Chromatography

Chromatography

Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases.

Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for more advanced use (and is thus a form of purification). Analytical chromatography is done normally with smaller amounts of material and is for measuring the relative proportions of analytes in a mixture.

Gas chromatography

Gas chromatography (GC), also known as gas-liquid chromatography, (GLC), is a separation technique in which the mobile phase is a gas. Gas chromatographic separation is always carried out in a column, which is typically “packed” or “capillary”. Capillary columns generally give far superior resolution and although more expensive are becoming widely used, especially for complex mixtures. Both types of column are made from non-adsorbent and chemically inert materials. Stainless steel and glass are the usual materials for packed columns and quartz or fused silica for capillary columns.

Gas chromatography is based on a partition equilibrium of analyte between a solid or viscous liquid stationary phase (often a liquid silicone-based material) and a mobile gas (most often helium). The stationary phase is adhered to the inside of a small-diameter (commonly 0.53 – 0.18mm inside diameter) glass or fused-silica tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column).

gas chromat

It is widely used in analytical chemistry; though the high temperatures used in GC make it unsuitable for high molecular weight biopolymers or proteins (heat denatures them), frequently encountered in biochemistry, it is well suited for use in the petrochemical, environmental monitoring and remediation, and industrial chemical fields.

Affinity chromatography

Affinity chromatography is based on selective non-covalent interaction between an analyte and specific molecules. It is very specific, but not very robust. It is often used in biochemistry in the purification of proteins bound to tags. These fusion proteins are labeled with compounds such as His-tags, biotin or antigens, which bind to the stationary phase specifically. After purification, some of these tags are usually removed and the pure protein is obtained.

Affinity chromatography often utilizes a biomolecule’s affinity for a metal (Zn, Cu, Fe, etc.). Columns are often manually prepared. Traditional affinity columns are used as a preparative step to flush out unwanted biomolecules.

However, HPLC techniques exist that do utilize affinity chromatogaphy properties. Immobilized Metal Affinity Chromatography (IMAC) is useful to separate aforementioned molecules based on the relative affinity for the metal (I.e. Dionex IMAC). Often these columns can be loaded with different metals to create a column with a targeted affinity.

Affinity

Ion exchange chromatography

Ion exchange chromatography (usually referred to as ion chromatography) uses an ion exchange mechanism to separate analytes based on their respective charges. It is usually performed in columns but can also be useful in planar mode.

Ion exchange chromatography uses a charged stationary phase to separate charged compounds including anions, cations, amino acids, peptides, and proteins. In conventional methods the stationary phase is an ion exchange resin that carries charged functional groups that interact with oppositely charged groups of the compound to retain. Ion exchange chromatography is commonly used to purify proteins using FPLC.

Ion exchange

Size-exclusion chromatography

Size-exclusion chromatography (SEC) is also known as gel permeation chromatography (GPC) or gel filtration chromatography separates molecules according to their size. Smaller molecules are able to enter the pores of the media and therefore, molecules are trapped and removed from the flow of the mobile phase. The average residence time in the pores depends upon the effective size of the analyte molecules. However, molecules that are larger than the average pore size of the packing are excluded and thus suffer essentially no retention; such species are the first to be eluted.

It is generally a low-resolution chromatography technique and thus it is often reserved for the final, “polishing” step of a purification. It is also useful for determining the tertiary structure and quaternary structure of purified proteins, especially since it can be carried out under native solution conditions.

size

Thin layer chromatography

Thin layer chromatography (TLC) is a widely employed laboratory technique and is similar to paper chromatography. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate.

Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents. For even better resolution and to allow for quantification, high-performance TLC can be used.

TLC

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