STOPPED FLOW METHOD & APPLICATION MURUGAVENI B.pptx

Stopped flow method

1. STOP FLOW METHOD PRESENDED BY; B.MURUGAVENI I MSC BIOCHEMISTRY ROLL NO: 23PSBCH11 REG NO: 23701511

2. CONTENT: INTRODUCTION HISTORY OF STOP FLOW METHOD STOP FLOW METHOD SUMMARY REACTANT SYRINGE MIXING CHAMBER DEAD TIME OBSERVATION CELL STOPPING SINGLE MIXING MULTI MIXING STOP FLOW APPLICATION OF STOP FLOW METHOD

3. INTRODUCTION: Stopped-flow is an experimental technique for studying chemical reactions with a half time of the order of 1 ms, introduced by Britton Chance and extended by Quentin Gibson. History of stopped flow method: The stopped-flow technique was invented in the 1950s by professor Britton Chance as a result of a need for instrumentation to follow enzymatic reactions in the tens of millisecond time range. In 1983 Yves Dupont from the French research agency, the CNRS (Centre National de la Recherche Scientifique) developed his own stopped-flow for his laboratory for fast fluorescence and radioactive labeling measurements.

4. STOP FLOW METHOD SUMMARY: Stopped-flow spectrometry allows chemical kinetics of fast reactions (with half times of the order of milliseconds) to be studied in solution. It was first used primarily to study enzyme-catalyzed reactions. Then the stopped-flow rapidly found its place in almost all biochemistry, biophysics, and chemistry laboratories with a need to follow chemical reactions in the millisecond time scale. In its simplest form, a stopped-flow mixes two solutions . Small volumes of solutions are rapidly and continuously driven into a high-efficiency mixer. This mixing process then initiates an extremely fast reaction.

5. The newly mixed solution travels to the observation cell and pushes out the contents of the cell (the solution remaining from the previous experiment or from necessary washing steps). The time required for this solution to pass from the mixing point to the observation point is known as dead time. The minimum injection volume will depend on the volume of the mixing cell. Once enough solution has been injected to completely remove the previous solution, the instrument reaches a stationary state and the flow can be stopped.

6. Depending on the syringe drive technology, the flow stop is achieved by using a stop valve called the hard-stop or by using a stop syringe. The stopped-flow also sends a ‘start signal’ to the detector called the trigger so the reaction can be observed. The timing of the trigger is usually software controlled so the user can trigger at the same time the flow stops or a few milliseconds before the stop to check the stationary state has been reached.

7. REACTANT SYRINGES: Two syringes are filled with solutions that do not undergo a chemical reaction until mixed together. These have pistons that are driven by a single drive piston or by independent stepping motors, so that they are coupled together and their contents are forced out simultaneously into a mixing device.

8. MIXING CHAMBER: Once the two solutions are forced out of their syringes they enter a mixing system that has to ensure complete mixing, with turbulent flow rather baffles than laminar flow, which would allow the two solutions to flow side by side with incomplete mixing.

9. DEAD TIME: The dead time is the time for the solutions to go from the mixing point to the observation point, it is the part of the kinetics which cannot be observed. So the lower the dead time, the more information the user can get. In older instruments this could be of the order of 1 ms, but improvements now allow a dead time of about 0.3 ms.

10. OBSERVATION CELL: The mixed reactants pass an observation cell that allows the reaction to be followed spectrophotometrically, typically by ultraviolet spectroscopy, fluorescence spectroscopy, circular dichroism or light scattering, and it is now common to combine several of these. Observation cuvette with a short light path (0.75 to 1.5mm) are usually preferred for fluorescence measurements to reduce self-absorption effects. Observation cuvette with longer light path (0.5 cm to 1 cm) are preferred for absorbance measurements. Modern stopped-flow can accommodate different models of cells and it is possible to change the cuvette between two experiments. For stopped-flow X-ray measurements, a quartz capillary with thin wall is used to minimize quartz absorption. Simultaneous x-ray and absorbance measurements are possible in the same capillary.

11. STOPPING: Once through the observation cell the mixture enters a third syringe that contains a piston that is driven by the flow to activate a switch to stop the flow and activate the observation.

12. SINGLE MIXING: In its simplest form, a stopped-flow mixes two solutions. Small volumes of solutions are rapidly and continuously driven into a high-efficiency Ball mixer, so mixing is completed in just a few microseconds. This mixing process then initiates an extremely fast reaction. The newly mixed solution travels to the observation cell and pushes out the contents of the cell (the solution remaining from the previous experiment or from necessary washing steps). The time required for this solution to pass from the mixing point to the observation point is known as dead time. The minimum injection volume will depend on the volume of the mixing cell.

13. MULTI MIXING STOPPED FLOW: A three syringe-stopped flow has 2 mixers that work consecutively. Syringes 1 and 2 mix first, then the mixture mixes with syringe 3. Two types of experiments can be performed with a multi-mixing stopped-flow. DOUBLE MIXING EXPERINMENT FOUR SYRINGE STOPPED FLOW

14. DOUBLE MIXING EXPERINMENT: A double mixing experiment (also called sequential mixing) is an application that generate one reactant by mixing two solutions because this reactant is not stable and could therefore not be loaded in a syringe for a single mixing application. So, in this scenario, a first reaction is generated in mixer 1. This mixture is allowed to age, from 2ms to several seconds, in a delay line before being mixed with a third sample. The second reaction is then followed optically. The delay line is also called the aging line, aging loop, or retardation loop but all of these terms refer to the same component. Such experiments are performed to generate an unstable reactant that could not be directly loaded into a two-syringe instrument.

15. The aging time is the time it takes for the solution to pass from mixer 1 to mixer 2. For short aging times (<200ms) the stopped-flow works in continuous flow mode, so the aging time is defined as the ratio of the volume of the delay line divided by the flow rate of solutions through the line. The user can change the delay line itself or can change the flow rate in the software. For longer aging times, the stopped-flow uses an interrupted flow method (push-pause-push) where the flow is stopped between the two mixing events.

16. FOUR SYRINGE STOPPED FLOW: A four-syringe stopped-flow has three consecutive mixers and four independent motors so it can carry out triple mixing experiments, for example by combining the double mixing and the concentration dependence.

17. APPLICATION OF STOP FLOW METHOD: Stopped-Flows are used across a wide range of academic and industrial applications including drug binding processes and protein stability. Stopped-Flows are key instruments used to increase researchers' understanding of rapid kinetics and are especially useful for chemists and biochemists. Stopped flow method allows the evaluation of the degradation kinetics in milliseconds and avoids the use of additional analytical methodologies; this method allows determining the reaction times on line.

18. REFERENCE: https://www.biologic.net/topics/what-are-stopped-flows-and-how-do-they-help-chemists-and-biochemists-understand-high-speed-chemical-reactions/ https://en.wikipedia.org/wiki/Stopped-flow