Chromatography

Principle and Definition The principle of chromatographic separation is very simple. The process is achieved by distributing the substances to be separated between a mobile phase and a stationary phase. Those substances distributed preferentially in the moving (mobile) phase pass through the chromatographic system faster than those that are distributed preferentially in the stationary phase.

Theoretical basis of chromatography These theoretical concepts are applicable to all types of chromatographic techniques. I- Distribution equilibria and rate of travel During chromatography a given solute finds itself either in the stationary phase, which acts as a "retarder" or in the mobile phase, which acts as a "carrier". This distribution is based on distribution equilibria and is expressed by the rate of travel.

1-Distribution equilibria The distribution of solutes between the two phases is governed by an equilibrium constant known as the distribution coefficient, K (or partition coefficient in certain types of chromatography). This allows quantifying the distribution of a compound between the stationary and mobile phases: K = Cstationary / Cmobile C stationary = concentration of solute in the stationary phase Cmobile = concentration of solute in the mobile phase

2-Rate of travel Factors limiting the rate of travel: The rate of travel of a solute in a chromatographic system is limited by: The velocity of the mobile phase (or carrier), that is the same for all components. The ratio of volume of the stationary phase to the volume of the mobile phase, that is also the same for all components. The value of the distribution coefficient that is characteristic for each component.

Classification of chromatographic techniques Different systems of classification may be adopted relying on criteria such as the separation mechanisms, the development procedure, the method of holding the stationary phase. i-According to the mechanism responsible for differential migration: The components will migrate through the system at different rates based on competition between the stationary and mobile phase for the solute molecules. The major types are:

1-Adsorption Chromatography 2-Partition Chromatography 3-Ion Exchange Chromatography (IEC) 4-Molecular Exclusion Chromatography ( Gel permeation or Gel filtration) 5-Affinity Chromatography

Adsorption Column Chromatography Stationary phase Selection of the adsorbent The ideal adsorbent should conform to the following specifications: It should be insoluble in the solvent used as mobile phase. It should be inert to the adsorptives (solutes), unless otherwise required. It should be colorless, especially when zones which contain colored substances are to be located visually. It should have a suitable particle size with great surface area to allow more efficient adsorption; but, not too fine to avoid slowing of the rate of percolation.

Types of adsorbents and applications of each type.

Mobile phase Selection of the solvent The choice of the suitable solvent (s) depends mainly on the elution power of the solvent and the relative adsorption of the class of compounds, which are to be separated. Strong eluents (more polar solvents) decrease adsorption, while weak eluents (less polar) increase it. Elution of the column can, therefore, be carried out with solvents of increasing polarity. A typical series is as follows: hexane < cyclohexane < benzene < chloroform < diethylether < ethyl acetate < acetone < ethanol < methanol < pure water.

Types of solvents as mobile phases in adsorption chromatography.

Factors affecting column efficiency and chromatographic separation 1- Particle size of the supporting medium: As the particle size of adsorbent decreases, separation is improved. However, an adsorbent of very small particle size will offer considerable resistance to flow. 2-Column dimensions: The column efficiency is improved as the length / width ratio of the column is increased. 3-Uniformity of packing of the column: If the column is not packed uniformly, uneven and irregular movement of the solvent front and less uniform zone formation will result. 4-Column temperature: As temperature is increased, the elution speeds up as the adsorption is generally reduced at higher temperatures.

This is carried by either the wet or dry packing techniques. Packing of the column This is carried by either the wet or dry packing techniques. Wet packing: A suspension (slurry) of the adsorbent, preferably the first solvent to be used in the separation, is prepared and gradually added to the column. The packing is allowed to settle between additions. The suspending liquid is allowed to flow out slowly but the liquid level in the column is maintained above the packing at all times.

Wet packing is most commonly used and results in more homogenous columns. Dry packing: In this method, the solid stationary phase is added in portions with vibrations between additions. The process is repeated until the column is adequately filled. The column is then washed carefully with the first solvent

Sample application Development Elution Detection.

Applications Adsorption Column Chromatography has been used for: Separation of alkaloids of Cinchona, ergot, opium and nux vomica, and cardiac glycosides from Digitalis and anthraquinones from senna species. Isolation and purification of vitamins and hormones. Examination of vegetable oil and pharmaceutical preparations. Purification of tincture of alkaloids from pigments before determination of their alkaloidal content.

Partition Chromatography The separation of the components of a mixture is, as in counter-current extraction, dependent on differences in the partition coefficients of the components between an aqueous and an immiscible organic liquid. The liquid stationary phase is adsorbed on an inert support, which may be either packed in a chromatographic tube (Column Partition Chromatography) or layered on a glass plate (TLC) or in the form of sheets of paper (PC).

Paper Chromatography The technique was introduced as a method of partition chromatography for the analysis of amino acid mixtures and has extended to all classes of natural products. It remains the method of choice for the fractionation of some groups of substances, e.g. flavonoids Principle and mode of action It is a type of planar chromatography in which the stationary phase is a specified type of paper. The paper is formed of cellulose, which has a great affinity for water and other polar solvents and holds them through formation of hydrogen bonds thus forming a polar stationary phase. Distribution of the solutes between the two phases is therefore mainly by partition.

Criteria for ideal PC separation Efficient separation is achieved by: 1-Using analytical grade chemicals and distilled water for preparation of the mobile phase (solvent-system). 2-Maintaining the composition of the mobile phase constant during development by using a sufficient amount of solvent and carrying the process in a well-closed chamber (chromatographic jar).

3-Maintaining a suitable and constant temperature through out the process. 4-Selecting the suitable solvent in which the components of the analyzed sample have a low but definite solubility. Highly soluble substances will appear at or very close to the solvent "front". Substances of very low solubility will remain near the point of application or " start line".

Procedure Steps followed to perform paper chromatography are: 1-Sample desalting The presence of inorganic salts generally causes streaking (tailing, trailing), discoloration and other distortions on the finished chromatogram. The removal of these salts can be achieved by ion exchange or precipitation.

2-Sample application (Loading) Spots are applied with micropipettes, at a suitable distance from each other, on the start line. They are kept as small as possible (less than 5 mm in diameter). In case of repeated applications, spots should be completely dried after each application. When large quantities of material are to be chromatographed, the sample is applied as a streak (e.g. in preparative paper chromatography (PPC) and quantitative chromatography).

3-Development of the chromatogram Several techniques may be adopted. These include the usual (or general) techniques such as the ascending, descending, radial and horizontal methods. Special techniques have also been devised in order to improve resolution, examples are the stepwise and multiple development, the continuous development and the two-dimensional development.

4-Drying of the chromatogram After development, the paper is withdrawn from the jar. The front of the solvent is marked immediately with a pencil and dried by hanging in open air at room temperature or by a current of cold or hot air (using a hair dryer). 5-Spot detection (location or visualization) Several methods are used for detection of the separated spots corresponding to the components of the analyzed sample. They include:

Thin Layer Chromatography (TLC) This is simple and fast and has now achieved a remarkable success in the separation of mixtures of all classes of natural products. Thin layer chromatograms often serve to identify drugs, plant extracts and biochemical preparations, or to detect contaminants or adulterants.

Analogy with Paper Chromatography Such as paper chromatography TLC is a type of planar chromatography. Instead of paper (in PC) a thin layer of finely divided adsorbent supported on a glass, plastic or aluminum sheet is used. Analogy with Column Chromatography TLC is also closely related to column chromatography and has often been called "Open-column chromatography". Both techniques are based on the same mechanisms of chromatographic separation: adsorption and partition. The major difference lies in the location of the stationary phase. In TLC, it is a thin layer spread on a glass plate, and in column chromatography, it is a column held in a glass tube.

Advantages of TLC over PC The method is more versatile, it allows the use of wide range of stationary phases and solvents. Fractionation is performed more rapidly, with smaller quantities of the mixture and is more reproducible. The separated spots are usually more compact and more clearly demarcated from one another. Concentrated sulfuric acid and other drastic reagents can be used for location of separated substance but not for the paper.

Applications Like with paper chromatography, TLC can be extended to, quantitative evaluation and to work that involves radioactive substances. Quantitative analysis can be performed by measuring the size of the spots with a planimeter or measuring the intensity of the color by photodensitometry. An alternative method is to scrap the bands and elute the components with the suitable solvent followed by colorimetric, spectrophotometric, fluorimetric, gravimetric or volumetric methods.