Chromatography is a physical method of separation in which the components to be Distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction .It is a collective term used for lab techniques used for separation of mixtures. It involves passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the analyte to be measured from other molecules in the mixture based on differential partitioning between the mobile and stationary phases. Chromatography may be preparative and analytical. The purpose of preparative chromatography is to separate the components of a mixture for further use (and is thus a form of purification). It is to separate the components of mixture for further 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. The two are not mutually exclusive.1
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TERMS RELATED TO CHROMATOGRAPHY
Chromatography is a physical method of separation in which the components to be separated are
distributed between two phases, one of which is stationary (stationary phase) while the other (the
mobile phase) moves in a definite direction.
A graphical or other presentation of detector response, concentration of analyte in the effluent or other quantity used as a measure of effluent concentration versus effluent volume or time. In planar chromatography "chromatogram" may refer to the paper or layer with the separated zones.
To separate by chromatography.
The assembly of apparatus for carrying out chromatographic separation.
The stationary phase is one of the two phases forming a chromatographic system. It may be a solid, a gel or a liquid. If a liquid, it may be distributed on a solid. This solid may or may not contribute to the separation process. The liquid may also be chemically bonded to the solid (Bonded Phase) or immobilized onto it (Immobilized Phase).The expression Chromatographic bed or Sorbent may be used as a general term to denote any of the different forms in which the stationary phase is used.
A stationary phase which is covalently bonded to the support particles or to the inside wall of the column tubing.
A stationary phase which is immobilized on the support particles, or on the inner wall of the column tubing, e.g., by in situ polymerization (cross-linking) after coating.
A fluid which percolates through or along the stationary bed, in a definite direction. It may be a liquid (Liquid Chromatography) or a gas (Gas Chromatography) or a supercritical fluid (Supercritical-Fluid Chromatography). In gas chromatography the expression Carrier Gas may be used for the mobile phase. In elution chromatography the expression Eluent is also used for the mobile phase.
To chromatograph by elution chromatography. The process of elution may be stopped while all the sample components are still on the chromatographic bed or continued until the components have left the chromatographic bed.
Note: The term "elute" is preferred to the term Develop used in former nomenclatures of
The mobile phase leaving the column.
The mixture consisting of a number of components the separation of which is attempted on the chromatographic bed as they are carried or eluted by the mobile phase.
The chemically pure constituents of the sample. They may be unretained (Le., not delayed) by the stationary phase, partially retained (Le., eluted at different times) or retained permanently. The terms Eluite or Analyte are also acceptable for a sample component. Solute is a term referring to the sample components in partition chromatography.
A term sometimes referring to the liquid stationary phase in partition chromatography.
Note: In liquid chromatography the term "solvent" has been often used for the mobile phase.
This usage is not recommended.
A region in the chromatographic bed where one or more components of the sample are located.2
Always on Time
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CLASSIFICATION ACCORDING TO THE PHYSICAL STATE OF THE MOBILE PHASE
Chromatographic techniques are often classified by specifying the physical state of both phases used. Accordingly, the following terms are in use:
Gas-liquid chromatography (GLC)
Gas-solid chromatography (GSC)
Liquid-liquid chromatography (LLC)
Liquid-solid chromatography (LSC)
The term Gas-Liquid Partition Chromatography (GLPC) can also be found in the literature.
However, often distinction between these modes is not easy. For example, in GC, a liquid may be used to modify an adsorbent-type solid stationary phase.
Gas Chromatography (GC)
A separation technique in which the mobile phase is a gas. Gas chromatography is always carried out in a column.3
diagram of gas chromatograph
Liquid Chromatography (LC)
A separation technique in which the mobile phase is a liquid. Liquid chromatography can be carried out either in a column or on a plane.
Note: Present-day liquid chromatography generally utilizing very small particles and a
relatively high inlet pressure is often Characterized by the term High-Pressure Liquid Chromatography, and the acronym HPLC.5
preparative HPLC apparatus
Supercritical-Fluid Chromatography (SFC)
A separation technique in which the mobile phase is a fluid above and relatively close to its critical temperature and pressure.
Note: In general the terms and definitions used in gas or liquid chromatography are equally
applicable to supercritical-fluid chromatography.4
CLASSIFICATION ACCORDING TO THE MECHANISM OF SEPARATION
Separation is based mainly on differences between the adsorption affinities of the sample
components for the surface of an active solid.
Separation is based mainly on differences between the solubilities of the sample components in the stationary phase (gas chromatography), or on differences between the solubilities of the
components in the mobile and stationary phases (liquid chromatography).
Separation is based mainly on differences in the ion exchange affinities of the sample
components. Present day ion-exchange chromatography on small particle high efficiency columns and usually utilizing conductometric or spectroscopic detectors is often referred to as Ion Chromatography (IC).6
Separation is based mainly on exclusion effects, such as differences in molecular size and/or shape or in charge. The term Size-Exclusion Chromatography may also be used when separation is based on molecular size. The terms Gel Filtration and Gel-Permeation Chromatography (GPC) were used earlier to describe this process when the stationary phase is a swollen gel. The term Ion-Exclusion Chromatography is specifically used for the separation of ions in an aqueous phase.
This expression characterizes the particular variant of chromatography in which the unique biological specificity of the analyte and ligand interaction is utilized for the separation.4
column and batch chromatography
An elution procedure used in liquid chromatography is the process in which the mobile phase is significantly more polar than the stationary phase, e.g., a microporous silica-based material with chemically bonded alkyl chains. The term "reverse phase" is an incorrect expression to be avoided. Normal-Phase Chromatography is an elution procedure in which the stationary phase is more polar than the mobile phase. This term is used in liquid chromatography to emphasize the contrast to reversed-phase chromatography.
The procedure in which the composition of the mobile phase remains constant during the elution process.
The procedure in which the composition of the mobile phase is changed continuously or stepwise during the elution process.
The elution process in which the composition of the mobile phase is changed in steps during a single chromatographic run.
A procedure in which parts or all of the separated sample components are subjected to additional separation steps. This can be done e.g., by conducting a particular fraction eluting from the column into another column (system) having different separation characteristics. When combined with additional separation steps , this may be described as Multi-Dimensional Chromatography. In planar chromatography two-dimensional chromatography refers to the chromatographic process in which the components are caused to migrate first in one direction and subsequently in a direction at right angles to the first one; the two elutions are carried out with different eluents.
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A procedure in which the temperature of the column is kept constant during the separation.
Programmed-Temperature Chromatography (Temperature Programming)
A procedure in which the temperature of the column is changed systematically during a part or the whole of the separation.
Programmed-Flow Chromatography (Flow Programming)
A procedure in which the rate of flow of the mobile phase is changed systematically during a part or the whole of the separation.
Programmed-Pressure Chromatography (Pressure Programming)
A procedure in which the inlet pressure of the mobile phase is changed systematically during a part or whole of the separation.
A technique in which the identities of the sample components are intentionally changed between sample introduction and detection. The reaction can take place upstream of the column when the chemical identity of the individual components passing through the column differs from that of the original sample, or between the column and the detector when the original sample components are separated in the column but their identity is changed prior to entering the detection device.
A version of reaction chromatography in which a sample is thermally decomposed to simpler fragments before entering the column.
A version of reaction chromatography in which the separated sample components eluting from the column are derivatized prior to entering the detector. The derivatization process is generally carried out "on-the-fly", i.e., during transfer of the sample components from the column to the detector. Derivatization may also be carried out before the sample enters the column or the planar medium; this is pre-column (preliminary) derivatizationâ€¦.7
IMPORTANT TYPES OF CHROMATOGRAPHIES
Note: may be this section discusses the types of chromatographies as discussed earlier but this section is specifically dealing with those important types of chromatographies that are used most importantly.
1. Column chromatography
Column chromatography is a separation technique in which the stationary bed is within a tube .The 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 the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube(open tubular column). Differences in rates of movement through the medium are calculated to different retention times of the samples.8
2. Planar chromatography
Planar chromatography is a separation technique in which the stationary phase is present as or on a plane. The plane can be paper, serving as such or impregnated by a substance as a stationary bed (paper chromatography) or a layer of solid particles spread on a support such as a glass plate (thin layer chromatography).9
Typical type of planar chromatography
3. Paper chromatography
Paper chromatography is a technique that involves placing a small dot or line of samples solution onto a strip of chromatography paper.
4. 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. 10
thin layer chromatography used to separate components of chlorophyll
5. Displacement chromatography
The basic principle of chromatography is: A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities. There are distinct differences between displacement and elution mode, substances typically emerge from a column in narrow, Gaussian peak.11
6 .Gas chromatography
Gas chromatography (GS), also sometimes known as Gas-Liquid chromatography, (GLC), is a separation technique in which the mobile phase is gas. Gas chromatography is always carried out in a column, which is typically "packed" or "capillary".
Gas chromatography (GS) is based on partition equilibrium of analyte between a solid 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 glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column). It is widely used in analytical chemistry ; though the high temperatures used in GC make it unsuitable for high molecular weight biopolymer or protein (heat will denature them), frequently encountered in biochemistry, it is well suited for use in petrochemical, environmental monitoring, and industrial chemical fields. It is also used extensively in chemistry research.12
A gas chromatograph with a headspace sampler.
7 .Liquid chromatography
Liquid chromatography (LC) is a separation technique in which the mobile phase is liquid. Liquid chromatography 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 high performance liquid chromatography (HPLC).
8. Affinity chromatography
Affinity chromatography is based on selective non-covalent interaction between an analyte and specific molecules. It is very specific, but not robust. It is often used in biochemistry in the purification of protein 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 removed and the pure protein is obtained. 13
9. Super critical chromatography
Supercritical fluid chromatography is a separation technique in which the mobile phase is a fluid above and relatively close to its critical temperature and pressure.
10. Chiral chromatography
Chiral chromatography involves the separation of stereoisomerism. In the case of enantiomers, these have no chemical or physical differences apart from being three-dimensional mirror images. Conventional chromatography or other separation processes are incapable of separating them. To enable chiral separations to take place, either the mobile phase or the stationary phase must themselves be made chiral, giving differing affinities between the analytes.
APPLICATION OF CHROMATOGRAPHY
Blood plasma is the liquid component of blood, which contains dissolved proteins, nutrients, ions and other soluble components. In whole blood, red blood cells, leukocytes, and platelets are suspended within the plasma. The goal of plasma purification and processing is to extract specific materials that are present in blood, and use them for restoration and repair. These are several components that make up blood plasma, one of which is the protein albumin. Albumin is a highly water-soluble protein with considerable structural stability.
The general approach to using chromatography for plasma fractionation for albumin is:
Recovery of supernatant I
Anion exchange chromatography
Cation exchange chromatography
Gel filtration chromatography
Albumin purification process consists of five steps:
Starting material is plasma that has been pretreated by centrifugation
A round of gel filtration is run
Ion exchange on DEAE SEpharose is run to bind the bind the albumin to the column
Albumin is eluted with a sodium acetate buffer
Final polishing with gel filteration.
The end result is a highly pure and safe batch of albumin that is 100% non pyrogenic, sterile, and free of active HIV-virus. The product purity is greater than 98% and the protein content is about 50 g/L. the next goal is to further modernize the facility by scaling down production costs and increasing capacity. 14