UV visible absorption spectrum

Principle of ultraviolet visible absorption spectrum

Both UV and visible absorption spectra belong to molecular spectra, which are produced by the transition of valence electrons. UV visible absorption spectrum analysis technology is a technology that analyzes, determines and infers the composition, content and structure of absorbing substances by using the UV visible spectrum generated by the absorption of UV and visible light by molecules or ions of substances.

Present in organic or molecular compounds σ Electrons, π electrons and lone pair n electrons, when molecules absorb radiant energy, these electrons will transition to higher energy levels. The transition forms include σ → σ*、 n→ σ*、 π → π * and N → π *, the energy required for various transition types decreases in the following order: σ → σ*> n→ σ*> π → π * >n → π *, this transition is closely related to the internal structure of the molecule, which is the theoretical basis for the use of UV visible absorption spectroscopy in the analysis of organic or molecular compounds.

In inorganic compounds, UV-Vis absorption spectra are mainly produced by charge transfer transition and coordination field transition.

Charge transfer transition refers to the transition form in which the atom in the molecule transfers the charge originally set on the metal M orbital to the ligand l orbital under radiation, or transfers in the opposite direction. The absorption spectrum produced by this transition is also called charge shift spectrum.

Coordination transition includes D-D coordination field transition, f-f coordination field transition, and π → π * transition in the ligand body under the influence of metal ions. D-D transition refers to the transition of transition metal ions after the d orbital absorbs radiation under the action of ligands; F-f transition is a transition produced by the splitting of F orbitals of lanthanides and actinides to absorb radiation; π → π * transition in coordination body refers to the coordination transition caused by the perturbation of metal ions. This transition is related to the bonding property. If it is combined by covalent bond and coordination bond, this change will be very obvious.

ultraviolet visible absorption spectrometer

The UV-Vis absorption spectrometer consists of the following four components:

(1) Radiation source: it must have a stable continuous spectrum with sufficient output power and can provide the wave band used by the instrument. Deuterium tungsten lamp is commonly used as the radiation source in the laboratory.

(2) Sample cell: also known as absorption cell, it is used to hold the test solution for absorbance measurement. There are two common sample cells: quartz cell and glass cell. The former is applicable to the ultraviolet to visible light region, and the latter is only applicable to the visible light region. This is because the glass has strong absorption in the ultraviolet region, which will cause great interference to the detection results of ultraviolet absorption. The measuring optical path of the sample cell is generally 0.5 ~ 10 cm.

(3) Detector: also known as photoelectric converter, commonly used are photocell, photomultiplier tube and electrical coupling device (CCD). CCD is more sensitive than the former two, especially suitable for detecting weak radiation. The detector used in this optical teaching kit is electrical coupling device.

(4) Display device: the spectrometer is usually connected to the computer to display the spectrum, data and operating conditions.

The ultraviolet visible absorption spectrometer built in this optical teaching kit includes: deuterium tungsten lamp, four-way liquid measuring cell, optical fiber and optical fiber spectrometer.

Application of ultraviolet visible absorption spectrum

UV visible absorption spectrometers are widely used in metallurgy, machinery, chemical industry, medical and health care, clinical testing, biochemistry, environmental protection, food, material science and other fields, especially suitable for quantitative and qualitative analysis of various substances.

In the metallurgical industry, ultraviolet visible absorption spectrometry can be used to analyze the element content of non-ferrous metal materials and ferrous metal materials. The analysis method based on this technology has been included in many national standard methods. For example, in gb/t 6730.9-2016 iron ore determination of silicon content, ammonium ferrous sulfate reduction - silicon molybdenum blue spectrophotometry, ammonium ferrous sulfate is used to reduce silicon molybdenum heteropoly acid to silicon molybdenum blue, and the absorbance is measured at the wavelength of 760nm, In order to determine the content of silicon in iron ore, in addition, there are many national standard detection methods that use ultraviolet visible absorption spectroscopy.

UV visible absorption spectroscopy is also very suitable for the analysis and detection of drugs, because most drugs are organic, so most drugs can produce absorption peaks in the UV region. For example, UV absorption spectrum can be used for qualitative analysis of drugs, and the consistency of drugs can be evaluated by comparing with the spectrum of standard samples.

The application of ultraviolet visible absorption spectroscopy is very extensive, so it will not be discussed here one by one. The characteristics of ultraviolet visible absorption spectrum analysis technology are:

  The sensitivity is high, and the lower limit of concentration that can be measured is generally 10-6g · ml-1, which is suitable for micro and trace analysis;

  Compared with other instrumental analysis methods, its relative error is generally 0.2% ～ 0.5%, which can meet the accuracy requirements of microanalysis;

  The price of the instrument is low;

  Simple operation, fast measurement, etc.

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Experiment 1: plotting and quantitative measurement of ultraviolet spectrum of phenol

Experimental principle

In this experiment, phenol was taken as the experimental object to show the construction method of UV absorption spectrum equipment and the quantitative analysis method of UV visible absorption spectrum.

Because the molecules with cyclic conjugated structure, such as benzene and phenol, are easy to produce π → π * transition, the molecules with this cyclic conjugated structure have strong characteristic absorption peaks in the UV spectrum. For example, benzene will produce three characteristic absorption bands in the UV region, which respectively appear at 180nm, 204nm and 255nm, while phenol has a characteristic absorption peak at 270nm. Since the absorbance of phenol in the UV region is in direct proportion to its content, which conforms to Lambert Beer law, the standard curve of phenol content can be drawn by UV visible absorption spectrometry.

Experimental instruments and reagents

The instruments and reagents required for this experiment are shown in the following table:

Ms11639 optical fiber spectrometer:

Ms11639 is an optical fiber spectrometer with a spectral range of 200nm-1100nm. The detector adopts Hamamatsu COMS detector. 16 bit a/d sampling and 75% quantum efficiency provide the spectrometer with high signal-to-noise ratio and large dynamic range. The spectrometer has good response performance in uv-vis-nis. Compared with common products, double blazed gratings are used to optimize the spectral response in UV and NIR bands, improve the sensitivity and efficiency of the spectrometer by 20%, and effectively reduce stray light by 50%. The ms11639 spectrometer equipped with double blazed gratings can effectively balance the full spectrum response, and can be widely used in physical and chemical analysis, biological samples, semiconductor material detection, optical detection, material detection and other fields.

Compared with traditional CCD detector, CMOS detector has better response in UV band. UV differential absorption spectroscopy is very suitable for the detection of nitric oxide and sulfur dioxide. Ms11639 has good thermal stability at 0-40 ℃, with spectral wavelength shift < 0.1nm, and can be applied to qualitative and quantitative detection scenarios.

Dh2000 deuterium halogen two in one light source:

Dh2000 deuterium halogen two in one light source is a desktop UV visible light source with a spectral range of 200-2500nm, covering the spectral curve from ultraviolet to near infrared. Deuterium halide beam combining output, controlled by separate switch, with optical path thermal switch. The deuterium lamp service time timer records the service time, and the halogen light source intensity is linearly adjustable. The service life of deuterium lamp is 2000 hours, and that of halogen lamp is 6000 hours. The light source output adopts SMA905 standard optical fiber interface.

Four way liquid spectrum measuring cell:

Cuvette spectrum measurement bracket, including four collimator cross-links, with limit card slot and filter slot, including shading cover.

Experimental process

1. preparation of phenol solution:

Prepare phenol solutions with concentrations of 5, 10, 16, 25, 35, 40, 45 and 50 (mg/l), of which 25 mg/l solution is used as the sample to be tested with unknown concentration.

2. construction of ultraviolet visible absorption spectrometer:

A UV resistant quartz fiber is used to connect the deuterium halide two in one light source and the four-way liquid measuring cell, and another fiber is used to connect the four-way liquid spectrum measuring cell and the optical fiber spectrometer; Connect the power supply to the spectrometer and light source; Connect the spectrometer and the computer with a USB cable; Open uspectralplus software, which completes the construction of the entire experimental equipment. The erection equipment is shown in the following figure:

Figure 1 Schematic diagram of absorbance experimental device

3. spectrum acquisition:

(1) Turn on the main switch of deuterium tungsten lamp, then turn on the sub control switch of deuterium lamp and tungsten lamp, and preheat for 3 to 5 minutes;

(2) Open uspectralplus software and select transmission mode;

(3) Save dark background spectrum: turn off the master switch of the light source, collect a spectrum, which is the dark spectrum, and then click "save dark spectrum";

(4) Save reference spectrum: in this experiment, pure water is used as the reference, pure water is injected into the cuvette, the cuvette is put into the four-way liquid measuring cell, the shading cover of the measuring cell is covered, the master switch is turned on, a spectrum is collected, and then click "save bright spectrum";

(5) Measurement of sample spectrum: inject phenol sample into quartz cuvette with a liquid transfer gun in turn, put the quartz cuvette into the four-way liquid spectrum measurement cell, click the "transmittance" button, then click the "transmission and reflection radiation", select the display mode as "absorbance", and click "save data", at this time, the absorbance data of the full spectrum is saved.

4. drawing of standard curve:

Select the maximum absorption wavelength of phenol, i.e. the absorbance at 276nm, and fill it in the table, and then make the absorbance concentration standard curve.

5. determination of unknown sample concentration.

5、 Experimental results

The absorption spectra of phenol solutions with different concentrations collected by the UV-Vis absorption spectrometer are shown in Figure 2:

Figure 2 Absorption spectra of phenol solutions with different concentrations

It can be seen from Figure 2 that phenol has three strong absorption peaks in the UV region, 194 nm, 219 nm and 276 nm respectively. With the increase of concentration, the intensity of the characteristic peak of the sample also increases. After consulting the literature, it is known that the characteristic peak at 276nm is often used as the basis for quantitative analysis. Fill in the absorbance at 276nm in Table 1.

Table 1: absorbance of phenol at 276 nm

Then, draw the standard curve for quantitative analysis of phenol, as shown in Figure 3 below.

Figure 3 Linear fitting curve between absorbance and concentration at specific peak intensity of phenol solution with different concentrations

According to the figure, the absorbance of phenol solution has a large linear correlation with its concentration. The linear fitting coefficient is 0.9961. The equation of the standard curve is:

C = 84.74A - 14.40

Finally, the absorbance 0.4196 of the unknown sample was substituted into the equation of the standard curve. It was found that the concentration of the unknown phenol sample was 24.97 mg/l, while the real concentration was 25.00 mg/l. The experimental measurement error

△ = - 0.03 mg/l.

6、 Experimental conclusion

Phenol has three strong absorption peaks in the ultraviolet region, which are 194 nm, 219 nm and 276 nm respectively; The standard curve of phenol concentration absorbance is obtained with the absorbance at 276nm, r2=0.9961, and the equation of the standard curve is: C = 84.74 a - 14.40; When the absorbance of the unknown sample to be tested is 0.4646, the concentration of phenol can be calculated to be 24.97 mg/l, and the measurement error △ = - 0.03 mg/l.