Chromatography
Chromatography is an important analytical technique used extensively in chemistry to separate, identify, and quantify components in a mixture. The principle of chromatography is based on differential partitioning between mobile and stationary phases. This method separates components between two phases: a stationary phase that has a large surface area and a mobile phase that moves through or over the surface of the stationary phase. Components in a mixture distribute themselves between the stationary and mobile phases, which varies from one component to another.
History and development
The technique of chromatography was first developed by Russian botanist Mikhail Tsvet in the early 1900s. He used the method to separate plant pigments by passing them through a column filled with calcium carbonate. The distinct colored bands that appeared gave rise to the term "chromatography" (chroma = color, graphe = writing). Since then, chromatography has evolved considerably and now includes many techniques, such as gas chromatography (GC), liquid chromatography (LC), and more specialized methods such as supercritical fluid chromatography.
Fundamentals of chromatography
At its core, chromatography involves a stationary phase and a mobile phase. The stationary phase is a solid or a liquid suspended over a solid, while the mobile phase can be a liquid or a gas. When a mixture is introduced into the chromatography system, it becomes distributed between the stationary and mobile phases. Separation occurs as a result of the different affinity of the components towards the two phases.
The distribution of a component between phases is often described by a partition coefficient:
K = (Concentration in stationary phase) / (Concentration in mobile phase)
If a component has a high affinity (large K
value) towards the stationary phase, it will move slowly. In contrast, components with a low affinity (small K
value) towards the stationary phase will move faster along the mobile phase. This difference in speed is what causes the separation.
Types of chromatography
1. Gas chromatography (GC)
Gas chromatography is commonly used for volatile compounds. In GC, the mobile phase is an inert gas such as helium or nitrogen, while the stationary phase is a liquid or solid base inside the column. When injected, the sample components vaporize and are carried through the column by the gas. Separation occurs by the interaction of each component with the stationary phase.
2. Liquid chromatography (LC)
Liquid chromatography is primarily used for non-volatile and thermally unstable compounds. The mobile phase in LC is a liquid, and the stationary phase can be a liquid on a solid or inert support. High performance liquid chromatography (HPLC), a type of LC, uses high pressure to push the solvent through packed columns, achieving rapid and efficient separation.
3. Thin layer chromatography (TLC)
Thin layer chromatography is a simple and quick method for analyzing mixtures. A flat glass, plastic, or aluminum plate is coated with a thin layer of an adsorbent material, usually silica or alumina. A drop of the sample is placed near the bottom of the plate, and it is then placed in a solvent. As the solvent rises through capillary action, it carries the components of the mixture at different rates due to different interactions with the stationary phase, resulting in separation.
Visualization of chromatography
Let us look at various aspects of chromatography with examples in vector graphics.
This figure shows a schematic illustration of a chromatographic separation, with three different components eluting at different times and distances along a column, shown as red, green, and blue circles.
Applications of chromatography
Chromatography is indispensable in a variety of scientific fields:
- Pharmaceuticals: Chromatography helps in purity testing, analyzing pharmacokinetics, and quality control.
- Environmental science: Detects pollutants such as pesticides or PCBs in water and soil samples.
- Clinical diagnosis: Analyzing biological fluids for diagnostic markers or toxins.
- Food and beverage: Ensures food safety and tests for contaminants or additives.
Example of chromatography process
Consider an example of HPLC analysis of caffeine in a beverage sample:
1. Preparation: Prepare the sample by filtering it to remove particulate matter.
2. Calibration: Inject standard caffeine solutions to calibrate the HPLC system.
3. Injection: Introduce the prepared sample into the HPLC instrument.
4. Separation: The mobile phase, often a mix of water and acetonitrile, separates caffeine as it passes through the column.
5. Detection: The detector, usually a UV detector, quantifies the amount of caffeine by measuring absorbance at a specific wavelength.
6. Analysis: Compare the sample's signal to the standard curve to determine caffeine concentration.
Factors affecting chromatography
Several factors can affect the quality and efficiency of a chromatographic separation:
- Flow rate of the mobile phase: Higher flow rates can decrease retention time but may affect resolution.
- Temperature: In GC, the column temperature can significantly affect the separation, and influence the volatility of the analytes.
- Column dimensions: The length and internal diameter of the column can affect the separation efficiency and time.
- Stationary phase properties: The choice of material in the stationary phase can affect the selectivity and interaction with analytes.
Conclusion
Chromatography is a powerful separation technique in analytical chemistry that provides invaluable information about the composition and quality of various materials. Its versatility in a variety of phases and detection methods makes it an important tool for research and industry applications. As technological and scientific advancements continue, the methods and applications of chromatography are likely to expand and become even more efficient and accurate.