Chromatography is a versatile and vital technique in the world of analytical chemistry. This method aids in separating and identifying substances in a mixture based on their movement through a stationary phase.
Principles of Chromatography
- Basic Principle: The principle underlying chromatography is that different components in a mixture will interact differently with a stationary phase and a mobile phase, resulting in the separation of these components. This interaction depends largely on the intermolecular forces between the substances.
- Stationary Phase: A solid or a liquid on a solid support. It's immobile and holds the substances temporarily.
- Mobile Phase: A liquid or a gas that moves over or through the stationary phase, carrying the mixture components with it.
- Equilibrium: Each component in the mixture achieves an equilibrium between the stationary and mobile phases. Those that interact more strongly with the stationary phase move more slowly, while those that interact more weakly move faster.
A basic procedure of chromatography.
Image courtesy of Biomedical and Biological Sciences.
Applications of Chromatography
- Purification: Chromatography is widely used for purifying samples by separating them into their individual components.
- Analysis: By analysing the separated components, one can determine the composition of a sample. This has numerous applications in forensics, quality control, and clinical labs.
- Biochemical Research: Used for DNA sequencing, drug discovery, and protein separation.
Retardation Factor (RF)
- Definition: RF value is the ratio of the distance travelled by the substance (solute) to the distance travelled by the solvent (mobile phase). It's a characteristic of each substance under specific chromatographic conditions.Equation: Rf = distance travelled by the solute/ distance travelled by the solvent
- Interpretation:
- If a substance has an RF value of 1, it has no affinity for the stationary phase and moves with the solvent front.
- An RF value of 0 indicates that the substance didn't move at all, showing a high affinity for the stationary phase.
- Different substances will have different RF values, allowing for their identification and separation based on these values.
Image courtesy of Chemistry Dictionary & Glossary
Paper Chromatography
- Basics: Uses a piece of paper, typically filter paper, as the stationary phase. The mobile phase (solvent) moves through the paper by capillary action.
- Procedure:
- A spot of the mixture is placed near the bottom of the paper.
- The paper is then placed in a solvent. The solvent moves up the paper, carrying the mixture components with it.
- As the solvent rises, the mixture components will move at different rates based on their interactions with the paper and solvent, leading to separation.
- Applications: Widely used in food science (to detect any adulterants in food), in the pharmaceutical sector (to detect chemicals in urine), and in teaching due to its simplicity.
Image courtesy of Watthana Tirahimonch
Thin Layer Chromatography (TLC)
- Basics: Uses a thin layer of silica gel or alumina on a flat, inert substrate. It's more sensitive and faster than paper chromatography.
- Procedure:
- A small amount of the mixture is spotted near the bottom of the plate.
- The plate is then placed in a suitable solvent. As the solvent rises, it separates the mixture based on each component's interaction with the stationary phase (silica or alumina) and the solvent.
- Detection: After separation, the compounds can be visualised using UV light or chemical staining, depending on the nature of the compounds.
- Applications: Used extensively in organic chemistry for separation and identification of compounds, in forensic labs for drug detection, and in manufacturing for quality control.
In the context of intermolecular forces, chromatography demonstrates the importance of understanding how different molecules interact. Such forces dictate how each component in a mixture moves and interacts with the stationary and mobile phases, allowing for their effective separation and identification.
(1) Lid (2) stationary phase (paper/silica gel or alumina on a plate) (3) Solvent front (4) Solvent
Image courtesy of Theresa Knott
FAQ
Detectors in chromatography identify and quantify the components of a mixture as they elute from the chromatograph. Common detectors include:
- UV-Visible detectors: These measure the absorbance of compounds at specific UV or visible wavelengths. They're effective for compounds that naturally absorb UV or visible light.
- Flame ionisation detector (FID): Used in gas chromatography, FID burns the eluted compounds, producing ions. The current generated by these ions is measured, and it's proportional to the concentration.
- Mass spectrometers: These provide information about the molecular weight and structure of the eluted compounds by ionising them and measuring their mass-to-charge ratio. Each detector has its advantages and is chosen based on the nature of the compounds being analysed and the information required.
Temperature can have a significant impact on the efficiency and results of a chromatographic process. It influences the viscosity of the mobile phase and the solubility of the components in the mixture. Generally, as temperature increases, the viscosity of the mobile phase decreases, leading to faster flow rates. However, higher temperatures might also increase the solubility of some compounds, affecting their retention times. Stability of the compounds at elevated temperatures must also be considered. It's essential to maintain consistent temperatures during chromatography to achieve reproducible results.
Not always. While chromatography is highly effective, some mixtures with components that have very similar intermolecular interactions with the stationary and mobile phases might not be entirely separated in a single run. In such cases, multiple chromatographic runs using different conditions, or a combination of different chromatographic techniques, might be necessary to achieve complete separation.
The choice of solvent, or the mobile phase, in chromatography is paramount because it determines how each component in the mixture interacts with the stationary phase. A good solvent should dissolve all the components of the mixture efficiently. Furthermore, the differences in solubility and interactions between the solvent and the individual components lead to their separation. The polarity, strength, and compatibility of the solvent with the mixture and stationary phase must be considered. An inappropriate solvent might not separate the components effectively, or at all, leading to inaccurate results.
The Rf (retardation factor) value represents the ratio of the distance travelled by a compound to the distance travelled by the solvent front. It can vary due to several reasons:
- Nature of the solvent: Different solvents can interact differently with the same compound, altering its movement on the stationary phase.
- Stationary phase characteristics: Variations in the thickness, amount, or type of stationary phase can influence Rf values.
- Temperature: As temperature affects solubility and viscosity, it can lead to changes in the rate at which a compound moves through the chromatographic medium.
- Sample application: Overloading or changes in the sample's application method might affect its migration. Consistency in experimental conditions is crucial to achieve reproducible Rf values.
Practice Questions
Chromatography operates on the principle that different components in a mixture interact differently with a stationary phase and a mobile phase, leading to their separation. The key to this separation is the varying intermolecular forces between the molecules of the mixture and the molecules of the stationary and mobile phases. Components with stronger interactions with the stationary phase will move slower as they're retained more, whereas those with weaker interactions will move faster. The differences in these interactions ensure that each component has a distinct movement, resulting in their separation over the chromatographic medium.
Paper chromatography employs filter paper as its stationary phase, utilising the absorbent nature of paper. The mobile phase moves via capillary action through the paper. It's commonly used in teaching, food science to detect adulterants, and in pharmaceuticals to detect chemicals in urine. On the other hand, thin layer chromatography (TLC) uses a thin layer of a solid substance like silica gel or alumina on an inert substrate as its stationary phase. TLC provides faster and more sensitive results compared to paper chromatography. It's extensively used in organic chemistry for separating and identifying compounds, in forensic labs for drug detection, and in manufacturing for quality control purposes.
