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8.3 Chromatographic Techniques

Chromatography is the collective term for a set of laboratory technique for separating mixtures. This involves passing a mixture dissolved in a "mobile phase" through stationary phase, which separates analyte to be measured from other molecules in the mixture and allows it to isolate.

Chromatography may be preparative or analytical depending on situation. The purpose of preparative chromatography is to separate components of a mixture for further use and is thus a form of purification. While analytical chromatography is done normally with smaller amounts of material and is for measuring the relative proportions of analytes in a mixture. These two types are not mutually exclusive though.

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Column chromatography is a separation technique in which stationary bed is within a tube and particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube/packed column or be concentrated on or along inside the tube wall leaving an open, unrestricted path for mobile phase in the middle part of the tube/open tubular column. Differences in the rates of movement through the medium are calculated to the different retention times of the sample.

chromatographic techniques

Paper chromatography is a technique that involves the placing of a small dot or line of sample solution onto a strip of paper chromatography. The paper is placed in a jar containing shallow layer of solvent and sealed. The solvent rises through the paper and meets the sample mixture which starts to travel up the paper with the solvent. This paper is made of cellulose, a polar substance, and thus compounds within the mixture travel farther if they are non-polar. More polar substances bond with the cellulose paper more quickly relatively, and thus do not travel as far.

column chromatography

Gas chromatography (GC), also sometimes designated as Gas-Liquid chromatography, (GLC), is a separation technique in which the mobile phase is a gas and is always carried out in a column, which is typically "packed" or "capillary". Gas chromatography (GC) is based on partial equilibrium of analyte between a solid stationary phase often a liquid silicone-based material and a mobile gas often Helium. The stationary phase is adhered to the inside of a small-diameter glass tube/capillary column or a solid matrix inside a larger metal tube/packed column. This is widely used though the high temperatures used in GC make it unsuitable for high molecular weight biopolymers or proteins as heat will denature them, it is well suited for use in petrochemical, environmental monitoring, and industrial chemical fields and is also used extensively in chemistry research.

Liquid chromatography (LC) is the separation technique in which mobile phase is a liquid and can be carried out either in a column or a plane. Present day liquid chromatography that generally utilizes very small packing particles and a relatively high pressure is referred to as HPLC or high performance liquid chromatography. In HPLC technique, the sample is forced through a column that is packed with irregularly or spherically shaped particles or a porous monolithic layer/stationary phase by a liquid/mobile phase at high pressure. HPLC is divided into two different sub-classes based on the polarity of the mobile and the stationary phases: technique in which the stationary phase is more polar than the mobile phase, say, toluene as the mobile phase, silica as the stationary phase is called normal phase liquid chromatography (NPLC) and the opposite, say, water-methanol mixture as the mobile phase and C18 = octadecylsilyl as the stationary phase is called reversed phase liquid chromatography (RPLC). The NPLC has fewer applications and RPLC is used considerably more.

Ion exchange chromatography uses ion exchange mechanism to separate the analytes and is usually performed in columns though it can also be useful in planar mode. Ion exchange chromatography uses a charged stationary phase to separate charged compounds including proteins, peptides and amino acids. In conventional methods the stationary phase is ion exchange resin carrying charged functional groups which interact with oppositely charged groups of the compound to be retained. Ion exchange chromatography is commonly used to purify proteins (keeping in mind the denaturing of protein) using FPLC.

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