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ajitreddy's picture

For the calibration of spectyrphotometer why blank should require what is the reason behind it.

Sami Tuomivaara
Sami Tuomivaara's picture


Let's say you want to measure the amount of substance X in buffer Y. If you know at what wavelength substance X absorbs strongly, you use that wavelength for quantification. However, buffer Y in the solution might absorb at the same wavelength as well.  If you used your measurement directly for quantification, you'd overestimate the amount of substance X because buffer Y would add to the measurement.

Now, you want to measure absorbance of the buffer Y without substance X (at the same wavelength), this measurement is called background or blank absorbance. You get the absorbance of substance X by subtracting the blank measurement from the original measurement.

For convenience, modern spectrohotometers let you measure the absorbance of the blank first, setting absorbance to zero, and then measure the absorbance of substance X in buffer Y. The reading you get from the spectrophotometer the same you'd get by measuring the absorbances of both solutions and doing the subtraction as mentioned above.