luminol

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luminol （Luminol） , also known as luminol. The chemical name is 3-amino-phenylenedicarbohydrazide. It is a pale yellow powder at room temperature and is a relatively stable synthetic powder Organic compound 。 The chemical formula is C eight H seven N three O 2。 For blood that cannot be observed by the naked eye at the crime scene, Luminol reagent can show a very small amount of blood stains（ Occult blood Reaction), At the same time, Luminol is a strong acid respiratory tract There are certain Irritant effect 。^{[4 ]} because hemoglobin Containing iron, which can catalyze hydrogen peroxide The decomposition of hydrogen peroxide turns hydrogen peroxide into water and monooxygen, which oxidizes luminol to make it glow. So Lumino is widely used in criminal investigation , bioengineering, chemical tracer and other fields. forensic medicine On, Luminol Reaction It can identify the blood stains that have been scrubbed for a long time. In biology, Luminol is used to detect copper, iron and cyanide The exists of.

Chinese name: luminol
Foreign name: Luminol
Alias: Luminescent ammonia, 3-aminobenzoyl hydrazide
chemical formula: C eight H seven N three O two
molecular weight: one hundred and seventy-seven point one six
CAS login number: 521-31-3
EINECS login number: 208-309-4
Melting point: 329 ℃

Boiling point: 1621.9ºC
Density: 1.433 g/cm3
Appearance: Light yellow powder
Flash point: 5 ℃
Application: Chemiluminescence reagent, commonly used in chemiluminescence immunoassay
Security description: S26-36/37-37/39-36
Hazard symbol: Xi,Xn
Hazard description: R36/37/38-22-20/21/22
Customs code: twenty-nine million three hundred and thirty-nine thousand nine hundred and ninety

catalog

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brief introduction

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name	luminol
alias	5-amino-benzoyl hydrazine; Luminol
English name	Luminol;5-Amino-2,3-dihydro-1,4-phthalazinedione
CAS	521-31-3
shape	Light yellow powder
content	≥ 98% ( HPLC )
melting point	＞ 300 ℃ (300 ℃~330 ℃ (319 ℃~322 ℃)
purpose	Chemiluminescent reagent, commonly used in Chemiluminescence immunoassay , e.g. metal cation And blood analysis
physical property	Soluble in Lye , soluble in dilute acid, almost insoluble in water, insoluble in alcohol. Neutral or light acid solution exposed to ultraviolet When it is medium, it shows strong bright blue fluorescence.
Luminosity testing	The optimum fluorescence wavelength is 425nm (at 60 mM K two S two O eight ，100 mM K two CO three , the chemiluminescence rate detected in pH11.5 solution)^[1]

Biological system Some enzymes such as Horseradish peroxidase HRP， And some Metal compound For example, iron in the blood can catalyze the decomposition of hydrogen peroxide to make hydrogen peroxide become water and monooxygen, wherein monooxygen can oxidize luminol, which can emit blue light after oxidation. This blue light can X-ray film Exposure development, fluorescence CCD Scanning imaging , or directly observe with naked eyes under weak light.

Lumino's Light

Lumino is a kind of chemistry Fluorescent molecule , can be converted into excited state Aminophthalic acid, which emits strong fluorescence. Hydrogen peroxide is the product of many biological oxidation reactions, so it is easy to link these biological oxidation reactions with light detection by introducing luminol. For example, use glucose oxidase / catalase The probe can detect hydrogen peroxide or glucose Concentration of, response time Only 0.5s (dynamic method).^[2]

synthesis

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3-Nitrobenzenedicarboxylic acid is obtained by cyclization and reduction. Add 3-nitrobenzoic acid into 8% hydrazine aqueous solution, heat to dissolve the solid. plus glycol , on evaporator The excess water in the solution is evaporated internally, heated at 215-220 ℃ for 2min to close the heterocycles, and cooled to yellow Nitro compound 。 Cooling, add 10% sodium hydroxide Solution, stirring, adding Sodium dithionate , heat it to boiling, keep it warm for 5min, and then add acetic acid , cool to bright yellow product. Leave overnight and filter the product.^[2]

Luminol reagent

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Chemiluminescence^[3]

The Luminol reagent we often say is Luminol and hydrogen peroxide（ hydrogen peroxide solution A mixture of Blood test 。 Lumino and hydroxide During the reaction, a double negative ion (Dianion) is generated, which can be oxidized by the oxygen decomposed from hydrogen peroxide, and the product is an organic peroxide 。 This peroxide (which is supposed to be a cyclic internal peroxide) is very unstable, and it immediately decomposes into nitrogen (Luminol is Organic oxidant as Dimethyl sulfoxide After oxidation, nitrogen is generated instead of nitrogen organic compound ）, generating excited state 3-Aminophthalic acid 。 Excited state to ground state During conversion, the released energy exists in the form of photons, and the wavelength is located in the blue part of visible light. Luminol glows only after being treated with oxidant. Usually, a mixed aqueous solution of hydrogen peroxide and a hydroxide base is used as an activator. Under the catalysis of iron compounds, hydrogen peroxide decomposes into oxygen and water: 2H two O two → O two ↑+2H two O Often used in the laboratory Potassium ferricyanide As a source of catalyst iron forensic medicine The catalyst on the hemoglobin Iron in. Enzymes in many biological systems can also catalyze the decomposition reaction 。 Luminol reagent is used to identify blood. Even if blood stains are wiped hemoglobin It will still remain. When Luminol reagent is sprayed on heme, it will active oxygen produce oxidation , emitting blue violet fluorescence. go by the name of Luminol Reaction 。 It is used to identify blood Organic matter 。^[1]^[3]

principle

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Lumino was synthesized as early as 1853. In 1928, chemists discovered for the first time that this compound has a wonderful characteristic that it can emit blue light when oxidized. A few years later, someone thought of using this property to detect blood stains. Blood contains hemoglobin, and the oxygen we breathe from the air depends on this protein Delivered to all parts of the body. Hemoglobin contains iron, and iron can catalyze the decomposition of hydrogen peroxide, turning hydrogen peroxide into water and monooxygen, which oxidizes luminol to make it glow. During the examination of blood stains, luminol reacts with heme (the auxiliary group responsible for transporting oxygen in hemoglobin), showing blue fluorescence. This detection method is extremely sensitive. It can detect only one millionth of the blood content. Even if a small drop of blood drops into a large vat of water, it can be detected. This shows how difficult it is for criminals to clean the scene.

Luminol luminescence is caused by oxidation, which means that there are many oxide And can afford Catalysis Luminol can also be made to glow by the metal, which includes hypochlorite bleach used daily. If criminals clean the scene with bleach, it may interfere with the use of Luminol. The two kinds of luminescence are slightly different. The luminescence caused by bleach is fast flashing, while that caused by blood stains is gradually emerging. Experienced detectives or policemen can usually distinguish between the two, but not always.

chemical reaction

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The method to perform the Luminol test is very simple, that is, spray the mixture to the place where there may be blood stains. If hemoglobin In contact with the mixture of luminol, the iron in hemoglobin accelerates hydrogen peroxide And Lumino. Lumino is here oxidation reaction Medium, lost Nitrogen atom and hydrogen atom And income Oxygen atom , the resulting compound is called 3- amino Phthalate 。 In the energized state, the 3 - amino o - left by the reaction is boosted to the oxygen atom of the electron in the higher orbit. The electron quickly falls back to a lower energy level and emits additional energy as a photon. When iron accelerates the reaction, the light emitted is enough to be seen in the dark room.

Researchers may use other Chemiluminescence Chemical substances, such as fluorescein , not to Lumino. The working principle of these chemicals is the same, but the process is a little different.

theory

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Even if the bloodstains at the crime scene have been wiped or cleared, forensic doctors can still use Lumino to find their location. In fact, the investigators sprayed Luminol and activator solution in the area to be investigated, and the iron in the blood immediately catalyzed Luminol's luminous reaction, making it produce blue light. The amount of catalyst required for this reaction is very small, so Luminol can be detected trace Of blood. The light lasts for about 30 seconds Long exposure The photos of surrounding environment It can't be too bright.

Practical significance

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Lumino shows obvious traces of blood, and researchers will film or video the crime Site records pattern. Normally, Lumino only shows the investigated area, possibly the blood. Because of other substances, including household bleaching agents , may also cause Lumino to glow. Experienced investigators can identify it according to the speed of its reaction, but they still need to run other tests to verify whether it is real human blood.

Lumino himself usually does not solve a murder case. This is only one step in the investigation process. However, it can show the necessary information to get the delayed investigation again. For example, the hidden blood splash mode can help researchers find the attack point, or even what kind of weapon to use (for example, bullets make the blood splash very different). Lumino may also show faint bloody shoe prints, which is valuable information after investigating the attackers' attacks.

In some cases, Lumino can give investigators more evidence. For example, if Lumino detects a trace of blood on the carpet, investigators can pull up the carpet and look for other blood stains on the floor below to see through the criminals' disguise of the crime scene.

shortcoming

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1. Lumino ferroalloy 、 horseradish Or emit fluorescence in the presence of some bleach. So if crime scene After being thoroughly treated with bleach, Luminol's fluorescence will strongly cover up the existence of any blood stains.

2. Luminol can detect a small amount of blood in animal blood and urine, so if the room to be tested contains urine or animal blood, the test results will be biased.

3. Lumino and Excreta The light emitted by reaction is the same as that emitted by reaction with blood.

4. Luminol may interfere with other tests, but Luminol does not interfere with DNA extraction.

5. Use Luminol in a dark environment, otherwise the fluorescence is difficult to recognize.

6. Lumino's luminous time is limited, so we should hurry to take photos.

Avoid interference

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1、 Let the site dry for a few days, bleaching agents Of Interference effect Will disappear, and even if the bloodstain passes many years, it can make Lumino shine.

2、 Use some kind of energy to restrain Hypochloric acid Interfering compounds. Obviously, it can't be used antioxidant , because that would also inhibit the reaction between the blood stain and Lumino. People's Chemical structure For example, suitable inhibitors have been found for the chlorine atoms it contains. The safest way is to use other methods to confirm that it is indeed a blood stain after the substance suspected of blood stain is detected by Lumino luminescence method.

Moreover, it was found that after the blood stain was treated by Lumino, it contained genetic material DNA It has not been destroyed and can be extracted for identification.

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