Simple Techniques for The Synthesis and Evaluation of Schiff Bases

Schiff bases are prepared by the condensation of primary amine and carbonyl compounds (aldehyde/ketone) discovered by a German chemist, Hugo Schiff. In the year 1864, he first synthesized Schiff’s base through azeotropic distillation by using aldehyde or ketone and primary amine to get azomethine group (>C=N). They can be regarded as a sub-class of imines with the general structure R1R2C=NR3 (R3 ≠ H). Depending on their structure, they can be regarded as either secondary aldimines or secondary ketimines. Schiff bases are imines in which R3 is an alkyl or aryl group (not hydrogen). R1 and R2 may be hydrogen. Schiff bases have a wide range of biological activities such as antimicrobial, anticancer, and antiviral.

Schiff base are macrocyclic or macro-acyclic chelating agents that contain both nitrogen and oxygen donor atoms and are often multidentate and have chelating ability. Schiff bases and their metal coordination compounds have immense applications as a ligand in the co-ordination chemistry even after their discovery over a century. ^{[1, 2, 3, 4]}

Formation of the Schiff base

The condensation reactions of aldehyde and amines are carried out under different conditions and solvents like methanol or ethanol.

The formation of Schiff bases takes place at room temperatures or at refluxing conditions. If it happens, then the purification step can be done by crystallization. The formation of Schiff base from aldehyde or ketone is a reversible reaction and takes place under acidic or basic conditions.

The reaction takes place through the formation of carbinolamine as an intermediate:

A Schiff base is a type of chemical compound where bonding to the alkyl or aryl group is through the Nitrogen atom and not through the Hydrogen atom. The other name for Schiff base is imine or azomethine because the carbonyl group is replaced by these groups. Schiff bases are the important mediators for the preparation of bioactive molecules. Schiff bases are significant not only in inorganic chemistry but also have vast applications in the organic, bio-organic, biochemical, and medicinal chemistry. ^{[5, 6, 7, 8]}

Chemistry of Schiff bases

A Schiff base is a compound that has a general chemical formula of R1N = CR2R3. The R1, R2, and R3 are either phenyl or alkyl groups, thus forming a stable imine compound. The Schiff bases are the condensation products of aldehydes and amines.

The Schiff bases can be formed either by acid or base catalysis or even in neutral solutions. The aryl Schiff bases have higher stability and can be formed easily compared to alkyl Schiff bases, which are less stable. The Schiff bases are considered to be subclasses of imines. They can be either secondary aldehydes or secondary ketimines based on their composition. The Schiff bases can be divided into two categories based on the number of C = N bonds formed, which are mono Schiff base and bis Schiff bases. ^{[9, 10]}

Physical Properties of Schiff Bases

The following are the general physical properties of Schiff bases. ^{[11, 12, 13]}

Table.1: Physical properties

Applications of Schiff Basses

Schiff bases are a class of compounds that have an imine group (-C=N-) and are applied in various ways in chemistry, pharmacy, and industry.

1.  Medicinal Applications

• Antibacterial agents – Applied to combat most of the bacterial infections.
• Antiviral agents – Applied to combat some specific viruses.
• Anti-inflammatory drugs – Applied to reduce inflammation.
• Antifungal agents – Applied to combat fungal diseases.
• Anticancer agents – Some Schiff bases are applied to combat cancers. ^{[14, 15, 16]}

2.  Analytical Chemistry

• Applied as analytical agents to detect metal ions.
• Form metal complexes with copper, nickel, and cobalt.

Applied in qualitative and quantitative analysis. ^{[17, 18, 19, 20,]}

3.  Coordination Chemistry

• Serves as a ligand and forms metal complexes that are stable.
• Applied to prepare transition metal complexes.
• Have applications in bioinorganic chemistry.

4.  Industrial Applications

• Applied in the dye and pigment industry.
• Applied in the synthesis of polymers.
• Applied as corrosion inhibitors.

5.  Catalysis

• Schiff base-metal complexes are applied as catalysts in organic reactions.

6.  Agricultural Applications

• Applied in the preparation of pesticides and herbicides. ^{[21, 22, 23, 24]}

Objective of the study:

• To synthesize Schiff bases using aromatic amines and carbonyl compounds.
• To purify the synthesized compounds.
• To determine the physical properties.
• To evaluate the biological activity.

1. MATERIALS AND METHODS
   1. Reagents and Solvents

O-phenylenediamine, Benzophenone, P-Dimethylaminobenzaldehyde, Aniline, Vanillin, Glacial acetic acid, Ethanol, Distilled water used for the synthesis of Schiff Bases.

1. 2. Synthesis of Schiff Bases

Fig.1: Synthesis of Sample Compounds

PHYSICAL PROPERTIES

1. 1. Solubility: Schiff bases are soluble in organic solvents like ethanol, methanol, chloroform, and so on. The solubility of Schiff bases varies depending on the substituents present on the imine functional group, especially polar and aromatic groups. The majority of Schiff bases are less soluble in water due to their nonpolar or weakly polar nature.

Table.1: Solubility of Synthesized Compounds

3.2Melting Point Determination: Melting point determination is a simple and significant technique for identification of Schiff bases. The melting point of Schiff bases depends on the type of starting materials (aldehyde or ketone and the amine) used in the preparation.

Fig.2: Melting Point Apparatus

Table.2: Melting Point Determination

4. CHARACTERIZATION AND EVALUATION

The Schiff bases can be characterized by the use of spectroscopic methods like IR, ¹H/¹³C NMR, UV-Vis, and mass spectrometry to ensure the presence of the imine group (–C=N–).

Evaluation of the Schiff bases will involve chemical tests (negative 2, 4-DNPH or Schiff reagent test) to ensure the absence of free carbonyl groups. Finally, the efficacy of the Schiff bases will be determined by the use of biological tests like antimicrobial, antifungal tests, among others.

1. 1. Spectral Analysis: Spectral analysis is the procedure of analysing a signal, waveform, or data set into its component frequencies to analyze its power spectrum and determine periodicities. Primarily founded on the Fourier theorem, it converts time-domain data into the frequency domain to detect, measure, and interpret frequency components. It is commonly employed in signal processing, engineering, physics, and medicine to detect oscillations or material composition.

Infrared (IR) Spectroscopy: Infrared (IR) Spectroscopy is an analytical method employed to identify and analyze chemical substances by measuring their interaction with infrared radiation. When IR radiation passes through a sample, molecules absorb specific frequencies that correspond to the vibrations of their chemical bonds. The IR spectrum, which is a graph of absorbance or transmittance at different wavelengths, can be analysed to identify functional groups and molecular structures.

 IR spectroscopy is a significant method for analysing Schiff bases by verifying functional group modifications. A distinctive absorption peak for the imine (–C=N–) group occurs around 1600-1650 cm?¹. ^{[25, 26, 27]}

Compound-1(S1) :

Compound-2(S2) :

Compound-3(S3) :

Compound-4(S4) :

Compound-5(S5) :

1. 2. Thin Layer Chromatography (TLC): Thin Layer Chromatography (TLC) is a rapid and economical method of analysis used for the identification of various compounds on the basis of their RF values. Various compounds give different RF values, making it easy to identify the compound. The Stationary phase and mobile phase used are as follows:

Stationary phase:  TLC grade Silica gel

Mobile phase: Hexane: Ethyl acetate (7:3) ^[28]

Table.3: Rf values of synthesized compounds

1. 3. Identification Test:

Schiff reagent test: The Schiff reagent test is used for the identification of Schiff bases. This test is employed to check the presence of aldehydes. The Schiff reagent gives a pink or magenta colour in the presence of free aldehyde groups. Hence, the pure Schiff bases give a negative test as the aldehyde group is changed to an imine group (-C=N-). The positive test result may indicate the partial formation of Schiff base or hydrolysis to the aldehyde.

2, 4-dinitrophenylhydrazine (2, 4-DNPH) test: In the case of Schiff bases prepared from ketones, the 2, 4-dinitrophenylhydrazine (2, 4-DNPH) test is negative, which confirms the absence of the ketone group. The positive test result yields a yellow, orange, or red precipitate, which indicates the presence of carbonyl groups.

Table.4: Identification tests for Schiff Bases

1. 4.  Antimicrobial activity:

Biological evaluation for synthesized compounds by in vitro studies.

Method

Determination of antimicrobial activity by Agar well diffusion method.

Microorganism Used

Lactobacillus species isolated from curd (Gram-positive bacteria)

E. coli (Gram-negative bacteria)

Standard: Metronidazole

Fig.3: Antimicrobial activity

The medium was prepared, sterilized by autoclaving at 121°C for 15 minutes, and poured into sterile Petri plates for solidification. Then we have inoculated the bacteria and after incubation, wells are made using cork borer. 100microlitre solutions of the compounds and standard drug (Metronidazole) are poured into wells and again incubated in inversed position for about 28-32 hours. ^{[29, 30, 31, 32, 33, 34, 35]}

We have observed the zone of inhibition around the wells in which the compound is poured.

T

Table.5: Antimicrobial activity of Synthesized Compounds with Standard