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!Synthesis!of!Chemiluminscent!Luminol!

!Samar!Almarzooqi!!!!!!!!!7/21/14!TA:!Mike!Banales!Chem!213!Section!1!!!!!!!!!!!!!!!!!!!!!!!!!!

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Introduction*!

Luminol!is!important!in!forensic!science!for!its!chemiluminscent!activity!when!in!

contact!with!an!oxidizing!agent.!The!luminol!will!cause!a!blue!light!to!be!emitted!that!is!

important!during!crime!scene!investigations!to!detect!blood!presence!since!blood!contains!

heme,!an!oxidizing!agent.1!Luminol!is!also!important!in!its!biological!use!to!detect!the!

presence!of!targeted!oxidizing!agents!like!neutrophils!in!the!blood.2!It!is!also!used!in!labs!to!

detect!the!presence!of!copper,!iron,!and!cyanides.3 Chemiluminscent reactivity works by

luminol activation in the presence of an oxidant that when in the presence of a catalyst,

decomposes to a very unstable product that loses its energy and as a result, emits a photon that is

a blue glow.4 In the blood, hemoglobin acts as that catalyst. Because luminol reacts in the

presence of any oxidizing agent, it can lead to false positives in crime scene investigations when

bleach or copper is present, leading to a blue glow that can be mistaken for blood.1!!

Recrystallization is used to purify the luminol product that is obtained through a series of

heating reactions of reactants 3-nitrophtalic acid and hydrazine followed by sodium dithionite.

Recrystallization is important in purifying solids by the selection of a solvent that when heated,

causes the desired product to dissolve and when cooled, makes the product insoluble. Through

filtration, the product is then separated as any impurities remained soluble in the solvent.5

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Scheme 1: Luminol Synthesis

In the first step, the nucleophilic nitrogen from the hydrazine adds into the electrophilic

carbonyl to yield an oxygen anion and nitrogen cation. Internal proton transfer occurs from the

positively charged amine to the hydroxide, forming water as a good leaving group. The oxygen

anion donates its electrons to reform a carbonyl, leading to loss of water as a leaving group. In

the second step, the nucleophilic amine adds into the carbonyl to again yield an oxygen anion

and nitrogen cation. An internal proton transfer occurs again as the carbonyl is reformed and

water is lost as a leaving group. The intermediate 3-nitrophthalyhydrazide and sodium dithionite

react in a final step to reduce the nitrogen dioxide group to a primary amine.

The purpose of the experiment was to synthesize luminol from toxic reactants via a series

of heating reactions followed by recrystallization. The product was tested for its

chemiluminesnce in an oxidant solution to determine if luminol was obtained. Final

identification of the product was also analyzed by IR and NMR.

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Experimental

Luminol. Water (10 mL, .56 mol) heated on a hot plate. 3-nitrophthalic acid (200 mg, .947

mmol) dissolved in 8% hydrazine (0.40 mL, 1.02 mmol) in a sand bath. Triethylene glycol (0.60

mL, 3.00 mmol), a boiling chip, and mercury thermometer placed in the reaction mixture and

heated to 110 °C then 215 °C for 5 minutes to a brown color, cooled to 100 °C, and heated with

hot water (3 mL, .167 mol). Solution cooled to room temperature followed by 20 minutes in an

ice bath. Vacuum filtration performed to obtain brown solid added to 10 % sodium hydroxide (1

mL, 5.32 mmol) to yield a dark red solution followed by sodium dithionite (600 mg, 3.45 mmol).

Solution was heated for 5 minutes followed by acetic acid addition (.40 mL, .699 mol). Solution

cooled to room temperature followed by 10 minutes on ice and vacuum filtration of the yellow

crystals that were dried overnight. Purification performed by recrystallization (5 water: 1 acetone

mixture) and placed in refrigerator overnight. Final vacuum filtration performed to collect yellow

crystalline product (.126 g, 70%) and chemiluminescence tested in sodium hydroxide (20 mL,

1.065 mol), hydrogen peroxide (3 mL, .03 mol), and bleach (3 mL, .0447 mol); 1H NMR (60

MHz, D6-DMSO) δ (ppm) 11.60 (s, 2H), 7.00 (d, 1H), 7.102 (d, 1H), 7.500 (dd, 1H), 3.345 (s,

6H), 2.000 (s, 2H); 1H NMR (400 MHz, D6-DMSO) δ (ppm) 11.2694 (s, 2H), 7.306 (d, 1H),

7.303 (d, 1H), 7.297 (dd, 1H), 3.43 (s, 6H), 2.117 (s, 2H); 13C NMR (400 MHz, D6-DMSO) δ

(ppm) 206.577, 150.643, 133.86, 124.79, 109.39, 102.49, 99.49, 95.3391, 95.34, 39.86; IR

(ATR) υmax (cm-1) 3451, 3008, 2914, 2690, 1706, 1652, 1598, 1490.

Results and Discussion

The synthesis of luminol utilized a series of heating reactions, vacuum filtrations, and

recrystallizations to obtain the chemiluminscent product. Luminol’s chemiluminescence was

tested in an oxidant solution of bleach to observe the blue glow emitted in a dark environment.

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The yellow crystalline product was identified through 1H 60 MHz, 1H 400 MHz, and 13 C

400 MHz NMR and IR as luminol but the most important identification tool was the

chemiluminscent test that showed a blue glow indicative of luminol presence. IR showed that

luminol was obtained but also showed the presence of impurities. As shown by the IR spectrum

(Figure 1), the peak at 3451 cm-1 was indicative of a secondary amine characteristic to the

luminol product and intermediate 3-nitrophthalythydrazide. To prove that luminol was obtained

instead of the intermediate or starting material, the peak at 3331 cm-1 was vital in proving the

luminol was obtain because the peak represented a primary amine functional group. If the

intermediate was obtained, there would be no peak in the 3000 cm-1 region other than the

secondary amine peak. The intermediate would have the same peaks since all other functional

groups are similar, making the primary amine peak essential in distinguishing the luminol

product from the intermediates. The peaks in the 2266-1959 cm -1 region were indicative of

overtones. The IR spectrum was important in distinguishing the product obtained as luminol

from the intermediate 3-nitrophthalythydrazide and starting material 3-nitrophthalic acid.

NMR (Figures 2, 3, 4) was not very important in identifying the luminol product and

instead, showed the presence of impurities. The 13C NMR (Figure 4) showed many underlying

peaks that made it difficult to distinguish between the different peaks. The 13C NMR showed

eight peaks characteristic of the eight chemically distinct carbons in the luminol product. The

most downfield peal at 206 ppm was indicative of the carbonyl carbons, the six peaks in the 100

ppm region indicative of the aromatic carbons, and the two upfield carbons indicative of the

carbon-nitrogen bond. The two peaks at 39 ppm were indicative of impurities water and acetone.

The 1H NMR (Figures 2 and 3) also showed acetone and water impurities in the 2.00-3.350 ppm

region. The 60 MHz NMR (Figure 2) and 400 MHz NMR (Figure 3) did show peaks indicative

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of the luminol. The distinctive peak was at 12 ppm, indicative of primary amine deshielded

further by the aromatic ring. It was expected to observe two singlet peaks around 4 ppm

indicative of secondary amines, but according to the instrument room TA, it is very hard for

these peaks to be observed.

The chemiluminescence test was the most important tool in validating the product

obtained as luminol as a blue glow was observed in the presence of oxidant bleach. IR was

helpful in confirming the presence of luminol with a distinctive primary amine functional group

over the nitrogen dioxide group, characteristic of the starting material and intermediate. The

NMR was not very helpful in distinguishing luminol as it showed many impurities and the

presence of starting material 3-nitropthalic acid. The percent yield obtained based on the

theoretical yield obtained in the prelab was 70%, but the purity of the product obtained was

shown to be contaminated with the presence of acetone, water, and starting materials. Therefore,

the 70% yield does not reflect successful luminol synthesis at a high yield. Though the NMR did

not show peaks distinctive of luminol, IR and chemiluminescence test showed some luminol was

synthesized successfully. For future experiments, more starting materials should be used to

ensure more luminol synthesis as there was room for error in the two recrystallization steps and

vacuum filtrations with loss of material. Furthermore, more care should be taken in the

recrystallization steps in a slower cooling process from a hot temperature to a cooler one,

ensuring that all impurities do not form in the crystalline structure of the luminol product.

Overall, luminol was synthesized in a difficult experiment, as was proven by the

chemiluminesnce and the IR spectrum, in a series of heating and recrystallization reactions.

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Resources

1 Williamson, Kenneth L. Macroscale and Microscale Organic Experiments. Toronto: D.C.

Heath., 1994. Print. 2 DeChatelet, Lawrence R., Gwynn D. Long, Pamela S. Shirley, David A. Bass, Michael J.

Thomas, Frederick W. Henderson, and Myron S. Cohen. The Journal of Immunology 129.4 (1982): n. pag. Web.

3 Steigmann, A Journal of the Society of Chemical Industry 61.2 (1942): 36. Web. 4 King, R., and G. Miskelly. Talanta 67.2 (2005): 345-53. Web.

5 Rummel, Sheryl. A., Lab Guide for Chemistry 2013: Introductory Chemistry Laboratory;

Hayden McNeil: Michigan, U.S., 2014