Chromatography: Principle and Theory of Chromatography, Types of Chromatography, Clinical significance/applications of Chromatography. HSBTE. DMLT. IVth Semester. Biochemistry.

Chromatography

“Chromatography is a laboratory technique used to separate components in a mixture based on their distribution between a stationary phase and a mobile phase”.

The stationary phase and mobile phase are two components of chromatography techniques.

Stationary Phase: The stationary phase is the immobile component of chromatography.

• · It is a solid or a liquid supported on a solid (such as a column packing material or a coated surface).

• · The sample molecules interact with the stationary phase as they move through it.

• · Separation occurs because different sample components have different affinities for the stationary phase.

• · Examples of stationary phases include silica gel, alumina, bonded phases in HPLC columns, and the paper or gel matrix in paper chromatography and gel electrophoresis.

Mobile Phase: The mobile phase is the moving component of chromatography.

• · It is a liquid or gas that carries the sample components through the stationary phase.

• · The choice of mobile phase depends on the specific chromatography and the properties of the sample being analyzed.

Principle/Theory of Chromatography:

Chromatography depends upon the equilibrium established between the mobile and the stationary phases. The sample is introduced into the stationary phase. The sample components interact with the stationary and mobile phases as the mobile phase (liquid or gas) flows over the stationary phase.

Partitioning: Components of the sample interact differently with the stationary and mobile phase. This leads to variation in the sample (analyte) component migration rate. Components with higher affinity for the stationary phase move more slowly. This leads to the separation of the sample components.

Types of Chromatography

Chromatography is an analytical technique used for separating and identifying components of a mixture. Its principle is based on components' differential distribution between a stationary and a mobile phase. There are several types of chromatography, each with its principles and applications. The following are some common types of chromatography

1. Thin Layer Chromatography (TLC): Principle: Separation based on the differential migration of components between a stationary phase coated on a thin layer (usually a plate) and a mobile phase.

2. Paper Chromatography: Principle: Separation based on the differential migration of components between a paper (stationary phase) and a liquid mobile phase.

3. Liquid Chromatography (LC): Principle: It has a liquid mobile phase and a solid or liquid stationary phase. Separation is based on the differential distribution of components between a liquid mobile phase and a solid or liquid stationary phase.

4. Gas Chromatography (GC): Principle: Gas Chromatography separates the volatile components based on their affinity with a liquid stationary phase and gaseous mobile phase

5. High-performance liquid Chromatography (HPLC): It is a type of liquid chromatography. It uses high pressure for faster and more efficient separation.

6. Column Chromatography Principle: Similar to liquid chromatography, but with a vertical column filled with the stationary phase.

8. Ion Exchange Chromatography: Principle: Separation based on the exchange of ions between the stationary phase and the mobile phase.

9. Size Exclusion Chromatography (SEC) / Gel Filtration Chromatography: Principle: Separation based on the size of molecules, with larger molecules eluting first.

Paper Chromatography

Paper chromatography is a technique used for separating and analyzing mixtures of chemical substances between a stationary phase and a mobile phase. In paper chromatography, the stationary phase is a piece of specialized chromatography paper and the mobile phase is a solvent or a mixture of solvents.

Principle (Theory): Adsorption, partition, and capillary action are involved in the paper chromatography.

1. Adsorption: The stationary phase in paper chromatography is the chromatography paper. The paper consists of cellulose fibers capable of adsorbing molecules from the sample mixture. Adsorption occurs when the sample molecules come into contact with the paper surface and adhere to it. The strength of adsorption varies depending on the nature of the molecules.

2. Partition: The mobile phase travels through the paper via capillary action. The mobile phase carries the sample components along with it. The sample molecules interact with the stationary phase (the paper) and the mobile phase (the solvent). The molecule partitions between the two phases depend on its solubility in the solvent and its affinity for the paper. Components are more soluble in the mobile phase and migrate quickly through the paper. Components with stronger interactions with the stationary phase move slowly.

3. Capillary action: The mobile phase, moves through narrow spaces in a solid material by capillary action. In paper chromatography, the cellulose fibers of the paper act as capillaries. This movement of the solvent carries the sample components along with it. It facilitates separation based on differences in partitioning behavior.

Procedure of Paper Chromatography:

1. Preparation of Chromatography Paper: Cut a strip of chromatography paper to a suitable size and shape. Mark a baseline near the bottom of the paper using a pencil or marker.

2. Preparation of Sample: Prepare the sample solution by dissolving the substances mixture in a suitable solvent. Apply a small volume of the sample solution to the baseline using a capillary tube, micropipette, or applicator. Allow the spot to dry.

3. Development of Chromatogram: Place the prepared chromatography paper in a suitable chamber or tank containing the mobile phase (solvent). The bottom edge of the paper should be in the solvent. However, the sample spot is above the surface of the solvent.

Cover the chamber to prevent evaporation and allow the mobile phase to move through the paper by capillary action. Allow the solvent to approach the top of the paper or reach a desired height. Remove the paper from the chamber.

4. Visualization of Chromatogram: Allow the paper to dry completely. Visualize the chromatogram under suitable lighting conditions. Components may be visible as colored spots. Sometimes UV light or chemical staining are used to visualize components.

Analysis of Chromatogram:

Measure the distances traveled by the separated components. Then calculate their Rf (retention factor) values. The Rf represents the ratio of the distance traveled by the component to the distance traveled by the solvent.

Compare the Rf values obtained with known standards or literature values to identify and characterize the separated components.

Clinical Significance/Application of Paper Chromatography:

Paper chromatography is not commonly used in clinical laboratories. The following are some of the clinical significances of paper chromatography:

1. Drug testing and Toxicology (Forensic technology): To test drugs and toxins in biological samples (such as urine or blood). This information is used to diagnose drug abuse, monitor medication compliance, and assess exposure to toxic substances.

2. Detection of metabolic disorders: Detection and diagnosis of various metabolic disorders of amino acids, organic acids, and carbohydrates. For example, abnormal patterns of amino acids in body fluids indicate disorders such as phenylketonuria (PKU), maple syrup urine disease, etc.

3. Identification of biomarkers: Identification of specific biomarkers present in biological samples. Biomarker identification (proteins, enzymes, or lipids) contributes to diagnosing and monitoring various medical conditions. For example, chromatographic separation of hemoglobin variants is essential for diagnosing hemoglobinopathies like sickle cell disease and thalassemia.

Thin Layer Chromatography

Thin Layer Chromatography (TLC) is widely used for separating mixtures into their components. The following are the principles, procedures, and clinical significance of TLC:

Principle:

TLC stationary phase is applied as a thin layer on a solid support such as glass, plastic, or aluminum foil. The stationary phase is silica gel, alumina, or cellulose. The sample mixture is spotted at the bottom of the TLC plate and allowed to migrate along the plate through capillary action. Sample component separation is based on their differential partitioning between the mobile phase (solvent) and the stationary phase.

The separation occurs because each component has a different affinity for the stationary and mobile phases. Components with a stronger affinity for the mobile phase will move faster up the plate. Components with a stronger affinity for the stationary phase will move slower. The components are visualized using various detection methods such as UV light, chemical staining, or fluorescence.

Procedure:

1. Preparing the TLC plate: The TLC plate is cleaned and activated by heating. The stationary phase is applied uniformly as a thin layer onto the solid support.

2. Spotting the sample: A small amount of the sample mixture is applied as a spot near the bottom of the TLC plate using a capillary tube or a micropipette.

3. Developing the plate: The TLC plate is placed in a solvent mixture present inside the developing chamber. The solvent acts as the mobile phase. The chamber is sealed to prevent evaporation of the solvent. The solvent rises up the plate through capillary action. This carries the sample components with it. The plate is removed from the chamber before the solvent front reaches the top.

4. Visualization: The plate is dried and then visualized. This can be done by exposing the plate to UV light if the compounds fluoresce, or by using chemical reagents to stain the spots.

5. Analysis: The Rf (retention factor) values of the spots can be calculated to identify the components. Rf is the ratio of the distance traveled by the compound to the distance traveled by the solvent front.

Clinical Significance TLC: TLC has several clinical applications.

1. Toxicology: To detect drugs and toxins in biological samples such as urine, blood, or saliva.

2. Clinical chemistry: To analyze lipids, amino acids, carbohydrates, and other biomolecules in clinical samples.

3.  Forensic analysis: For drug analysis, unknown substance identification found at crime scenes, and trace evidence analysis.

4. Quality control: Quality control of pharmaceuticals, food products, and herbal supplements to ensure compliance with regulatory standards.

5. Pharmaceutical analysis: TLC is used for analyzing drug purity, identifying impurities, and monitoring the progress of reactions in drug synthesis.

Clinical significance/applications of Chromatography

Chromatography plays an important role in medical laboratories. It is used for the separation, identification, and quantification of various compounds. The following are some clinical significance and applications of chromatography:

1. Drug Monitoring: To monitor therapeutic drug levels in patient samples. It ensures that drugs are present in the right concentration for effective treatment.

2. Clinical Toxicology: Drugs and toxins Identification and quantification of biological samples (blood, urine, or tissues) are essential for diagnosing cases of poisoning or overdose.

3. Clinical Chemistry: Separation and analysis of blood components, including amino acids, lipids, and various metabolites.

Chromatography is employed in the identification of disease markers and diagnostic biomarkers in biological fluids.

4. Hormone Analysis: Quantification of hormones in blood and urine samples to diagnose endocrine disorders.

5. Therapeutic Drug Monitoring: Monitoring the levels of drugs used in long-term therapy to ensure therapeutic efficacy and prevent adverse effects.

6. Protein Analysis: Separation and analysis of proteins in clinical samples for diagnostic purposes.

7. Chromatography is used in the identification of specific proteins related to diseases, such as cancer markers.

8. Genetic Testing: Chromatography techniques, such as capillary electrophoresis, are employed in the separation of DNA fragments for genetic testing and analysis.

9. Clinical Microbiology: Identification and characterization of microorganisms by analyzing their metabolic byproducts using chromatography.

10. Vitamin and Nutrient Analysis: Chromatography is utilized for the quantification of vitamins and essential nutrients in biological samples, contributing to nutritional assessments.

Dr. Pramila Singh