How to measure absorbance quantitatively?

Absorbance spectrometer can be used to qualitatively identify "fingerprint" substances or quantitatively measure the concentration of colored substances (hair color subgroups) in transparent solutions. In some cases, it is necessary to know the absolute absorbance or extinction coefficient of the unknown substance. In other cases, the concentration of the unknown relative to the known concentration of the standard solution can be determined by the formula. In both cases, the derivation of absorbance is known as Beer-Lambert's law.

A spectrometer measures absorbance by passing a wavelength parallel light through a flat, parallel substance and measuring the beam that passes through it. For liquid samples, we have the option of placing it in a colormetric dish. Because part of the energy is absorbed by the molecules in the sample, the incident light (l0) of the sample is stronger than the light passing through (l) of the sample.

Transmittance (T) (experessed as a percentage) is a ratio relation: T=l/l0.

The transmittance of the opaque sample was 0%, while that of the transparent sample was 100%. The transmittance will be less than 100% if there are molecules with light absorption capacity in the optical path. The number of molecules interacting with light depends on the optical path (l) and the molecular concentration (C). The attenuation effect also depends on the ability of the molecule to absorb light of that wavelength, expressed as the extinction coefficient or molar absorption coefficient.

T=l/l0=e-ε^(lc)

Absorbance (A) is the logarithm base 10 of the ratio of the intensity of light incident before passing through A solution or substance to the intensity of light transmitted after passing through the solution or substance. The relationship between concentration, optical path and absorbance is as follow: Aλ=-log(l/l0)=εcl.

The absorbance of a completely transparent saml=ple is 0 (T=100%), and the absorbance of a compelety opaque sample is infinite (T=0%). The value of ε depends in the units of C. The units of C are usually molality (mole/liter), l in centimeters, and ε in liters (mole/centimeter).

Light reflection at the air/sample interface and sample/colorimeter interface as well as light absorption of the solution will weaken the light intensity. These factors can be quantitatively analyzed separately, but the effects of these factors can be eliminated by defining I0 as light passing through blank or control samples.

To eliminate the effect of the absorbance of the blank solution, there are two options: you can measure the absorbance of the blank solution first, then subtract this value from the sample absorbance, or set the instrument to 100%T or 0 absorbance units before starting the measurement.This is very similar to the tare of a scale.

There are many factors that influence the effectiveness of Beer-Lambert's law. The linearity of a color subgroup is usually confirmed by measuring the absorbance of a range of criteria (see luminescent subgroup sampling in Table 1). This calibration also eliminates errors caused by experiments, equipment, and reagent batches (such as colorimetric dishes with unknown optical paths).