What is Chromatography?


Chromatography is a laboratory technique that helps separate mixtures. It involves mixing a mixture in a solvent (the mobile phase) and passing it through a system to separate the different components. The mixture then passes through a stationary phase that consists of fixed materials. Chromatography can be used to analyze a variety of samples.

Column chromatography

Column chromatography uses stationary and mobile phases to separate the mixture into distinct components. The separation is performed by removing the more significant components than the mobile phase. The eluted fractions are collected in different containers and analyzed for desired components. The mixture is either preadsorbed on the sorbent or inert media or can be directly added to the column.

Column chromatography involves the separation of compound mixtures by their polarity. The most polar compound will travel first through the column, while the most minor polar compound will move last. The green compound will collect in a clean test tube near the end of the column. The red and blue compounds are then collected separately.

The two types of solvent systems used in column chromatography are isocratic and gradient systems. Isocratic chromatography uses one solvent, while the gradient system uses two solvents with predefined ratios. These systems separate the components of plant extracts.

Ion-exchange chromatography

Ion-exchange chromatography is a method that relies on the difference in charge between solutes. Resins in the stationary phase are charged, and the resin’s charge displaces solute species. For example, negatively charged proteins displace chlorine anions from the stationary-phase resin since the negatively charged protein has a higher negative charge at the pH of the mobile phase. In addition, adjusting the pH of the mobile phase can manipulate the charge of solutes.

The ion exchange column is made of stainless steel, titanium, glass, or inert plastic. The diameter can vary from 2mm to 5 cm. After loading the sample in the column, the polar analyte is electrostatically bound to the oppositely-charged ions. The number of interactions between the target analyte and the stationary phase determines the retention time. The bonded analytes are then eluted using an aqueous mobile phase from the column. The eluent is pH-controlled to make sure that the sample is stable.

Ion-exchange chromatography is a widely used technique for biomolecule purification. Its principle is simple: molecules are separated according to their charges. The interaction between charged molecules in a sample and oppositely charged moieties in a stationary phase makes it possible to remove only those negatively charged molecules. Ion exchange chromatography is a highly versatile technique that can separate large biomolecules.

Thin-film chromatography

Thin-film chromatography is a method to separate mixtures of non-volatile organic compounds using a thin layer of adsorbent material. It is a speedy and straightforward method that can be used to separate various types of compounds. The sample and reference compounds are dissolved in the appropriate solvent and applied to the bottom of the chromatography plate. The mobile phase (solvent) is then allowed to run up the plate, separating the compounds of interest.

Using this technique, you can distinguish between different kinds of compounds by their differences in Rf values, measuring their distances. These values are measured in cm. For example, the Rf values of two different types of compounds are different when they are separated using thin-film chromatography. Therefore, it is essential to consider these differences when identifying different compound types using this method.

Thin layer chromatography is a valuable technique for separating multi-component pharmaceutical formulations. This technique utilizes a glass plate coated with silica gel to separate the components. Its advantage is that it is inexpensive and takes only a few minutes. This method is sometimes used as a preliminary analytical method before HPLC. In addition, it is an effective way to analyze impurities in various substances.

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