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Spectrophotometric Study on the Effect Of Cetyltrimethylammoniumbromide (CTAB) In The Presence Of Eosin Blue Dye In Methanol-Water Mixed Solvent System

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FC13320 | 57-09-0 | Cetyltrimethylammonium bromide


Eosin Blue dye, which is sensitive to changes in the polarity of its surroundings,Eosin blue is a synthetic, water soluble dye that is often used in the fields of chemistry, biology and medicine.it is also a commonly used dye in microscopy.

The interaction between the dye (Eosin Blue ) and the surfactant  which was cetyltrimethylammoniumbromide (CTAB) was studied spectrophotometrically in order to determine the effect of  (CTAB) in the presence of Eosin blue dye in the methanol-water mixed solvent system in 0.1, 0.2, 0.3 and 0.4 volume fraction of methanol in water.

In the research or experiment, there were preparations of co solvents (methanol and water), preparation of the surfactant (CTAB) stock solution and then the preparation of the dye (Eosin B) stock solution, which at the end of the experiment itwas discovered that there was a  bathochromic shift (Red shift) in the monomeric form of Eosin B in 0.1 fraction of methanol, the peaks were seen from 524nm to 526nmand  there was also a shift in the peaks of the 0.2 volume fraction which were seen from 525nm to 527nm, then for the 0.3 volume fraction the wavelengths were from 525nm to 528nm and finally for the 0.4% volume fraction, the wavelengths were observed from 526nm to 529nm respectively. 

The maximum wavelength absorbance of 0.1% to 0.4% shifted from 526nm to 529nm respectively and a bathochromic shift (Red shift) was observed and it was also observed that with increase in the concentration of the surfactant, there was a decrease in the maximum wavelength of the dye.

TABLE OF Contents






1.0        INTRODUCTION.. 1

1.1        Background of the study. 1


1.3        Chemical properties of CTAB.. 7

1.4        Uses of CTAB.. 7

1.4.1         Biological Uses. 7

1.4.2         Medical Uses. 7

1.4.3         Deoxyribonucleicacid (DNA) Extraction. 8

1.5        Nanoparticle synthesis. 8

1.6        Toxicological Studies on CTAB.. 8

1.7        Surfactant studies. 10

1.7.1         Structure of Surfactants. 11

1.8        EOSIN BLUE DYE.. 13

1.9        Properties of Eosin B. 14

Properties of Eosin Blue dye. 14

1.10.1       Uses. 15        Use in histology. 15

1.11      Co- solvent (WATER & METHANOL) 17

1.12      AIM… 17

1.13      OBJECTIVES. 18



2.0        LITERATURE REVIEW… 19





3.1        MATERIALS. 29

3.2 REAGENTS. 30

3.3 Sample preparation. 30


3.3.3 PREPARATION OF STOCK SOLUTION OF CTAB(0.0002) mol\dm-3 . 30


3.3.5 Spectrum analysis: 31



Figure 4.1. Shows the interaction EB and CTAB  in 0.1 vol fraction of methanol and water at various concentration. 35


5.0        Conclusion. 41

References. 42




1.1       Background of the study

Spectroscopy is the study of the interaction between matter and electromagnetic radiation. It is a powerful tool used in science and engineering to investigate the properties of materials, determine their composition and identify their structure. (Pavia et al.,2015)

Spectroscopy has many applications in fields such as chemistry, physics, astronomy and biology.The history of spectroscopy began with the discovery made in 1859 by the German physicist Gustav R. Kirchhoff, that each pure substance has its own characteristic spectrum. Another German physicist, Joseph von Fraunhofer, repeating more carefully an earlier experiment by a british scientist, William Wollaston, had shown in 1814 that the spectrum of the sun’s electromagnetic radiations does not grade smoothly from one color to the next but has many dark lines, indicating that light is missing at certain wavelength because of absorption. These dark lines sometimes called Fraunhofer lines, are also collectively referred to as an absorption spectrum. The spectra of materials that were heated in flames or placed in electric-gas discharges were studied by many scientist during the 18th and 19th century. These spectra were composed of numerous bright discrete lines, indicating that only certain wavelength were present in the emitted light. They are called brightline, emission or spectra. (Chu, 2023)

There are many types of spectroscopy, each with its own set of applications and techniques. Some of the most common types of spectroscopy include; (Hussain & Mustansar, 2019.)

  • Atomic absorption spectroscopy: This method is used to determine the concentration of metal ions in a sample by measuring the absorption of infrared radiation.
  • Infrared spectroscopy: This method is used to study the vibrations of molecules in a sample by measuring the absorption of infrared radiations.
  • Ultraviolet-visible spectroscopy: this method is used to study the electronic transitions of molecules by measuring the absorption or reflection of UV or visible light.
  • Raman spectroscopy: This method is used to study the vibrations of molecules in a sample by measuring the scattering of light.
  • Nuclear magnetic resonance: This method is used to study the interactions between the atomic nuclei and a magnetic field by measuring the absorption of radio waves.
  • X-ray crystallography: This method is used to determine the structure of crystals by measuring the diffraction of x-rays.

Surfactants are chemical compounds that decrease the surface tension or interfacial tension between two liquids, a liquid and a gas, or a liquid and a solid. Surfactants may function as emulsifiers, wetting agents, detergents, foaming agents, or dispersants. (Rosen et al., 2012). 

The role of co-solvents in the process of formation of micelles in surfactant solutions is of considerable interest both from the fundamental and applied view points since application of surfactants in many physio-chemical and interfacial phenomena largely depends on it. (Kabedev, 2023).

Amphiphiles having a polar hydrophilic head group and a stretchy hydrophobic hydrocarbon chain which on impulse forms aggregates in aqueous solution results in a supra-molecular assemblies simply known as micelles at a definite concentration called critical micelle concentration (CMC) .(Zana, (2002). This amphiphilic nature of such molecules is accountable for their distinctive properties like self-aggregation, adsorption at the interfaces and dissolving of hydrophobic molecules that are responsible for their extensive utilize in the food, pharmaceutical, petrochemical, agrochemical, textile, paint and coating industries as wetting and foaming agents, emulsifying and suspension stabilizers. (Torchilin, 2001). The amphiphilic compounds have a unique feature of self-aggregation forming micelles that result due to the hydrophobic attraction by hydrocarbon chains of tail and electrostatic repulsion of hydrophilic groups. They have a unique property such as CMC, a necessary criteria for micelle formation to be used in drug delivery systems. The surfactants are usual amphiphiles having two core parts i.e., the hydrophilic part providing a highly polarized environment and other is the hydrophobic part that provides a low polarity environment (H. Schott et al (1985)).

Cetyltrimethylammonium bromide (CTAB), sometimes called cetrimonium bromide, is a quaternary ammonium salt with surface-active and antiseptic properties. It was mentioned in the chemical literature as early as 1936, when G. S. Hartley, B. Collie, and C. S. Samis at university College London studied transport numbers of paraffin-chain salts in aqueous solutions. (ACS, 2023). CTAB is a cationic surfactant, that in addition to its potential use as a drug solubilizer, also has intrinsic antibacterial properties. As a first example for CTAB, MD simulations were used to investigate the behavior of CTAB micelles. (Kabedev, 2013).

Solvents are substances that can dissolve other substances to form a homogeneous solution. They are used in many industries and applications, including in the production of chemicals, pharmaceuticals, and consumer products. They are ordinarily a liquid, in which other materials dissolve to form a solution. (Augusrtyn, 2023)

There are many different types of solvents, including polar and non-polar solvents.Polar solvents, such as water and alcohols, have a partial positive and negative charge and can dissolve polar compounds like salts and sugars. Non-polar solvents, do not have a charge and can dissolve non-polar compounds like oil and fats. (Mondal, 2020)

In chemistry, co-solvents are substances added to a primary solvent in small amounts to increase the solubility of a poorly-soluble compound. Their use is most prevalent in chemical and biological research relating to pharmaceuticals and food science, where alcohols are frequently used as co-solvents in water often less than 5% by volume (Shi & John, 2007) to dissolve hydrophobic molecules during extraction, screening, and formulation.


Dyes are colored substances that are used to impart color to a variety of materials including fabrics, paper, leather and even food. They are usually soluble in water or other solvents and have a high affinity for the materials they are applied to. They can be derived from natural sources such as plants, animals and minerals or they can be synthesized chemically. Natural dyes were commonly used before the advent of synthetic dyes in the mid-19th century, but synthetic dyes have largely replaced natural dyes due to their superior color fastness, stability and ease of use. (Groeneveld et al., 2023)

EOSIN B DYE                                                                                                                            

Eosin is a class of fluorescent red dye. It is an artificial derivative of fluorescein consisting of two closely related compounds, eosin Y and eosin B. Eosin Y is far more commonly used. It is a tetrabromo derivate of fluorescein and has a slightly yellowish cast (so is also known as Eosin Yellowish). Eosin Y can be further divided into water-soluble and ethanol-soluble eosin Y. Eosin is the name of several fluorescent acidic compounds which bind to and form salts with basic, or eosinophilic, compounds like proteins containing amino acid residues such as arginine and lysine, and stains them dark red or pink as a result of the actions of bromine on eosin. In addition to staining proteins in the cytoplasm, it can be used to stain collagen and muscle fibers for examination under the microscope. Structures that stain readily with eosin are termed eosinophilic. In the field of histology, Eosin Y is the form of eosin used most often as a histologic stain.(Lai et al., 2012).

Eosin B is a synthetic, water soluble dye that is often used in the fields of chemistry, biology and medicine. It is a derivative of fluorescein, a commonly used dye in microscopy.

The chemical structure of Eosin B has a diphenylmethane backbone, with two sulphonate groups and an ethylene bridge. Its molecular formula is (C20H8Br4N2Na205) and its molecular weight is 691.89 g/mol.

Eosin B is often used as a histological stain particularly in the staining of eosinophillic granules in cells. It is also used as a dye in the production of textiles, paper and leather.

In chemistry, Eosin B has been used as a fluorescent dye. It has been investigated for its application in the detection of metal ions, such as mercury and copper, as well as for its use as a photosensitizer in photodynamic therapy.

Eosin B has also been used as a colorimetric sensor in the detection of anion and cations. It forms a complex with the target analyte resulting a change in color or fluorescence properties that can be detected by spectrometric methods. This makes it a promising tool for environmental monitoring and clinical diagnostics.

In summary, Eosin Blue is a versatile dye that has found numerous applications in chemistry, biology and medicine. Its unique chemical property makes it a promising tool for various research fields.


Cetyltrimethylammonium bromide (CTAB), sometimes called cetrimonium bromide, is a quaternary ammonium salt with surface-active and antiseptic properties. It was mentioned in the chemical literature as early as 1936, when G. S. Hartley, B. Collie, and C. S. Samis at university College London studied transport numbers of paraffin-chain salts in aqueous solutions. (ACS, 2023). CTAB is a cationic surfactant, that in addition to its potential use as a drug solubilizer, also has intrinsic antibacterial properties. As a first example for CTAB, MD simulations were used to investigate the behavior of CTAB micelles. (Kabedev, 2013). The cetrimonium (hexadecyltrimethylammonium) cation is an effective antiseptic agent against bacteria and fungi. It is also one of the main components of some buffers for the extraction of DNA. (Clarke & Joseph, 2009).

Figure 1.1: Structure of Surfactant(Cetyltrimethylammoniumbromide) (CTAB)

1.3       Chemical properties of CTAB

Molar mass: 364.45 g/mol

Formula: C19H42BrN

Appearance: White powder

ATC code: D08AJ02 (WHO)

Melting point: 237 to 243 ᵒC (459 to 469 ᵒF; 510 to 516 K)

1.4       Uses of CTAB

1.4.1    Biological Uses

Cell lysis is a convenient tool to isolate certain macromolecules that exist primarily inside of the cell. Cell membranes consist of hydrophilic and lipophilic endgroups. Therefore, detergents are often used to dissolve these membranes since they interact with both polar and nonpolar endgroups. CTAB has emerged as the preferred choice for biological use because it maintains the integrity of precipitated DNA during its isolation. (Azmat et al., 2012). Cells typically have high concentrations of macromolecules, such as glycoproteins and polysaccharides that precipitate with DNA during the extraction process, causing the extracted DNA to lose purity. The positive charge of the CTAB molecule allows it to denature these molecules that would interfere with this isolation. (Clarke & Joseph, 2009).

1.4.2    Medical Uses

CTAB has been shown to have potential use as an apoptosispromoting anticancer agent for head and neck cancer (HNC). [6] Thus, using this approach, CTAB was identified as a potential apoptogenic quaternary ammonium compound possessing in vitro and in vivo efficacy against HNC models. CTAB is also recommended by the World Health Organisation (WHO) as a purification agent in the downstream vaccine processing of polysaccharide vaccines.

1.4.3    Deoxyribonucleicacid (DNA) Extraction

CTAB serves as an important surfactant in the DNA extraction buffer system to remove membrane lipids and promote cell lysis. Separation is also successful when the tissue contains high amounts of polysaccharides. . (Clarke & Joseph, 2009). CTAB binds to the polysaccharides when the salt concentration is high, thus removing polysaccharides from solution. A typical recipe can be to combine 100 mL of 1 M Tris HCl (pH 8.0), 280 mL 5 M NaCl, 40 mL of 0.5 M EDTA, and 20 g of CTAB then add double distilled water (ddH2O) to bring total volume to 1 L.

1.5       Nanoparticle synthesis

Surfactants play a key role in nanoparticle synthesis by adsorbing to the surface of the forming nanoparticle and lowering its surface energy. (Mehta et al., 2009). Surfactants also help to prevent aggregation (e.g. via DLVO mechanisms).

1.6       Toxicological Studies on CTAB

CTAB has been used for applications from nanoparticle synthesis to cosmetics. Due to its use in human products, along with other applications, it is essential to be made aware of the hazards this agent contains. The Santa Cruz Biotechnology, Inc. offers a comprehensive MSDS for CTAB and should be referred to for additional questions or concerns. (Santa, 2011). Animal testing has shown ingestion of less than 150 g of the agent can lead to adverse health effects or possibly death by CTAB causing chemical burns throughout the esophagus and gastrointestinal tract that can be followed by nausea and vomiting. (Santa, 2011). If the substance continues through the gastrointestinal tract, it will be poorly absorbed in the intestines followed by excretion in feces. (IJT, 1997).Toxicity has also been tested on aquatic life including Brachydanio rerio (zebra fish) and Daphnia magna (Water flea). Zebra fish showed CTAB toxicity when exposed to 0.3 mg/L for 96 hours, and water fleas showed CTAB toxicity when exposed to 0.03 mg/L for 48 hours. (Sigma, 2008).  CTAB along with other quaternary ammonium salts have frequently been used in cosmetics at concentrations up to 10%. Cosmetics at that concentration must only be used as rinse-off types such as shampoos. Other leave-on cosmetics are considered only safe at or below 0.25% concentrations. Injections into the body cavity of pregnant mice showed embryotoxic and teratogenic effects. Only teratogenic effects were seen with 10 mg/kg doses, while both effects were seen at 35 mg/kg doses. Oral doses of 50 mg/kg/day showed embryotoxic effects as well.(IJT, 1997).  Similar tests were completed by giving rats 10, 20, and 45 mg/kg/day of CTAB in their drinking water for one year. At the 10 and 20 mg/kg/day doses, the rats did not have any toxic symptoms. At the highest dose, the rats began experiencing weight loss. The weight loss in the male rats was attributed to less efficient food conversion. The tests showed no microscopic alterations to the gastrointestinal tract of the rats.(Isomaa, 1996).  Other toxicity tests have been conducted using incubated human skin HaCaT keratinocyte cells. These human cells were incubated with gold nanorods that were synthesized using seed-mediated, surfactant-assisted growth of gold nanoparticles. Gold nanoparticles are shown to be nontoxic, however once the nanoparticles are put through the growth solutions, the newly formed nanorods are highly toxic. This large increase in toxicity is attributed to the CTAB that is used in the growth solutions to cause anisotropic growth. Experiments also showed the toxicity of bulk CTAB and the synthesized gold nanorods to be equivalent. Toxicity tests showed CTAB remaining toxic with concentrations as low as 10 μM. The human cells show CTAB being nontoxic at concentrations less than 1 μM. Without the use of CTAB in this synthesis, the gold nanorods are not stable; they break into nanoparticles or undergo aggregation. (RAY, 2009).  The mechanism for cytotoxicity has not been extensively studied, but there has been possible mechanisms proposed. One proposal showed two methods that led to the cytotoxicity in U87 and A172 glioblastoma cells. The first method showed CTAB exchanging with phospholipids causing rearrangement of the membrane allowing β-galactoside to enter into the cell by way of cavities. At low concentrations, there are not enough cavities to cause death to the cells, but with increasing the CTAB concentration, more phospholipids are displaced causing more cavities in the membrane leading to cell death. The second proposed method is based on the dissociation of CTAB into CTA+ and Br− within the mitochondrial membrane. The positively charged CTA+ binds to the ATP synthase not allowing H+ to bind stopping the synthesis of ATP and resulting in cell death. (Schachter & David, 2013).

CTAB is also used in research as a surfactant to aid in the solubilization of proteins and other biomolecules, and as a cationic stabilizer for nanoparticles. It has also been investigated for its potential use in gene therapy and drug delivery applications.(Azmat et al., 2012).

As with any medication or chemical, it is important to use CTAB according to the instructions and to avoid excessive exposure or ingestion .Consult your healthcare provider or a qualified expert for medical service(S. De et al., 2005).



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