spectrophotometry is the science of measuring the light-absorbing and light-transmitting characteristics of a substance. Many substance absorb light and transmitlight of specific wavelengths within the ultraviolet (200-400)ม visible (400-700) and near-infrared (700-1000) regions of the electromagnetic spectrum These light-absorbing/light-transmitting characteristics of a substance are useful in determining the presence and concentration of that substance in a sample
the instument used to measure the amount of light of a specific wavelanght absorbed or transmitted by a substance is called a spectrophotometer in a spectrophotometer a sample of the substance is placed across the path of a light beam of a chosen specific wavelenght the spectrophotometer determines the intensity of the light entering the sample and the intensity of the light leaving the sample then calculates the amount of light transmitted and absorbed by the substance
a diagram of the light path through a visible-light spectrophotometer is shown to the right a beam of light emerges from its source and passes through a prism which dissects the light into a continuous spectrum of wavelengths the user can select which single specific wavelenght of light passes to the sample with the monochromator light of a single wavelenght is called monochomatic light the monochomatic light that passes to the sample is the know as incident light its intensity is represented the value. as the incident light passes through the sample a certain amount of the light will be absorbed by the sample the monochromatic light that is not absorbed emerges from the sample it is called transmitted and its intensity is represented by the value l the intensity of the transmitted might is detected by a photodetector
once the transmitted light is detected the instrument calcultes the fraction of the incident light transmitted by the sample a value known as transmittance from the transmittance value the instrument will calculate the amount of the monochromatic light absorbed by the sample a value known as absorance A, using the formula A=-log10 T both the transmittance and absorbance are displayed on the display screen of the instrument