Chromatography Techniques & Key Components

Chromatography Techniques & Key Components

Chromatography: Overview

Chromatography is an analytical technique used in labs to separate different components individually from a sample mixture. The separation of the components involves distributing them into two phases

  • Mobile phase: The phase comprises a gaseous or liquid component, where molecules move in a definite direction.
  • Stationary phase: It’s a solid phase or a liquid layer adsorbing on a solid surface. Here components remain stationary with no movement. 

The factors that influence the separation of the components include different molecular characteristics such as partition (liquid-solid), adsorption (liquid-solid), and affinity or molecular weight differences. 

They determine whether molecules will stay longer in the stationary phase, move slower, or move faster in the systems. The time each component takes in its elution from the stationary phase is known as the retention time. 

There are different types of chromatographic separation techniques available, including column chromatography, high-performance liquid chromatography (HPLC), gas-liquid chromatography, and thin-layer chromatography (TLC). 

They all serve different purposes, as gas-liquid chromatography is used to separate esther, alcohol, lipid, and amino groups from a complex mixture, while the molecular-sieve chromatographic method has applications in determining the molecular weights of proteins.

The applications of chromatography include drug development, detecting trace amounts of chemicals or components of a mixture, protein purification, leaf pigment separation, and testing the quality of water and air.

In this article, we will review more about the different types of chromatographic separation methods.  

Key Components of Chromatography Systems

Different chromatographic techniques involve different instrumentation based on their working mechanisms. However, two common systems in each system include:

  • Stationary Phase: It's either an immobile solid or liquid phase and serves as a substrate for the separation of components of the mixture. 
  • Mobile Phase: It’s either a liquid or gaseous phase in which components get separated based on their physicochemical properties. The flow rates of components in the phase are determined by the difference in their chemical properties. 
Figure: An illustration of different chromatographic phases. 
  • Chromatography column: Columns are the space matrix where the separation of analytes takes place. They come in different lengths and diameters based on chemical analysis applications. For example, the HPLC column is typically 25 cm long, has a 4.6 mm diameter, and is packed with silica beads. Gas chromatography involves two types of columns—capillary and packed columns—with both having different lengths and widths.   
  • Sample Injection System: In this system, the sample to be separated is added. It also acts as a controller to switch between different mobile phases. The system can be controlled manually or automated. 
  • Detectors: It’s used for the detection of analytes after separation. The three types of detectors used include variable wavelength detectors, fixed wavelength detectors, and diode array detectors.

The Types of Chromatography Techniques

Various types of chromatographic techniques have been developed to serve a range of applications. 

Gas Chromatography

It is also known as gas-liquid chromatography. It involves equipment like packed or capillary columns, a chromatogram, an autosampler, and a detector. It has a liquid stationary phase and a mobile phase composed of inert gas, which are passed through the chosen column at high pressure for effective separation of components. 

The technique is used in the food industry for the quantification of contaminants and compounds, such as carbohydrates and vitamins, in food samples. Further, it’s also used in air quality testing, drug testing, pesticide detection, and product safety testing.

Paper Chromatography 

The technique is also known as liquid-liquid chromatography. In this method, a cellulose-made thick filter paper acts as a solid support, water drops settled in its pores act as a stationary phase, and a fluid placed in the developing tank acts as a mobile phase. 

The technique is extensively utilized in the separation of plant pigments (such as photosynthetic pigments) and proteins from a complex mixture. 

Figure: Paper chromatography illustration.

Thin-Layer Chromatography 

It’s also known as solid-liquid chromatography. Here, the stationary phase is a glass plate coated with a solid adsorbent substance, such as silica gel, alumina, or cellulose. It’s a versatile technique used in labs to simultaneously separate multiple samples. 

The sample mixture placed in the stationary phase gradually starts moving upward through capillary action driven by the organic solvent used in the process. The movement rate of components depends on the solid phase, the polarity of the material, and the solvent

The TLC technique has uses in identifying natural products, such as fixed oil and waxes, and the separation of metabolites from a  range of samples, including blood plasma and serum. 

Figure: Thin-layer chromatography

Partition Chromatography

The chromatographic technique works on the principle of separation of molecules based on the difference in their partition coefficient between two liquid phases. The other chromatographic technique that works on similar principle includes Reverse phase partition, Liquid-liquid partition, and Thin-layer chromatography.

These methods are crucial for separating and identifying various biomolecules, such as fatty acids, amino acids, and carbohydrates. 

Affinity Chromatography 

The separation of biomolecules from a sample mixture based on their macromolecular binding interaction with the substrate is known as affinity chromatography. The interactions widely exploited in labs for research workflows include interaction between:

  • Enzyme and substrate
  • Protein and nucleic acid
  • Antigen and antibody
  • Receptor and ligand

The technique is used to purify many biomolecules, including hormones, nucleic acids, antibodies, and specific proteins. 

Size-Exclusion Chromatography 

The technique is also known as gel filtration. During the process, the molecules are separated based on their size by filtration through the gel. The small molecules move into pores, while large molecules remain outside and get washed out in the column's empty space. Therefore, they are separated and eluted based on their decreasing molecular weight. 

It’s an effective technique for analyzing different proteins, studying the properties of biomolecules, and checking the quality of samples. 

Ion-Exchange Chromatography 

The basic principle of ion exchange chromatography is the exploitation of the interaction between solid support matrix and charged protein groups. The proteins are separated either based on the ionic strength or pH change of a buffer solution. The two types of ion-exchange chromatography include Cation exchange chromatography and anion-exchange chromatography.

The technique is widely used in labs to separate biomolecules such as nucleotides, amino acids, and peptides from a sample mixture. 

Figure: Ion Exchange Chromatography

Enhance Chromatography Precision With Excedr

Chromatography is an analytical technique used in labs to separate and study individual components from a mixture of the sample. The technique involves solid support and a stationary and mobile phase for the separation of biomolecules based on their different properties. 

Different types of chromatography include affinity, column exchange, and gas chromatography techniques. They all have different work mechanisms and serve different purposes. If you’re looking for an affordable option to enhance chromatography precision, consider leasing chromatography equipment through Excedr.

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Leasing through Excedr not only saves you time but also more money at hand to invest in other lab operations. How? As it doesn’t require you to pay expensive upfront costs or repair and maintenance charges. Everything is covered under our leasing program.

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