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Chromatography

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Chromatography

Chromatographic separation analysis, also known as chromatographic analysis, is an efficient physical separation technique that separates multi-component mixtures, analyzes chemical components and their contents by providing appropriate detection means. Chromatography has a wide range of applications and can be used in almost all organic compounds. It can also separate inorganic and macromolecules, and even biomacromolecules. In addition, chromatography provides fast analysis, low sample usage, and easy operation. Chromatographic separation methods can be divided into gas chromatography, high performance liquid chromatography and gel permeation chromatography.

Gas chromatography (GC)

Gas chromatography is a chromatographic technique using an inert gas such as nitrogen as a mobile phase, a solution or a fixed adsorbent as a stationary phase. After the sample is introduced into the inlet for high-temperature gasification, it is evaporated onto the column. This can be done using a packed or capillary column. The column is the stationary phase and the inert gas is the mobile phase. The compounds are separated on the column by how they interact with these phases; heat also moves the compounds along the column. After leaving the column, the compounds can be detected by various detectors. Based on the sequence of being introduced into the detectors, each component can be qualitatively analyzed and the content can be calculated according to the peak height or the peak area. Commonly used detectors include thermal conductivity detectors, flame ionization detectors, helium ionization detectors, ultrasonic detectors, photoionization detectors, electron capture detectors and mass spectrometer detectors.

The principle of GC is to separate the gas sample based on the difference in the distribution of mobile phase and stationary phase in the column. Therefore, GC is only used for the analysis of gases and low-boiling compounds. For high-boiling solids and liquids, high performance liquid chromatography (HPLC) can be used.

High performance liquid chromatography (HPLC)

The separation principle of high performance liquid chromatography and gas chromatography is similar, except that the mobile phase is changed to liquid, and a high pressure infusion system is added.

HPLC relies on pumps to pass a pressurized liquid and a sample mixture through a column filled with adsorbent, leading to the separation of the sample components. The active component of the column, the adsorbent, is typically a granular material made of solid particles (e.g., silica, polymers, etc.), 2–50 μm in size. The components of the sample mixture are separated from each other due to their different degrees of interaction with the adsorbent particles. The pressurized liquid is typically a mixture of solvents (e.g., water, acetonitrile and/or methanol) and is referred to as a “mobile phase”. Its composition and temperature play a major role in the separation process by influencing the interactions taking place between sample components and adsorbent. These interactions are physical in nature, such as hydrophobic (dispersive), dipole–dipole and ionic, most often a combination.

HPLC can be classified as liquid-solid adsorption chromatography, liquid-liquid partition chromatography, ion exchange chromatography, ion-pair chromatography and size exclusion chromatography. As long as the sample is completely soluble in the mobile phase, it can be tested in HPLC.

Gel permeation chromatography (GPC)

Gel permeation or size exclusion chromatography (GPC/SEC) is a highly valuable tool for characterizing natural and synthetic polymers and proteins. The stationary phase of GPC is microsphere with various pores and channels on the surface and inside. It could be composed of polystyrene with high degree of crosslinking, polyacrylamide, gel of glucose and agarose, or porous silica gel.

In GPC, separation is mainly based on the hydrodynamic size of molecules. When a polymer solution is introduced into the column, the solute molecules are trying to penetrate into the micropores inside the filler due to the difference in concentration. Smaller molecules have longer travel distance since they can enter both small pores and large pores, while larger molecules only enter large pores. Due to the difference in elution time, after multiple permeation-diffusion equilibriums the largest polymer molecules first elute, followed by smaller molecules. A curve of the polymer size as a function of retention time is obtained.

GPC has been widely used to determine the molecular weight of polymers. Basic GPC/SEC systems that have only a UV or refractive index (RI) detector provide only relative molecular weight measurement. Light scattering detectors, on the other hand, directly measure the absolute molecular weight of the sample as it elutes from the column. So the combination of GPC and LS results in the measurement of absolute molecular weight which is independent from the column calibration. Through GPC it is also possible to monitor the synthesis process of the polymer, explore the polymerization mechanism and study branched polymers.

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