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Stopped Flow Manual

1. Absorbance O an 0 50 100 150 23 SFM 4 USER S MANUAL bio V logje The same curve shows that contamination is still present after the first washing and that the delay line needs to be washed with at least 100 ul to be reasonably free of contaminating reactant A similar experiment with a delay line of 100 ul is shown in figure 4 3 b It shows that the total dynamic volume of this delay line is close to 120 ul a volume very close to the 122 ul indicated in table 1 6 2 Optical density 0 150 300 450 Total purge volume ul 4 4 Recovery of the material contained in the delay line In the interrupted mode the reaction mixture is transiently stored in the delay line During this incubation period unwanted mixing occurs at both ends of the delay line so that only a fraction of the mixture can be recovered from the delay line The experiment described below is intended to give an estimate of that fraction PHASE N 1 2 3 A f TIME ms 50 50 300 0 Tp 20 0 SYRINGE NUMBER VOLUMES ul Not used DNPA 1 5 mM NaOH 100 mM HC1 240 mM Collect 24 SFM 4 USER S MANUAL ae logie This experiment is designed to test delay line N 2 In the first phase DNPA and NaOH are pushed through the delay line and wasted The second phase is used to wash the last mixer with HCI The reaction mixture is then allowed to age f
2. nM u A ee Transfers parameters and sets MPS 51 in auto mode Each time parameters are entered in the table a window opens on the right side of the screen and indicates the flow rate per syringe at the time and the total flow rate in the cuvette at the time of the current phase and the total volume injected during this phase To eliminate this window type Tab 20 SEM 4 USER S MANUAL gto V logie 4 9 Acceleration phases It is possible that your instrument will be able to push solutions at a faster rate than 6 ml s but this is not guaranteed in all cases and eventually one of the motors can get stalled giving a completely flawed reaction mixture To program flow rates up to 8 ml s before the actual fast flow rate you must enter an acceleration phase An example is given below BIO LOGIC SFM 4 S V3 10 Help lt F1 gt Syringe Vol Stopped Flow Parameters 3 4 5 6 50 0 0 0 400 0 0 0 2995 pl 0 3200 pl 0 1795 pl 3200 pl Transfers parameters and sets MPS 51 in auto mode We recommend that you test the sequence on inexpensive buffer solutions of same viscosity as that which will be used in the real experiment 4 10 Programmable synchronizing pulses The MPS 51 power supply can be programmed to deliver a synchronizing pulse trigger This pulse is delivered from connector 4 see section I of this manual at an
3. coe p qu i SFM 4 USER S MANUAL fegie TABLE OF CONTENTS INTRODUCTION AND SPECIFICATIONS cece ese were to 4 I General description EES ms es ar ed Es al 4 5 a 00S 8949 Modes ot Operation 1 2 7 ESE EE lt Ed 1705506015166505 L4 Principle Of Operation 626 deesses em es ORS 8 1 5 Description of the mechanical design eere 9 1 6 The ageing lO0DS amu AA Gea eee ee eS 9 J 2 GENERAL INSTRUCTIONS FOR INSTALLATION _ o ooooooooooo o 10 2 Operating features 3 55 39 4 tao rey eee tbe does E 10 22 AC power and Connections 5 4 doe quee tu ad asi QU a ai cete 12 ds 12 ne rand Water cIFrCulatioli a sac eeces 2 9 2 4 Installation of the software on the hard disk 12 JAN SFM 4 USER S MANUAL 1 INTRODUCTION AND SPECIFICATIONS 1 1 General description The Bio Logic stopped flow module SFM 4 consists of a mechanical subsystem and a power supply MPS 51 4 The mechanical sub system consists of four machined syringes one valve block with four 3 way valves the possibility to include one to three mixers and up to two ageing loop The SFM 4 syringes valves delay lines and cuvettes are enclosed i
4. auum Uus FE dams s Manual Speed Reference Limit N 4 Stop M From low speed N 1 to high speed N 5 At this point it 1s possible to Control the manual speed of the motors Drive the motors from the keyboard Initialize the syringes counters The motors can be controlled by the front panel of the MPS 51 except when the automatic mode is turned on They can also be controlled by the computer keyboard when the Manual menu is turned on To select the motor use the vertical arrow Use lt PageUp gt or lt PageDown gt to raise or lower the piston of the syringe respectively The speed of the manual movement can be selected by typing lt S gt by selecting the Manual Speed function with the Space Bar and typing lt Enter gt or by using the horizontal arrows 3 6 Initialization of the syringes The microprocessor of the SFM 4 integrates the movements of the 4 syringes so that the actual residual volumes can be displayed at all times on the screen When the instrument is turned on the counters show a nonsense value and have to be 14 SFM 4 USER S MANUAL ble initialized Select the syringe to be initialized with the vertical arrows Use the keyboard or the manual switch of the MPS 51 power supply to raise the plunger of the syringe to be initialized UNTIL THE SYRINGE REACHES ITS UPPERMOST P
5. De t pep d 7 2 4 CT xe VOLUMES dears cate add uec be ees b pea 9 2 biguld DUES St i aa ate udo ER dne o Vie ee Suas 9 DES SIN CCOO SS SIC tom sonata ves Sacs e ap 9 DS DE XII CS VINE ta cd ea ese Es d erepta ore 10 25 3 DIVEMING valve OUIDUL sustos is ee a AIR Rar 10 a SOFTWARE OPERATION 5 53 5425 dia EV TIE orari ph sea deer petet Sad 11 S T InttodUetloll dutes sie e Has ee ee EUR uis d e 11 3 2 Installation of the SFM 4 Q version of the software 11 3 3 SFM 4 Q driving software configuration u e eee LL Lee 11 3 3 1 SyripednstallatoD soto et is que OC PROPIO Ned eee es 12 3 3 2 Installation OF ihe exit Val VE o d aca ROO ES ea a 12 53 3 3 110851311511011 0 7 1172 597131 pol esse veda eee PY D EA e elt 12 3 4 SFM 4 driving software 4 25 caused todos nee chee cases mis 13 3 5 Manual control of the Syringes erus terere niaaa Vei e b Re 14 3 6 Titialzatonoft the a A PEE ESO ed erba dide 14 Ss PAIS ESV NEES erie wa O Gant eee Bs Sag ae SEE 15 3 8 Creating d drive Sequence ae had e bec SG vor st oe Von a wees 17 3 D Acceleration phases uua sores da QUIE eae eee 17 SLE Tneubation Se QUE CE oce vci OETA dietes 18 0 3 12 10110656101 6131 suivant
6. Way ke SUD OUO A RO eade 21 7 LES REACTIONS 1424 19 AAA 29 TT EValualion of the dead time ated 29 7 2 Evaluation of the cuvette washing and quality of stop 30 7 41130516 73110 TUNING oe ee NC Creo Re epe aoa de e POST 31 7 3 1 Experiments using DCIP and ascorbic acid 32 7 3 2 Measurement of variable ratio mixing by following alcohol 061070702620956 pa du rca a co EE RARUS 34 7 4 Mixing solutions of unequal density and viscosity 35 SFM 4 USER S MANUAL 1 INSTALLATION OF THE OPTICAL SYSTEM The Bio Logic stopped flow module can be adapted with any good quality optical system However higher performance can be achieved with the Bio Logic optical and light detection components Examples of installations and equipment are listed in the table below Table I SFM 4 fibre optics for illumination OK if connected to a spectrophotometer for fibre optics for light detection a host absorbance measurements oe OOO BioKine software Main applications advantages or limitations Probably insufficient for fluorescence Except in case of very high fluorescence signal connection to a fluorometer will lead to an insufficient sensibility in detection Since no BioKine is in the package data analysis will have to be provided by
7. ed E aa 18 3 13 Saving or loading the experimental parameters 19 3 14 Runtimp the automatic MO CES tre ewe n 19 4 CALIBRATION OF THE INSTRUMENT cera added Y Eee ERR PESO P arg 21 41 Description of the test redolo ea eae hauls aed Reg Mug 21 4 2 HOW to pet varous ageing UES au wants ARTS va educa Ei ES 22 4 3 Measurement of flow lines volumes and of efficiency of washing 22 4 4 Recovery of the material contained in the delay line 24 4 5 General checking of the instrument a a koto edet teres 25 4 6 Dead Nine ofthe 110300077020 are nS eats hae esp an dba 26 4 7 Unequal Tatio MIXING els ve dba EORR P dd deer at Ee dao d 27 48100101 1 01 Ros US e eat 28 4 9 General guide lines for creation of the drive sequences 29 SFM 4 USER S MANUAL del V VJ OGG 1 GENERAL INSTALLATION 1 1 Introduction This section of the manual contains information on the installation and operation of the SFM 4 Q It is recommended that the content 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 1 2 Installation The b
8. SFM 4 User s Manual eU RX SFM 4 USER S MANUAL os 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 IN THE EVENT OF FAILURE OF A PRODUCT COVERED BY THIS WARRENTY THE PRODUCT MUST BE RETURNED TO AN AUTHORIZED SERVICE FACILITY FOR REPAIR AND CALIBRATION AND TO VALIDATE THE WARRANTY THE WARRANTOR MAY AT THEIR DISCRETION REPLACE THE PRODCUT IN PLACE OF REPAIR WITH REGARD TO ANY INSTRUMENT RETURNED BECAUSE OF DEFECT DURING THE WARRENTY PERIOD ALL REPAIRS OR REPLACEMENTS WILL BE MADE WITHOUT CHARGE IF THE FAULT HAS BEEN CAUSED BY MISUSE NEGLECT ACCIDENT OR ABNORMAL CONSITIONS OF OPERATION REPAIRS WILL BE BILL AT NORMAL COST IN SUCH CASES AN ESTIMATE WILL BE SUBMITTED BEFORE WORK IS STARTED IN CASE ANY FAULT OCCURS NOTIFY BIO LOGIC OR NEAREST SERVICE FACILITY GIVING FULL DETAILS OF THE DIFFICULTY AND INCLUDE THE MODEL NUMBER TYPE NUMBER AND SERIAL NUMBER UPON RECEIPT OF THIS INFORMATION SERVICE OR SHIPPING INSTRUCTIONS WILL BE FORWARDED TO YOU EXCEPTION ARC LAMPS SOLD BY BIO LOGIC ARE ONLY WARRENTIED FOR A PERIOD OF 3 MONTHS FROM DATE OF PURCHASE
9. E Es a d a a Qu uU uu ci T ee v m _ a a co oa aaa a o O O 2 o EE a p oa i o o p go o o o i o CA E o da E n 0 0 a 0 D D p a e e D cs ss 0 A i la a T A Le ee 0 da oL O 0 T d o de Se 0 a y ae a ee Ce a a lt a o E o a de d 0 0 00 i d 0 0 a 0 T i 00 Dr 4 7 s a da i DC um de i a o f y o Ween 1 S e i 7 225 SES 20020 i ce A A dd de E it i Cea Co es Ca d E 0 1 0 a E g a 0 Ae EG m 0 T 1 0 0 i 20 E ue e aes ca a e 1 ij Ut i e 0 a a e a Tr 00 0 s Ur i i y pe 0 MT 0 Da m Ur TU 0 PU sae Po qm OU E 2 i O a o a 1 O i a 0 ee 0 D T D e uu t 7 oa 00 Du De 0 0 2 E e 7 e i gt 20 1 La i eu 1 200 cs jj Gs v a La 1 E T j 0 M a 0 j a a o E c p Se ee c 0 g a a a a 7 ce 0 RIT S FOR SFM 4 N 165 HEAD NUT N 157 BRACE PTFE CUVETTE N 887 HEAD CAPS ADAPTOR FOR APTICAL FIBER COLLIMATOR WITH OR WITHOUT LENS Un de QE PE N 1067 PM FIXATION A
10. Stopped Flow Parameters 2995 pl 3201 pl 1795 pl 3200 pl Transfers parameters and sets MPS 51 in auto mode The sample experiment shown utilizes two phases The first phase is used solely _ to trigger data acquisition so that the shot may be observed The second phase defines the shot Over fifty milliseconds syringe 2 and syringe 4 each push 200 microliters through the mixer Syringe 1 remains dormant in this experiment Once the experiment is designed the MPS 51 is programmed with the experimental parameters Experimental design is passed to the MPS 51 over the serial RS 232 cable Next during the actual experiment the MPS 51 controls the syringes and the computer is used to collect data using Bio Kine software and the the Data Translation Card inside the computer 6 3 General advice 6 3 1 Achievement of fastest dead times lhe dead time of a stopped flow experiment is the time before which observation of the mixture is impossible The dead time depends on a number of factors only some of which the experimenter can control Ideally the dead time depends only on the rate of flow of the mixture from the mixer and the volume of the cuvette As the flow rate increases the dead time falls As the cuvette volume falls so does the dead time Nevertheless an effective stopped flow experiment depends on a number of other inter related factors such as adequate signal complete washing of the 26 SFM 4 USER S
11. 540 0 045 10 000 20500 4 540 0 045 10 000 20500 Serial port COM1 1 Exit valve lead 3ms Type CTRL P to change serial port number Type lt CTRL S gt to change hard stop lead Type lt ESC gt when installation is done 3 3 1 Syringe installation Unless otherwise specified the SFM 4 stopped flow module is equipped with standard 18 ml syringes 917 mm giving 4 54 ul per mechanical step The flow rate is limited by the minimum duration of the drive impulses giving a maximum flow rate of 6 ml s with the standard 18 ml syringes The 4ml syringes 28 mm give 1 005 ul per mechanical step 3 3 2 Installation of the exit valve 2 lt CTRL S gt will select the exit valve lead from 1 to 5 ms This lead is used to compensate for the electromagnetic lag that may affect the valve The default setting is 3 ms 3 3 3 Installation of the serial port The program detects the number of available serial ports Type lt CTRL P gt to select which serial port you would like to use rico SFM 4 USER S MANUAL 3 4 SFM 4 driving software By validating Driving Software the SFM 4 Q menu will soon appear on the screen BIO LOGIC SFM 4 Q 0 Help F1 NONAME QF4 Syringe Vol Quenched F low Parameters 3 4 0 0 0 Waste Coll W Drive Sequence 1 1 9 Load Save An action can be performed by placing the cursor of the desired function and validating with the lt Enter gt key Displacement of th
12. and the other through the valve block and observation head 2 4 Installation of the softwareon the hard disk Note at this point we assume that you are familiarwiththe DOS filesstructureSee your DOS manual for more details Make a backup copy of your original diskette set and put it in a safe place Note The followingprocedureis available if you orderedtherapid kineticsoftware BIORINE softwaretogetherwith SFM 4 QS if it is not the case then do not read the instructionsconcerning BIOKINE Insert disk 1 in your diskette drive and run INSTALL Follow the instructions displayed Afterthe INSTALLATION is done a BIO BAT program is copied onto the root directory of the hard disk and the following directories are created gt X BIOLOGIC BIOKINE x BIOLOGIC gt X BIOLOGIC SFM4S gt X BIOLOGIC SFM4Q Where X is the name of the disk drive C D E 12 EV d SFM 4 USER S MANUAL eto V If you wish the software to run automatically when your computer is turned on use the DOS editor EDLIN or any other TEXT EDITOR to add the following line to the AUTOEXEC BAT file BIO BAT THE NEXT INSTALLATION STEPS ARE SPECIFIC OF THE VERSION OF THE SFM 4 VERSION USED PLEASE CONSULT THE PART OF THE MANUAL CORRESPONDING TO THE VERSION THAT HAS BEEN ORDERED OR INSTALLED 13 ao a n O a a E a e a cee 0 e a o e oo oe oe rr 0 0
13. condition arises in which the age of the mixture tracks IM cuverrE linearly with distance along the flow path during the steady state condition at any particular point in the flow path the mixture is of a particular age Furthermore the age of the mixture in the cuvette during the shot is the theoretical dead time which is the time before which Observation of the mixture 1s impossible The second stage of the experiment begins when the flow is stopped At this point the mixture in the cuvette and elsewhere becomes stationary and continues to age Observation of the mixture in the cuvette after the stop therefore shows a timecourse of the reaction from the dead time onward The figure below shows an example reaction in which reagent A in the presence of B reacts to form product C A has a strong absorbance while B and C do not Therefore as a reaction proceeds the absorbance of a mixture of A and B should fall as A is diminished 24 SFM 4 USER S MANUAL JAN A eae STAGE 1 SHOT STAGE 2 REACTION tal e La c eo a 7 6 2 Design and execution of stopped flow experiments Experiments are designed using the Bio Kine SFM 4 driver software on the computer This is accomplished by filling in elements of a spreadsheet like table which describes the actions of the device A sample experiment is shown here 25 SFM 4 USER S MANUAL JA BIO LOGIC SFM 4 s V3 10 Help lt Fl gt Syringe Vol
14. 0 0 0 0 0 0 0 0 ms SES Susa 0 0 0 0 0 0 0 0 0 yl 5277 uiis 0 0 0 0 0 0 0 0 0 yl Soe 0 0 0 0 0 0 0 0 0 yl 54 0 0 0 0 0 0 0 0 0 yl Synchro 1 0 0 0 0 0 0 0 0 0 Synchro 2 0 0 0 0 0 0 0 0 0 Manual Speed Reference Limit N 4 lt Stop gt From low speed N 1 to high speed N 5 At this point it is possible to Control the manual speed of the motors Drive the motors from the keyboard Initialize the syringes counters The motors can be controlled by the front panel of the MPS 51 except when the automatic mode is turned on They can also be controlled by the computer keyboard when the Manual menu is turned on To select the motor use the vertical arrow Use lt PageUp gt or lt PageDown gt to raise or lower the piston of the syringe respectively The speed of the manual movement can be selected by typing lt S gt by selecting the Manual Speed function with the Space Bar and typing lt Enter gt or by using the horizontal arrows 17 07 bie V SFM 4 USER S MANUAL logia 4 6 Initialization of the syringes The microprocessor of the SFM 4 integrates the movements of the 4 syringes so that the actual residual volumes can be displayed at all times on the screen When the instrument is turned on the counters show a nonsense value and have to be initialized Select the syringe to be initialized with the vertical arrows Use the keyboard or the manual switch of the MPS 51 power supply to rais
15. A 3 2 Dimensions of the head Front view 4 00 6 2 00 4 50 00 SFM 4 USER S MANUAL JAN fiegis 3 3 Front view references 5 161 ie 1228 5 a 149 SAI 1264 EES ryt 949 Y 332 1261 1227 1220 m 1236 i 623 139 DN l 18 119 1233 738 Io w 20 A Pr 23 b T GRE IL t LILI puce 168 1230 SFM 4 USER S MANUAL de gt m 3 4 Side view references 1 T 3 124 17 113 1262 28 1234 PER T r sd SFM 4 USER S MANUAL eto V X ede 3 5 Top view references 1084 1082 169 97 166 422 887 2 RR j 2 423 H ah 9 37 ES Ep I eil A 1265 En LA 1 886 1 167 1279 1280 i E 1229 1433 bie V SFM 4 USER S MANUAL 4 SCHEMATIC DIAGRAMS 9 1 CPU schematics 9 2 CPU components 9 3 CMP02 logic part schematics 9 4 CMP02 logic part components 9 5 CMP02 Analogic part components 9 6 CMP02 Analogic part schematics 9 7 Front panel schematics 9 8 Front panel components 9 9 Power supply schematics 9 10 RS 232 Pinout 9 11 Serial port configuration of MPS 51 power supply S SFM 4 USER S MANUAL 9 11 Serial port configuration of MPS 51 power supply The serial port of MPS 51 uses standard 25 pin RS 232C data line configuration It can be connected to the serial communication card of the microcomputer via a 25
16. USER S MANUAL Ne logie 4 6 Minimum ageing time The minimal ageing time for the SFM 4 is obtained using the shortest intermixer volume no additional delay line and MIX 0 block operating in the continuous mode The total intermixer volume using this delay line has been evaluated above to be 22 ul The minimum possible ageing time depends on the achievable flow rate With the valve a maximum flow rate of 15 ml s is recomended So that the flow that can be reached in the second delay line can be in the range of 12 ml s giving an ageing time in the range of 2 ms An example is shown below PHASE N 1 2 3 4 5 6 TIME ms 10 40 20 0 0 0 olf 0 0 0 0 0 0 SYRINGE NUMBER VOLUMES ul Not used Reactant 1 60 240 0 0 0 0 Reactant 2 60 240 0 0 0 Quencher 30 120 0 Collect Delay line N 1 0 ul Delay line N 2 0 ul In case the valve is not used and direct collection is performed higher performances can be reached If two syringes can be used to push one solution flow rates can reach higher values An example 1s shown below 6 0 ee _ sme wes mue 0 0 0 0 0 0 180 Reactant 1 Reactant 1 Reactant 2 Quencher Collect 27 ATPase EGTA Calcium ATP 32P PCA SFM 4 USER S MANUAL Bie V Flow rate will reach 18 ml in the flow line between the last two m
17. ates 100 100 S3 re 0 S4 Collect Sequence Start Stop Esc 22 shots to go Start or Stop according to the status of unit When ready type lt S gt to run The run can also be activated by depressing button 5 on the front panel of MPS 51 or from an external trigger signal In case of necessity the sequence can be terminated by typing the lt S gt key again IMPORTANT The power supply has been designed to deliver high power to the motors over a short time low duty cycle it is turned off shortly after completion of the run Permanent activation of the power supply may result in components overheating The red LED 2 on the front panel indicate when at least one of the motor power supplies is activated Reset the program button 3 if this LED remains permanent turn off the power supply in case the trouble persists Start the sequence again This type of trouble should never occur under normal operation This warning has nevertheless been included here to prevent failure under abnormal environmental conditions Type F1 to obtain more details about the syntax used to enter and correct the drive sequence Identification On the left of the screen a field is reserved to enter four letters to identify the contents of the three syringes Set the cursor in this field by depressing the Tab key Return to the data field by the same action lege SFM 4 USER S MANUAL bio W 4 CALIBRATION OF THE
18. by depressing the lt Tab gt key Return to the data field in the same way With ordinary aqueous solutions the motor can drive the syringes up to at least 6 ml s without acceleration phase This means that motors will not stall if the following sequence is programmed BIO LOGIC SFM 4 Q 0 Help F1 n ooo e aa EEUU e TEST Quenched F low Parameters Phase 2 Flow ml s Phase 1 2 3 4 5 6 7 8 9 rime 100 50 0 0 0 O0 0 0 0 ms Tot 10 000 LS Seeds 030 0 0 0 0 0 0 0 ulf S1 6 000 S2 030 0 0 0 0 0 0 0 u S2 0 000 LS usns 0 0 0 0 0 0 0 0 0 ul S3 0 000 S4 0 0 0 0 0 0 0 0 0 ul S4 6 000 Tot Vol ul Waste Co11 WN WN NW MW W W W W MW 600 Drive sequence 1 1 9 Transfers parameters and sets MPS 51 in auto mode Each time parameters are entered in the table a window opens on the right side of the screen and indicates the flow rate per syringe at the time and the total flow rate in the cuvette at the time of the last phase and the total volume injected during this phase To eliminate this window type Tab 3 9 Acceleration phases It is possible that your instrument will be able to push solutions at a faster rate than 6 ml s but this is not guaranteed in all cases and eventually one of the motors can get stalled giving a completely flawed reaction mixture To program flow rates up to 8 ml s before the actual fast flow rate you must enter an acceleration
19. can be adjusted at will by the user In case the volume collected is not very large as compared with the tube volume it is recomended to wash the old solution out of the tube during two shots SFM 4 USER S MANUAL bio A 9 2 5 2 Exit in a syringe In this method a syringe 1s plugged at the SFM 4 exit This may be used in case the delay line volume is small as compared to the volume collected When using this method it is recomended to wash the delay lines and the mixers with buffer between two shots using one of the syringes During the actual run this buffer contained in the delay line will dilute the collected sample and will have to be taken into account during sample evaluation This mode of operation will be used mostly for short ageing times when the delay line volumes are small The shortest distance betwee mixers are in the range of 20 ul The maximum flow rate of the SFM 4 instrument is 6 ml s without acceleration and 8 ml s with acceleration Thus with two syringes flowing at the same time through a delay line ageing times in the range of 1 5 ms may be reached This time can come very close to 1 ms with three syringes flowing at the same time 2 5 3 Diverting valve output This is the classical quenched flow mode It may be used when the delay line volume becomes too large as compared to the volume collected This will be usually the case for long ageing times in continuous flow mode and in the interupt mode The
20. diverting valve can operate correctly up to a certain pressure Because of this its operation is not guarantied above 15 ml s 10 SFM 4 USER S MANUAL TA 3 SOFTWARE OPERATION 3 1 Introduction This section of the manual contains operating instructions and a description of the experimental procedure It is assumed that you have run the installation softawre as described in section I 3 2 Installation of the SFM 4 Q version of the software Dos yansi m Run BIO from Root directory You will get the following screen Use the arrow keys to select the menu option you wish to run P re SFM 4 S SFM 4 Q Exit Driving Software Config Driving Software Application Driver Software V3 0 Copyright AEF 1991 Bio Logic Company All right reserved Z A de FONT RATEL 38640 CLAIX FRANCE Pit 76 98 6684 32 Fax 76 98 69 09 3 3 SFM 4 Q driving software configuration The SFM 4 Q configuration program is allready set at the default values It can be used to install or change the syringe model set different flow rate limitations enable or disable the hard stop mode and select serial port number After validating Config Driving software the following screen will appear 11 SFM 4 USER S MANUAL bio V LOGIE Syringe and RS232 installation V3 02 Volume per step ul Flow Rate ml s Syringe Vol ul Lower limit Higher limit 4 540 0 045 10 000 20500 4 540 0 045 10 000 20500 4
21. diverting valve for waste and collect panel N 2 Alternatively an external flow line can be connected for directinjection of the mixture into a quenching solution This mode may be used with or without an additional delay line as shown in panel N 3 and 4 below It can be used in a simple 3 syringe mode and direct collectionin a syringe as described in panel 5 In another mode the mixture can be injected onto a filterat the same time as it is mixed with a flow of washing buffer panel N 6 Flash quenching with a photoreactive reagent is also a mode that can be easilly implemented to the SFM 4 Many other configurations are possible you are invited to inquire about the feasibility The commercial reference SFM 4 QS has all the components for the two applications A SFM 4 S or a SFM 4 Q can be updated to SFM 4 QS Light Detection Mixer 2 motor 1 motor 2 Mixer 2 motor 1 motor 2 motor 1 motor 2 E poe Collect Mixer 1 I Mixer 2 motor 1 motor 2 motor 1 motor 2 SFM 4 USER S MANUAL 1 3 Specification Table A SPECIFICATIONSorSFM 4 Numberof syringes Drivingmechanism one steppingmotor per syringe Steppingmotor driveoperatingat 6400steps per motorturn 1 to 3 25 to 100011 Numberof mixers Ageingline betweenthe two m
22. entry If these precautions are not taken and for example if a long continuous tube is connected to the outlet a long column of liquid will be pushed during the flow At motor stop the inertia of this liquid column will inevitably generate underpressure in the cuvette and leads to cavitation 2 5 2 Hard stop system In this mode flow will be immobilized by a combination of two mechanisms firstly from the stepping motors stop secondly by a high speed electrovalve which closes the output of the SFM 4 cuvette This electrovalve is actuated by the programmable power supply of the SFM 4 No overpressure is developed in the observation cuvette because of the perfect synchronization with the motor halt The result is the elimination of the stop and overpressure artifact giving an incomparable quality of the fastest stopped flow traces Operation of the valve is as follows 1 SFM 4 is in manual mode The valve is always open 2 SFM 4 is in automatic mode Between two shots the valve is always closed During a run a the valve opens at the beginning of the flow b the valve closes at a designated number of miliseconds before the flow stops 2 5 3 Installing the hard stop valve Install first the appropriate cuvette in the observation head tighten the upper Screw Connect the hard stop assembly to the MPS 51 power supply see general instructions section I Note Connect a small tube to the horizontal exit of the hard stop devic
23. executed once in series if one of the phases has a time value of 0 it will just be skipped in the sequence Other type of sequences can be created as in the following examples A subsequence as 2 2 5 indicates that phases 2 to 5 will be executed two times as follows 2 3 4 5 2 3 4 5 A sequence as 2 2 5 6 8 indicates that phases 2 to 8 will be executed as follows 2 3 4 5 2 3 4 5 6 8 3 13 Saving or loading the experimental parameters The created data can be saved on files A series of experimental conditions can thus be prepared before the experiment and subsequently recalled vvhile the program is being run At the command level type lt F gt for Files Save or load the parameters by following the instructions displayed on the screen For help at any point during operation type lt F1 gt The data on the screen can be erased by typing lt C gt 3 14 Running the automatic mode The sequence created in the microcomputer will be transferred to the MPS 51 unit to be executed in the automatic mode Verify that the manual valves corresponding to the active syringes are set in the right position i e pointing to C Type lt A gt or select Auto to enter the automatic mode The screen will show the following messages SFM 4 USER S MANUAL bio logie BIO LOGIC SFM 4 Q V3 10 Help F1 Quenched F low Parameters Syringe Vol 2 3 4 5 6 Time 20 200 20 500 100 Su 100 0 0 SCS
24. fast reaction to calibrate the dead time of the SFM 4 instrument then we use the neutral pH slow reaction to check the quality of the stop to evaluate the washing of the observation cell and to test the variable ratio mixing capabilities Syringe 1 2 or 3 10 mM Ascorbic Acid AA Syringe 4 5 mM Dichlorophenolind Total absorbance ophenol DCIP change 0 23 The decoloration of DCIP was followed by measuring the transmittance at 524 nm We used 0 6 1 0 2 D 3 04 0 5 0 6 0 7 Ge Og 4 the TC 50 10 transmittance cell The reaction was followed at pH 9 0 and pH 2 0 Reaction at pH 9 0 is shown in the figure above Data acquisition was made using BioKine software and acquisition was started at the beginning of the drive phase 29 SFM 4 USER S MANUAL e Jegie Collected data show a transition with a half time around 100 ms Using the slow reaction conditions ensures that 100 of the reaction is observed At the same time smooth kinetics Observed absorbance enable an easy detection ee of any stop artifact The reaction at pH 2 0 is shown in the figure of this page it gives a much faster transition with a half time close to f Time Cms 2 ms Measurement of the rate constant and amplitude of the transition at pH 2 0 permits an evaluation of the dead time of the instrument Calculations are described below Results of the analysis using the BioKine software pH Total f
25. great care permutation is not recomended 2 3 Plunger disassembly and reassembly Lower the plunger with the manual control down to its lowest position Unscrew the two screws holding the plunger onto its two vertical guides Remove the plunger and its holder Wash and or exchange the teflon head Reassemble in the reverse order Note Drive syringe leakage A slight amount of leakage past the teflon syringe tips is normal and necessary and increases with the speed of the syringes This slight leakage is the result of design trade offs regarding the sealing quality and rigidity of the syringe tips Excessive leakage can often be alleviated by a number of remedies The amount of leakage which is considered excessive is an operational definition depending 4 SFM 4 USER S MANUAL V 6 on the nature of the experiments being performed etc Examples of experiments which put high demands on the sealing quality would be those with higher flow rates and or those using more viscous solutions The following are tips to help minimalize syringe leakage 1 In general the SFM 4 should be stored between usages i e overnight or longer with the drive syringes dry and in the fully withdrawn position such that the white teflon syringe heads are outside of the drive syringe barrel This allows the teflon to expand slightly between uses allowing a better seal during experiments 2 If leakage occurs which e
26. kinetic of enzyme renaturation is to be measured As a test reaction we used cytochrome c denatured in 5 5 M guanidine This solution was mixed with buffer in large dilution ratio using the SFM 4 stopped Fluorescence Carbitrary scale 0 100 200 300 400 00 Time ms flow so that rapid decrease of guanidine concentration result in refolding of the enzyme The kinetics of renaturation was followed by monitoring the cytochrome c intrinsic fluorescence Conditions were Syringe 1 100 mM NaCl and 20 mM MOPS pH 7 5 Syringe 2 not used filled with buffer Syringe 4 50 uM cytochrome c in 5 5 M Guanidine Cl and 20 mM MOPS pH 7 5 Excitation wavelength was at 290 nm and emission was recorded at right angle through a low pass cut off filter with 50 transmission at 320 nm Temperature was 25 C SFM 4 USER S MANUAL eto V Various mixing ratio vvere used the result shovvn above have been obtained for a 1 to 10 dilution of the enzyme solution contained in syringe 4 giving a final enzyme concentration of 5 uM and a final guanidine concentration of 550 mM At this concentration of salt the enzyme shows rapid renaturation The experimental curve was fitted with two exponentials of rate constants 83 si and 9 s and amplitudes of 38 90 and 62 of the total transition respectively The fit is shown as a dotted line under the experimental trace Convection artifact These artifacts are due to the slow rise o
27. pin 25 pin cable or via 25 pin 9 pin the later being used in most AT computers Pin number Signal name 25 pin connector 9 pin connector Chassis ground TxD transmit data RxD Receive data RTS Request to send CTS Clear to send DSR Data set ready SG Signal ground DCD Data carrier detect DTR Data terminal ready RI Ring indicator 1 2 3 4 5 6 7 8 2 2 CO I O July 1992 16
28. syringe 2 is mixed in the second mixer with alkali from syringe 3 the solution flows through DL and the hydrolysis reaction is stopped in mixer 3 with excess acid flowing from syringe 4 The grid below shows a sample SFM 4 program any other choice of phase number 22 SFM 4 USER S MANUAL COAN oque being valid Phase N 2 is used to purge the delay line during which time all solution is evacuated into waste After phase N 2 the mixture is collected in phase N 4 and the amount of DNP formed during the flow through DI assayed SYRINGE NUMBER VOLUMES ul Not used DNPA 1 5 mM NaOH 400 mM HC1 500 mM Valve Delay Le Delay line The purge volume is the volume flowing into the delay line before collection and is equal to 2xV Figure below shows the results as a function of purge volume Absorbance for zero and infinite time of incubation respectively were measured independently It is obvious that the reaction products oe collected from the delay line after using a purge volume of zero are completely contaminated by the old reaction mixture standing in the delay line before the experiment was started The curve shows a clear break around 25 pl It is at this point that DI has been vvashed once This first experiment Volume of purge ul confirms that the real volume between mixer 2 and 3 when no delay ligne is installed is around 24 ul as indicated in table of chapter 2 4
29. C XT AT or compatible microcomputer Various screens permit the user to know the volumes of solution contained in the four syringes perform manual or automatic movement of the syringes create a sequence of reaction with complete control of time and volume delivered by the four syringes save or recall the sequences program the synchronization pulse used to trigger the acquisition system load the data acquisition software Bio Kine 1 2 Modes of operation The SFM 4 has two main operating mode that are briefly described below More detail can be found in other sections of this manual i SF mode commercial reference SFM 4 S In this configuration the SFM 4 is a full stopped flow instrument with an optical observation chamber This is described in the panel N 1 in figure 1 below In this configuration the SFM 4 has unique features for a stopped flow instrument Four solutions can be mixed and injected in the cuvette two delay lines can be installed with three mixing chambers The speed capability of the SFM 4 instrument with its 4 syringes running gives a dead time below 1 ms in the observation cuvette ii QF mode commercial reference SFM 4 Q In this configuration the SFM 4 featuresa complete quench flow instrument In this mode of operation various mode of operations are possible possible as described in the figure 1 below It can be used as a four syringe quench flow instrument with two delay lines three mixers and a
30. EE seva doth ed ah 8 2 0 Exchange Of MIXEIS abe eat REGERE eee ee Es 9 3 MECHANICAL DRAWINGS usem tes thee ens ata ii 9 3 1 Generar dimensions ces A UE RE 10 32 Dimensions ofthe head ee cam A Kes 11 3 3 Front VIEW TELETENCES 23 9 e EE TE Re ed 11 A Side VIEW TelClenCeS uos eoe E qon eR EE 13 0 TOD VION TSterencEs quia DOR a De lt a es se De ou ie 14 SFM 4 USER S MANUAL CAUTION THESE INSTRUCTIONS ARE INTENDED FOR QUALIFIED PERSONNEL ONLY TO AVOID ELECTRIC SHOCKS DO NOT PERFORM ANY SERVICING OTHER THAN THAT DESCRIBED BELOW UNLESS YOU ARE QUALIFIED TO DO SO 1 INTRODUCTION This section of the manual contains information about factory service maintenance Careful cleaning with distilled water is required at the end of a series of experiments or before a change of buffer 2 GENERAL MAINTENANCE 2 1 Disassembly of the SFM 4 water jacket This operation permits an overhaul of the entire upper part of the SFM 4 This part contains the many important components of the instrument the syringe body the valves and the first mixer Empty the coolant Set the syringe plugers at their lowermost position Unscrew the upper cover Lift the upper cover lf necessary lift out the lower part of the water jacket The syringe bodies can be removed and unscrewed at that point Note syringe removal can
31. INSTRUMENT This part of the manual describes some test reactions which may be used to calibrate and test the performance of the SFM 4 quench flow Details about the manipulation of this instrument are given in the previous chapters of the user s manual please refer to it for instructions There are a large number of references that may be used to help the user to understand more about the basics of rapid mixing and quench technique We recommend three of them as well as the many references that they include 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 4 1 Description of the test reaction The test reaction described here 1s the Alkaline hydrolysis of 2 4 dinitrophenyl 2222000000000 E acetate DNPA Gutfreund 1969 Later mno we describe the study of sarcoplasmic Bellum reticulum Ca ATPase phosphory lation dephosphorylation to illustrate an experiment of real biochemical interest Courtesy of P Champeil and S Orlowsky CEN Saclay France Optical Density Alkaline hydrolysis of 2 4 dinitrophenyl acetate DNPA The absorbance spectrum of DNPA in HCI is shown in figure 4 1 After a short exposure to Na
32. M 4 USER S MANUAL oto f egie Ratio V1 4 V2 V4 was kept constant and equal to 1 Varying V1 and V2 give a final dilution of ADH equal to V2 V1 V2 V4 The advantage of this technique is that the final concentration of NAD 1s constant whatever the final ADH concentration Ratio of 1 2 to 1 120 were used PNE Absorbance Transmittance was measured at 340 nm with a 5 mm cuvette and the rate of formation of NADH was observed by following the decrease of the transmitted light Here are shown some of the traces obtained The initial rate of the reaction was measured for each of the traces These rates are plotted as function of the dilution factor on a log log scale This plot shows a reasonable alignment on a line of slope 1 indicating a linear relationship between the initial rate and the dilution factor gt Initial Rate relative scale The horizontal dotted line shows the remaining ADH activity which was measured after two washings of the cuvette with V2 0 no enzyme following a shot with V2 V4 1 2 dilution of ADH Further washing would have reduced this contaminating activity which corresponds to a concentration of 1 1000 of ADH This however sets a limit for the reasonable dilution which can be obtained with the stopped flow Final Dilution 35 SFM 4 USER S MANUAL CTA A eae 7 4 Mixing solutions of unequal density and viscosity This situation is commonly found when the
33. MANUAL ee V EY logic cuvette prevention of cavitation and prudent use of valuable reagents The relationships between these factors requires careful consideration and experimentation balanced trade offs are often necessary to achieve successful stopped flow experiments 6 3 2 Washing It is necessary to completely wash the cuvette during the shot so that observation after the shot is only of the recently mixed samples To accomplish this sufficient volume of mixed samples needs to pass through the cuvette during the shot This volume varies with flow rate and viscosity of the sample 6 3 3 Cavitation Cavitation occurs when turbulence creates regions of low enough pressure in a liquid that a cavity is formed which fills with the liquid s vapour These cavities collapse incompletely leaving behind small bubbles of vapour 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 at a given flow rate De gassing of solutions decreases the probability of cavitation by lowering the total vapour pressure available to fill the cavities 6 3 4 Signal amplitude The signal amplitude is generally proportional to the path length of the cuvette and the construction of signal generating agent A required increase in signal could be accomplished by an increase in path length an increase in the c
34. MPONENTS 2 1 The observation chamber The observation chamber has three observation windows allowing measurements of transmittance single or double wavelength fluorescence and light scattering or fluorescence polarization without adding reflecting or beam splitting elements The two windows at right angles to the incoming light can be equipped with lenses to increase the efficiency of light detection The observation head can be equipped with several types of cuvettes described in the next page 3 Cuvettes TC xx 10 have a 1x1 mm section cuvettes TC xx 15 have a 1 5x1 5 mm section Replacement of the observation cuvette is described in section xxx The FC type cuvettes have blackened edges to reduce light scattering in fluorescence configuration FC 15 and FC 20 are the best choices for CD experiments in the far UV Their large aperture facilitates low noise recording at these wavelengths The TC 100 models have been primarily designed for absorbance however both sides of the light path are transparent and can be used for fluorescence This will be a primary choice for fluorescence experiments using dilute samples and excitation with a laser or any other low divergence source SFM 3 Cine The cuvettes available for the SFM 3 unit have continually evolved to fulfill the requirements of our users Here is a list of presently available cuvettes You are invited to inquire about any special need Light p
35. ND ITS BRACE SFM 4 USER S MANUAL JAN Fog 1 INSTALLATION OF THE OPTICAL SYSTEM e UR HERE 2 INSTALLATION OF THE STOPPED FLOW COMPONENTS 2 1 The observation chamber ia oia RJ e 2 2 Installation of the mixer blocks and of the delay lines 2 5 Volume of tlie HOW INS test teca aO Za Intermixer VOIMMES sua desena EE ERASERS 25 LIO oule SUENA se sa exe dn ob pa en o aed ae 25 IEree DONES IO lS SIA ye der a CR e ed 252 JT NS ea ope SUE RC da eiut GE 29 8 installing tbe hard stop Valve 1 39 wor iaa 254 ERENT SVEIDBE o dus ea GE eerie ee Sale iom ao a 3 SPECIAL ACCESSORIES OR INSTALLATIONS ess 31 Small INE SVMS ua lon P ORC RES dades 32 A mixer for high density solutions lt lt lt 321106501101101 a deg ERE To SE eoa e dosi 322 111512113110117 ae aaa EE dm ee DPI 3 3 Observation head with separate cooling part A SOFTWARE OPERATION dice bos LIC OCU COM ES PDR DI ES et 4 2 Installation of the SFM 4 S version of the software 4 3 SFM 4 S driving software configuration 231 5ynnge mnstalldDO
36. OH 2 4 dinitrophenol Av BGER DNP is produced The spectrum of DNP under various pH conditions 1s also shown revealing an isobestic point at 325 nm The test reaction vvill be as follows DNPA in acidic conditions gt exposure to NaOH for time t leading to formation of DNP gt Quenching of the hydrolysis reaction with excess HCI The quantity of DNP formed during the exposure to alkali will be followed at 325 nm In acid the molar extinction at this wavelength is 5 6 10 M em At 20 C the reaction DNPA OH gt DNP has a second order rate constant in water of 56 M s Conditions can easily be set to make the concentration of OH 21 SFM 4 USER S MANUAL bio V LOGIE sufficiently larger than that of DNPA to create a pseudo first order hydrolysis reaction of apparent rate constant kapp 56 OH s These properties make this reaction a very useful tool for calibration of any quench flow instrument and the reaction can simultaneously be checked in a conventional optical stopped flow 4 2 How to get various ageing times The SFM 4 instrument can be operated in the continuous or interrupted mode In the continuous mode the delay between two mixers is adjusted by the delay line volume and the flow rate The use of stepping motors enables setting a flow rate which is independent of the solution viscosity This is not feasible with an instrument based on a pneumatic drive The continuous mode is used
37. OSITION There is no real danger in raising the plunger to the very end Once approaching the end of its course the motor will oscillate and vibrate as it becomes out of phase with the driving pulses it will completely lose its torque Nevertheless there is no reason to unnecessarily prolong this treatment either Type lt R gt or select Reference to set to zero volume the counter of the corresponding syringe CAUTION measurement of the residual syringe volume is made by counting the logic pulses from the controller Thus if for any reason a syringe is blocked during a run the value measured may become erroneous This may occur in the case of incorrect positioning of the valves The Limit function if this function is set to Stop the motor s plungers cannot be driven from the keyboard above their upper limit less than zero volume This function is disabled by setting it to Pass Type lt L gt or validate the function Limit to enable or disable this limitation 3 7 Filling the syringes Utmost care should be exercised during this operation Normal operation of the system requires that no bubbles are present in the injection syringe should this occur the buffer flow through the observation chamber will not be correctly controlled by the plunger movement The four syringes of the SFM 4 stopped flow module can be driven and refilled independently in the manual mode Filling may be performed with disposable plastic syringes i
38. Se o oo e 0 c t P o V 2 Qn oe 0 ee 0 Cs o 4 a c o D vc 2 0 e a 0 o d d a EGG P 0 e e 7 d 0 0 0 a 5 EG a n a ep a Mes 00 ad 7 v Coe ee T ee Be e 0 0 0 0 0 E yr 00 O 0 4 noc e a Ne 9 fi 0 7 S 2 wee 0 Boy a Lo v 0 Ze c o O ss m 7 cs 0 1 j i ane 0 7 9 UD A E m o 7 a wae i a n oo _ p CO O du N A 7 Da i T 2 Ne i D A a o o o o O E M as Go p i Ze Wee es A a 0 a eL Ds ae jo 0 0 Cl a o S Ze x 1 o De gee o Le a v pru p SFM 4 USER S MANUAL f egie INTRODUCTION conesa CELESTE deditio eee SER 3 2 GENERAL MAINTENANCE 5 134 re teed aet o a ded ub abbates 3 2 1 Disassembly of the SFM 4 water jacket 3 2 2 Valve inspection or replacement cases eee 4 2 3 Plunger disassembly and reassembly ooooooooooo 4 2 4 Syringe disassembly and reassembly vu di esde ates 5 2 5 Disassembly and reassembly of the observation head or delay lines DOCKS P DEU ek OS ED OE OT ee El 6 DO TUDICOSOB sonrisa rata dao 8 2 7 Replacement Of opservation CUV
39. ameters Syringe Vol Phase 3 4 5 6 0 0 0 0 0 0 0 0 Load Save Note the second Sync line vvill be absent vvhen the hard stop mode is enabled An action can be performed by placing the cursor of the desired function and validating with the lt Enter gt key Displacement of the cursor is performed by using the space bar Alternatively execution of a function can be obtained by typing the first letter of the function The Syringe Volume box on the right side of the screen indicates what volume is being displayed in the syringe counters If no number is shown this indicates that the communication with the MPS 51 power supply is not established Turn the power supply on check the connection or eventually reset the power supply by pressing the reset button on its front panel Type esc or Exit to exit from the SFM 4 program NOTE In the absence of connection with the computer or with the PC turned off the MPS 51 unit is automatically set in the manual mode It is therefore possible to drive the syringe and wash the instrument without being connected 16 nnt SFM 4 USER S MANUAL YAS eae 4 5 Manual control of the syringes From the main menu control line type lt M gt or type lt Enter gt with the cursor on the Manual function This results in display of the Manual menu BIO LOGIC SFM 4 S V3 10 Help F1 NONAME SF4 Stopped Flow Parameters Syringe Vol Phase 1 2 3 4 5 6 7 8 9 Time 0
40. asic equipments are A stable surface to ensure correct vertical positioning of the SFM 4 Q A PC or compatible microcomputer and one RS 232 serial interface If radioactive reaction mixture is collected through the upper collect port with the aid of a disposable plastic syringe make sure that the syringe is inserted tight to avoid projection of any material 1 3 Water circulation The SFM 4 Q module may be connected to a circulating water bath for temperature regulation 1 4 AC power and connections Before connecting the SFM 4 to the local AC line verify that the setting of the instrument matches the local line voltage The connections of the SFM 4 Q are described in the general section of this manual Prepare the SFM 4 for operation by connecting the mechanical sub system to the MPS 51 unit Connect the MPS 51 to the serial port of the micro computer SFM 4 USER S MANUAL bio V 0 2 INSTALLATION OF THE SFM 4 Q COMPONENTS 2 1 Introduction This section of the manual contains operating instructions and a description of the experimental procedure Please read the previous section of this manual before attempting any operation of the apparatus Before shipping the unit has been thoroughly checked and all movable parts lubricated and so can be used immediately 2 2 Installation of the mixer block and of the delay lines In the quenched flow mode the four syringes of the SFM 4 can be used to perform several goals It is diff
41. ath Aperture Volume of Dead Time at Holder Part N Drawing mm mm cuvette 10 ml s total Model Application No a I b W1 flow rate ms 3 rc o8 5 Sig f 0 9 1 4 fae ur Fluorescence FC 15 i Wo 3 e 3 1 i RDL Absorbance CD al FC 20 2 0 4 7 5 4 TC 50 10 5 0 1 5 Absorbance CD TC 50 15 1 5 0 5 358 TC 100 10 i a A 2 3 1 1 Absorbance TC 100 15 lt AS O 0 1 A11 cuvettes are in suprasil transparent from 185 to 2500 nm 2 From mixer to center of the cuvette 3 Dead time is inversely proportional to the flow rate Black Quartz Transparent Quartz The FC type cuvettes have blackened edges to reduce light The TC 100 model has been primarily designed for scattering in fluorescence configuration FC 15 and FC 20 absorbance however both sides of the light path are d are the best choices for CD experiments in the far UV transparent and can be used for fluorescence This will Their large aperture facilitates low noise recording at be a primary choice for fluorescence experiments using these wavelengths dilute samples and excitation with a laser or any other low divergence source 9 SFM 4 USER S MANUAL Biol 2 2 Installation of the mixer blocks and of the delay lines In the stopped flow mode the four syringes of the SFM 4 can be used to performe several goa
42. b ria de Pre nee tie t a es 4 3 2 Installation of the hard stop s a 3e Yew ss bot wa bins ra 4 3 3 Installation of the serial port o 4 4 SFM 4 driving software tes Ach wou emesa deci aet 4 5 Manual control of the syringes usas sace e delegat YE S eA 4o Initialization ofthe Syringes socer vacuo eddie E acaba dre 4 5 Eme TBE syrin eS ex eot err Eug d od udis a aE Ao Creatine a drive Sequence diarreas 490 Acceleration phases snis dua qu at RAEE 4 10 Programmable synchronizing pulses eee LL LL LL 4 11 Saving or loading the experimental parameters 3 12 Running the automate MODE spice eee bw S DATA ACOUISITION SOFTWARE 9 tree Soom os 6 A SHORT STOPPED FLOW PRIMER ceres 6 1 The general Principle of Experiments with the SFM 4 Stopped Flow dise ep 6 2 Design and execution of stopped flow experiments O Gemera ady CE cies ad dere dad o e dene edo ai SFM 4 USER S MANUAL ee AS 05010857 Sene SESE See cepa d een 21 6033 850112118101 a Vac AR Rh dod el ee 27 6 24 SIP Mah ANID INNS persiana pecs aie ach aie 21 OSS LOW Tale ne ES LE
43. be performed without opening the water jacket see chapter 24 below Follow the instructions of chapter 24 below To prevent corrosion of the water jacket two magnesium blocks are screwed one on each part of the water jacket It is normal that these magnesium blocks get rapidly corroded They have to be exchanged only before they become completely destroyed Note The upper part can be screwed back onto the SFM 4 frame without the lower 3 SFM 4 USER S MANUAL V fes part of the water jacket The instrument will be functional except for the temperature regulation Reassemble in the reverse order 2 2 Valve inspection or replacement After prolonged and extensive use of the Sfm 3 instrument the SFM 4 valves may need to be inspected or replaced To this use the special flat wrench delivered with the instrument 1 Remove coolant from the device 2 Use the wrench to loosen the stainless steel nut which surrounds the valve shaft and plugs the bulkhead of the device In this operation keep the handle attached to the valve shaft Remove the entire assembly consisting of the valve the spring the handle and the nut 3 Inspect the valve check for the presence of scratches Wash and reinstall in the reverse order 4 If replacement is felt necessary replace with an entirely new assembly Return the used one to the factory or to your distributor NOTE although the valve have been machined with extreme
44. can be used two ways i to store the mixture between two flow period This is the interupt mode it will be used for ageing time larger than 100 ms ii in the continuous flow mode In this case the ageing of the mixture is given by the time it takes to flow from one mixer to the other The age will be given by the formula below Ageing time between two mixers Intermixer volume flow rate The mechanical volume between two mixers is given below Delay line used volumes in ul 500 Flow line Mixer 1 to 2 Mixer 2 to 3 External tube Two models provided 0 76 mm dia 4 54 ul cm 1 58 mm dia 19 61 ul cm In the No delay line mode the MIX 0 mixing block has to be used in the Delay line mode the MIX DL mixing block has to be used The volumes indicated above are the mechanical volumes The hydrodynamical volume may vay slightly around these values It is recommended to calibrate these volumes with known reactions using procedures described in the chapter 4 3 Also similarly to this mode two ageing mode may be used here continuous flow or interrupt mode 2 5 Liquid outlet system Two mode of liquid ejection can be used free flow system of diverting valve system 2 5 1 Free flow system In this mode the outlet is continously open and connected to a tube The mixture is ejected into a test tube or a becher containing a quenching solution This tube forms the last delay line and its length
45. ctallow optical recording during the drive sequence These features greatly facilitatethe determination of the initial phase of the reaction being monitored and make the equipment suitable for very accurate continuous flow experiments SFM 4 USER S MANUAL bio 1 5 Description of the mechanical design The observation chamber and the syringesof the SFM 4 stopped flow module are mounted vertically facilitatingpurging off bubbles which are evacuated during refillingby a few up and down movements of the drive syringe The maximum tilt angle of the instrument is 20 from the vertical The syringes valves and observation chamber are very carefully thermo regulated This thermo regulation prevents the occurrence of temperature artifactson a very wide temperature range and permits rapid kinetic studies even at temperatures below 0 C 1 6 The ageing loops The SFM 4 instrument has ageing loops permitting various delay to be obtained between two mixers The ageing loops are machined into stainless steel spacers These spacers can be inserted between two mixer blocks or between one mixer block See INSTALLATION OF THE STOPPED FLOW COMPONENTS in SFM 4 S and SFM 4 Q sections for full description of ageing lines installation and calculation of volumes Replacement of the ageing loops is an easy operation which usually takes only a few minutes Ageing loops of nominal volumes up to 1000 ul are available Standard equipement of SFM 4 S ve
46. e cursor is performed by using the space bar Alternatively execution of a function can be obtained by typing the first letter of the function The Syringe Volume box on the right side of the screen indicates what volume is being displayed in the syringe counters If no number is shown this indicates that the communication with the MPS 51 power supply is not established Turn the power supply on check the connection or eventually reset the power supply by pressing the reset button on its front panel Type lt esc gt or Exit to exit from the SFM 4 program NOTE In the absence of connection with the computer or with the PC turned off the MPS 51 unit is automatically set in the manual mode It is therefore possible to drive the syringe and wash the instrument without being connected 13 SFM 4 USER S MANUAL TAN logie 3 5 Manual control of the syringes From the main menu control line type lt M gt or type lt Enter gt with the cursor on the Manual function This results in display of the Manual menu BIO LOGIC SFM 4 Q V3 10 Help F1 NONAME QF4 Quenched Flow Parameters Syringe Vol Phase 1 2 3 4 5 6 7 8 9 Time 0 0 0 0 0 0 0 0 0 ms Sii dues 0 0 0 0 0 0 0 0 0 ul S2I 0 0 0 0 0 0 0 0 0 ul SS 24904 0 0 0 0 0 0 0 0 0 ul 545 ausus 0 0 0 0 0 0 0 0 0 ul Waste Co11 W W W W W W W W W Drive sequence 1 1 9 dms dee um aum auum aum dams WEM ues duum
47. e for waste The functions of the hard stop valve are programmed from the Installation menu of the software see section 4 3 Hard stop may be turned on or off see section 4 3 When on the current pulse used to close the hard stop valve is actuated with a few milliseconds lead to compensate for electrical and mechanical delays Correct value for this lead is around 4 ms 11 CIAN SFM 4 USER S MANUAL 2 5 4 Exit in a syringe In this method a syringe is plugged at the SFM 4 exit The linear momentum of the liquid flowing out of the cuvette will be dissipated in the liquid contained in the syringe This procedure gives clean stop signal For a better result it is recomended to use high quality 10 to 20 ml glass 1 Syringes with teflon piston Hamilton Plastic syringes are too soft and do not give good results 1 to carefully eliminate the bubbles 12 SFM 4 USER S MANUAL JAN 3 SPECIAL ACCESSORIES OR INSTALLATIONS 3 1 Small drive syringe The SFM 4 stopped flow works with a high driving speed range In particular different speeds can be programmed on each of the syringes leading to a mixing ratio different from 1 to 1 in each of the mixers Ratios as high as 1 to 20 can be obtained by programming different flow rates in the syringes without syringe exchange For continuous operation with high dilution ratio we advise installation of a syringe of smaller volume for injecting the solution to be diluted This w
48. e the plunger of the syringe to be initialized UNTIL THE SYRINGE REACHES ITS UPPERMOST POSITION There is no real danger in raising the plunger to the very end Once approaching the end of its course the motor will oscillate and vibrate as it becomes out of phase with the driving pulses it will completely lose its torque Nevertheless there is no reason to unnecessarily prolong this treatment either Type lt R gt or select Reference to set to zero volume the counter of the corresponding syringe CAUTION measurement of the residual syringe volume is made by counting the logic pulses from the controller Thus if for any reason a syringe is blocked during a run the value measured may become erroneous This may occur in the case of incorrect positioning of the valves The Limit function if this function is set to Stop the motor s plungers cannot be driven from the keyboard above their upper limit less than zero volume This function is disabled by setting it to Pass Type lt L gt or validate the function Limit to enable or disable this limitation 4 7 Filling the syringes Utmost care should be exercised during this operation Normal operation of the system requires that no bubbles are present in the injection syringe should this occur the buffer flow through the observation chamber will not be correctly controlled by the plunger movement The four syringes of the SFM 4 stopped flow module can be driven and refilled
49. eaction time s 4 9 General guide lines for creation of the drive sequences All of the parameters below are for 1 1 mixing ratio CONTINUOUS MODE SYRINGE NUMBER VOLUMES ul Not used Reactant 1 Reactant 2 Quencher Collect Delay line N 1 0 ul Delay line N 2 variable 29 Me ES SFM 4 USER S MANUAL Phase 2 is the purge phase Tp must be varied to give the same flovv rate as in collection phase N 4 at least up to about 6 ml s Above this an acceleration phase has to be included between the purge and the collect phase Ageing time will be calculated according to T7 dl To 2 Vc The collection volume is set according to what should be obtained at the collection port dl is the measured volume of the delay line The purge volume depends on the delay line used as indicated below Delay line Purge Volume Nominal Volume ul Vp ul 40 to 90 60 to 100 150 to 200 200 to 300 INTERRUPTED MODE The 190 ul delay line and above are generally used with this mode Here is an example sequence many others may be created PHASE N t 2 RE we TIME ms 0 150 Ta 15 40 0 ol SYRINGE NUMBER VOLUMES ul O 0 0 0 0 0 Of Not used 1 Reactant 1 Reactant 2 Quencher Collect This corresponds to the collection of 180 ul of solution The reaction age is T Ta dl 40 2 60 Ta 73 ms 30 4 c 0 o 7 0 ss 0 E
50. eless been included here to prevent failure under abnormal environmental conditions 5 DATA ACQUISITION SOFTWARE With the BioKine software the same microcomputer may be used both to control the SFM 4 stopped flow and perform data acquisition analysis Once the parameters have been loaded in the memory of the MPS 51 unit switch to the automatic mode by typing lt A gt and return to the Bio Kine software by typing lt E gt to exit The Bio Kine menu will soon appear and will then be ready for data acquisition Direct access to the data acquisition software from the main menu will be obtained by validating kinetic software see section 4 2 Please refer to the BioRine Manual for Software Operation 23 SFM 4 USER S MANUAL AS 6 A SHORT STOPPED FLOV PRIMER 6 1 The general Principle of Experiments with the SFM 4 Stopped Flow Module There are many variations on the stopped flow experiment such as multiple mixes continuous flow and accelerated flow However the simplest stopped flow experiment occurs in two Stages In the first stage flow is initiated by two plungers forcing liquid through a mixer and along a flow path into an observation cuvette The mixture ages as it travels along this path the rate of ageing depending on the flow rate of the mixture and the volume of the flow path In this first stage the mixer flow path and cuvette are initially washed by the constantly refreshed mixture until a steady state
51. f light buffer from the last mixer and subsequent entry into the observation chamber after mixing This entry is detected by a sudden and reproducible change in absorbance or fluorescence at 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 a large dilution ratio was used so that the final mixture has a density not too different from that of the NaCl buffer As a consequence no convection artifact was visible when data acquisition was prolonged for more than 100 seconds On the other hand if a 1 1 mixing was used the high concentration of guanidine in the cuvette 2 75 M would have resulted in the formation of a large gradient of density at the last mixer Under these conditions if no precautions are taken rapid rise of NaCl buffer in the observation cuvette can be observed about 20 s after mixing A method to completely eliminate the convection artifact has been proposed by Blond Elguindi et al 1988 in their work referenced at the end of this manual These authors used heavy water in the light buffer for it to match the density of the mixture in the cuvette However the best solution is to use the high density mixer model HDS developed by Bio Logic This mixer is described in section 3 2 37 e o D or o e 4 Po e et
52. h does not necessitate removal of the observation head Disconnect the exhaust tube Unscrew and remove the black cover of the observation head Slowly pull and remove the upper teflon cylinder Remove the observation cuvette Replace the new observation cuvette Reassemble in the reverse order Replace and tighten the black head cover by hand SFM 4 USER S MANUAL 2 8 Exchange of mixers Exchange of Mixer 3 Mixer position Exchange of Mixer 1 Remove parts 887 and 886 Unscrew the 4 screws of the head Unscrew part 165 166 Remove ageing loop Remove parts 164 165 157 UP Unscrew part 162 and the cuvette Remove O Ring 2 2 x 1 6 of part Unscrew and remove part 162 160 lop o Remove Mixer 1 Remove O Ring 2 2 x 1 6 of uni part 160 screw part 162 Remove mixer 2 Insert new Mixer groove upside E Insert O Ring 22 x 1 6 Screw part 160 put again ageing loop and head nsert new mixer groove DOWN 166 and screw it again upside Insert O Ring 2 2 x 1 6 Insert cuvette and parts 157 165 164 MIXER 2 ANN rn Miss SS SS 162 b SS VRE HIXER SFM 4 USER S MANUAL V 3 MECHANICAL DRAWINGS 3 1 General dimensions 81 00 81 00 n3 co uw e o i gt e ce n un co o en o e o 2 e 126 00 51 50 51 50 a TE ca 00 91 SFM 4 USER S MANUAL
53. hat the manual valves corresponding to the active syringes are set in the right position i e pointing to C Type lt A gt or select Auto to enter the automatic mode The screen will show the following messages 810060 SFM 3 0 Stopped Flow Parameters 2 3 4 5 6 7 8 9 10 0 0 80 0 0 56 EAU 0 0 0 0 0 0 0 0 0 yl 3 Eds 0 0 0 0 0 0 0 0 0 pl 54 DCIP 0 15 30 80 0 0 0 0 0 yl Start Stop Esc Exit 22 shots to go start or Stop according to the status of unit When ready type S to run The run can also be activated by depressing button 5 on the front panel of MPS 51 or from an external trigger signal In case of necessity the sequence can be terminated by typing the lt S gt key again 22 15 Syringe Vol 4001 pl 3200 yl 2801 pl 3200 pl SFM 4 USER S MANUAL JAN IMPORTANT The power supply has been designed to deliver high power to the motors over a short time low duty cycle it is turned off shortly after completion of the run Permanent activation of the power supply may result in components overheating The red LED 2 on the front panel indicate when at least one of the motor power supplies is activated Reset the program button 3 if this LED remains permanent turn off the power supply in case the trouble persists Start the sequence again This type of trouble should never occur under normal operation This warning has neverth
54. icult to list all the possibilities here The most usual being described below It can be used as a four syringe quench flow instrument with two delay lines three mixers and a diverting valve for waste and collect This assembly is illustrated in the figure of page 5 The diverting valve is mounted on the SFM 4 body through three spacers consisting of one delay line mixing block labeled MIX DL and a second delay line For very short dead times the delay lines can be omitted permitting millisecond ageing time to be reached In this case the intermediate mixer block to use is the one labeled MIX 0 See figure page 5 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 as shown in the figure page 6 Exit valve Mix DL SFM 4 USER S MANUAL 2 3 Volume of the flow lines The numbers in the table below refer to the numbers on the figure of the next page With 3 mixers and exit valve With 2 mixers and direct exit Without delay With delay Without delay With delay line lines MIX 0 line MIX 0 MIX DL Line number Delay line oe oe ca ains SSS SSS SSS 13 S1 S2 S3 S4 SFM 4 USER S MANUAL TAN gt gt oye 2 4 Intermixer volumes The ageing lines
55. ill enable the motor pushing this syringe to run at a faster and smoother rate Syringes of 4 ml are available from stock Other volumes may be designed on customer request This model of syringe is installed in the SFM 4 module as the standard version Please refer to the technical section of this manual for syringe disassembly and reassembly See section 4 3 for software modification associated with this syringe replacement 3 2 A mixer for high density solutions 3 2 1 Description Mixing solutions of different densities proffers a formidable challenge for stopped flow instruments In typical folding unfolding experiments heavy solutions of urea or guanidine chloride are mixed with pure aqueous buffers The result is an unavoidable convection reaching the observation cuvette 10 to 30 seconds after mixing creating a massive artefact ruining definitively the kinetics being recorded The SFM 4 module can be equipped with a specially designed mixer model HDS that includes an internal siphon like frame that allows complete 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 3 2 2 Installation Installation procedure is identical to that of a standard mixer Please refer to the technical section for description 13 SFM 4 USER S MANUAL A ede 3 3 Ob
56. independently in the manual mode Filling may be performed with disposable plastic syringes inserted into the four cylindrical receptacles The thermostat jacket allows equilibration of the buffer before the filling sequence Filling is performed with the valves pointing to the R labels While refilling exert a slight manual pressure on the plunger of the reservoir syringe this will prevent a negative pressure in the reservoir during pumping which could result in bubble formation 18 SFM 4 USER S MANUAL Bubbles in the drive syringes may be eliminated simply by driving up and down several times the drive syringe when they are connected to the reservoir One or two shots in the observation cuvette will then be sufficient to definitively eliminate any bubbles remaining in the valves and the mixer In all cases it is strongly recommended that buffers be degazed and filtered IMPORTANT Precautions when only two syringes are used in particular in SF mode The mixing has to occur in the last mixer so use of syringe 4 is mandatory Use Syringes 1 and or 2 and or 3 with the syringe 4 The unused syringes and their flow lines should be filled with buffer as carefully as the other syringes Turn the valve of the unused syringe to the C position cuvette and make a few manual pushes in the upward direction to ensure that the flow line between the valve and the mixer is correctly filled with buffer Turn the valve back to the R po
57. ine volumes were obtained according to the procedure described above 25 SFM 4 USER S MANUAL eto Delay line Volume to Flow rate per volume ul use ul ime frame ms syringe ml s Absorbance Continuous to Continuous to Continuous to Continuous 9 to Continuous 17 to Interrupted 100 to Interrupted 250 to Interrupted 500 to The important point here is to observe agreement on one hand between experiments using different delay lines and on the other hand between experiments using the same delay lines but operated under different modes continuous or interrupted The results show that the data obtained under these various conditions are reasonably consistent The apparent rate constant for the DNPA DNP reaction is 2 3 sec which yields a second order rate constant of 46 s M In chapter 4 10 below are listed some parameter guidelines for the various delay lines and modes These parameters were used in the experiments described in this section and in section below 7 100 ul Continuous mode 200 ul Interrupted mode 50 ul Continuous mode X 200 ul Continuous mode A 20 ul Continuous mode POPU 1 1101 1010 81011 00 8 1 SR NR 1 1 11 180 0 Deo bo ZO Reaction time s 26 SFM 4
58. ixers Programmablariggerfor data acquisitiorand synchronizationf accessories 50011 to 15ml 2011 Fillingrangeof the drivesyringes Minimuminjectiorvolumeper shot 0 05to 6 ml s syring amp up to 8 ml s with accelerationphase Flow rateange 1 1to 1 20 largerthan 1 100 withdouble dilution Variableratiorange 0 7 ms at 12 ml s total flowrate withFC 08 cuvettein SF mode 1 0msin QF mode withthe minimalvolume delayline and 20 ml s Minimaldead time Material stainlesssteel or Kel Fon specialorder Syringevolume 18 ml other volumeson specialorder Volumeper step 0 1411 Durationof flow adjustabldrom1 ms to 9999ms per phase Powerrequirement 300 Watt 110 220Volt 50 60Hz Total weight 12 kg gas SFM 4 USER S MANUAL CTA A eas 1 4 Principle of operation The syringes of the SFM 4 are driven by four independent stepping motors The stepping motors are of hybrid technology of 200 steps per revolution and 4 phases each phase being powered by a constant current supply 2 9 A per phase The power supply of each motor is microprocessor controlled A complex impulse sequence enables micro positioning of the motor s rotor with an accuracy equivalent to 1 32 of the mechanical step This gives an effectivenumber of steps of 6400 per revolution or a volume quantificationof 0 14 ul per micro step when standard syringes are used With the damping produced by the rotor inertia this results in an almost continu
59. ixers giving an ageing of around 1 2 ms 4 7 Unequal ratio mixing One of the major advantages of the SFM 4 instrument is the ability to vary the mixing ratio by simply modifying the stepper motor program This does not require any syringe change The quasi continuous movement of the motor down to very low speeds permits efficient mixing to large ratios 4 8 Double mixing We performed a double mixing experiment to measure the rate constant of the calcium induced transition in the absence of ATP The enzyme incubated in EGTA is mixed with calcium in the first mixer of the instrument after which the solution is allowed to age in the first delay line interrupted mode After this incubation the enzyme is mixed with ATP in the second mixer and allowed to age 23 ms in the second delay line continuous mode Finally the reaction is quenched with perchloric acid PCA in the last mixer The SFM 4 parameters are set as follows TIME ms 0 420 SYRINGE NUMBER VOLUMES ul Delay line 200 ul Delay line 7 N N 0 ul 1 92 The amount of phosphorylation as a function of calcium incubation time is shown in the figure below These results indicate that the rate constant of the change induced by calcium alone is slower than that induced by calcium in the presence of ATP 28 SFM 4 USER S MANUAL bie N Double mixing experiment 2 x gt E E 1 E o Lu 0 4 0 2 4 10 R
60. low rate ml s Obs Absorbance Rate constant s 1 Dead time was evaluated according to DT 1 11 Ln A0 A1 13 ms Theoretical minimal dead time with this cuvette and this flow rate is 0 9 ms see section 2 1 of the manual Transmittance SFM 4 USER S MANUAL A J egie 7 2 Evaluation of the cuvette washing and quality of stop Reaction used is as described in section 7 1 Experiments described in the figure below show one method of evaluating of the quality of the cuvette washing This experiment again enables one to check the absence of stop artifact The data acquisition was started 100 ms before initiation of the drive sequence Only syringes 1 and 4 were used Syringe 1 contained Ascorbic Acid at pH 9 0 and syringe 4 was filled with 100 uM DCIP the mixing ratio was 1 to 1 The cuvette used has a 5 mm light path and total volume of 15 pl Three experiments are shown using the following parameters Duration of drive Volume pushed Total flow sequence ms per syringe 11 rate ml s 31 SFM 4 USER S MANUAL V v ege The results shown indicate first the absence of a stop artifact and second how washing is improved by increasing the volume of mixture passing through the cuvette 7 3 Variable ratio mixing The possibility to obtain variable mixing ratio by a simple programming of the instrument i e without changing the syringes is one of the major advantages of the Bio L
61. ls It is difficult to list all the possibilities here Some are described below a To load up to three reagents and to mix it in different shots with the content of the syringe N 4 b To vary the concentration of one or two substate and mix the result with the content of syringe N 4 c To perform sequential mixing and delays between up to 3 reagents before they are mixed with the content of syringe N 4 For the first two applications the observation head will be installed on the SFM 4 body through one mixing block labeled MIX 0 and with no additional delay lines see figure on the next page For the last application the observation head will be mounted on the SFM 4 body through three spacers consisting of one delay line mixing block labeled MIX DL and a second delay line see figure on the next page Uma 7 l SFM 4 USER S MANUAL 2 3 Volume of the flow lines i A 2 51 EL 29 34 The numbers in the table below refer to the numbers on the figure above Flow line volume e 1 80 1 P Cuvette Cuvette SFM 4 USER S MANUAL 2 4 Intermixer volumes The table below summarizes the mechanical volumes between the mixers The ageing time in each of these flow line will be given by Ageing time between two mixers Intermixer volume flow rate Mixer 2 to 3 Mixer 3 to Dependent on the cuvette model center of the See chapter 2 1 cuvette In the No delay line m
62. n a water E jacket to allow temperature regulation of the reactants containers The syringe I plungers of the stopped flovv are driven by four stepping motors via four ball screws THE MECHANICAL DESIGN The mechanical part of the SFM 4 module is carefully constructed the parts in contact with the biological sample and the buffers are all machined out of materials selected for their inert characteristicsstainless steel teflon Kel F and quartz Millisecond dead time can be achieved with the SFM 4 instrument due to the combined effectsof high performance control of the stepping motors and a drastic reduction of the dead volumes Ageing lines of various volumes can be used The ageing line of the instrument can be replaced and made tightin a few minutes INTELLIGENT POWER SUPPL Y The high performance of the SFM 4 unit and the high speed of the stepping motors can be achieved only because of the quality of itspower supply MPS 51 4 The MPS 51 4 unit contains four independent constantcurrent power supplies all driven independently by their own microprocessors The sequence of impulses to be sent to the stepping motors are stored in the memory of each board One main microprocessor board synchronizes the four power supplies and performs the communication with the microcomputer via serial interface ES logie SFM 4 USER S MANUAL MICROCOMPUTER COMMANDS The SFM 4 module is controlled by the user with the keyboard of a P
63. ng software the following screen will appear Syringe and RS232 installation V3 02 Volume per step yl Flow Rate ml s Syringe Vol pl Lover limit Higher limit 4 540 0 045 10 000 20500 4 540 0 045 10 20500 4 540 0 045 10 00 20500 Serial port COM1 Hard stop lead 4 ms Hard Stop status Enabled Type lt CTRL P gt to change serial port number Type lt CTRL S gt to change hard stop lead Type lt ALT S gt to enable disable Hard Stop Type lt ESC gt when installation is done 4 3 1 Syringe installation Unless otherwise specified the SFM 4 stopped flow module is equipped with standard 18 ml syringes o 17 mm giving 4 54 ul per mechanical step The flow rate is limited by the minimum duration of the drive impulses giving a maximum flow rate of 6 ml s with the standard 18 ml syringes The 4ml syringes 28 mm give 1 005 ul per mechanical step 4 3 2 Installation of the hard stop Typing lt ALT S gt will enable or disable the hard stop mode in SF CTRL S gt will select the hard stop lead from 1 to 5 ms 4 3 3 Installation of the serial port The program detects the number of available serial ports Type lt CTRL P gt to select which serial port you would like to use 15 eto V SFM 4 USER S MANUAL 4 4 SFM 4 driving software By validating Driving Software the SFM 4 menu will soon appear on the screen BIO LOGIC SFM 4 S 60 Help F1 NONAME SF4 Stopped Flow Par
64. nserted into the four cylindrical receptacles The thermostat jacket allows equilibration of the buffer before the filling sequence Filling is performed with the valves pointing to the R labels While refilling exert a slight manual pressure on the plunger of the reservoir syringe this will prevent a negative pressure in the reservoir during pumping which could result in bubble formation Bubbles in the drive syringes may be eliminated simply by driving up and down several times the drive syringe when they are connected to the reservoir One or two shots in the observation cuvette will then be sufficient to definitively eliminate any bubbles remaining in the valves and the mixer In all cases it is strongly recommended that buffers be degazed and filtered IMPORTANT Precautions have to be taken to fill all the flow lines of the usnused syringes if any The SFM 4 Q Module is now ready for operation 15 d e SFM 4 USER S MANUAL bie LOSE 3 8 Creating a drive sequence This is performed from the main menu The drive sequence has been divided into 9 phases In each phase the operator has to enter the duration of the phase in milliseconds and the volume in microliters delivered by the four drive syringes in each of the phases Selection is made by displacing the cursor with the arrow keys In the left of the screen a field 1s reserved to enter four letters identifying the contents of the four syringes Enter in this field
65. o E o o E 7 oe o 0 ee A 2 ce Se Coe Ce coe y e v n nu c n Uu C c Do e 2 C oe EEN o c ee e e o c e a o ge c gt a o 4 o a c o o we p 0 a mu 0 0 0 Ce a _ oS Be o o o o O ee o s o o os 2 o c i 0 E ss a c oe o E pe 1 7 o ay m se Des 7 1 P Coy oe j e 00 0 7 T Si i E Bes 0 D du p dd 0 a o Ge 0 O CER e ee po a o e n oe ee d T onc e T T D 0 Ge Qu c e a p po a j oa 0 ae ee n a E a a 0 SFM 4 USER S MANUAL bie V LOGIC GENBRAEJINST ALIA TION ocx Re hac oec dE RE dE ee 3 IT INtOdUC ON A ri lt ta ta he eae A 3 L3 Water CCU OM 6d y CEN PR CUP EE ESE AR NS 3 hed AC power and Connections suaus it eu Na Data 3 2 INSTALLATION OF THE SFM 4 Q COMPONENTS eere 4 ZA 11150011161611 te ead hae a o Sera Me NOD eae 4 2 2 Installation of the mixer block and of the delay lines 4 2 3 1 01011767071118 110717111725 oli
66. ode the MIX 0 mixing block has to be used in the Delay line mode the MIX DL mixing block has to be used The volumes indicated above are the mechanical volumes The hydrodynamical volume may vay slightly around these values It is recomended to calibrate these volumes with known reactions using procedure similar to that used in the Quenched flow mode Also similarly to this mode two ageing mode may be used here continuous flow or interrupt mode j 2 5 Liquid outlet system During the injection phase the liquid in the cuvette can reach linear velocities greater than 20 meters per second At the flow stop the liquid column has to be immobilized in a fraction of millisecond Depending on the stop mode this can result in overpressure or underpressure that are potential sources of stop artefact In the SFM 4 there are several procedures for the output flow 2 5 1 Free flow system In this mode the outlet is continously open and connected to a tube This procedure may to be used in case of pressure sensitive organelles or in case of the existence of pressure artefact on the cuvette material as in CD Care should be taken to allow the liquid column to be interrupted as close as possible to the SFM 4 exit 10 SFM 4 USER S MANUAL To do this an exit tube is provided in the standard equipment This tube has a vent permitting entry of air It is recomended to connect this tube to a larger PVC tube to permit further air
67. ogic stopped flow instruments The microprocessor control of the stepping motors giving 6400 steps per revolution of the motor gives a smooth and quasi continuous movement of the syringe over a very large range of flow rate A few exemplary experiments using the instrument are described below 7 3 1 Experiments using DCIP and ascorbic acid Contents of the syringes syringe 1 Ascorbic acid 20 mM syringe 2 Buffer TE Syringe 4 Dichlorophenolindophenol 100 uM a During the drive sequence the four syringes are programmed to deliver equal volumes to V1 V2 and V4 respectively The SFM 4 stopped fl w is programmed so that the volume of DCIP V4 is constant as is V1 V2 V1 V2 V4 is then constant and equal to 1 1 giving a constant final DCIP concentration 1 2 that of the DCIP in syringe 4 In a series of experiments V1 and V2 are varied giving a changing final concentration of ascorbic acid equal to V1 V1 V2 V4 By this method a Transmittance increase Time CS SEDIS A pere er Era ente de enfoc i ere pego 0 0 Z 0 4 05 08 1 12 2 4 16 1 8 SFM 4 USER S MANUAL glo V N este high dilution ratio of one component here AA can be obtained without changing the concentration of the other DCIP Resulting data are shown in the figure above where dilutions up to 1 24 were used The rate constants measured using the BioKine software show a satisfactory linear relationship as a function of AA concentrati
68. on Numbers Rate of DCIP decoloration in parentheses represent Var lation with AA concentration obtained by dilution the degree of dilution of 7 AA o Final concentration of DCIP was 50 uM i Dilution factors of 1 50 or higher can be k s 1 6 obtained This is B demonstrated in the next experiment where we mixed 1mM DCIP in syringe 4 with buffer in 2 syringe 1 There is no reaction to follow in this case the only goal of i this experiment is to test final absorbance after mixing AA mM The results shown in the figure below indicate a satisfactory linear relationship between the absorbance measured after mixing in the SFM 4 stopped flow and the final DCIP concentration which was calculated according to the ratio of the volumes delivered by syringes 1 and 4 SFM 4 USER S MANUAL eto V logie Dilution of 1 mM DCIP in water n m C ul 1 0 0 20 Absorbance 0 050 4484 AAT ADE o APS ge 1 Dilution factor 7 3 2 Measurement of variable ratio mixing by following alcohol dehydrogenase activity Buffer used was 100 mM Tris Cl 1 mM EDTA 5 g l of semicarbazyde Cl and 25 mM ethanol Contents of the syringes 51 Buffer S2 Buffer 1 mg ml alcohol dehydrogenase ADH 54 Buffer 1 mM NAD As described for the DCIP experiment above each experiment is a mixture of volumes V1 V2 and V4 delivered at the same time by the three syringes 34 2 gi SF
69. oncentration of agent However the experimenter is limited by practical concerns such as value of sample viscosity of sample dead times and inherent limitation of signal such as inner filter effect 6 3 5 Flow rate The flow rate is limited by the speed with which the stepping motors can push At the design nominal flow rate of 6ml s with all four syringes and using the smallest cuvette sub millisecond dead times may be accomplished However solutions of increased viscosity will lower the obtainable syringe speed Also at colder thermostated temperatures the speed often falls The limitation to syringe speed may sometimes be defeated by the use of ramping which allows each syringe to come to some intermediate speed for a short time before jumping to the higher speed Ihe dead time may be decreased by decrease of cuvette volume which may have adverse impact on signal amplitude The dead time may also be decreased by increased flow rate Increased flow rate beyond the use of two syringes at their natural limit 6ml s may be achieved in two ways 27 SFM 4 USER S MANUAL eto V legis a Use two or three syringes to simultaneously push the first reagent through the first mixer and or into the second mixer with each syringe at its maximum flow rate Syringe 4 also pushes at its maximum rate generating mixing in mixer 3 at a total flow rate of 18 or 24 ml s b Use ramping to try to exceed the natural limit to the
70. or several seconds in the delay line phase 3 Phase N 5 corresponds to the purge of the delay line the solution being pushed and evacuated Purge volume is again equal to 2xV After this purge 60 ul of the reaction mixture are 0 120 240 360 collected and measured Tp Pesetas Wi is the purge duration selected to give a constant flow rate during the purge phase of 1 5 ml s for each syringe Absorbance Results of this test are shown above as a function of purge volume Due to the long ageing time in phase N 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 demonstrate that for a delay line of 200 ul nominal volume corresponding to a total approximate volume of 220 ul the first 20 to 30 ul and the last 30 to 40 ul should be discarded to avoid contamination 4 5 General checking of the instrument Experiments shown below have been obtained under the following conditions Syringe 1 not used Syringe 2 1 5 mM DNPA in 2 mM HCI Syringe 3 100 mM NaOH Syringe 4 240 mM HCI Reaction times were obtained by varying the delay line volume the flow rate and the operation mode The actual delay l
71. ous 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 four syringes can be driven independently and their speed adjusted using the microcomputer with a very simple menu see SOFTWARE INSTRUCTIONS in SFM 4 S and SFM 4 Q sections for more details The automatic mode is used in the actual stopped flow 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 upper position of the syringe and zeroing can be done using the keyboard of the microcomputer The movements of the syringes being completely controlled by the microprocessor there is no need for a stop syringe Thus the stop artifact present in most conventional stopped flow systems is absent in the SFM 4 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 51 4 power supply The independence of the four syringes allows a higher versatilityof the injection sequence e g injection of one syringe only unequal filling of the syringes variable ageing times variable concentration variable ratios etc The reproducibility and regularityof the linear translation of the syringes and the absence of pressure artifa
72. phase An example is 17 SFM 4 USER S MANUAL Go leg given below BIO LOGIC SFM 4 Q V3 20 Help F1 Syringe Vol Quenched Flow Parameters 2 3 4 5 6 10 50 0 0 0 50 0 0 O 50 Waste co11 W W Drive sequence 1 1 9 Transfers parameters and sets MPS 51 in auto mode We recommend that you test the sequence on inexpensive buffer solutions of same viscosity as that which will be used in the real experiment 3 10 Programming the exit valve The exit valve action 1s to divert the exit flow either to waste or to collect The waste mode will be operated during all the washing phases that are preliminary to the collection of the usefull sample The programming is performed by entering a label on the 6th line of the control screen If a W is entered the valve will not be activated during this phase and the flow during will be eliminated through the Waste port of the valve If a C is entered on a given phase the valve will be activated and the liquid will flow out of the collection port of the valve 3 11 Incubation sequence An incubation phase between two flow phase is simply programmed by typing the time of incubation in the first line and typing zero in all of the syringe volumes 3 12 Drive sequence The programmed phases will be executed according to the drive sequence The default 18 SFM 4 USER S MANUAL N edie sequence is 1 1 9 which indicates that all phases 1 to 9 will be
73. rsion does not include ageing lines as described in INSTALLATION OF THE STOPPED FLOW COMPONENTS section SFM 4 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 the entire volume between the two mixers has to be taken into account This volume includes the ageing line plus the dead volumes volumes between the both sides of the delay line and the mixers The complete description of the volumes are described in the sections VOLUME OF FLOW LINES SFM 4 S and SFM 4 Q SFM 4 USER S MANUAL 0 2 GENERAL INSTRUCTIONS FOR INSTALLATION This section of the manual contains information on the installation and preliminary operation of all versions of SFM 4 It is recommended that the contents of this section be read and understood before any attemptis made to operate the instrument In case of difficultiesplease contact Bio Logic or its nearest representative 2 1 Operating features Figure 2 2 5 6 7 4 4 8 10 SFM 4 USER S MANUAL aS NN 2 Table B FUNCTION AME LCD DISPLAY Used to displaymessages selected syringeauto mode SYRINGE SELECTOR Selectsthe syringefor the manual control 5 Inputforan externalsignalto trigger the drivesequence TRIGGER INPUT l SYNCHROPULSEOUTPUT Pulse outputto triggerthe recording system or any electronicdeviceto be synchronizedvi
74. servation head with separate cooling part This feature permits a temperature regulation of the observation head different from the main body of the instrument This may be used in cases where mixing of the solution produces a temperature change of the solution flowing into the cuvette 4 SOFTWARE OPERATION 4 1 Introduction This section of the manual contains operating instructions and a description of the experimental procedure It is assumed that you have run the installation softawre as described in section I 42 Installation of the SFM 4 S version of the software Run BIO from Root directory You will get the following screen pee rd SFM 4 S SFM 4 Q Exit SSS SS SSS SSS SSS Driving Software Kinetic Software Config Driving Software Config Kinetic Software TRUM E as aa Application Driver Software V3 0 1 Copyright c 1991 i Bio Logic Company All right reserved 5 Z A de FONT RATEL 38640 CLAIX FRANCE Ph 6 98 68 31 Fax 76 98 69 09 ji o id DO SFM 4 USER S MANUAL 4 3 SFM 4 S driving software configuration The SFM 4 S configuration program is allready set at the default values It can be used to install or change the syringe model set different flow rate limitations enable or disable the hard stop mode and select serial port number After validating Config Drivi
75. sition The Stopped Flow Module is now ready for operation 19 Mod SFM 4 USER S MANUAL eto A ede 4 8 Creating a drive sequence This is performed from the main menu The drive sequence has been divided into 9 phases In each phase the operator has to enter the duration of the phase in milliseconds and the volume in microlitersdelivered by the four drive syringes in each of the phases Selection is made by displacing the cursor with the arrow keys In the left of the screen a field is reserved to enter four letters identifying the contents of the four syringes Enter in this field by depressing the Tab key Return to the data field in the same way If SFM 4 is used with Bio Kine software all the informations entered will be saved with acquired data With ordinary aqueous solutions the motor can drive the Syringes up to at least 6 ml s without acceleration phase This means that motors will not stall if the following sequence is programmed BIO LOGIC SFM 4 S V3 10 Help F1 NEN Phase f 2 Stopped F lom Parameters Phase 1 2 3 4 5 6 7 8 9 Flow ml s 100 0 0 0 Tot 000 0 0 batai S 6 000 S2 0 000 0 000 porch 6 000 Vol p1 600 anas ee ee ee ee PI ee ee ee em ee PADS a Ku ee anas dans
76. stepping motor velocities Again careful trade offs are required to balance each of these factors against each other in order to accomplish fully successful stopped flow experiments stalled motors E lower dead times increase flow rate inadequate washing decrease cuvette volume loss of signal overuse of reagent increase pathlength increased dead time 1 inadequate washing increase signal overuse of reagent increased viscosity causing cavitation increased viscosity leading to stalls increase reagent increased viscosity leading to inadequate washing SFM 4 USER S MANUAL JAN 7 TEST REACTIONS 7 1 Evaluation of the dead time The reaction used is the reduction of 2 6 Dichlorophenolindophenol DCIP by ascorbic acid AA A complete description of this reaction and its use can be found in Tonomura et al Analytical Biochemistry 1978 84 370 383 DCIP has a strong absorbance at 524 nm and reduction by ascorbic acid results in a nearly complete decoloration The second order reduction rate constant is highly dependent on pH and varies from about 10 34151 at pH 2 0 to 10 311 51 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 We use the acid pH
77. the spectrometer used As above BioKine Same as above for the optics BioKine can be included only if one can solve the problem of compatibility between the host spectrophotometer and BioKine This option is feasible for most cases SFM 4 fibre optics for illumination The host spectrophotometer is just used as a host spectrophotometer detection light source As the detection 15 made directly on channel PMS 200 PM tube Bio Kine the SFM with Bio Logic detection channel light computer A D board will be sufficient to enable absorbance and fluorescence experiments This configuration only saves the price of the light source O O DOO HEHE 0 AD COP OEE BO COOP PRO CU OU PA VOS CCOO RD CV OHS GOSSOS DG OUS SERRE OE DOOR EE Illumination may be a little weak for low intensity fluorophores Complete set up SFM 4 MOS 200 or Completely independent set up for fluorescence MOS 200 S Bio Kine A D board and absorbance Very high intensity light source computer with MOS 200 S Complete set up SFM 4 MOS 400 CD As above with CD capability in addition Bio Kine A D board computer Complete set up SFM 4 MOS 400 DW As above with dual wavelength absorbance or Bio Kine A D board computer dual wavelength excitation fluorescence Please refer to the specific brochure for optical system installation SFM 4 USER S MANUAL JAN AN regis 2 INSTALLATION OF THE STOPPED FLOW CO
78. ththe instrument MANUALMOVEMENT Manualcontrolof the syringes MOTORON INDICATOR Lightswhen at least one of the motors is activated START STOP Initiates or stops the programmed sequencein the automaticmode The instrumentmay also be startedand stoppedusingthe keyboardof the PC PROGRAMRESET Resetsthe MPS 51main program MAINPOWER FUSE ACLINECONNECTOR MAINPOWER SWITCH 3 A for220V or6A forll5V c sendsthe powerpulsesto thestepping motors MOTOR POWER CONNECTOR LOGIC CONNECTOR Connectsthe MPS 51controllerto the JI MOTOR FUSES J O gt mM DN 5 HARDSTOP SF or OUTPUT VALVE QF BNCCONNECTOR 11 5 1 ge AN SFM 4 USER S MANUAL 2 2 AC povver and connections Before connecting the SFM 4 to the local AC line verifythat the setting of the instrument matches the local line voltage Prepare the SFM 4 for operation by connecting the mechanical subsystem to the power supply unit and the power supply unit to the controller Plug the controller into the appropriate AC line Prepare the SFM 4 for operation by connecting the mechanical subsystem to the MPS 51 unit Connect the MPS 51 to the serial port of the microcomputer 2 3 Water circulation The SFM 4 module may be connected to a circulatingwater bath for temperature regulation The coolant flows through two internal circuits one around the injection and reservoir syringes
79. to study reactions from one to one hundred or so milliseconds In the interrupted mode the reaction mixture 1s held in the delay line for a programmed time and then mixed with quencher in the last mixer This mode is used for incubation times of a few ten milliseconds to several seconds or more The critical decisions in the use of the quench flow instrument are the choice of delay lines flow rates and operational mode described above The calibration experiments which follow are intended to give some examples of these choices 4 3 Measurement of flow lines volumes and of efficiency of washing Knowledge of the exact volume V between the two mixers 1s critical In the continuous mode the age of the reaction T is equal to T V F F being the programmed flow rate Because of the use of stepping motors there 1s no need to calibrate the flow rate it will be as specified by the user In the table 1 6 2 above an estimation was given about the total volume V for each of the delay lines used It 1s recommended these volumes to be checked and calibrated by the user Below 1s an example of the calibration of the smallest delay between mixers 2 and 3 This will be obtained by inserting no delay line between mixer 2 Block labelled MIX 0 and mixer 3 This is supposed to give a total inter mixer volume of 22 ul when inserted in position No delay will be inserted between mixer 1 and mixer 2 The reaction proceeds as follows DNPA in
80. xceeds several drops per syringe run empty the guilty syringe then withdraw it completely from the drive syringe barrel Inspect the white teflon tip both visually and with your fingers Scratches and other defects which allow leakage will often be invisible in the white teflon due to low contrast but apparent by touch If there are defects in the tip order new ones If the tip is dirty clean it with an ethanol dampened wiper 3 If leakage persists after observing steps 1 and 2 above the tips may be removed and boiled to expand the teflon Teflon will contract upon cooling slightly less than it expands upon heating allowing a better seal with the syringe barrel To remove the tip fully withdraw the syringe shaft from the barrel loosen the Allen screw at the base of the syringe shaft and lift the shaft from the crossbar Then remove the teflon tip by unscrewing it from the steel shaft Boil the tip for ten to fifteen minutes making sure the boiling flask does not go dry When the tips are cool replace them by reversing the removal procedure described above 4 If leakage persists order new tips 2 4 Syringe disassembly and reassembly Remove the syringe plunger and its holder as described above Unscrew the syringe from the bottom of the water jacket using a fork wrench or pipewrench dimension 24 mm Pull and remove the syringe body from below Wash or replace Check the Kel F sealing on top of the syringe Carefull
81. y stage of the drive sequence The default value is 0 for all the phases which means that 0 Volt is always present on the synchro out BNC connector Use the horizontal arrows to place the cursor on the phase to be used to trigger the recording device type any number to validate the number 1 will appear at this location Type gt 0 lt to reset More than one phase can be active for triggering during these phases the MPS 51 controller will deliver a 5 Volt signal If Bio Kine software is being used acquisition will start on the falling edge i e at the end of the active phase For example with the sequence shown in section 4 12 acquisition will start at the end of phase 1 beginning with phase 2 In case the hard stop is desabled a second trigger pulse can be programmed for another application 21 se 4 11 Saving or loading the experimental parameters The created data can be saved on files A series of experimental conditions can thus be prepared before the experiment and subsequently recalled while the program is being run At the command level type lt F gt for Files Save or load the parameters by following the instructions displayed on the screen For help at any point during operation type F1 The data on the screen can be erased by typing lt C gt 4 12 Running the automatic mode The sequence created in the microcomputer will be transferred to the MPS 51 unit to be executed in the automatic mode Verify t
82. y wash the outer surface of the syringe body 5 SFM 4 USER S MANUAL ee logie Grease the O ring with silicon grease Screw the syringe back in place by hand Tighten with the fork wrench 2 9 Disassembly and reassembly of the observation head or delay lines blocks Stop the coolant circulation Unscrew the four screws holding the head Pull the head out Replacement of the cuvette and of the mixer are described in 2 Replace 48001 Replace the two O rings see next page for O ring description and dimensions Reassemble Kit O Rings SFH 4 5 2 2x1 6 RN B SS 020 1 0 6 02 6x1 9 d 0 ji 069 x Kit O Rings SFM 4 Q S os 00 9 Qty 14 0 1 6 x 1 25 10 O 2 2 x 1 6 24 26 x 006 27032 02 6x1 9 02 2x1 9 02 6x1 9 Kit O Rings SFM 4 9 Qty Model 0 0 25 12 0 1 6 x 1 25 2 6x1 9 8 22 16 20 2 6 89 02 6x1 9 Qty Model B 3 SFM 4 USER S MANUAL a LOGIC 2 6 Lubrication Clean the instrument periodically to remove moisture dust and grease Unscrew and clean the valves when the instrument is not to be operated for a long period of time Lubricate the driving screw with mineral oil periodically Access to it is obtained by removing the front cover 2 7 Replacement of observation cuvette Replacement of the observation cuvette is a routine operation whic

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