Principle of separation of different components: Chromatography Chromatography involves a sample (or sample extract) being dissolved in a mobile phase (which may be a gas, a liquid or a supercritical fluid). The mobile phase is then forced through an immobile, immiscible stationary phase. Principle of separation of different components:  Differential affinities (strength of adhesion) of the various components of the analyte towards the stationary and mobile phase results in the differential separation of the components. Affinity, in turn, is dictated by two properties of the molecule: ‘Adsorption’ and ‘Solubility’. The phases are chosen such that components of the sample have different solubilities in each phase. A component which is quite soluble in the stationary phase will take longer to travel through it than a component which is not very soluble in the stationary phase but very soluble in the mobile phase. As a result of these differences in mobilities, sample components will become separated from each other as they travel through the stationary phase.

Chromatography Distribution of analytes between phases An analyte is in equilibrium between the two phases: Partition coefficient

Chromatography The time between sample injection and an analyte peak reaching a detector at the end of the column is termed the retention time (tR ). The time taken for the mobile phase to pass through the column is called tM.

Chromatography Ideally 1<k’<5 Retention factor or capacity factor, k', is often used to describe the migration rate of an analyte on a column. k’< 1 elution is so fast that accurate determination of the retention time is very difficult k’ > 20 elution takes a very long time Ideally 1<k’<5 Selectivity factor, α , describes the separation of two species (A and B) on the column; Species A elutes faster than species B. The selectivity factor is always greater than one.

Chromatography Band broadening and column efficiency The Theoretical Plate Model of Chromatography Τhe chromatographic column contains a large number of separate layers, called theoretical plates. It is important to remember that the plates do not really exist. They serve as a way of measuring column efficiency: either by stating the number of theoretical plates in a column, N (the more plates the better) or by stating the plate height; the Height Equivalent to a Theoretical Plate, HETP, (the smaller the better).

Chromatography The number of theoretical plates that a real column possesses can be found by examining a chromatographic peak after elution: where w1/2 is the peak width at half-height. ☞ columns behave as if they have different numbers of plates for different solutes in a mixture.

Chromatography where u is the average velocity of the mobile phase. A, B, and C are factors which contribute to band broadening. A - Eddy diffusion The mobile phase moves through the column which is packed with stationary phase. Solute molecules will take different paths through the stationary phase at random. This will cause broadening of the solute band, because different paths are of different lengths. B - Longitudinal diffusion The concentration of analyte is less at the edges of the band than at the center. Analyte diffuses out from the center to the edges. This causes band broadening. If the velocity of the mobile phase is high then the analyte spends less time on the column, which decreases the effects of longitudinal diffusion. C - Resistance to mass transfer The analyte takes a certain amount of time to equilibrate between the stationary and mobile phase. If the velocity of the mobile phase is high, and the analyte has a strong affinity for the stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase. The band of analyte is broadened. The higher the velocity of mobile phase, the worse the broadening becomes.

Chromatography Van Deemter plots A plot of plate height vs. average linear velocity of mobile phase. Such plots are of considerable use in determining the optimum mobile phase flow rate.

Chromatography Resolution Baseline resolution is achieved when R = 1.5 A measure of how well species have been separated is provided by measurement of the resolution, R ☞ The selectivity factor, a, describes the separation of band centers but it does not take into account peak widths. Baseline resolution is achieved when R = 1.5

Chromatography Resolution To obtain high resolution, the three terms must be maximized: ☞ An increase in N, the number of theoretical plates, by lengthening the column leads to an increase in retention time and increased band broadening- which may not be desirable. Instead, to increase the number of plates, the height equivalent to a theoretical plate can be reduced by reducing the size of the stationary phase particles. ☞ By controlling the capacity factor, k', separations can be greatly improved. This can be achieved by changing the temperature (in Gas Chromatography) or the composition of the mobile phase (in Liquid Chromatography). ☞ When a is close to unity, optimising k' and increasing N is not sufficient to give good separation in a reasonable time. In these cases, k' is optimised first, and then a is increased by one of the following procedures: Changing mobile phase composition, changing column temperature, changing composition of stationary phase, using special chemical effects (such as incorporating a species which complexes with one of the solutes into the stationary phase)

Chromatographic techniques Stationary phase Mobile phase Basis of separation Notes Paper chromatography solid (cellulose) liquid polarity of molecules compound spotted directly on a cellulose paper Thin layer chromatography (TLC) solid (silica or alumina) glass is coated with thin layer of silica on which is spotted the compound Liquid column chromatography glass column is packed with slurry of silica on-exchange chromatography solid (cationic or anionic resin) ionic charge of the molecules molecules possessing the opposite charge as the resin will bind tightly to the resin, and molecules having the same charge as the resin will flow through the column and elute out first.

Technique Stationary phase Mobile phase Basis of separation Notes Size exclusion chromatography solid (microporous beads of silica) liquid size of molecules small molecules get trapped in the pores of the stationary phase, while large molecules flow through the gaps between the beads and have very small retention times. So larger molecules come out first. In this type of chromatography there isn’t any interaction, physical or chemical, between the analyte and the stationary phase.

Technique Stationary phase Mobile phase Basis of separation Notes Affinity chromatography solid (agarose or porous glass beads on to which are immobilized molecules like enzymes and antibodies) liquid binding affinity of the analyte molecule to the molecule immobilized on the stationary phase if the molecule is a substrate for the enzyme, it will bind tightly to the enzyme and the unbound analytes will pass through in the mobile phase, and elute out of the column, leaving the substrate bound to the enzyme, which can then be detached from the stationary phase and eluted out of the column with an appropriate solvent.

Technique Stationary phase Mobile phase Basis of separation Notes Gas chromatography liquid or solid support gas (inert gas like argon or helium) boiling point of the molecules samples are volatilized and the molecule with lowest boiling point comes out of the column first. The molecule with the highest boiling point comes out of the column last.

 Gas chromatograph