How to detect short-lived free radicals?
Updated 2023-02-21

Spin trapping technique has been widely used in biology and chemistry because it can achieve the detection of short-lived radicals. For spin trapping experiments, many factors such as the time of trapping agent addition, trapping agent concentration, system solvent and system pH can affect the experimental results. Therefore, for different radicals, it is necessary to select the trapping agent and design the experimental scheme reasonably to achieve the best experimental results.


  1. 1.Trapping Agent and Solvent Selection

  3. The common O-center radicals are hydroxyl radicals, superoxide anion radicals, and singlet oxygen.
  • Hydroxyl radicals (∙OH)


For hydroxyl radicals, they are usually detected in aqueous solutions and captured using DMPO, which forms adducts with DMPO with half-lives of minutes to tens of minutes.


  • Superoxide anion radicals (∙O2-)

For superoxide anion radicals, if DMPO is chosen as the trapping agent, the detection needs to be performed in a methanol system. This is because the binding ability of water and DMPO is higher than that of superoxide radicals to DMPO. If superoxide radicals are detected in water, the binding speed of water to DMPO will be greater than that of superoxide radicals to DMPO, resulting in superoxide radicals not being easily captured. Of course, if the superoxide radicals are produced in large amounts, they may also be captured by DMPO. If one wants to trap superoxide radicals in aqueous solution, BMPO needs to be chosen as the trapping agent because the half-life of adducts formed by BMPO trapping superoxide radicals in aqueous solution can be up to several minutes.


  • Single-linear state (1O2)

For single-linear state oxygen detection, TEMP is usually selected as the capture agent, and its detection principle is shown in Figure 1. Single-linear state oxygen can oxidize TEMP to form TEMPO radicals containing single electrons, which can be detected by electron paramagnetic resonance spectrometry. Since TEMP is easily oxidized and prone to background signal, TEMP needs to be tested before detecting single-linear state oxygen as a control experiment.

Figure 1 Mechanism of TEMP for detecting singlet oxygen



Table 1 Common O-center radical detection trapping agent and solvent selection


2、Addition Time of Trapping Agent


In photocatalytic reactions, when light irradiates the catalyst, the valence band electrons are excited to the conduction band, producing electron/hole pairs. Such experiments generally require the addition of the trapping agent before the light irradiation, and in combination with the in situ light system, the variation of the radical signal with the light irradiation time can be studied, as shown in Figure 2, with different light irradiation times, the ∙OH content generated varies.


Fig. 2 Results of CIQTEK in-situ illumination experiments


In the warming reaction, if the reaction temperature is lower than the tolerance temperature of the trapping agent, the trapping agent can be added before the reaction. If the reaction temperature is higher than the tolerance temperature of the trapping agent, the trapping agent should be added after the reaction for rapid sampling.


  1. 3、Free Radical Detection and Precautions


(1) DMPO capture radicals formed by the adduct life is generally short, for the generation rate is slow and need a long time to accumulate chemical reactions, BMPO can be used as a capture agent, such as BMPO and ∙OH formation of the addition product life can reach several days, so the signal can be continuously captured and accumulated.


(2) When DMPO is used as a trapping agent, it will reorganize or decompose itself in different oxidation environments. This can be avoided by reducing the catalyst concentration, changing the pH of the system, and taking quick samples for testing.


Figure 3 EPR spectrum of DMPO autoxidation products in different environments

(Data tested by CIQTEK EPR series products)


(3) In order to accurately determine the type of free radicals generated, the results can be secondarily verified by eliminating the free radicals through chemical means. For example, for ∙OH, ethanol and DMSO are scavengers of ∙OH, and these scavengers can be added so that further verification of ∙OH can be performed. As shown in Figure 4, the intensity of ∙OH signal becomes lower and CH3∙CHOH signal appears after the addition of ethanol. For superoxide anion radicals, a secondary verification can be performed by adding superoxide dismutase (SOD).


Figure 4 Secondary verification using ethanol to scavenge ∙OH

(Data obtained from the testing of the State Instrument EPR series)


  1. 4、CIQTEK Continuous Wave Electron Paramagnetic Resonance Spectroscopy 


With the in-situ light system, high/low temperature system and electrochemical system, GOKE Quantum EPR Spectrometer can be used to study various types of paramagnetic molecules, such as organic radicals, metal complexes, doped materials, defective materials, metalloproteins, etc., which can meet the demand of free radical detection in multiple scenarios.

CIQTEK EPR Series Products