Why is it important to use standards or develop a standard curve in spectrophotometry?

Answer 1

Hello Affar,

There are several reasons.

1) You need a standard curve to ensure precision and accuracy of your measurement.

2) It is necessary when you are trying to quantify the concentration of an unknown. For example, let's say you are trying to quantify the concentration of chlorophyll in a leaf sample extract. If you take your sample and place it into a spectrophotometer, it will spit out a value; typically the absorbance. This value is mostly meaningless unless you have developed a standard curve from a serial dilution of purified chlorophyll. Each sample in your serial dilution will have an absorbance value associated with its concentration. If you graph your concentration versus absorbance on an x-y plot, you can run a trendline through your points and develop a line equation --> y=mx+b. Once you have your line equation, you can simply plug in your absorbance value in for y in the line equation and solve for x (i.e. concentration).

3) From a practical point of view, having standard helps minimize instrument, reagent and user error.