Application of UV-Visible Spectrophotometers in Absorbance Measurement

1. Principle of Absorbance Measurement

When the frequency of the incident light matches the vibrational frequency of the molecules in a substance, or when the incident light causes electronic transitions in the molecules, optical absorption occurs. The higher the concentration of the solution, the more molecules are available to absorb the light passing through the solution. Consequently, the less light is absorbed, the more light passes through the solution, and vice versa.

In 1852, Beer established the relationship between absorbance, solution concentration, and path length, formulating the fundamental law of light absorption known as the Lambert-Beer Law.

The Lambert-Beer Law is the fundamental principle of absorbance measurement, describing the relationship between the strength of light absorption at a specific wavelength and the concentration of the absorbing substance and its path length. When a parallel beam of monochromatic light passes through a homogeneous, non-scattering, colored solution with a path length of

$bb$

b and a concentration

$cc$

c of the absorbing substance, the absorbance of the solution is directly proportional to both the concentration of the solution and the path length.

A=kcb=lg(I₀/I)

A: Absorbance

k: Molar absorptivity (commonly in units of L/(mol*mm))

c: Concentration (commonly in units of mol/L)

b: Path length (commonly in units of mm)

I₀: Incident light intensity

I: Transmitted light intensity

Figure 1: Absorbance Principle Diagram

2. Introduction to the Application System

(1) Light Source:
The light source should be capable of outputting stable power and a continuous spectrum. For the ultraviolet range, laboratories commonly use pulsed xenon lamps or deuterium lamps. For the visible range, tungsten-halogen lamps are frequently used.

(2) Sample Cell:
The sample cell is used to hold the sample to be tested. The commonly used container for directly holding the sample is a quartz cuvette, typically with a thickness of 10mm, suitable for the ultraviolet to visible light spectrum range.

(3) Detection Equipment:
Also known as a spectrophotometer, this integrates optical dispersion devices and detectors that can perform photoelectric conversion. The SR50C fiber optic spectrometer from JINSP is used in this measurement application. The spectrometer has a built-in synchronous trigger function for the pulsed xenon lamp. In addition to being used with a cuvette holder for testing as shown in the figure below, it can also be paired with an immersion fiber optic probe or a flow cell for sampling according to actual needs.

(4) Display:
The display connects the spectrometer to a laptop, showing the data during the measurement process. The measurement application uses proprietary upper computer software developed by JINSP.

 

 

Figure 2: Absorbance Detection System Using Pulsed Xenon Lamp

3. Experimental Example

JINSP has developed a complete system for spectroscopic absorbance measurement and related accessories. In this experiment, a KNO₃ solution was used, and the test was conducted at room temperature using the SR50C miniature fiber optic spectrometer from JINSP. The experimental results are shown in the table below:

Spectrometer Model: SR50C (200-400 nm)
Wavelength Range (nm)	Resolution (nm)	Customizable Options Based on Customer Needs: Wavelength Range, Resolution, Spectrometer Size
200-400	0.5
Path Length of Cuvette	KNO3 Concentration (mg/L)	Absorbance at 220 nm	Absorbance at 275 nm	Correlation Coefficient (R2)
10mm	0.2	0.043278	0.044611	0.9978
	0.3	0.067225	0.065858
	0.4	0.087306	0.08754
	0.5	0.115057	0.108142
	0.8	0.166477	0.161765
	1.0	0.207256	0.20099

 Table 1: Absorbance of KNO₃ Solution at 220 nm and 275 nm

Figure 3: Linear Relationship Between KNO₃ Solution Concentration and Absorbance

Conclusion:

From the figure, it is evident that the absorbance of the potassium nitrate solution has a strong linear correlation with its concentration. The linear fit coefficient

$R2=0.9978R^2\; =\; 0.9978$

R²=0.9978, and the equation of the standard curve is:

$A=1985.74C+0.0048A\; =\; 1985.74C\; +\; 0.0048$

A=1985.74C+0.0048 Using the fitted standard curve, the absorbance of an unknown concentration sample can be substituted into the equation to determine its concentration. Therefore, the JINSP UV-Visible spectrophotometer can provide good measurement results in absorbance measurements to meet customer needs.