QFM-4000 User s Manual Version 2.0

Transcription

1 QFM-4000 User s Manual Version 2.0

2

3 Table of contents 1. INTRODUCTION AND SPECIFICATIONS General description The mechanical design Intelligent power supply Microcomputer commands Driving system QFM-4000 innovations One single ageing line Sample collection system Sample storage lines Description of the flow lines hardware Specifications QFM-4000 temperature regulation INSTALLATION Control unit MPS-70/ AC power and connection of MPS USE INSTRUCTIONS Operational modes Storage line installation Filling ports Filling valves Drive syringes filling Storage line filling Final mixture exit Collection device Air purge SOFTWARE Device installation Device configuration Stopped-flow status area INSTRUMENT OPERATION Manual syringe control Hardware control Software control Syringe initialization Filling the syringes Creating a driving sequence SFM options Sequence edition Setting the reaction time Running a shot TEST EXPERIMENT Practical recommendations Filling the instrument Shot procedure Description of the test reaction A test experiment in standard mode (reactants in drive syringes) A test experiment in volume optimized mode (reactants in storage line) INSTRUMENT SERVICE

4 7.1. Flow line and intermixer volumes (76)-(35) QFM-4000 cleaning and storage Long-term storage of the QFM Pulsed-flow parameters

5 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. 3

6 1. INTRODUCTION AND SPECIFICATIONS 1.1. General description Each Bio-Logic stopped-flow module (QFM & SFM), consists of a mechanical subsystem and a motor power supply (MPS). All QFM syringes, valves and flow lines are enclosed in a water jacket to allow temperature regulation of the reactants containers. The syringe plungers of the QFM are driven by stepping motors via ball screws The mechanical design The mechanical part of the QFM module is carefully constructed. The parts in contact with the sample and the buffers are all machined out of materials selected for their inert characteristics: stainless steel, Teflon, Kel-F, VITON, EPDM, PEEK and quartz Intelligent power supply The high performance of the QFM and the high speed of the stepping motors can be achieved only because of the quality of its power-supply. The MPS unit contains independent constant current power supplies for each syringe, all driven independently by their own microprocessor. The sequence of impulses to be sent to the stepping motors is stored in the memory of each motor board. One main microprocessor board synchronizes all the power supplies, and performs the communication with the microcomputer via a serial interface Microcomputer commands The QFM module is controlled by the MPS software program running on a PC or compatible microcomputer under Windows XP or later versions. Various menus and windows permit the user to know the volume of the solution contained in each syringe perform manual or automatic movement of the syringes create a reaction sequence with complete control of time and volume delivered by the syringes save or recall the sequences 1.5. Driving system The syringes of the QFM-4000 are driven by independent stepping-motors. The stepping-motors are of hybrid technology with 200 steps per revolution and 4 phases, each phase being powered by a constant current supply (2.9 A per phase). The power supply of each motor is microprocessor controlled. A complex impulse sequence enables micro-positioning of the motor's rotor with an accuracy equivalent to 1/32 of the mechanical step. This gives an effective number of 6400 steps per revolution, or a volume quantification of 0.14 µl per micro-step (µ-step), when standard (10 ml) syringes are used. With the damping produced by the rotor inertia, this results in an almost continuous, linear movement of the syringe even at very low flow rates. The motors can be activated manually or automatically. The manual mode is mainly used to refill or wash the syringes; the syringes can be driven independently and their speed adjusted using the microcomputer with a very simple menu. The motor impulses are counted in the positive direction (refilling), or negative direction (emptying), so that the contents of each syringe can be continuously displayed. Zero volume corresponds to the uppermost position of the syringe and referencing the zero volume position can be done using the keyboard of the microcomputer. An observation system can be synchronized with the syringe "start" or "stop" by using the trigger pulses available on the front panel of the MPS unit. The independent control of each syringe allows a high versatility in the injection sequence. It is possible to make an injection of one syringe only, unequal filling of the syringes, variable ageing times, variable concentration, variable mixing ratios and other complicated actions with only a few keystrokes. The reproducibility and regularity of the linear translation of the syringes and the absence of pressure artifact allow optical recording during the drive sequence. This feature greatly facilitates the determination of the initial phase of the reaction being monitored and makes the equipment suitable for very accurate, continuous flow experiments. 4

7 1.6. QFM-4000 innovations The syringes of the QFM are mounted vertically. This allows easy purging of bubbles, which are evacuated during refilling by a few up and down movements of the drive syringe. The syringes, valves, and observation chamber are very carefully thermoregulated. This thermoregulation prevents the occurrence of temperature artifacts on a very wide temperature range. A QFM-4000 instrument can be purchased as such (quenched-flow only) and modified later to give a SFM-4000 optical stopped-flow instrument, the reverse also being true. The QFM-4000 instrument is designed to exploit the outstanding µvolume specification and the extreme precision of the drive of the SFM In optimal condition this instrument will allow quenched-flow reactions to be measured with minimal volumes in the range of 10 to 20 µl per shot for the two reacting solutions. The QFM-4000 incorporates a series of innovation that makes it the most efficient quenched-flow instrument. This includes the following : One single ageing line Reaction times from a few milliseconds to seconds can be obtained with only one single ageing line. The QFM-4000 achieves such a large time range with a single ageing line by operating in pulsed flow mode. This new pulsed flow mode generates turbulent flow even in the case where the mean flow rate would just produce laminar flow which would require exceeding washing volumes (see Figure 1). The individual pulses sizes vary from 0.3 to 2 µl and are automatically set by the instrument to adapt to the flow line geometry and to the ageing conditions. Value of the mean flow rate is achieved by varying the frequency of the pulses. The pulsed flow mode also ensures a correct mixing of the reactants on the whole range of ageing time. For instruments using continuous flow modes, the mixers cease to operate correctly at decreasing flow rates (increasing ageing times). This necessitates switching to interrupted mode which is an unfavorable mode regarding sample economy. This situation is eliminated by the pulsed-flow mode since the instantaneous flow rate in the pulses ensures a correct mixing of the reacting solutions. Laminar flow (low flow rate) Turbulent flow (high flow rate or pulsed flow) Figure 1 : laminar and turbulent flow rate Absorbance Turbulent Volume Laminar Absorbance at the exit of the tube as a function of washing volume Thus this new pulsed flow mode has many advantages: a) Extension of the range of use of delay lines b) Extension of the range of use of the continuous flow mode c) Since a much lower mean flow rate can be used, it allows a drastic reduction of the delay line volumes. This ageing line is the core of the flow system, because of its low volume (3.5 µl default value but the volume of delay line is factory calibrated for each unit) and because it does not have to be exchanged, the QFM-4000 instrument has unique sample economy and sample recovery specifications. In optimal conditions this instrument will allow quenched-flow reactions to be measured with minimal volumes in the range of a few microliters per shot and for minimal total quantities few tens of microliter for each of the reacting solutions Sample collection system The QFM-4000 specification would have been useless without an efficient sample recovery principle. To do this, an air purge is used in the second mixer that allows full sample recovery after the mixing 5

8 sequence. This also allows cleaning of the exhaust tube before the next shot. This allows a dead volume of less than one µl after the last mixer Sample storage lines For the µvolume operation it is possible to install in the QFM-4000 instrument sample storage lines allowing the use of very low total volumes of reactants. It is thus possible to execute a full kinetics with total reactant volumes in the range of 100 µl Description of the flow lines hardware (see Figure 2) The QFM-4000 instrument consists of two reagent syringes (S1 and S2), one syringe used for delay line washing (S3) and one syringe for the quench solution (S4). Four independently programmable motors are used to actuate S1, S2, S3 and S4 respectively. The S3 syringe may or may not be used (depending on the operational mode) Each drive syringe is set with four-way L-valves (V1 to V4) of very small dead volume (a few µl) Flow lines are easily replaceable PEEK or TEFZEL tubing whose length and diameters may be adapted to the operational mode and ageing time. The lines L1 and L2 may used as solution micro-reservoir for expensive solutions. (L1 and L2 volumes may be varied from a few 10 µl to 200 µl).. These lines are easily replaced and adjusted by the user Solutions in S1 and S2 are mixed in the mixer M1 which can be either a T mixer or an X mixer with an input for S3. S1 + S2 mixture is allowed to age in the delay line DL. Volume of the DL is fixed to a default value of 3.5 µl but it may be easily replaced if necessary. Ageing in DL is set by adjusting the mean flow rate of S1 + S2 + S3 Ageing times can be varied from a few milliseconds to several seconds. (depending on motor programming and syringe size). The maximum flow rate that can be obtained is also dependent on flow line diameters. Reaction product is mixed with the quenching solution in the mixer M2. Final mixture is accumulated in the exit line EL. Collection of the reaction product out of EL is made manually by an air purge. The air purge is made through a fourth port of the last mixer. This line is a narrow tube of mm diameter. This is a 1/64 ratio with the delay line cross section. Because of the ratio, a very low fraction of the liquid will enter the purge tube. The tiny amount that will enter this tube will in any case be flushed away during the air purge. The air purge can also be done through a 0.76mm diameter tube equipped with a check valve. Temperature regulation is made by water circulation around the drive syringe and in the flow line compartment 6

9 S1& S2 = reagents S3 = buffer S4 = quencher EL Exit purge L1 M1 DL L2 M2 Purge / injection port Syringe filling port V1 V2 V3 V4 L port R port S3 S4 S1 S2 M1 M2 M3 M4 Figure 2 : QFM-4000 flow line hardware [note that the valve N 3 is represented in the position that connects the L port to the flow line. All other valves are shown in the position that connects the drive syringes to the flow lines. This is the position used for the shots. 7

10 1.8. Specifications The general specifications of QFM-4000 are listed in Table 1 below. Table 1 QFM-4000 Specifications GENERAL QFM-4000 SPECIFICATIONS Number of syringes 4 Driving mechanism One stepping motor per syringe (6400 steps per motor turn) Number of mixers 2 to 3 Ageing line between the two mixers Factory calibrated: typical volume 3 µl Ageing time range 4 ms to 10 s Trigger Programmable trigger for data acquisition and synchronization of accessories Filling range of the drive syringes 100µL to 1.9 ml (up to 3.6 ml for the S3 syringe) Syringe size and speed 1.9 ml syringe: ml/s 3.6 ml syringe : ml/s Minimum flow rate for efficient mixing 1 ml/s in continuous flow, (total flow rate through each mixer). Unlimited in pulsed flow mode Minimum reactant volume per shot 10 µl/syringe Variable ratio range By customization Delay line purge Air or buffer Exit line purge Air Material PEEK (stainless steel or Kel/F on special order) Size of standard syringe 1.9 ml standard syringes for S1, S2 and S4 3.6 ml for S3 (6.8 and 10 ml syringes are also available) Reactant volume in storage line 20 to 200 µl Volume per µ-step 0.03 µl for the 1.9 ml syringe 0.06 µl for the 3.6 ml syringe Duration of flow adjustable from 1 ms to 10 s per phase Power requirement 300 Watt, 110/220 Volt, 50/60 Hz Total weight 16 kg 1.9. QFM-4000 temperature regulation The QFM module may be connected to a circulating temperature bath for temperature regulation. The coolant flows through two internal circuits: one around the injection and reservoir syringe ports and the other through the valve block and observation head. 8

11 2. INSTALLATION This section of the manual contains information on the installation and preliminary operation of the QFM-4000 instruments. It is recommended that the contents of this section be read and understood before any attempt is made to operate the instrument. In case of difficulties please contact Bio-Logic or its nearest representative Control unit MPS-70/4 The QFM-4000 instrument is powered by the MPS-70/4 unit.operating features of this unit are shown in table 2 below and described in Figure 3 and Figure 4 Figure 3: MPS-70 front panel Figure 4: MPS-70 rear panel 9

12 Table 3: MPS-70 panel description AC power and connection of MPS-70 Before connecting the SFM to the local AC line, verify that the setting of the instrument matches the local line voltage. Prepare the QFM for operation by connecting the mechanical subsystem to the MPS-70 unit. Connect the MPS-70 to the USB port of the microcomputer. Finally, plug the MPS-70 into the appropriate AC line. 3. USE INSTRUCTIONS The QFM-4000 instrument is easy to use. However some practice is necessary to exploit its full performance. The instruction below gives the most important recommendations. We recommend a careful reading of this part of the manual before attempting to use the instrument Operational modes The operational modes described here are just a few examples among many more. Classical quenched-flow mode. This uses only S1, S2 and S4. The reactants are set in the syringes S1 and S2 10

13 The solutions in S1 and S2 are used for washing old solution or aged solution out of the delay line. This mode is the simplest to use and will be preferred when about 200 µl to 1 ml of each of the S1 and S2 solutions are available. Volume optimized quenched-flow mode The principle of this mode is to fill either the syringe S1 or S2 (or both) with inexpensive buffer and use the flow lines L1 or L2 (or both) for reactants storage. Flow lines of various volumes can be constructed from TEFZEL or PEEK tubes of calibrated cross section and length (Figure 5). The reactants are introduced in the lines via the valves V1 and/or V2. During the shots, these solutions are pushed through the mixer and delay line by the buffer contained in S1 and/or S2. Figure 5 : flow line For all modes, the sample economy will be further improved by using the syringe N 3 to push the aged mixture out of the delay line at the end of the driving sequence. The syringe N 3 can also be used to wash the delay line before the actual shot and S1 + S2 mixing Storage line installation Storage lines are set between the valve and the mixer. Access to the line is made by removing the front panel of the instrument. (Note : first unscrew and release the exit tube (as shown in Figure 9)). Figure 6 shows the instrument with 2 different size of storage lines. This delay line can be constructed either with PEEK or TEFZEL tubes. Figure 6 : storage line (25ul and 100µl) 3.3. Filling ports The QFM-4000 includes two filling ports per syringe. A syringe filling port (light brown R port for reservoir) and a line filling port (red L port, for collect). The R port is used to fill the syringe with the reactant or with buffer. The L port is used to fill the storage line during volume optimized quench flow mode. Each of these ports accepts Female Luer Adapter to be used when syringes are used for filling. These ports can also accept Check Valves or Injection Port Adapters. Injection port adapters are a good choice for injecting the reactants in the storage lines with minimal dead volume losses Filling valves Each syringe is fitted with one four way L-valve. This valve has four positions as indicated in figures 7 and 8. These two positions are termed by reference of the flat part of the handle. Vertical front (Figure 7) : in this position the flat part of the handle is viewed from the front of the instrument. In this position termed C the drive syringe is connected to the storage lines (L1 or L2) and to the mixer. It is the operative position during the shots. It is recommended to check that the valves of all active syringes are in this vertical position before attempting a shot. Horizontal down (Figure 8) : in this position the flat part of the handle is down. In this position termed R the drive syringe is connected to the R port. This position is used to fill the drive syringe. 11

14 Horizontal up : here the flat part of the handle is up. In this position termed L, the flow line between the valve and mixer is connected to the L port. This position is used to wash this line or to fill the storage line installed there. Vertical back : in this position the flat part of the handle is vertical but turned to the rear side of the instrument. In this position termed W, the L and R port are connected together. This position may be used for washing the lines between the port and the valve. Figure 7 : valve position for shooting Figure 8 : valve position for filling the syringe 3.5. Drive syringes filling This is executed from the R port with the valve in the horizontal-down R position. Pull the solution by operating the drive syringe. Execute a few up and down movement to evacuate the bubbles. A spacer may be inserted under the syringe drive assembly to limit its movement and thus preventing the plunger to get out of the syringe body. This may not happen when the syringe movement is controlled from the software. There is however no limit for the plunger movement when driven from the power supply Storage line filling The most efficient way for line storage filling is first to flush the line with air. This is done by setting the valve in the horizontal-up L position and injecting air with a syringe through the L port. Do not omit to set a tube at the exit port to collect the purged solution. When done set the injection syringe on the port (via Luer adapter or Injection Port Adapter). Caution : When filling one flow line be sure to have all other valves set on R or W positions but not on L or C. With any other valves on L the risk will be that the solution remaining in the lines may flow out of the corresponding L port. A valve in C position may also be a risk of cross contamination between syringes Final mixture exit QFM-4000 does not have an exit electrovalve. This is another source of dead volume reduction and sample economy. After the last mixer, the final solution is flowing downstream the last mixer in an exit line (EL) whose length and diameter may be modified. This tube is used to collect the final mixture. It is connected to the last mixer and pass though front panel of the instrument. This passage is then made firm and tight with a nut and ferrule (see Figure 9). 12

15 At low total flow rates (< 0.2 ml/s) this line may be used for temporary storage of the final solution provided its volume is larger than the total volume to be collected. At the end of the shot the solution used for prewashing (if any) has been eliminated through the open end of the line. The line content is now collected in a tube (e.g. an Eppendorf tube) by flushing air manually via the purge port (see figure 10). At higher flow rates (low ageing times) this procedure cannot be used. Because of its inertia the liquid column in the tube will have a tendency to fragment at the moment of stop. Part of it will then be expelled out of the exit tube. In this case it is recommended to use a sealed tube or a syringe at the exit port to eliminate the risk of solution loss (see figure 11). Figure 9 : Exit tube 3.8. Collection device The collection device can be an Eppendorf tube (Figure 10), a syringe (Figure 11) or any other system that may be preferred by the user. Caution : An open tube is not adequate at high flow rates (low ageing times). Most of the solution will be flushed out of the tube because of its high speed. In this case use a syringe or closed tube with a pierced cover. Figure 10 : Eppendorf collecting device Figure 11 : syringe collecting device 3.9. Air purge The two mixers may be connected to the air purge. This air purge is made though the M1 and M2 ports on top of the instrument. M1 purge is not connected in standard mode. This may be used for flushing the delay line between two shots. In the standard modes, a liquid purge from the S3 syringe is preferred. 13

16 The M2 purge is connected to the last mixer via a tube of thin cross section. (see Figure 12).The operation is very simple. Connect a 1 or 2 ml syringe full with air. After a shot exert a pressure on the syringe, this will result in flushing the exhaust tube. Release the pressure before the next shot. Figure 12 : air purge 4. SOFTWARE The QFM-4000 is controlled by computer and it is delivered with the Biokine32 software that is common to all Bio-Logic rapid-kinetics instruments. This section briefly describes the configuration of 14

17 the software. Please note that these procedures and examples have been generalized and configuration choices should be made based upon the equipment purchased and intended experiments. To install Biokine insert the CD supplied with the instrument and run setup.exe. Follow the instructions. The biokine.exe file is created into the c:/bk32 repertory. Run Biokine Device installation Once Biokine loaded, choose Install, device installation in the main menu. The stopped-flow communication is established from this window by checking the stopped-flow device box and choosing the USB communication mode. Accept the parameters using OK button. Figure 13 : Device installation window 4.2. Device configuration Once the stopped-flow device and its serial port selected in the device configuration menu (refer to section 4.1) choose the Install, stopped-flow configuration menu (see Figure 14). Figure 14 : stopped-flow configuration The device to be installed should be configured according to the instrument purchased and mode chosen for use. The QFM-4000 comes equipped with 1.9 ml syringes in S1, S2 and S4 and a 3.6 ml syringe in S3. These are the default syringes installed in Biokine software. 15

18 The active syringe is displayed in yellow, select the nature of the syringe that have been installed in each position of the SFM by clicking on the right one. Use the CUSTOM button to enter syringe specifications if you have a custom syringe. In this condition the window shown in Figure 15 is displayed, it is then necessary to enter volume, piston diameter and screw pitch of the custom syringe to add it to the standard ones. Figure 15 : custom syringe Warning : incorrect syringe configuration will cause volume and flow rate calculations to be incorrect 4.3. Stopped-flow status area A vertical menu bar on the left of the screen is dedicated to the stopped-flow device. This menu bar gives access to the syringe control window using the button (refer to section 5.1). 5. INSTRUMENT OPERATION 5.1. Manual syringe control The syringes of the QFM-4000 can be controlled either manually or automatically. Automatic control of the syringes is strictly used only for experiments. The manual control of the syringes is used for initialization, filling and emptying the syringes. The manual movement of the syringes can either be made directly from the MPS or though the Biokine software. Both methods are described in the following sections Hardware control Syringe control directly from the MPS hardware is made through the use of the buttons on front panel of the MPS (see Figure 4 (MPS-70)). The (+) and (-) buttons are used to select the syringe to be moved. The (up) and (down) buttons are used to empty and fill the syringes respectively Software control Syringe control from the MPS software is made through the «Syringes Command:Load» window available under the Syringes Command menu ( Figure 16). The syringe to be moved is selected by clicking on the corresponding frame, or pressing the <Left> or <Right> arrows keys on keyboard. The new selected syringe will be surrounded with a red rectangle (Figure 16). Syringes are emptied or filled using the,, and buttons or with the <Up> arrow, and <Down> arrow. The button and <Up> arrow move a syringe upwards by one elementary movement and the button and <Down> arrow move a syringe downwards by one elementary movement. The button arrow move the piston upwards by 10x elementary movements and the button move the piston downwards by 10x elementary movements. The size of the elementary steps and syringe movement speed is controlled in the Manual Speed section of the «Syringes Command:Load» window (Figure 16). The and buttons change the manual speed. The display shows the speed in flow rate based on the syringe installed and moved. 16

19 Figure 16 : syringe control window 5.2. Syringe initialization The MPS module that controls the QFM-4000 syringes follows the movements of the syringes so that the actual residual volumes are displayed at all times in the MPS software «Syringes Command:Load» window (Figure 16). When the MPS is turned on, the software started and Syringe Command turned on, the syringe volume counters show and have to be initialized (Figure 16). The syringes are initialized by setting the syringes to their uppermost (empty) position and resetting the syringes in the MPS software. The syringes can be selected and moved to their uppermost positions either directly with the MPS or through the MPS software. Once a syringe has reached its uppermost position, the syringe motor will oscillate and vibrate as it becomes out of phase with the driving pulses. There is no danger to the QFM-4000 or syringe motors when this occurs, but there is no reason to unnecessarily prolong this treatment. The syringes can be reset individually by pushing the button for each syringe or all at once by pushing the button in the software «Syringes Command:Load» window (Figure 16).!CAUTION!: Measurement of residual syringe volume is made by counting the logic pulses from the controller for each syringe. If, for any reason, a syringe is blocked during a run, the pulses will not correspond to the true volume delivered and the value displayed may become erroneous (e.g. in the case of incorrect positioning of a valve). In this case, it is advisable to reinitialize the syringes. If, by accident, a syringe is returned to its uppermost position the syringe volume counter will again show and the syringe must be reinitialized. To avoid such accidents, the Up and Low Limits checkbox may be checked. When this box is checked, the MPS software will not allow the syringes to be driven beyond their upper and lower limits. This also avoids accidentally pulling the syringe plunger completely from the syringe and spilling solution onto the QFM syringes.!warning!: The Up and Low Limits only applies to control of the syringe from within the MPS software. These limits can be bypassed by manual control of the QFM directly from the MPS Filling the syringes!warning!: Utmost care should be exercised during this operation. Normal operation of the system requires that no bubbles are present in the injection syringes. Should this occur, the buffer flow through the QFM flow lines will not be correctly controlled by the plunger movement and artifacts may be observed. For best results it is recommended that all solutions be degassed and filtered before filling the QFM

20 The syringes can be emptied and filled manually. The filling of the syringes follows the steps below. Attach a syringe (disposable plastic syringes may be used) containing sample or buffer to the R port Set the syringe valve to its horizontal position and fill the syringe manually while exerting a slight pressure on the reservoir syringe. The pressure exerted on the reservoir syringe prevents any vacuum from occurring in the reservoir syringe which could result in bubble formation. Eliminate any bubbles in the QFM syringe by driving the QFM syringe up and down several times while it is connected to the reservoir syringe. Turn the syringe valve vertical. Empty one or two elementary movements of the syringe to definitively eliminate any bubbles remaining in QFM flow lines. Repeat the above process for the other syringes.!important!: ALL SYRINGES MUST BE FILLED EVEN IF THEY WILL NOT BE USED FOR AN EXPERIMENT! The valve handles of all (used or unused) syringes should be left in the vertical position Creating a driving sequence SFM options Experiments are performed with the QFM-4000 through the use of a driving sequence. Driving sequences are created in the window shown in Figure 17, this window can be reached from the Mixing sequence button in the stopped-flow status area. Figure 17 : driving sequence You have the option to repeat the driving sequence automatically by checking repeat sequence. Figure 18 : repeat sequence window 18

21 In the example shown in Figure 18, the times of the phase 2 will change automatically between each shot, and will be successively 5000, 8000 and 10000ms, with a waiting time of 5s between each shot First operation should be to check the configuration of the stopped-flow, it is made by clicking on the button (refer to Figure 19). Figure 19 : SFM options The cuvette, HDS mixer, lead time and acceleration phases are not available with this instrument. Overheating protection : not applicable for QFM-400 Pulse mode : that enables the pulse mode that is specific for QFM Default mode is selected Delay line : enter the delay line volume according to the line installed in the QFM-4000 (this value is given at the installation and remains the value by default). Warning : incorrect delay line configuration will cause ageing time calculations to be incorrect Sequence edition A driving sequence is entered in the program grid in the «Quenched Flow Program» window. Each column of the grid represents a driving sequence phase. Each phase contains actions for the QFM to perform. A complete driving sequence may contain from 1 to 20 phases. Although only 5 phases are shown initially, additional phases may be inserted using the Insert Phase command under the Edit menu. Figure 20 shows an expanded view of the program grid. The duration of a phase is entered (or shown) in ms ( ms/phase) on the first line of the program grid. The volume in µl delivered by each of the syringes during a phase is entered on the line next to the appropriate syringe. The Lock control is set for each phase. If Lock is set to On duration of the phase is automatically controlled (Locked) according to the desired ageing time and volumes delivered in each of the phase. If Lock is set to Off, the phase duration is free and has to be entered by the user. To enter the phase duration and syringe volumes delivered, click on the corresponding cell or use the keyboard arrows keys to navigate between cells. The BACKSPACE key can be used for correction and the DEL key to clear a value. The Lock control is set by pressing o for On and f for Off on the keyboard. Selected values entered in the program grid can be cut, copied and pasted using the Cut, Copy and Paste functions available under the Edit menu. To perform a cut, copy, or paste operation, select the area of the grid desired by dragging the mouse with the left mouse button pushed in and then choose the Cut, Copy or Paste functions desired under the Edit menu. The values will be stored in the Windows clipboard for the Cut and Copy functions. Values will be pasted from the Windows clipboard for the Paste function. If copy area is bigger than paste area, the operation is done only for values that can fit inside paste area. 19

22 Phase duration Syringe Volumes Time lock Synchro pulse Figure 20 Program Grid!CAUTION!: Blank and non-numeric values entered in the program grid are considered as zero values. A phase duration of 0 ms will cause the phase to be skipped in the execution of the drive sequence. The contents of the syringes can be entered in the Syringe Contents frame of the «Quenched Flow Program» window. The text is entered from the keyboard and the BACKSPACE and DEL keys can be used for corrections.!important!: It is strongly recommend that users take advantage of this feature of the Biokine software to keep track of the samples loaded into the QFM syringes. Each time a program grid cell value is changed, information about the current syringe, current phase and driving sequence is updated and displayed below and to the right of the grid (Figure 21). This information indicates: 1. Current phase number and total number of phases in the driving sequence. 2. Volume delivered by the current syringe during the current phase or current phase total volume (if an entire phase is selected). 3. Flow rate of the current syringe during the current phase or current phase total flow rate (if an entire phase is selected). 4. Total volume delivered by each syringe during the driving sequence Figure 21: Driving Sequence Information Once the sequence ready, click on the or button to activate the shot control window in the stopped-flow status area. 20

23 Standard operations can also be made from the same window: Create a new sequence using the button Load a sequence using the button Save a sequence using the button Close a sequence using the button 5.5. Setting the reaction time The ageing time is set in the driving sequence window as shown in Figure 22. Flow time of each Locked phase will be calculated automatically by the software according to the volumes to be delivered in each of these phases and according to the delay line installed. Figure 22 : ageing time setting 5.6. Running a shot Once a driving sequence has been entered or loaded, it is transferred to the MPS by pushing the Single or Multiple button (in classic mode). The MPS is now in automatic mode and the shot control window will be displayed in the stopped-flow status area (as shown in Figure 23). The Shot control window shows the number of shots possible based the current volumes in the QFM syringes. It also indicates whether the SFM is running a driving sequence or ready for the next shot. The driving sequence is executed by pushing the button or the start-stop button on the front panel of the MPS. The button can be used to stop an experiment prematurely if necessary. If the Single button was used to transfer the driving sequence to the MPS, only a single shot can be made. The button must then be pushed to return to the driving sequence and the Single button must be pushed again to re-transfer the driving sequence to the MPS for a subsequent shot. If the Multiple button was used to transfer the driving sequence to the MPS, the used to execute shots until the shot window shows that 0 shots remain. The pushed to return to the driving sequence. button can be button is then Figure 23: shot control window 21

24 6. TEST EXPERIMENT 6.1. Practical recommendations It is recommended to become familiar with the QFM-4000 instrument before attempting any use with expensive sample. We recommend an extensive exercise with test reactions such as described here and to use the advice given in this section. It is a good practice to use this test reaction before attempting any new configuration or reaction protocol Filling the instrument In this part the user can find few advices to fill the instrument and to avoid cross contamination Turn all valves in position R and do few up and down moves to get rid of the bubbles. Once driving syringe 1 filled turn valve 1 in C position and make sure the other valves are still in R position. It is the way to avoid cross contamination during the filling of the line. Reduce the speed of the driving syringe in the MPS software (speed n 3 is slow enough) to fill the storage lines. If the flow from syringe 1 is too fast a part of storage line 2 could be contaminated. Once all the storage lines filled, flush the exit line with air, the instrument is now ready for a shot Shot procedure The driving sequence given in section 6.3 is a standard shot procedure. A first phase is used to rinse the delay line, a second phase is a waiting phase used to flush the exit line, and the third phase is corresponding to the quenched-flow experiment. When working with such a sequence it is advised to leave valves 1 and 2 in R position during the washing phase to avoid water to enter in storage line 1 and 2 because of elasticity phenomenon. So during phase 2 it is necessary to turn valves 1 and 2 in position C and to flush the exit line with air through M2. Phase 3 is used to collect the quenched solution, once the motors stopped, flush the exit line with air to collect the whole solution Description of the test reaction Alkaline Hydrolysis of 2,4-Dinitrophenyl Acetate (DNPA) A complete description of the alkaline hydrolysis of 2,4-dinitrophenyl acetate (DNPA) can be found in: Gutfreund, H. (1969), Methods in Enzymology, 16, DNPA can be hydrolyzed by OH - to 2,4-dinitrophenol (DNP). At 20 C the reaction has a second order rate constant in water of 56 M -1 s -1. Conditions can easily be set to make the concentration of OH - sufficiently larger than that of DNPA so that the reaction occurs under pseudo first-order conditions with an apparent rate constant, kapp, of 56 s -1 [OH - ] (NOTE: The [OH - ] is the concentration of OH - after mixing with DNPA). The reaction can be quenched at any time by addition of excess acid and the amount of DNP produced determined by absorbance at 325 nm. Figure 24 shows the absorbance spectrum of DNPA and DNP under various conditions of ph. It can be seen that the absorbance spectrum of DNP changes with ph, but there is a clear isobestic point at 325 nm. These properties make the alkaline hydrolysis of DNPA a useful tool for the testing of a quenched-flow instrument. The reaction can also be followed by the stopped-flow technique, omitting the acid quench. 22

25 Absorbance Figure 24- DNPA/DNP Absorbance Spectra 1.5 DNPA + HCl DNP ph 7.0 DNP + HCl 2mM DNP + HCl 100mM Wavelength (nm) Note : it is recommended to dissolve first DNPA in DMSO then dilute 1/100 in water A test experiment in standard mode (reactants in drive syringes) Delay line used : 3.5 µl Solution in S1 : 1 mm DNPA N HCl Solution in S2 : 1 N NaOH Solution in S3 : H20 Solution in S4 : 2 N HCl The sequence used will be as follows: Time t V V V V Lock Off Off On This corresponds to a 1/1/1 mixing ratio between the two reactants and the quencher. In the first phase 100 ul of water are pushed into the delay line to wash the line and get rid of the old solution from the previous shot. In phase two, a 10 second pause is chosen to have time to flush the exit line and to prepare the collection device. In the third phase, 50 µl of each reactants will mix and will begin to flow out of the exit line. The t values are calculated by the software for each of the desired reaction times. The reaction time range used for this experiment was 4ms to 1000 ms After each shot the exit line is flushed with air so that all the mixture is collected. 150 µl are collected at each shot in Eppendorf tubes. Absorbance is measured at 325 nm in a microcuvette without further dilution. For comparison solutions are hand mixed separately in a tube with the same volume ratio. In one case DNPA is added first and allowed to react for a few seconds in the tube before addition of HCl; this give the final reaction absorbance (t > 2 s). In a second case DNPA is first mixed with HCl before addition of NaOH; this gives the initial (t = 0) absorbance value. 23

26 Figure 25 shows the result of this experiment. The data are fitted with an exponential of reaction rate of 56 M -1 s -1 *[OH - ] Reaction progress (%) time (ms) Figure 25 : DNPA hydrolysis 6.4. A test experiment in volume optimized mode (reactants in storage line) To simulate the use of an expensive solution available in limited amount, a storage line of 200 µl was set in L1. This was used to store 200 µl of the same DNPA solution as that used in the fast reaction test above. Condition were : Delay line : 7.5 µl Solution in S1 : H2O Solution in L1 : 1 mm DNPA N HCl Solution in S2 : 1 N NaOH Solution in S3 : H20 Solution in S4 : 2 N HCl The sequence used will be as follows : Time t1 V V V V Lock Off Off On This corresponds to a 1/1/1 mixing ratio between the two reactants and the quencher In the t1 phase, 15 µl of each reactant will mix and will begin to flow out of the exit line. Using this sequence 12 shots can be done to empty the storage line With such a sequence the reacting mixtures contained in S1 and S2 will be collected at a final dilution of

27 Because this sequence includes lower washing volume, a pre-washing phase was included. 100 µl of water is used to flush the delay line. After this first phase the solution contained in 110 the exit line is evacuated by the air purge 100 and a new tube set at the exit line for 90 collecting the sample during the t1 phase. 80 The intermediate 10 s phase is set here 70 to allow sufficient time for purge and tube exchange. 60 The reaction time range used for this 50 experiment was 4 to 350 ms. FFigure 26 shows the result of this experiment. 11 data points were collected. The data are fitted with an exponential of reaction rate 56 M -1.s - 1 *[OH - ] Reaction progress (100%) time (ms) F Figure 26 : DNPA hydrolysis 25

28 7. INSTRUMENT SERVICE 7.1. Flow line and intermixer volumes DEFAULT FLOW LINES installed in the instrument. Tube diameter Length (mm) and volume (µl) (mm)(inches) S1 S2 S3 S4 Top of syringe to valve (0.51) (0.02) (76) - (15) (76) - (15) (76) - (15) (0.76) (0.03) (76)-(35) TEFZEL R filling port to valve (0.51)(0.02) TEFZEL (70) - (14) (70) - (14) (80) - (16) (80) - (16) L filling port to valve (0.51)(0.02) TEFZEL (50) - (10) (50) - (10) (55) - (11) (55) - (11) Tube diameter (mm)(inches) Length (mm) and volume (µl) Valve 1 to mixer 1 (L1) (0.76) (0.03) TEFZEL (50) - (23) Valve 2 to mixer 1 (L2) (0.76) (0.03) TEFZEL (50) - (23) Valve 3 to mixer 1 (0.76) (0.03) PEEK (130) - (59) Valve 4 to mixer 2 (0.3) (0.016) PEEK (150) - (10) Exit line (EL) (0.76) (0.03) PEEK (100) (45) M1 air purge Not connected M2 air purge (0.76)(0.03)PEEK+ check valve indifferent DELAY LINE VOLUME The standard delay line installed is a 0.3mm diameter line with a 36mm length. It corresponds to a volume of 3.5µl in standard (but could be different according to factory calibration). This volume is used for the ageing time calculation by the software. STORAGE LINES The line between the valves 1 & 2 and mixer 1 can be replaced by longer tubes for reactants storage. The Figure 6 and Figure 6 show the instrument with the default line installed or with a storage line L2 of 100 µl. FITTINGS AND NUTS The QFM-4000 flow lines are constructed with Low Pressure connection parts from UPCHURCH Co ( We recommend all modifications and replacements be made with components from this company. All connections are on ¼ -28 standard, all tubes are 1/16 external diameters References of parts used in QFM-4000 are indicated below : Part description Upchurch reference X-mixer P-722 T mixer P-632 Short nuts (natural) P-235X Fitting ferrules for P-235X (blue) P-200X Fitting ferrules for valves (transparent) P-240X Female luer adapters P-624 Injection port adapters P-295 Check valves CV-3301 Plug P PEEK tube (green) PEEK tube (blue) PEEK tube (natural) Tefzel tube

29 0.03 Tefzel tube QFM-4000 cleaning and storage Each day s experiments the QFM-4000 should be cleaned. A thorough cleaning will ensure that it has a long functional life and diminish any chance of sample contamination for the next user of the instrument. The procedure below is the recommended daily cleaning procedure to be done before shutting off the instrument. Step 1. Step 2. Step 3. Step 4. Remove any remaining samples or buffer from the syringes. Wash the syringes and flow lines 2 3 times with water. This is done by filling each syringe with water to a volume at least equal to the sample volume used for experiments. With the syringe valve handles set to vertical, empty the syringes completely. Since the liquid will exit via the exit valve, it will wash the flow lines and exit valve as well as the syringes. Wash the syringes and flow lines one time with % ethanol. Set the valve to horizontal position and wash with water and ethanol all flow lines from the C port. When finish flush air in the lines. Step 5. Dry the syringes, flow lines and cuvette with a single wash of air. Use the same procedure as in step 2. The syringes should be emptied in reverse numerical order so that all liquid is pushed out of the syringes, flow lines and exit valve. Step 6. Set all syringe valve handles to horizontal position and move all syringes to their lowermost positions. The syringe plungers should exit the QFM-4000 so that the plunger tips are completely visible. If this is done using the MPS software it will be necessary to uncheck the Up and Low Limits checkbox in the software «Syringes Command:Load» window Note: You may observe a few drops of liquid that fall from the syringes when the syringe plungers are completely out of the QFM instrument. This is normal as a small amount of liquid is always trapped between the plunger tip and the syringe barrel to make a tight seal.!important!: Do not forget this step! The syringe plunger tips are made of Teflon. Pulling the syringe plungers out of the QFM each night allows the tips to expand and make a tight seal during use, minimizing any chance of leaks. Step 7. Step 8. Turn all syringe valve handles to vertical position. Turn off the MPS Long-term storage of the QFM-4000 If the QFM will be not used for a long period of time (more than several weeks), it should be cleaned as above. In addition, if the QFM is connected to a circulation temperature bath, the temperature bath should be disconnected from the QFM and the QFM drained completely of all cooling liquid. Afterwards, it is recommended that the QFM cooling circuits be flushed with ethanol followed with air. The QFM is now ready to be stored Pulsed-flow parameters These parameters are set as default. Here is shown as to access these parameters for verification only. Do not attempt to change any of theses values as this will result in incorrect functioning of the instrument. Enter the "Limits" menu, then type "CTRL L". A window will open that shows the set of default parameters. Default parameters for the pulsed-flow are as shown below : Flow limit : 250 Pulse duration : 1000 Volume :