CHROMATOGRAPHY I 2024

CHROMATOGRAPHY

Chromatography is largely used laboratory technique employed to separate, identify, and analyze complex mixtures of substances. Thus plays a crucial role in various scientific researches like in chemistry, biology, pharmaceuticals and environmental findings. In these techniques a sample mixture is introduced to carry out certain processes to obtain individual constituents of the sample mixture with the aim of carrying out certain quantitative and qualitative analysis.

COMPONENTS

It is a complex analytical technique that involves several key components work together to separate and analyze mixtures. Here we are going to introduce each components.

SAMPLE – The sample is the mixture of substances that you want to separate and analyze. It consist of various compounds that would be separated and analysed.

Stationary Phase – The stationary phase play a crucial role in chromatography. It is a crucial component during the process. As the name suggest it remains stationary while the process carried out and used in various forms depending on the type of chromatography being used. It is a material that is either packed into a column (in column chromatography) or coated onto a surface (in thin-layer chromatography). Stationary phase interact with sample to carry out the processes.

CHROMATOGRAPHY

Mobile Phase -The mobile phase is a fluid that flows through the stationary phase, along with sample components with it. Like , In liquid chromatography, the mobile phase is a liquid like hexane , heptane etc, whereas in gas chromatography, it will be a gas like hydrogen, helium , nitrogen etc. The choice of mobile phase depends mainly on the chromatographic techniques and the properties of the sample.

Column– In many techniques, column is used to fill the stationary phase then sample is passed through it to carryout its separation. The column provides a controlled environment for the separation process. column chromatography is common in techniques like liquid chromatography (LC) and gas chromatography (GC).

Injector– The Injector is a component that introduces the sample into the chromatographic system. It ensures that a accurate amount of the sample is introduced into the system for separation.

Detector -The detector is a component that monitors the elution (exit) of separated components from the chromatographic system. Different types of detectors are used depending on the technique, including UV-visible detectors, mass spectrometers, flame ionization detectors (FID), and more.

Chromatogram -The detector generates signals that are used to create chromatograms, which are graphical representations of the separation and further these graphs are analyzed to get required results.

Data Analysis System – Modern chromatographic systems are often have data analysis software that interprets the signals from the detector, identifies peaks corresponding to sample components, and quantifies their concentrations. This softwares are essential for obtaining meaningful analytical results.

Temperature Control – In some chromatographic techniques, temperature control of the column or detector is important for achieving specific separation conditions or improving sensitivity.

All these components work in harmony to execute the separation and analysis of complex mixtures in chromatography.

Principals

The main principles of chromatography revolve around the separation of components in a mixture based on their differential interactions with a stationary phase and a mobile phase. This separation process is fundamental to all chromatographic techniques and is governed by several key principles like :

Selective Partitioning – Chromatography work on the properties of the varying affinities of different substances for two phases: the stationary phase (solid or liquid) and the mobile phase (gas or liquid). Components in the mixture partition between these phases to different extents based on their chemical and physical properties of each individual substance.

Equilibrium -Chromatographic separation is an Equilibrium process. As the mobile phase moves through the stationary phase, each component in the sample reaches a dynamic Equilibrium between being adsorbed onto the stationary phase and being carried along by the mobile phase.

CHROMATOGRAPHY

Retention Time – The time it takes for a component to travel the chromatographic system and come out from the column or plate is known as its retention time. Retention time is a characteristic property of each component and is influenced by factors such as the affinity for the stationary phase and the speed of the mobile phase.

Separation Based on Differences – Components with stronger interactions with the stationary phase take more time in the column or on the plate and thus have longer retention times. Conversely, components that interact more weakly with the stationary phase come out earlier. This separation is important for analyzing and identifying the individual substances within a mixture.

Detection and Analysis – A detector at the end of the chromatographic system records the elution of components as peaks in a Chromatogram. The area under each peak provides quantitative information about the amount of each substance, while the position of the peak in the Chromatogram identifies the substance based on its retention time.

Optimization – Chromatography conditions can be varied to optimize separation. Variables such as the type of stationary phase, mobile phase composition, flow rate, and column temperature can influence separation, efficiency and selectivity.

Column Packing -In column chromatography, the stationary phase is typically packed into a column. The quality and properties of the packing material are critical for efficient results.

Types of chromatography

Chromatography is a separation technique of different types, each designed for specific applications and analytical purposes. Some of the most common types of chromatography are as follows:

Liquid Chromatography

Liquid chromatography involves the separation of components in a liquid sample using a liquid mobile phase. It is further classified into below subtypes-

High-Performance Liquid Chromatography (HPLC): HPLC uses high-pressure pumps to increase separation efficiency and is commonly used for analyzing a wide range of compounds, including pharmaceuticals, chemicals, and biomolecules.

Ion Chromatography (IC): IC specializes in the separation of ions, making it ideal for analyzing inorganic and organic ions in solutions. The ion exchange chromatography consists of positively and negatively charged ions.Ion-exchange chromatography separates molecules based on their respective charged groups.

Gas Chromatography

Gas chromatography separates volatile compounds in a gaseous sample using a gas as the mobile phase. GC is frequently utilized in the analysis of volatile organic compounds, such as those found in environmental, forensic, and petrochemical applications. For example , analysis of Diesel sample.

Thin-Layer Chromatography

In Thin-Layer Chromatography or TLC, a thin layer of a stationary phase is coated on a glass or plastic plate. Liquid samples are spotted on the plate, and separation occurs as a solvent (the mobile phase) moves up the plate by capillary action. It’s often used for quick qualitative analyses of various mixture compounds in the field of pharmaceutical analysis and many others.

Paper Chromatography

Similar to TLC, paper chromatography uses paper as the stationary phase. It is a cost-effective method for separating and identifying components in mixtures, commonly used in educational and simple analytical experiments.

Size-Exclusion Chromatography (SEC)

SEC is a type of liquid chromatography that separates molecules based on their size and shape. It’s valuable for determining the molecular weight and size distribution of polymers and biomolecules.

Affinity Chromatography

This specialized technique is used to purify and separate biomolecules based on their specific interactions with ligands or antibodies immobilized on the stationary phase. It’s crucial in the isolation of proteins, enzymes, and antibodies

Chiral Chromatography

Chiral chromatography is employed to separate enantiomers, which are mirror-image isomers of molecules. It’s important in pharmaceutical research and the production of optically pure compounds.

Supercritical Fluid Chromatography (SFC)

SFC uses supercritical fluids, which exhibit both gas-like and liquid-like properties, as the mobile phase. It is used for separating compounds that are not easily analyzed by other chromatographic methods.

Flash Chromatography

Flash chromatography is a rapid form of liquid chromatography typically used for preparative work to separate and purify larger quantities of compounds in a shorter time frame.

These are just a few examples of the many chromatography techniques available. Scientists choose the appropriate type of chromatography based on the specific properties of the sample and the information they aim to obtain, making chromatography a versatile tool in analytical chemistry and various scientific fields.

Applications  

Drug Purity and Quality Control: Chromatography ensures the purity and quality of pharmaceutical compounds by analyzing raw materials, intermediates, and final products.

Pharmacokinetics: It’s used to study how drugs are absorbed, distributed, metabolized, and excreted in the body, aiding in drug development and dosing.

Pollutant Analysis: Chromatography helps identify and quantify pollutants in air, water, and soil, aiding in environmental monitoring and regulatory compliance.

CHROMATOGRAPHY

Pesticide Residue Testing: It’s used to detect pesticide residues in food and agricultural products to ensure safety.

Food Safety Testing: Chromatography is crucial for detecting contaminants, additives, and preservatives in food products, ensuring consumer safety.

Protein Purification: Chromatography separates and purifies biomolecules like proteins, enzymes, and antibodies, essential in biopharmaceutical production.

DNA Sequencing and Analysis: It’s used to separate and identify DNA fragments in genetics and genomics research.

Clinical Diagnostics: Chromatography plays a role in diagnosing diseases by analyzing blood, urine, and other bodily fluids for biomarkers and metabolites.

Drug Testing: It’s used for drug screening in clinical and forensic toxicology

Polymer Characterization: Chromatography determines the molecular weight and distribution of polymers, aiding in material development and quality control

Forensic Science :Chromatography helps identify and quantify drugs in forensic samples, providing crucial evidence in criminal investigations and analyze biological samples for toxins and poisons.

 Plant Compound Analysis: Chromatography identifies and quantifies bioactive compounds in plants, aiding in natural product research and drug discovery.

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FAQS:

What is Chromatography ?

Chromatography is largely used laboratory technique employed to separate, identify, and analyze complex mixtures of substances. Thus plays a crucial role in various scientific researches like in chemistry, biology, pharmaceuticals and environmental findings. In these techniques a sample mixture is introduced to carry out certain processes to obtain individual constituents of the sample mixture with the aim of carrying out certain quantitative and qualitative analysis.

What is the basic use of Chromatography?

chromatography is mainly used for the separation, isolation and purification of complex sample mixture into separate individual substance.

Types of Chromatography ?

There are many types of chromatography techniques , commonly used are Liquid chromatography , High-Performance Liquid Chromatography , Ion Chromatography , Gas Chromatography , Thin-Layer Chromatography (TLC) , Paper Chromatography , Size-Exclusion Chromatography , Affinity Chromatography , Chiral Chromatography , Supercritical Fluid Chromatography , Flash Chromatography etc.

What is the principle of chromatography ?

The main principles of chromatography revolve around the separation of components in a mixture based on their differential interactions with a stationary phase and a mobile phase.. This separation process is fundamental to all chromatographic techniques and is governed by several key principles like Selective Partitioning , Equilibrium and Retention Time.

Full form of TLC ?

TLC stands for THIN LAYER CHROMATOGRAPHY, a thin layer of a stationary phase is coated on a glass or plastic plate. Liquid samples are spotted on the plate, and separation occurs as a solvent (the mobile phase) moves up the plate by capillary action. It’s often used for quick qualitative analyses of various mixture compounds in the field of pharmaceutical analysis and many others.

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