Tomography Concept, Principle, and Classification
First, the concept:
Chromatography is a separation technique that relies on the differences in physical and chemical properties of components in a mixture, such as adsorption, molecular size, shape, polarity, affinity, and partition coefficient. This method allows for the effective separation and analysis of complex mixtures.
The technique was first introduced in 1903 by Russian botanist Mikhail Tsvet, who used it to demonstrate that plant leaves contain not only chlorophyll but also other pigments. He applied adsorption chromatography, which laid the foundation for modern chromatographic methods. Today, chromatography is widely used in biochemistry, molecular biology, and analytical chemistry as a powerful tool for purification and identification.
Second, the principle:
Chromatography works based on the differential interaction between the analyte and two phases: the stationary phase and the mobile phase. The sample is carried through the stationary phase by the mobile phase, and the different affinities of the components result in their separation.
Common substances separated using this technique include sugars, organic acids, amino acids, and nucleotides.
Third, classification:
There are several ways to classify chromatography:
1. Based on the physical state of the two phases:
- Gas chromatography (GC): includes gas-liquid and gas-solid chromatography.
- Liquid chromatography (LC): includes liquid-liquid and liquid-solid chromatography.
2. Based on the method of operation:
- Column chromatography: involves packing the stationary phase in a column and moving the sample through it.
- Thin-layer chromatography (TLC): uses a thin layer of stationary phase on a plate.
- Paper chromatography: uses filter paper as the stationary phase.
3. Based on the separation mechanism:
- Adsorption chromatography: separation based on adsorption strength.
- Partition chromatography: depends on the distribution coefficient (Kd) between two immiscible phases.
Kd = Ca / Cb, where Ca is the concentration in the mobile phase, and Cb is the concentration in the stationary phase.
- Ion exchange chromatography: separates ions based on their charge interactions with an ion exchanger.
- Gel filtration chromatography (size-exclusion): separates molecules based on their size.
- Affinity chromatography: uses specific ligand-receptor interactions for selective binding.
- Hydrophobic interaction chromatography: separates based on hydrophobicity.
- Reversed-phase chromatography: uses a non-polar stationary phase and polar mobile phase.
Fourth, gel chromatography (also known as gel filtration or size-exclusion chromatography):
Gel chromatography is a technique that separates molecules based on their size as they pass through a porous gel matrix. The gel acts like a sieve, allowing smaller molecules to enter the pores and elute later, while larger molecules are excluded and elute earlier.
Principle:
The stationary phase consists of a three-dimensional network of pores. Common gels include agarose, polyacrylamide, and dextran. For example, Sephadex G series gels vary in crosslinking degree (G10 to G100), with the number indicating the water content. Different gels are selected depending on the molecular weight of the target compounds.
Separation process:
As the sample moves with the eluent, the components separate based on their size and ability to penetrate the gel pores.
Main factors affecting gel chromatography:
1. Column selection and packing: The column size should match the sample volume and desired resolution. The gel must be evenly packed without air bubbles.
2. Mobile phase: A buffer solution containing salt is often used to prevent gel adsorption. The choice depends on the sample’s solubility and stability.
3. Sample loading: Typically, 1–5% of the gel bed volume is loaded for general separations.
4. Gel regeneration: Dextran gels can be regenerated using NaOH and NaCl solutions.
Fifth, ion exchange chromatography:
Ion exchange chromatography separates ions based on their interaction with charged groups on the stationary phase. The mobile phase contains an electrolyte solution that facilitates reversible ion exchange.
Principle:
The stationary phase contains ion-exchange groups that attract oppositely charged ions from the mobile phase. This technique is widely used for separating proteins, nucleic acids, and other charged molecules.
Types of ion exchangers:
- Anion exchangers: positively charged, bind anions.
- Cation exchangers: negatively charged, bind cations.
Common materials include ion exchange resins and cellulose-based matrices.
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