Basics of Chromatography Operation

What is Chromatography?

• Chromatography is a versatile and powerful laboratory technique used to separate and analyze the mixtures of various compounds based on their physical and chemical properties. 
• Chromatography is an essential technique in many scientific and industrial settings that permits separation      and analysis of complex mixtures with high precision.
• Various parameters such as stationary phase, mobile phase composition, flow rate, and temperature influence the separation in chromatography.
• Optimization of these parameters is essential for successful separations.
• Chromatography is a widely used separation technique in chemistry and analytical science including chemistry, biochemistry, pharmaceuticals, and environmental science. 
• The chromatogram provides information about the number of components, their identities (based on retention times), concentrations, and purity. Researchers can interpret the data to draw conclusions about the composition of the sample.
• Chromatography can take many forms, such as gas chromatography (GC), liquid chromatography (LC), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and many others. Each type of chromatography has its own specific applications and requirements, making it a versatile tool for a wide range of analytical and preparative tasks.

Principles of Chromatography:

• Chromatography works on the principle of differential partitioning.
• The basic principle of chromatography is that different compounds in a mixture will move at different rates through a medium, allowing for their separation. 
• It is used to separate, identify, and quantify a mixture into its individual components based on their different interactions with a stationary phase and a mobile phase. 
• Here, the stationary phase is a solid or liquid support.
• The mobile phase is a fluid that conveys the sample or mixture via the stationary phase.
• In chromatography system, a detector records the elution times and generates a chromatogram, a graphical representation of signal intensity versus time for each compound. This data offers valuable information about the composition and concentration of the sample.

Components of Chromatography 

Chromatography is classified into two phases 

1. Stationary Phase: 

• This is a solid or liquid material that is fixed only in one place and doesn’t move during the process. 
• The sample is applied to the stationary phase, and separation occurs as components interact differently with this phase. 
• A common stationary phase consists of silica gel, alumina, and paper for column, thin-layer, and paper chromatography, respectively.

2. Mobile Phase: 

• This is a fluid-type material such as liquid or gas that moves through the stationary phase, carrying the sample components with it. 
• The choice of mobile phase depends on the kind of chromatography being used. 

For example, in 

1. Liquid Chromatography (LC): The mobile phase is typically a liquid solvent.
2. Gas Chromatography (GC): it’s a carrier gas like helium or nitrogen.

Basics of Chromatography Operation

Sample Application: 

• The mixture which is required to be segregated is applied to the stationary phase. 
• This is often done by injecting a small amount of the sample onto a column in case of column chromatography and spotting it onto a plate in case of thin-layer chromatography.

Separation Process: 

• As the mobile phase flows through or over the stationary phase, the components of the sample will interact differently. 
• Some components will be more attracted to the stationary phase, causing them to move more slowly, 
• While the remaining components will be carried along more quickly by the mobile phase.

Detection: 

• A detector is used to monitor the components as they come out from the column or move across the stationary phase. 
• Various kinds of detectors are being used based on the type of chromatography and the compounds being analysed. 
• Some common detectors are shown below.

1. Ultra-violet visible spectrophotometers, 
2. Mass Spectrometers, and 
3. Refractive Index Detectors.

Data Analysis: 

• The data generated by the detector is used to produce a graph of detector response versus time or volume known as a chromatogram.
• Peaks in the chromatogram represent separated compounds. 
• The area under each peak can be used to quantify the amount of each compound.

Retention Time: 

• The time taken by the compound to elute from the column or move through the stationary phase is called its retention time. 
• Each compound has a characteristic retention time in a given chromatographic system making it useful for identification.

Classification of Chromatography

Chromatography is classified into various techniques based on its own specific principles and applications.

Some common types of chromatography techniques are discussed below

Liquid Chromatography (LC): 

Liquid Chromatography uses a liquid mobile phase and is commonly used for compounds that are dissolved in a liquid.

1. High-Performance Liquid Chromatography (HPLC): A form of liquid chromatography that uses high pressures to enhance separation efficiency.
2. Ultra-High-Performance Liquid Chromatography (UHPLC): It is similar to High-Performance Liquid Chromatography, but it functions at even higher pressures to furnish faster separations.
3. Reverse-Phase Chromatography (RP-HPLC):  Separates compounds based on their hydrophobicity.
4. Ion-Exchange Chromatography (IEC):  Separates ions based on their charge.
5. Size-Exclusion Chromatography (SEC): Separates molecules based on their size and shape, with larger molecules eluting faster since they don’t penetrate the porous stationary phase commonly used for biomolecules.

Gas Chromatography (GC): 

Uses a gaseous mobile phase and is ideal for volatile compounds.

1. Gas-Liquid Chromatography: Uses a liquid stationary phase coated on a solid support.
2. Gas-Solid Chromatography: Employs a solid stationary phase, such as silica or alumina.
3. Capillary Gas Chromatography: Uses very narrow columns for improved separation efficiency.

Thin-Layer Chromatography (TLC): 

• Utilizes a thin layer of adsorbent material on a solid support for separation.
• Involves applying a sample to a thin layer of adsorbent material on a solid support (usually a glass plate). 
• The sample is then separated as the mobile phase travels through the stationary phase.

Affinity Chromatography

Utilizes a stationary phase designed to bind specifically to a target molecule or class of molecules, allowing for highly selective separations.

Column Chromatography: 

• Involves packing a column with a stationary phase and passing the sample through it.
• This technique utilizes a column packed with a stationary phase (e.g., silica gel or alumina). 
• The sample is applied to the top of the column, and as the mobile phase is passed through, the components are separated based on their interactions with the stationary phase.

Paper Chromatography

• Involves the use of filter paper or cellulose paper as the stationary phase. 
• It is simple and often used in educational settings.

Chiral Chromatography:

Separates enantiomers (mirror-image isomers) of compounds, which are important in pharmaceutical and chemical industries.

Supercritical Fluid Chromatography (SFC):

• Uses supercritical fluids as the mobile phase, typically carbon dioxide with a co-solvent. 
• Supercritical Fluid Chromatography combines properties of both liquid and gas chromatography.

High-Throughput Chromatography:

A rapid method used in drug discovery and combinatorial chemistry to screen large numbers of compounds quickly.

Paper Electrophoresis:

A variant of chromatography that uses an electric field to separate charged molecules on paper.

Planar Chromatography:

Includes techniques like thin-layer chromatography (TLC) and paper chromatography that are conducted on flat surfaces rather than in columns.

Flash Chromatography:

A faster form of column chromatography is used for purification, often in organic chemistry laboratories.

Separation techniques of Chromatography

Applications of Chromatography: 

• Chromatography is used in a wide range of fields, including chemistry, biochemistry, pharmaceuticals, food analysis, environmental analysis, and more. 
• It can be used for quantitative and qualitative analysis.

Frequently asked Questions

What is Chromatography?

• Chromatography is a versatile and powerful analytical tool that helps scientists separate and analyze complex mixtures efficiently and accurately. 
• The specific details of how chromatography is performed can vary depending on the type of chromatography and the goals of the analysis.

What are the two phases in Chromatography?

Stationary Phase, Mobile Phase

Define the term Optimization in Chromatography 

The choice of stationary phase, mobile phase, and other conditions (such as temperature and flow rate) can be optimized to achieve the desired separation and analysis.

What is a chromatogram?

Chromatogram is a data generated by the detector that is used      to produce a graph of detector response versus time or volume.

What are the steps of chromatographic analysis?

Chromatographic analysis consists of four steps

1. Sample Collection, 
2. Sample Injection, 
3. Sample Separation, 
4. Sample Detection.

What are the types of chromatography?

Chromatography is classified into 

1. Liquid Chromatography, 
2. Gas Chromatography, 
3. Thin-Layer Chromatography 
4. Paper Chromatography

What is the main objective of chromatography?

The main objective of chromatography is to segregate the various substances that form a mixture.

What are the five mechanisms of chromatography?

Chromatography is divided into five mechanisms based on their separation mode

1. Adsorption,
2. Distribution, 
3. Exchange, 
4. Exclusion 
5. Affinity.

What is RF in chromatography?

In chromatography, the term RF is defined as Retention Factor

List out common liquid solvents used in chromatography.

Common liquid solvents used in chromatography, water, methanol, isopropanol, acetonitrile and formic acid,