Continuous Flow is a type of assay used to easily measure the progress of a reaction at discrete time points and is commonly used for determining initial rates and inhibition values.
In a Continuous Flow experiment, the composition of the reaction is measured continuously, normally by absorbance, while the reactants flow and mix continuously. This is in contrast to Stopped Flow, where the reactants are mixed in a chamber and then measured, or Batch Reactions, where a number of reactions are performed and stopped at various times by the use of a compound which will halt the reaction.
The apparatus used for continuous flow assays will have reactants initially separated in chambers. They are pumped through a mixing chamber and then out a long tube. At various points along this tube, measurements can be taken - generally by spectrophotometers at various points along. Since the flow rate of the reactants is constant, each point on the tube corresponds to a discrete point in time. Reactants which have absorbance values which change during the course of the reaction are therefore necessary to perform a traditional continuous flow assay.
Inhibitors can be added and ratios of reactants can be changed at will to gather data. By taking readings at different points along the output tube, discrete time points can be measured with a high degree of accuracy.
Advantages and Disadvantages
The continuous flow assay offers a high degree of accuracy by allowing what amounts to a large number of readings at each time point, since the reactant mixture at one point is measured multiple times as it passes the sample point. It is very useful for measuring inhibition because reliable data can be obtained and the inhibitor then added upstream while continuously measuring.
Continuous flow assays require a large amount of reagent, since it needs to flow continuously. If the experiment uses expensive or difficult to produce reactants, it is therefore not the best technique.
- If your apparatus has a 3 meter long tube for measurement with an internal diameter of 1 centimeter and fluid flows through it 100ml/1min, assuming there is no lag time in the mixing chamber, what time point of the reaction will a measurement taken 1m along the tube represent?
- If the conditions are the same as the above scenario, assuming that you use two reagents and the reaction volume is 1 part of A:2 parts B, how much of reactant A will you need to run the assay for 30 minutes continuously flowing?
- Describe the technique (including equations) that you would use to turn a transmittance value from a point on the reaction tube into meaningful data about the progress of the reaction.
- If your intial conditions are 5m substrate with no product present and you are running an enzyme whose speed is limited only by diffusion, where would you expect to find 50% reaction completion using the apparatus described in problem 1?
- If the overall flow of reactants from the storage tanks to the mixing chamber is 5 ml/ml MAX for both, the flow through the tube is 1ml/min MAX, the volume of the mixing chamber is 15 ml, and the tube has an internal diameter of 0.25 cm and a length of 10 meters, using two reactants, how long from starting the reaction will it take for the first products to reach the end of the tube?
- Atkins, Peter and de Paula, Julio. Physical Chemistry for the Life Sciences. New York, N.Y.: W. H. Freeman Company, 2006. (241).
- Segel, Irwin. Enzyme Kinetics. Wiley-Interscience Publishing, 1993 (80-89)
- Kasidas, GP, and GA Rose. "Continuous-flow Assay for Urinary Oxalate Using Immobilised Oxalate Oxidase."Annals of Clinical Biochemistry. 22 (1985): 412-9. Print.
- Yagminas, AP, and DC Villeneuve. "An Automated Continuous-Flow Assay for Serum Sorbitol Dehydrogenase Activity and Its Use in Experimental Liver Damage." Biochemical Medicine. 18.1 (1977): 117-25. Print.
- Tammy Nguyen, Matthew Austin