How to Calculate Calibration Plot
- 1). Use your micropipette to add 100 microliters of the substance of interest (for example, sodium hydroxide) to each well on one row of your assay plate. Each row will, in general, have 12 wells within it.
- 2). Leave the first well untouched. Use your micropipette to add 100 microliters to the second well. Pump your micropipette lever up and down to mix the water with your solution, then remove 100 microliters from the second well and mix it with the solution in the third well. Continue down the line of solutions. This results in the concentration of each well in the row being half of the concentration of the well before it.
- 3). Follow the instructions of your assay kit and add the appropriate amounts of reactants to tint the solutions within your assay plate.
- 4). Place your assay plate in the spectrophotometer. Allow the machine to read the levels of tint within the different wells. For each concentration, your machine will provide a numerical tint reading.
- 5). Graph, on a set of x and y axes, the concentration versus the tint reading provided by your machine. This is a calibration curve. In future, when you are reading unknown quantities, you will be able to refer to it and translate the tint reading into the concentration of the solution.
Source...