THE METHODS
The Stopped-Flow
An experiment using a basic stopped-flow apparatus is quite simple in principle. This apparatus uses the drive motor to rapidly fire two solutions, contained in separate drive syringes, together into a mixing device. The solutions then flow into the observation cell displacing the previous contents with freshly mixed reactants. A stop syringe is used to limit the volume of solution expended with each experiment and also serves to abruptly stop the flow. The flow of solution into the stop syringe causes the plunger to move back and trigger data collection. The fresh reactants in the observation cell are illuminated by a light source and the change, as a function of time, in many optical properties (Absorbance, Fluorescence, Light Scattering, Turbidity, Fluorescence Anisotropy etc.) can be measured. The measurement of these optical properties is performed by the system's detectors, which can be mounted either perpendicular or parallel to the path of incoming light depending on in which optical property you are interested. The SF-2004 series of stopped-flows allow the mounting of three detectors (two perpendicular to and one in line with) the incident light. Data from all three detectors can be collected simultaneously.
A double mixing stopped-flow system is capable of mixing three reactants in sequential fashion to perform Double Mixing Experiments. In this more advanced system there are three drive syringes. The first two drive syringes drive two solutions together through a mixer and into a delay line. The length of the delay line along with how long it takes the solutions to pass through the delay line determines the length of the first reaction. After passing through the delay line the first two solutions (now mixed) enter a second mixer where they are mixed with a third reactant and then the mixed solutions pass into the flow cell. The reactants displace the previous contents of the observation cell and trigger data collection as described for the basic stopped-flow system. The members of the SF-2004 family of Stopped-Flows are all capable of performing both single and double mixing experiments in their base configuration.
Regardless of which configuration the stopped-flow uses, the time resolution of this method is limited by the the time required for the reactants to flow from the final point of mixing to the observation cell. This time is referred to as the dead time of the instrument. The SF-2004 series of Stopped-Flows have guaranteed dead times of less than 2 milliseconds. For all stopped-flows, the volume of the mixer, observation cell and flow lines all affect the volume of reactants that must be pushed to obtain a satisfactory reaction. The SF-2004 series of stopped-flows utilizes micro volume flow cells, lines and mixers. These micro volume devices allow the SF-2004 series of stopped-flows to push as little as 30 microliters of reactant per shot! If you wish to perform single mixing stopped-flow experiments using your own spectrofluorimeter or spectrophotometer, KinTek Corporation provides the SF-MiniMixer Stopped-Flow.
The Chemical Quench-Flow
Stopped-flow techniques are limited by the fact that one does not always have an optical signal for the reaction of interest and the optical signals cannot be interpreted rigorously if the extinction coefficients of intermediates or products are not known. In these cases direct measurement of the conversion of substrate to product is required. Chemical quench-flow methods allow such a measurement.
A basic chemical quench-flow allows the mixing of two reactants, followed (after a specified time interval) by quenching with a chemical agent (usually acid or base). A drive motor is used to force reactants contained in drive syringes together into a mixer after which the mixed reactants pass into a reaction delay line. After passing through this delay line the reaction passes through another mixer where chemical quench is added to terminate the reaction. The quenched sample is then collected and analyzed to quantitate the conversion of product to substrate (usually the substrate is radiolabeled and product formation is determined by either gel electrophoresis or standard chromatography methods). The duration of the reaction is determined by the volume of the reaction delay line and the flow rate of the reaction through the delay line. In practice, the reaction time is varied by changing the length of tubing in the reaction loop and to a certain extent the flow rate through the delay line. The apparatus is then flushed and a new reaction delay line is selected to obtain a different reaction time. By selecting various delay lines reaction time of 2 to 100 milliseconds can be obtained. This simple design limits the maximum attainable reaction time by the maximum delay line size. Longer reaction times cannot be obtained by using slower flow rates because it is necessary to use rapid rates of flow to maintain turbulent flow necessary for efficient mixing. A 40 cm reaction loop (which contains 200 microliters) can provide a 100 millisecond reaction time (very long delay lines are unwieldy). This design also wastes precious reactants, by using the reactants to push themselves though the delay line. This means that after each time point the delay line is full of reactants. Therefore after the delay line is changed, it contains unused reactants (as much as 200 microliters at a time), which must be discarded.
To minimize sample volumes without any wasted reactants between different reaction times and to easily achieve reaction times longer than 100 milliseconds, KinTek Corporation designed the RQF-3 Rapid Chemical Quench-Flow. In our RQF-3 Rapid Chemical Quench-Flow the two reactants are loaded into small loops of tubing containing 15 microliters solution. A three-way valve is then used to put the loaded sample loop in line with the drive syringe containing the buffer. The drive syringes are then used to force the reactants together through the delay line to the point of mixing with the quenching solution, followed by sample collection. The delay line size in the RQF-3 is controlled by our innovative eight-way valve, which allows the user to put the correct loop in position by the simple twist of a handle. Using the delay lines provided on the eight-way valve reaction times from 2 milliseconds to approximately 100 milliseconds. For longer reaction times (from 100 milliseconds to infinity) the RQF-3 utilizes the computer-controlled stepped motor to operate in push-pause-push mode. The first push is used to mix the reactants in the delay line. There the motor pauses, allowing the reaction to continue, and then the pause is followed by a second push which mixes the reactants with the quench solution. In this fashion, the RQF-3 makes it simple to perform quench-flow experiments for any time course the user desires.
With our computer-controlled servo motor design the RQF-3 can perform sophisticated double mixing experiments. In this type of mixing experiment the first mixing occurs as described above, however when the mixed reactants exit the delay line into the exit line they are mixed with a third reactant rather than a quench solution. The computer then pauses the stepping motor, which holds the reactants in the exit line for a time entered by the user. The computer then fires the motor again and the reactants are pushed into a collection tube containing a chemical quench. This methodology has been used many times to perform experiments that require double mixing, such as pulse-chase experiments.
Another of our instruments (described below), the RPL-3 Rapid Optical Photolysis Chamber is based on the KinTek Corporation world leading Quench-Flow and mounts to the same drive system. This allows the customer to have both a RQF-3 Rapid Quench Flow and a RPL-3 Rapid Photolysis Chamber at a very affordable price.
The Optical Photolysis Method
The technique of optical photolysis is fairly simple and is summarized in this simple diagram. This instrument has similar properties to both the stopped-flow and the quench-flow. In our RPL-3 Rapid Optical Photolysis Chamber the reactants are forced by a drive motor into an optical flow cell where they irradiated from a light source. This source can be almost anything. We have built photolysis chambers utilizing many light sources, including flash lamps, lasers and even synchrotron radiation! The timing of the reactions is achieved by using the computer controlled servo motor in push-pause-push mode as described for the Quench-Flow. The computer first pushes the drive syringes, which mixes the reactants and fills the optical flow cell with mixed solution. Once the optical flow cell is filled the motor pauses for a predetermined amount of time and then triggers the light source, which then irradiates the flow cell. After the flow cell has been irradiated the motor pushes again and either clears the reactants completely from the instrument for collection, or optionally it mixes the irradiated samples with a third solution for further reaction followed by collection. The amount of time the third solution reacts with the irradiated solution is controlled in the same fashion as the second mixing of a double mixing experiment in the RQF-3 above. Our RPL-3 Rapid Optical Photolysis Chamber is based on our world leading Quench-Flow and mounts to the same drive system. This allows the customer to have both a RQF-3 Rapid Quench-Flow and a RPL-3 Rapid Photolysis Chamber at a very affordable price.
