow menu bar 31 SFM 300 400 User
130. ure 63 Dead time In Ao A Figure 63 dead time evaluation Configuration of Bio Kine Load Bio Kine Enter the Install device menu and select the stopped flow device and its serial port Configure the stopped flow device and the syringe sizes Push some water into the cuvette and do the absorbance reference Initialise the syringes and then fill the syringes V v Vv v v Slow reaction at pH 9 gt Click on the button then on the SFMDp ons button select the 100 10 cuvette and a 2 ms lead time Validate by clicking the OK button gt Edit the driving sequence shown in Figure 64 gt In your acquisition software choose to perform one measurement every 1 ms during 3 seconds gt Run the sequence gt The data obtained is shown in Figure 65 Fast reaction at pH 2 gt Click on the 599 button then on the SFMOPlens button select the TC 100 10 cuvette and a 2 ms lead time Validate by clicking the OK button Edit the driving sequence shown in Figure 66 In your acquisition software choose to perform one measurement every 50 us during 0 15s gt Run the sequence gt The data obtained is shown in Figure 67 gt gt 71 SFM 300 400 User s Manual ver 2 7 Dead time calculation From the kinetics at pH 9 we get 0 787 A U From the kinetics at pH 2 we get A 0 151 U and k 445s Therefore the real dead time is 3 7 ms The estimated dead time given by the MPS softwa
131. ve process for the other syringes 7 It is recommend that the syringes be filled in reverse numerical order to best remove bubbles from the SFM and cuvette 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 Stopped Flow Module is now ready for operation 96 SFM 300 400 User s Manual ver 2 7 Figure 92 SFM Syringe Filling Procedure Panel 2 97 SFM 300 400 User s Manual ver 2 7 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 recommended daily cleaning procedure to be done before shutting off the instrument 1 Remove the 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 waste tube it will wash the flow lines and waste part of the electrovalve as well as the syringes To clean the collection port the best is to run a washing sequence where water is collected i
132. vette 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 1 2 2 QUENCHED FLOW QF MODE commercial reference SFEM X00 Q In this configuration the SFM functions as a complete quench flow instrument This configuration allows for various modes of operation as described in Figure 1 SFM 300 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 it is mixed with a flow of washing buffer SFM 400 Q It be used as 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 Alternatively 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 a
133. with air The SFM is now ready to be stored 12 6 Creating a quenched flow 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 activating the hard stop and triggering data acquisition Driving sequences are created in the window shown in Figure 93 This window can be reached from the Mixing Sequence button in the stopped flow status area 98 SFM 300 400 User s Manual ver 2 7 n Quenched Flow Program time ms Syr 1 Syr 2 Syr 3 Syr 4 Valve Synchro 1 Collect Waste Off Off Waste Off Remove Phase Phase 3 5 Volume pl Flow Rate ml s Syringes contents Load Save As Comments Print Shots Drive Sequence Ageing Times 14 20 DL1 DL2 EDL SFM Options Close Figure 93 example of quenched flow sequence The first operation should be to check the configuration of the stopped flow This is done by clicking on the _ SFM Options Stopped flow options button refer to Figure 94 Cuyette Hard Stop Acceleration Phases Auto Automatic C Manual m None C Manual Valve Lead d ms V HDS Mixer X Overheating Protection Default DK Cancel Delay Lin
134. xer 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 9 1 The flow rate through the intermixer volume is modified by changing the flow rate of the syringes in the driving sequence The use of stepping motors in the SFM allows a large range of syringe flow rates to be programmed and many tage values achieved with minimal changes 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 101 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 experiment DL2 is Delay line n 3 the intermixer volume for mixers 2 and 3 is then 116 7 ul and the total flow through the intermixer volume is 4 ml s syr2 syr3 which indicates 1167ul 29 2 ms i 4ml s The continuous flow method is generally used to study reactions from 1 to 200ms It is generally the most economic ageing method with respect to sample consumption within this time range To get correct mixing a minimum flow rate of 1 ml s through each mixer is required 104 SFM 300 400 User s Manual ver 2 7 ere time ms Insert Phase Syr 1 nl Remove Phase
135. ypes of mixing experiments It is difficult to list all the possibilities here A few common types 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 mix 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 All SFM Q instruments are shipped standard with a quench exit valve 1054 to simplify sample collection The exit valve and delay line s are installed in the SFM body differently depending on how many syringes are present and the type of experiment performed SFM 300 The exit valve and delay line are installed as shown in Figure 80 SFM 400 The exit valve and delay line s are installed as shown in Figure 81 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 Table7 Figure 79 Exit Valve Table7 SFM 400 Exit Valve Installation MIXING BLOCK COMMENTS 0 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 w
136. zation 47 SFM 300 400 User s Manual ver 2 7 Stopped flow options r Cuvette Hard Stop Acceleration Phases p Delay Line 1 ul f Auto n t 17 47 4 FC 15 Manual FC 20 O Manual 50 10 None Delay Line 2 ul TC 50 15 TC 100 10 Valve Lead m 100 15 23 ms Ejection delay Line pl 3 HDS Mixer Hard Stop closed between shots 4 X Overheating Protection igger Default DK Cancel Figure 37 stopped flow options Once all parameters are fixed from the stopped flow option window click on the OK button Clicking on hntos opens a new window Figure 38 giving informations on the double mixing experiments The theory the description of the methods is explained double mixing also called double jump experiment can be described as follows and are mixed first mixer after a defined incubation time in a delay line the mixture is mixed with C The second reaction is then monitored in the cell It is done using 3 syringes and 2 mixers according to the following diagram The aim of this mode is to propose automation of double mixing experiments by changing automatically the ageing time between shots the dead time of the second reaction remains constant in each experiment Cuvette Configuration Samples and B must be installed before first mixer M1 into syringes 1

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