CHROMATOGRAPHY.doc.pdf

CHROMATOGRAPHY Chromatography is a flexible laboratory method used to separate components in a mixture according to how differently they flow between a stationary phase and a mobile phase. The blend is dissolved in a mobile phase and then passed through a stationary phase. The mixture's constituent...

CHROMATOGRAPHY Chromatography is a flexible laboratory method used to separate components in a mixture according to how differently they flow between a stationary phase and a mobile phase. The blend is dissolved in a mobile phase and then passed through a stationary phase. The mixture's constituent parts separate from one another to form unique bands or spots as they move through the stationary phase at varying rates. The unique properties of the mobile and stationary phases, such as molecular properties linked to adsorption, partition, and variations in molecular weights, influence this separation and result in varying retention times for each component, also referred to as retention time. The key components of chromatography include: 1. Stationary Phase: consisting of a liquid layer adsorbed on a solid substrate or a solid phase, where molecules interact differently according to their properties. 2. Mobile Phase: made up of a gaseous or liquid component that propels the mixture through the stationary phase. 3. Separated Molecules: The mixture's components interact with the phases, causing them to separate according to their affinities. This separation results in unique bands or spots on the stationary phase, which is referred to as the chromatogram. There are many different kinds of chromatography, including paper chromatography, gas chromatography, thin-layer chromatography, column chromatography, high performance liquid chromatography, gel-permeation chromatography, ion-exchange chromatography, and affinity chromatography. According to their underlying theories and techniques of separation, each kind has particular uses and instrumental configurations. Paper Chromatography Paper chromatography is a sort of liquid chromatography that uses a liquid solvent as the mobile phase and a piece of paper as the stationary phase. It is a low-cost, very effective analytical instrument that uses minuscule amounts of material. Principle of Paper Chromatography The fundamental principle of paper chromatography can be based on partition chromatography or adsorption chromatography. In partition chromatography, the components of the stationary phase—water contained in the paper pores—and mobile phase—solvent that moves up the paper—differ in solubility and distribution. This allows for effective separation. Differential component adsorption onto the paper's solid surface (the stationary phase) and solubility in the solvent (the mobile phase) are the basis for separation in adsorption chromatography. The mixture's constituent parts separate according to their respective affinities for the stationary and mobile phases when the mobile phase moves up the paper as a result of capillary action. Examples of materials used in the mobile and stationary phases in paper chromatography are as follows: Mobile Phase: Solvents: - Isopropanol: ammonia: water (9:1:2) - Methanol: water (4:1) - N-butanol: glacial acetic acid: water (4:1:5) - Dimethyl ether: cyclohexane - Kerosene: 70% isopropanol - Various other pure solvents, buffer solutions, or mixtures of solvents depending on the nature of the substance to be separated. Stationary Phase: Papers: - Whatman filter papers of different grades like No.1, No.2, No.3, No.4, No.20, No.40, No.42, etc. - Acid or base washed filter paper - Glass fiber type paper - Hydrophilic papers modified with methanol, formamide, glycol, glycerol, etc.  - Hydrophobic papers achieved through acetylation of OH groups for reverse phase chromatography - Papers impregnated with silica, alumina, or ion exchange resins. Procedure of Paper Chromatography The basic steps involved in paper chromatography are: 1. Selection of the paper: Pore size and sample quality are among the considerations that go into choosing the paper. 2. Sample preparation: To create the mobile phase, the sample is dissolved in an appropriate solvent. 3. Spotting the sample: With a capillary tube, the sample is spotted onto the paper. 4. Chromatogram development: The components are separated when the paper is submerged in the mobile phase, which moves up the paper as a result of capillary action. 5. Drying and detection: After the paper has dried, the separated components are identified by spraying it with a detecting solution. Types of Paper Chromatography The main types of paper chromatography are: 1. Ascending chromatography: The mobile phase travels up the paper. 2. Descending chromatography: The mobile phase travels down the paper. 3. Ascending-descending chromatography: The mobile phase first travels up and then down the paper. 4. Two-dimensional chromatography: The paper is developed in two perpendicular directions using different solvents. 5. Radial or circular chromatography: The paper is circular, and the components separate into concentric circular zones. Applications of Paper Chromatography Paper chromatography has a wide range of applications, including: 1. Amino acid, organic acid, alkaloids, polysaccharides, protein, and pigment separation and identification. 2. Assessment of pollutants, drugs, and food colours. 3. Monitoring chemical processes and compound purification. 4. Drug and its metabolite forensic analysis. 5. Investigating the ripening and fermentation processes. Thin Layer Chromatography Thin-layer chromatography (TLC) is one chromatography method for separating elements in non-volatile mixtures. TLC works on the basis of component distribution between a mobile phase and a stationary phase. A thin layer of an adsorbent substance, such as alumina or silica gel, placed on a plate serves as the stationary phase, while a solvent that rises the plate by capillary action serves as the mobile phase. Due to the analytes' varied affinities for the stationary phase, separation results when they ascend the TLC plate at different speeds. Different spots form on the plate as a result of components that have varying affinities for the stationary phase. Components with higher affinities move slower and those with lower affinities move faster. The retention factor (Rf), which is used to identify and characterise the components, is the distance travelled by each component in relation to the total distance travelled by the solvent front. The components of a TLC system include ready-made TLC plates with a stationary phase, a TLC chamber for plate development, and a mobile phase consisting of a solvent mixture. The basic TLC process involves: 1. Spreading a small portion of the sample mixture onto a stationary phase-coated TLC plate. 2. Immersing the plate in a chamber with the solvent used for the mobile phase, which rises up the plate via capillary action. 3. Based on their relative polarities and interactions with the stationary phase, the mixture's constituents separate into separate spots as the solvent travels along the plate. 4. After the separation, the separated spots can be further visualized, identified, and examined. Application of TLC 1. Biochemical Analysis: In biochemical analysis, thin layer chromatography (TLC) is widely used to isolate or separate biochemical metabolites from bodily fluids such as serum, urine, blood plasma, and body fluids. It is useful for determining the identities of natural compounds such as alkaloids, glycosides, waxes, volatile oils, fixed oils, and essential oils. 2. Pharmaceutical Applications: With more than half of its applications going towards pharmaceutical and medical research, TLC is essential in these areas. It provides simplicity in the separation procedure, making it possible to monitor coloured compounds with ease, separate several samples in parallel, and employ sensitive and particular colour reagents for spot detection. Because of its unique development and detection methods, disposable plates, and convenience of use, TLC is used for pharmaceutical preparations. 3. Clinical Analysis: TLC is used in clinical settings to separate mixtures into their constituent chemicals in order to identify individual substances or evaluate the purity of the mixture. Its affordability, ease of use, short development time, great sensitivity, and repeatability make it popular. Applications for TLC include the manufacturing of pharmaceuticals, clinical analysis, 4. Forensic Studies: Forensic investigations use Thin Layer Chromatography to check bodily fluids like blood and urine for drug residue. In forensic investigations, it is an invaluable instrument for analysing neutral and acidic drugs. 5. Among many other domains, TLC is utilised in industrial chemistry, food chemistry, environmental toxicity, water analysis, pesticide analysis, cosmetics, plant materials, and herbal analysis. COLUMN CHROMATOGRAPHY The basic principle of column chromatography is the differential adsorption of compounds in a mixture onto a stationary phase as they move through a column at different rates due to differences in their affinity for the stationary phase. The components with lower adsorption travel faster through the column compared to those with higher adsorption. The rate of movement of each component is expressed as the retardation factor (Rf), which is the ratio of the distance traveled by the solute to the distance traveled by the solvent. Operation of Column Chromatography 1. The column is packed with a stationary phase, typically silica gel or alumina. 2. The sample mixture is introduced at the top of the column. 3. The mobile phase, consisting of solvents, is added to the column. It acts as a solvent, developing agent, and eluting agent. 4. The mobile phase carries the sample mixture through the column at different rates based on the affinity of each component for the stationary phase. 5. The separated components are collected as fractions at the bottom of the column. Components of Column Chromatography 1. Stationary phase: A solid material with good adsorption properties, such as silica gel or alumina. 2. Mobile phase: Solvents or a mixture of solvents used to carry the sample mixture through the column. 3. Column: A cylindrical glass or plastic tube packed with the stationary phase. 4. Sample mixture: The mixture of compounds to be separated. Applications of Column Chromatography 1. Isolation of active constituents 2. Separation of compound mixtures. 3. Removal of impurities or purification of compounds. 4. Isolation of metabolites from biological fluids 5. Estimation of drugs in formulations or crude extracts Column chromatography is a versatile technique used for the purification and separation of both solids and liquids. GAS CHROMATOGRAPHY (GC) Gas chromatography (GC) is an analytical technique used for separating, identifying, and quantifying components of a mixture of organic compounds. It works by selectively partitioning the compounds between a stationary phase and a mobile gas phase inside a column, followed by sequential elution of the separated components. The main principle behind GC is that components in the mixture distribute themselves between the stationary phase and the mobile gas phase based on their relative vapor pressure and affinities for the stationary phase. Substances that interact more with the stationary phase are delayed and thus separated from substances that interact less. The key components of a gas chromatograph include: 1. Mobile phase (carrier gas): Helium, nitrogen, or hydrogen that carries the sample through the column 2. Sample injector: Vaporizes the sample and injects it into the carrier gas stream 3. Column: Packed with stationary phase for separation, enclosed in a temperature-controlled oven 4. Detector: Measures the quantity of components eluting from the column, such as Flame Ionization Detector (FID) or Mass Spectrometer (MS) 5. Data system: Captures and analyzes the detector output to generate chromatograms Operation The operation involves injecting the sample into the heated injector, where it vaporizes and enters the carrier gas stream. The components separate inside the column based on their interactions with the stationary phase and elute at different times. The detector measures the quantity of each component as it exits the column. GC has a wide range of applications in various industries: 1. Environmental monitoring for volatile organic compounds, pesticides, and industrial chemicals 2. Pharmaceutical research and manufacturing 3. Food and flavor analysis 4. Petrochemical and fuel analysis 5. Forensic toxicology 6. Metabolomics and lipidomics research The technique offers high separation efficiency, sensitivity, and selectivity, making it a powerful tool for analyzing complex mixtures of volatile organic compounds.