Chromatography

Chromatography is an analytical technique used to separate a given mixture into its components. The technique is based on the principle that when a mixture and a mobile phase are allowed to flow over a stationary phase, the separation occurs based on the differential affinities of the components for these 2 phases. Chromatography is a commonly used technique in clinical laboratories for diagnosing inborn carbohydrate, protein, and lipid metabolism errors. The parameters quantified are vitamins, hormones, metabolites, tumor markers, and drugs in body fluids. In pharmacology, chromatography is used to estimate the purity and potency of drugs. Chromatography can analyze environmental samples for drugs, toxins, and pollutants and help discover many new biomolecules, providing insights into disease mechanisms and biomarker discovery. The chromatography phases refer to the stationary and mobile phases involved in the separation process. Here are the main phases of chromatography: The mobile phase is a liquid or gas that carries the sample and propels the compounds through the stationary phase, resulting in separation. By adjusting the composition of the mobile phase, the migration rate can be controlled, leading to efficient separation. The mobile phase can be isocratic or gradient, polar or nonpolar, based on the nature of the analyte. The stationary phase is a crucial component of chromatography that interacts with analytes as they pass through, leading to separation. Solid, liquid, or gas can be held over a supporting medium. The stationary phase interacts with various mixture components based on the polarity, affinity, size, and charge. Thus, different analytes have varying degrees of interaction with the stationary phase, leading to differential retention times and elution profiles. Different types of chromatography use combinations of stationary and mobile phases to achieve separation. Refer to image 1 for the classification of chromatography.  The stationary phase is spread as a thin layer on a flat surface. The sample is added as a small spot or a band over it, and the entire plane is kept in a slanting position over a liquid mobile phase. The mobile phase moves over the stationary phase due to capillary action carrying the sample, resulting in separation. When a sheet of paper is used, it is called paper chromatography. If a thin layer of absorbent material like silica, aluminum, or cellulose coated over a glass plate is used, it is called thin-layer chromatography. After separation, the colorless compounds are identified using fluorescence, radioactivity, or by producing visible color. The position of each molecule is identified, and the distance traveled is measured. The retention factor or Rf value for each of the molecules is expressed, and the identification of the molecules is compared with the standard Rf. The Rf value is calculated by dividing the distance a solute travels by the distance the solvent travels. A number between 0 and 1 represents the Rf value and is affected by several factors, including the type of stationary phase, the polarity of the solvent, the temperature, and the solvent concentration. Refer to image 2 (planar chromatography).  This is the most commonly used type, where a column of fiberglass or steel filled with silica particles acts as a stationary phase. The mobile phase is gas or liquid. In normal-phase column chromatography, a polar stationary phase separates non-polar compounds; in reversed-phase chromatography, the nonpolar stationary phase and the polar mobile phase are used.  Here, the separation is based on the differential adsorption of the analytes onto the solid stationary phase. Silica, charcoal, and calcium hydroxyapatite are the common absorbents packed as stationary phases onto the columns. The interactions between the compounds and stationary phase can be Van der Waals, hydrogen bonding, dipole-dipole, and hydrophobic interactions. These interactions cause analytes to be retained on the stationary phase to varying degrees.  Separation is based on differences in partition coefficients, allowing selective elution and separation of compounds based on their affinity for the 2 phases. Also known as gel permeation chromatography, the separation is based on the size. The column is packed with gels of controlled pore size, which acts like a molecular sieve, and by steric effects, the compounds are classified based on molecular size. This is useful for separating proteins, viruses, and nucleic acids. This relies on the reversible interaction between charged analytes (ions) and oppositely charged groups immobilized on a solid stationary phase. This interaction allows ions to be selectively retained and then eluted from the stationary phase by manipulating the composition of the mobile phase. Cation and anion exchange resins are used in the stationary phase to isolate anionic and cationic substances, respectively. The separation is based on the interaction of the proteins and the ligands. The gel with bound ligands interacts with the proteins and retains them, enabling the separation of desired proteins, which can be eluted. This separates enzymes, vitamins, hormones, antibodies, etc. Here, inert gasses like nitrogen, helium, or argon or a low-mass gas such as hydrogen are used in the mobile phase to separate volatile compounds or substances that can become volatile after derivatization. The separation occurs based on the differences in vapor pressure after converting them into volatile compounds. Gas chromatography (GC) estimates lipids, drugs, and vitamins. There are several ways of classifying GC methods based on the type of stationary phase present. These categories include gas-solid chromatography (GSC), gas-liquid chromatography (GLC), and bonded phase gas chromatography. In liquid chromatography (LC), the mobile phase is a liquid in which the sample with ions and molecules is dissolved. The 2 most common forms of LC used for analysis are liquid chromatography-mass spectrometry (LC-MS) and high-performance liquid chromatography (HPLC). Liquid chromatography (LC), when combined with MS, is highly efficient with low detection limits. MS further sorts and identifies the compounds eluted from LC in the electric and magnetic fields according to the charge mass ratio. This is a highly sensitive and efficient technique among different chromatography techniques, also known as high-pressure liquid chromatography. Here, the solvent mixture can pass through columns containing stationary phases under high atmospheric pressure of 10 Pa to 400 Pa. This high pressure creates a high flow rate in a sample of 0.1 cm/sec to 5 cm/sec, allowing separation in a few minutes. High pressure ensures high resolution and better separation of closely related compounds; it also enables using MS as detectors, which require high flow rates. The eluents are detected using UV absorption and fluorescence. The basic instrumentation setup is given in image 3.