What is chromatography and how does it work?

Scientists have discovered and refined ways to unravel the mysteries of complex mixtures, separating components with precision and accuracy. This is the fascinating realm of chromatography – which has developed into a powerful analytical and preparative technique for analyzing substances and purifying process streams. From the vibrant pigments in plants to the intricate compounds in pharmaceuticals, chromatography plays a crucial role in various fields, offering insights that drive innovation, discovery, and commercial production of many types of therapeutics.

In this article, we will explore the meaning of chromatography, its principles, methods, benefits, types, applications, and more. Whether you're a curious learner or a seasoned professional, this comprehensive guide will provide you with a deeper understanding of chromatography and why it’s important in the scientific world.

History of chromatography

Chromatography was first developed by Russian botanist Mikhail Tsvet in 1903 while studying plant pigments. Tsvet discovered that by passing a solution of pigments through a column packed with calcium carbonate, he could separate the pigments based on their different affinities for the column packing, the stationary phase. This technique, named "chromatography" from the Greek words for "color" and "to write," laid the foundation for modern chromatography.

Despite its early promise, chromatography only gained widespread recognition in the mid-20th century. Major advancements were made by Archer John Porter Martin and Richard Laurence Millington Synge, who co-developed partition chromatography and earned the Nobel Prize in Chemistry in 1952. Thin layer chromatography (TLC) was developed in the 1930s to separate nonvolatile components but wasn’t broadly used until the 1950s. The introduction of gas chromatography (GC) in the 1950s and high-performance liquid chromatography (HPLC) in the 1960s further expanded the technique's capabilities, allowing for the separation of compounds with high resolution and sensitivity.

Liquid chromatography has expanded beyond HPLC to include ultra-high pressure liquid chromatography (UHPLC) and fast performance liquid chromatography (FPLC). Stationary phases for FPLC have evolved to include including resins, membranes, fibers, and monoliths using a variety of chemistries. These advances have broadened chromatography’s applications across many fields, including pharmaceuticals, environmental science, food and beverage, forensics, and biotechnology. Today, it’s an indispensable tool in preparative and analytical chemistry as well as biologic production.

Principles of chromatography

Now, let’s have a look at some of the basic principles of chromatography:

• Stationary phase: A liquid or solid phase that remains fixed in place and interacts with the components of the mixture.
• Mobile phase: A liquid or gas phase that moves through the stationary phase, carrying the components of the mixture.
• Separation mechanism: Components interact to different degrees with the stationary phase due to differences in their physical or chemical properties, such as size, charge, solubility, or affinity.
• Differential partitioning:
Based on these differences, components in a mixture separate within the stationary phase or flow through with the mobile phase.
• Detection: Separated components are detected and quantitated using various methods including UV-vis spectroscopy, mass spectrometry, or fluorescence detection.
• Resolution: The effectiveness of separation is determined by the resolution, which measures how distinctly the components are separated from each other.

How does chromatography work?

Chromatography works by exploiting the differences in the chemical or physical properties of the components in a mixture. These differences can include charge, solubility, affinity, or size. The process involves:

1. Sample introduction: The mixture is introduced into the chromatography system, typically through an injection port or application onto a stationary phase.
2. Separation: The components are separated as they travel through the stationary phase. This separation is driven by the interactions between the components and the stationary phase, as well as the flow and composition of the mobile phase.
3. Detection: The separated components are detected and may be quantitated using various detection methods, such as UV-vis spectroscopy, mass spectrometry, or fluorescence detection.
4. Collection: For preparative purposes, different portions can be collected to allow processing of fractions identified as containing the molecule of interest.

Types of chromatography

Here are the main methods of chromatography explained in detail:

Kinds of chromatography	Description
Thin layer chromatography (TLC)	Uses a thin layer of stationary phase (usually silica gel or alumina) coated onto a flat, inert substrate like glass or plastic. Sample applied as a small spot near the bottom of the plate. Plate placed in a solvent; solvent travels up the plate by capillary action. Different components travel at different rates, resulting in separation. Commonly used for monitoring reactions, identifying compounds, and checking the purity of samples.
Gas chromatography (GC) Explore gas chromatography with Leica Microsystems	Uses a gaseous mobile phase (carrier gas) with a liquid or solid stationary phase within a column or a simple capillary. Sample vaporized and injected into the column. Carrier gas transports the sample through the column. Components interact with the stationary phase and separate based on volatility, size, and affinity. Highly effective for analyzing volatile and semi-volatile compounds, such as environmental pollutants, essential oils, and flavors.
Liquid chromatography (LC) Explore liquid chromatography with Cytiva	Uses a liquid mobile phase to separate components of a mixture. FPLC, HPLC and UHPLC employ pressure to push the mobile phase through a column or other housing packed with the stationary phase, which is usually particles. In addition to chromatography resins, FPLC may use membranes, monoliths, or fiber-based matrices. Sample applied manually to or injected into the column or other housing; components separated based on interactions with the stationary phase. Widely used as an analytical technique in pharmaceuticals, biochemistry, and environmental science for high resolution and sensitivity. Preparative liquid chromatography is a critical part of most biopharmaceutical production schemes.
Paper chromatography	This variant of partition chromatography uses paper as the stationary phase. Small spot of the sample applied to the paper. Paper placed in a solvent; solvent moves through the paper by capillary action. Different components travel at different rates, resulting in separation. Often used in educational settings and for separating pigments, amino acids, and other small molecules.

Applications of chromatography

Chromatography is used in various industries, including:

• Pharmaceuticals:

  - Chromatography is essential in drug discovery, development and quality control.
  - It is used to purify active pharmaceutical ingredients (APIs), analyze the composition of drug formulations, and help protect the safety and efficacy of medications.
  - Techniques like HPLC and GC are commonly employed to detect impurities, degradation products, and to quantitate active compounds in pharmaceutical products.

• Environmental science:

  - Chromatography plays a crucial role in monitoring and analyzing environmental pollutants. It is used to detect and quantitate contaminants in air, water, and soil samples. For example, GC is used to analyze volatile organic compounds (VOCs) in air, while HPLC is used to detect pesticides and herbicides in water and soil.
  - Chromatography helps in assessing environmental health and ensuring compliance with regulatory standards.

  Food and beverage:

  - Chromatography is used for quality control and safety testing in the food and beverage industry.
  - It helps in detecting contaminants, such as pesticides, mycotoxins, and additives, ensuring that products meet safety standards.
  - HPLC and GC are commonly used to analyze flavors, colors, preservatives, and nutritional content in food and beverages, ensuring product consistency and quality.

• Forensics:

  - Chromatography is a valuable tool in forensic science for analyzing evidence from crime scenes.
  - It is used to detect and identify drugs, toxins, and other substances in biological samples, such as blood, urine, and hair.
  - GC and HPLC are frequently used in toxicology to determine the presence of drugs and poisons, providing critical information for criminal investigations and legal proceedings.

• Biotechnology:

  Chromatography is used in biotechnology for the purification and analysis of biomolecules, such as proteins, nucleic acids, and metabolites.
  - In FPLC, chemistries including affinity chromatography and ion exchange chromatography are used to isolate and purify specific biomolecules from complex mixtures. Size exclusion chromatography is used to analyze size.
  - Chromatography is an essential tool in research, diagnostics, and the production of biopharmaceuticals.

Conclusion

Chromatography is a versatile and essential technique in both analytical chemistry and bioprocessing. Its ability to separate and quantitate components in a mixture makes it invaluable across numerous industries.

Understanding the principles, methods, and applications of chromatography can provide insights into its critical role in scientific research and industry.

Frequently asked questions (FAQs)

1. What is chromatography and what is it used for?

Chromatography is a technique used to separate and quantitate components in a mixture. It is widely used in pharmaceuticals, environmental science, food and beverage, forensics, and biotechnology.

2. How does chromatography work?

Chromatography works by exploiting differences in the physical or chemical properties of components in a mixture. These differences can include size, charge, solubility, or affinity for the stationary phase. The process involves introducing the sample, separating the components, and detecting them.

3. What are the main types of chromatography?

The main types include thin-layer chromatography (TLC), gas chromatography (GC), liquid chromatography (LC), and paper chromatography. Each type has unique characteristics and applications.

4. What are the benefits of chromatography?

Chromatography offers the ability to analyze complex mixtures, versatility in applications, and both quantitative and qualitative analysis. It’s essential for trace analysis and detailed composition studies. Liquid chromatography supports high resolution and sensitivity.

5. What are some examples of chromatography uses?

Examples include drug development and quality control in pharmaceuticals, pollutant analysis in environmental science, quality control and safety testing in food and beverages, forensic testing in criminal investigations, protein and DNA analysis in biotechnology, and purification and analysis of biomolecules in bioprocessing.