applications of chemiluminescence

Atomic Spectroscopy

Applications of Chemiluminescence

Analytical Chemistry

/ Instrumentation

Paper No. : 06 Atomic Spectroscopy

Module :23 Applications of Chemiluminescence

Principal Investigator: Dr.NutanKaushik, Senior Fellow The Energy and Resouurces Institute (TERI), New Delhi

Co-Principal Investigator: Dr. Mohammad Amir, Professor of Pharm. Chemistry, JamiaHamdard University, New Delhi

Paper Coordinator: Dr. MymoonaAkhtar, Associate professor, Dept. of Pharm. Chemistry, JamiaHamdard, New Delhi.

Content Writer: Dr. MymoonaAkhtar, Associate professor, Dept. of Pharm. Chemistry, JamiaHamdard, New Delhi.

Content Reviwer: Dr. Gita Chawla, Associate Professor of Pharm.

Chemistry, JamiaHamdard University, New Delhi

Atomic Spectroscopy



/ Instrumentation

Description of Module

Subject Name Analytical Chemistry / Instrumentation

Paper Name Atomic Spectroscopy

Module Name/Title Applications of Chemiluminescence

Module Id 13

Pre-requisites

Objectives Application of Chemiluminescence in food industry,

Identification of adulterants,

Immuno Assays based on Chemiluminescent reactions, Reducing the time of

Immuno assays,

Use of luminol in forensic sciences,

Keywords Chemiluminescence, Isoluminol, Luminol, Chemiluminescent Immunoassay

Paper Coordinator: Dr. MymoonaAkhtar, Associate professor, Dept. of Pharm. Chemistry, JamiaHamdard, New Delhi.

Content Reviwer: Dr. Mohammad Amir, Professor of Pharm. Chemistry, JamiaHamdard University, New Delhi

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Introduction

Chemiluminescence is the production of light from a chemical reaction. Two chemicals react to

form an excited (high-energy) intermediate, which breaks down releasing some of its energy as

photons of light. CL is generally defined as the emission of light (ultraviolet, visible,or infrared)

as a result of a chemical reaction without using an externallight source. It can be used

therefore used for study of various reaction kinetics. Cl has been used in identification of

chemicals in biological sample (forensics), dynamics of proteins, study of DNA and adulteration

in processed foods

Application of CL in various fields

Fig 1: Application of Chemiluminescence in different field of science

APPLICATION OF CL IN FOOD ANALYSIS

Chemiluminescence (CL) detection has become quite a useful tool in the last years due to

its simplicity, low cost and high sensitivity. Moreover, no external light source is needed.

CL is often described as a dark-field technique: the absence of strong background light

level reduces the background signal and leads to improved detection limits

For nitrogen detection by CL technique

The chemiluminescent nitrogen species is nitric oxide

And it is NO+O3 reaction that is CL.

Determination of N-nitrosoamines

N-nitrosoamines are known as potent carcinogens

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Method 1

In meat and sauces, nitroamine detection is based on some means of chemical

denitrosation.The produced NO reacted with ozone to form electronically excited

nitrogen dioxide which then decayed back to the ground state with the emission of

photon.The CL emission is then detected and measured by a photomultiplier tube.

R2N-NO → R2NH + NO

NO + O3→ NO2∗ +O2

NO2∗→ NO2 + hν

Method 2

N-nitroso compounds (NOC) are treated with sulfamic acid to destroy nitrite, Whereas N-

nitroso compounds and N-nitroso precursors (NOCP) are treated with 110mM nitrite first

and then sulfamic acid. Both the products obtained after sulfamic acid treatment are

N-Nitrosamines

Volatile

nitrosamines Non-Volatile

nitrosamines

Removed from a food matrix by distillation

and separated by GC

Removed by extraction and

derivatization

as methyl esters

Determine has been done on the basis of ability to make a complete breakdown of the bond between the

nitroso compounds and the amino part of the molecule.

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decomposed to NO by refluxing with HBr/HCl/HOAc/EtOAc .The NO produced is

measured by CL

Table 1: Analytical application of common CL systems in food analysis

Determination of Nitrites

Chemulumincence is used to detrmine nitrites in tinned beef, sausage and vegetables. The

method is based on the strong CL of luminol in alkaline and iodine produced on-line by nitrite

and iodide in acid. A microflow injection analysis (FIA)system on a chip for the determination of

nitrite in some vegetables and fruits has also been developed (figure on next slide).The chip was

made by using two transparent poly(methylmethacrylate) (PMMA) slices (50mmx40mmx5mm),

and the microchannels etched by CO2 laser were 200m wide and 100m deep with the volume of

reaction area about 1.8μL. Nitrite was sensed by the CL reaction of luminol with ferricyanide

that was the product of the reaction of ferrocyanide and nitrite in acidic medium. The linear

range of the nitrite concentration was 8–100gL−1 and the detection limit was 4gL−1 (S/N = 3).

Food additives Analytes CL systems LOD Samples

Nitrogen containing

components

Sulfonamides bis[4-Nitro-2-(3,6,9-

trioxadecyloxycarbonyl)phenyl]

oxalate/ H2 O2

1 J.Lg L− 1 Milk

N-nitroso compounds Peroxyoxalate 0.04 J.Lg mL− 1

N-nitrosodimethylamine Ru(bipy)3 2+ / peroxydisulfate 0.29 ng mL− 1 Meat, beer, beverage

Luminol/ NO2 − / H+ 0.06 J.Lg L− 1 Food

Luminol/ K3 Fe(CN)6 4 J.Lg L− 1 Food N-nitrosamines Luminol/ peroxynitrite 1.5 ng L− 1 Drink

Sugars Glucose Luminol/ glucose oxidise 4 J.Lmol L− 1 Drink, honey

Lactose Luminol/ [Cu(HIO6 )2 ]5− 20 J.Lg mL− 1 Milk

Luminol/ K3 Fe(CN)6 6 J.Lg mL− 1 Sugar “Chemical”

perservatives

Parabens Ce(IV)/ Rhodamine 6G 34 ng mL−1 Soy sauces

Sulfite Rh 6G/ Tween 80/ SO3 2− 0.03 mg L− 1 Beverage

Ru(bipy)3 2+ / K2 S2 O8 41 nmol L− 1 Sugar

Ru(bipy)3 2+ / KMnO4 25 nmol L− 1 Sugar

Na2 CO3 / NaHCO3 / Cu2+ 0.05 nmol L− 1 Wine KMnO

4 / riboflavin 8 ng mL− 1 Beer, wine

Some metals Cr(III) Luminol/ H2 O2 / OH− 1.6 × 10− 7 nmol L− 1 Drinking Co(II) Luminol/ O2 4 fg mL− 1 Egg yolk

Others DDT Tween 20/ BSA/ MAbs 0.06 J.Lg L− 1 Fish meat

NMCs Peroxyoxalate/ CTMAB 4 J.Lg L− 1 Fruit juice

Carbofurn Luminol/ KMnO4 / OH− 0.02 J.Lg mL− 1 Lettuce

DDVP Luminol/ H2 O2 / CTMAB 8 ng mL− 1 Vegetable

Propanil KMnO4 / H+ 8 J.Lg L− 1 Drinking

Carbaryl Ce(IV)/ Rh6G/ H+ 45.6 ng mL−1 Grain

Luminol/ KMnO4

/ OH− 4.9 ng mL− 1 Cucumber

Luminol/ KMnO4 / OH− 4.9 ng mL− 1 Cucumber

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Fig : 2 Schematic diagram of the micro-FIA on-chip system used for the CL-determination

ofnitrite: (a) the features on the plates of chip system, (b) dimensions of the micro-flow channel.

(Source: M. Liu et al. Analytica Chimica Acta 670 (2010) 1–10)

Determination of Sugar

The sugars are important ingredient of some food and the most commonly found sugars are

glucose, sucrose, maltose, lactose and fructose. Excessive consumption sugars have been

associated with increased incidence of obesity and decay.

Source: M. Miro, Anal. Chim. Acta 54 (2005) 57

Method

Sugars (glucose, fructose and lactose in ternary mixtures) can be determined by a simple

continuous-flow CL system without separating them consisting of luminol and K3Fe(CN)6. The

method is based on the different kinetics of the individual sugars in the oxidation reaction with

+

+

Glucose

Glucose Oxidase

Molecular Oxygen Hydrogen Peroxide

Luminol

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potassium ferricyanide. The CL intensity is measured and recorded every second from 1 to 300 s.

The data obtained is processed using an artificial neural network.

• The method has number of advantages like

– Simultaneous detection of sugars

– High reproducibility,

– simplicity and rapidity

Parabens

Chemulumincence is used to analyse the concentration of parabens. Parabens include

methylparaben (MP), ethylparaben (EP), propylparaben (PP), and butylparaben (BP) are used as

preservatives to prevent foods from microbial and fungal attack. The maximum permitted

concentration of parabens in foods is 0.1%. Detection of parabens is based on reaction of

parabens and Ce(IV)–rhodamine 6G CL in sulfuric acid medium. The method shows higher

sensitivity than most of the reported methods.

The possible mechanism is that the Ce(IV) was reduced by the products hydrolyzed by parabens

to the excited-state Ce(III) in sulfuric acid medium andthen energy was transferred from the

Ce(III)* to rhodamine 6G to form the excited-state rhodamine 6G, emitting light.

Chemiluminescent Immunoassay

Another application ofChemiluminescenceis in Enzyme assay. During Enzyme Immuno Assay

(EIA) the process uses enzyme labeled antibodies and antigens to detect the small biological

molecules required. Based on basic immunology concept that an antigen binds a specific

antibody. Such antigen molecules, which can be identified in a fluid sample, include molecules

such as peptides, hormones and proteins. The enzymes used in CLIA convert a substrate to a

reaction product, which emits a photon of light instead of developing a particular colour. The

particular luminescence indicates the presence of the particular antigen and /or particular

biological molecule.

Traditional immunoassay always needs a long incubation time, which in turn causes the whole

analysis to take several hours for completion, so the throughput and application range are largely

limited..Researchers have designed different methods to shorten the analysis time by improving

mass transport and reaction kinetics. The rapid immunoassay has expanded the applications of

CLIA.

CLIA is a method to determine the concentration of samples according to the intensity of the

luminescence that the chemical reaction emits. CLIA combines the CL systems and the

immunoreactions.

The most popular CL substrates are

luminol,

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isoluminol and their derivatives,

acridinium ester derivative,

peroxidase and

alkaline phosphatase (ALP).

The most commonly used labeling enzymes are

horseradish peroxidase (HRP) and

ALP

Fig 3: Micro-bubble-accelerated immunoreaction for fast flowinjection immunoassay.

Source: Yang Z J, et al. Biosens. Bioelectron.,2008, 24(1): 35–40

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Fig.4: Channel and substrate Channel and substrate zone two-dimensional resolution for

multiplex immunoassay of tumor markers. Source:Fu Z F, et al. Anal. Chem., 2006,

78(19): 6999–7005


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Atomic Spectroscopy


Highly sensitive chemiluminescent immunoassay

Fig. 5: Ultrasensitive enhanced chemiluminescence enzymeimmunoassay amplified

by double-codified goldnanoparticles labels.

Chemiluminescence detection of biological samples can be determined by

1. Western blotting

2. DNA hybridization

WESTERN BLOTTING

Western blotting (WB), is also known as protein blotting. It is evolved from DNA (Southern)

blotting and RNA (Northern) blotting.WB allows the transfer of proteins from a sodium

dodecyl sulfate (SDS) polyacrylamide gel to an adsorbent membrane . The blotted proteins

form an exact replica of the gel and have proved to be the starting step for a variety of

experiments. The subsequent employment of antibody probes directed against the

nitrocellulose bound proteins has revolutionized the field of immunology .


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Atomic Spectroscopy


Fig 6: Western blotting and detection. Reference: www.sciencedirect.com

DNA HYBRIDIZATION DETECTION

The method employes a biotin-streptavidin system which binds an enzyme specifically to a

target DNA and upon exposure to substrate, the enzyme catalyzes a chemiluminescent

reaction. The image is captured within seconds by a Polaroid or X-ray film. The method is

capable of detecting DNA in the hundred atto mol range. Hybridization analysis is based on

the principle that two polynucleotides will form a stable hybrid by base-pairing if their

nucleotide sequences are wholly or partly complementary.A specific restriction fragment in a

Southern blot can therefore be detected if the membrane is probed with a second, labelled

DNA molecule that has the same, or similar,sequence as the fragment being sought

PRINCIPLE OF SOUTHERN BLOT

In these early techniques the immobilized DNA was unfractionated, simply consisting of total

DNA that was bound to nitrocellulose powder or spotted onto a nitrocellulose sheet. The

introduction in the early 1970s of gel electrophoresis methods that enable restriction

fragments of DNA to be separated on the basis of their size prompted the development of

techniques for the transfer of separated fragments masses from gel to nitrocellulose support

Fig 7: Sothern blotting..(ref:www.discoverbiotech.com)

APPLICATION OF CL IN FORENSC SCIENCE

The luminol chemiluminescence reaction is responsible for the glow of lightsticks. The

reaction is used by criminalists to detect traces of blood at crime scenes. In this test, luminol

powder (C8H7O3N3) is mixed with hydrogen peroxide (H2O2) and a hydroxide (e.g., KOH) in

a spray bottle. The luminol solution is sprayed where blood might be found. The iron from

the hemoglobin in the blood serves as a catalyst for the chemiluminescence reaction that

causes luminol to glow, so a blue glow is produced when the solution is sprayed where there

is blood.Only a tiny amount of iron is required to catalyze the reaction. The blue glow lasts

for about 30 seconds before it fades, which is enough time to take photographs of the areas so

they can be investigated more thoroughly.

http://www.sciencedirect.com/


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Atomic Spectroscopy


Fig 8: Footsteps made visible by use of Chemiluminscent reaction

Fig 9 A trail of blood made visible with the use of luminol reagent

reference: www.thoughtco.com

Procedure

The procedure requires

Luminol stock solution (2 g luminol + 15 g potassium hydroxide + 250 mL water)

3% hydrogen peroxide in water (common over-the-counter concentration)

http://www.thoughtco.com/


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Atomic Spectroscopy


potassium ferricyanide or a sterile blood lancet and sterile alcohol pad

In a clear test tube or cup, mix 10 ml of the luminol solution and 10 ml of the peroxide

solution. Activate the glow either by adding ~0.1 g of potassium ferricyanide to the solution

or with a drop of blood. The blood must be on the alcohol pad. The forensic test is for dried

or latent blood, so the reaction between the alcohol and fresh blood is necessary.

HOW THE LUMINOL TEST WORKS

The iron in the hemoglobin found in blood catalyzes an oxidation reaction in which the

luminol gains oxygen atoms while losing nitrogen and hydrogen.This produces a compound

called 3-aminophthalate. The electrons in the 3-aminophthalate are in an excited state. Blue

light is emitted as energy is released when the electrons return to the ground state

Summary

In this module we have learned about the application of chemiluminescence in food industry,

how identification of various adulterants can be done. We have also learned about

immunoassays based on Chemiluminescent reactions and how it helps in reducing the time of

assays. The forensic applications of chemiluninescent reactions was also discussed, including

the use of luminol in forensic sciences for blood stain visibility and DNA analysis.

applications of chemiluminescence

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