Analytical Chemistry Section D Separation Technique

Contents 1. Principles of Chromatography 2. Thin-layer Chromatography

1. Principles of Chromatography Chromatographic Separation Chromatography is the process of separating the components of mixtures(solutes) that are distributed between a stationary phase and a flowing mobile phase according to the rate at which they are transported through the stationary phase. Solute Migration and Retention Solutes migrate through a stationary phase at rates determined by their relative affinities for each phase, and are characterized by defined retention parameters. Sorption Processes During a chromatographic separation, solute species are continually transferred back and forth between the mobile and stationary phases by the process of sorption followed by desorption. Several mechanisms by which this occurs give rise to different modes of chromatography.

Chromatographic Separations(1) * Elution - The placing of a sample onto a liquid or solid stationary phase and passing a liquid or gaseous mobile phase through or over it. * Differential Rate of Migration - It will lead to their separation over a period of time and distance. 1. Principles of Chromatography

Chromatographic Separations(2) 1. Principles of Chromatography Table1. A classification of the principal chromatographic techniques

Solute Migration and Retention(1) *Distribution Ratio, D ⇒ D = C S / C M (C S : the total solute concentration in the stationary phase, C M : the total solute concentration in the mobile phase) - Large values of D lead to slow solute migration, Small values of D lead to rapid solute migration. ⇒ Solutes are eluted in order of increasing distribution ratio. *Retention (or Retardation) - the interaction of solutes with the stationary phase slows down their rate of migration relative to the velocity of the mobile phase. 1. Principles of Chromatography

Solute Migration and Retention(2) *Planar Separations(PC and TLC) - Retardation Factor, R f (0≤ R f ≤1) ⇒ R f = 1 / (1+k) (The maximum value of R f is 1 → solute migrating at the same velocity as the mobile phase. The minimum value for R f being zero → solute spends all of the time in the stationary phase and remains in its original position on the surface.) (Distance Moved by Solute) (Distance Moved by the Solvent Front) 1. Principles of Chromatography ⇒ R f =

Sorption Processes *Sorption - Solute species are transferred from the mobile to the stationary phase (↔ Desorption ) *Sorption Mechanism ① Adsorption ② Partition ③ Ion-exchange ④ Exclusion 1. Principles of Chromatography

Sorption Processes *Adsorption - Electrostatic Interactions (Hydrogen-Bonding, Dipole-Dipole and Dipole- Induced Dipole Attractions) - Adsorbent of which Silica Gel is the most widely used. (Its surface comprises Si-O-Si and Si-OH(Silanol) groups, the latter being slightly acidic as well as being polar.) 1. Principles of Chromatography

Sorption Processes 1. Principles of Chromatography Table 2. Adsorbents for chromatographic separations (listed in order of decreasing polarity.)

Principles and Procedures Thin-Layer chromatography is a technique where the components of mixtures separate by differential migration through a planar bed of a stationary phase, the mobile phase flowing by virtue of capillary forces. The solutes are detected in situ on the surface of the thin-layer plate by visualizing reagents after the chromatography has been completed. Stationary Phase A variety of finely divided particulate sorbents are used as thin-layer stationary phases. These include silica-gel, cellulose powder, ion-exchange resins, restricted pore-size materials, and chiral selectors. Mobile Phase Single solvents or blends of two or more solvents having the appropriate overall polarity necessary to achieve the required separation are used as mobile phases. They range from nonpolar hydrocarbons to polar alcohols, water, and acidic or basic solvents. 2. Thin-layer Chromatography

Solute Detection Methods of visualizing solutes include spraying the surface of the thin-layer plate with a chromogenic regent, or viewing it under a UV lamp if the sorbent has been treated with a fluorescent indicator. Alternative TLC Procedures Alternative development procedures aimed at improving chromatographic performance have been introduced, and new stationary phases are becoming available. Applications of TLC Thin-layer chromatography is used primarily as a qualitative analytical technique for the identification of organic and inorganic solutes by comparisons of samples with standards chromatographed simultaneously. Quantitative analysis is possible but precision is relative poor. 2. Thin-layer Chromatography

Principles and Procedures(1) *Distribution Ration, D ⇒ D = C S / C M (Those with the largest values moving the least, whilst those with the smallest values moving with the advancing mobile phase, or solvent front.) 2. Thin-layer Chromatography Figure 1. TLC plates

Principles and Procedures(2) *TLC Procedure(1) ① Sufficient mobile phase to provide about a 0.5cm depth of liquid is poured into a development tank, or chamber, which is then covered and allowed to stand for several minutes to allow the atmosphere in the tank to become saturated with the solvent vapor. ② Small volumes of liquid samples and standard, or solutions, are spotted onto the sorbent surface of a TLC plate along a line close to and parallel with one edge (the origin). The plate is then positioned in the tank with this edge in contact with the mobile phase and the cover replaced. 2. Thin-layer Chromatography

Principles and Procedures(3) *TLC Procedure(2) ③ The mobile phase is drawn through the bed of sorbent from the edge of the plate, principally by capillary action, and this development process is halted shortly before the solvent front reaches the opposite side of the plate. Sample components and standards migrate in parallel paths in the direction of flow of the mobile phase, separating into discrete zones or spots. ④ The plate is removed from the development tank, dried in a current of warm air, and solute spots located by appropriate methods. ⑤ Each solute is characterized by the distance migrated relative to the solvent front, i.e. its R f value, which will lie between 0 and 1, and unknowns are identified by comparisons with standards run simultaneously. 2. Thin-layer Chromatography

Stationary Phase(1) *Microparticulate sorbents - particle diameters of between 10 and 30 μm. - The smaller the mean particle size and the narrower the size range, the better the chromatographic performance in terms of band spreading (efficiency) and resolution. 2. Thin-layer Chromatography

Stationary Phase(2) 2. Thin-layer Chromatography Table 3. Stationary phases (sorbents) for thin-layer chromatography

Mobile Phase *General Guidelines in selecting and optimizing the composition of a Mobile Phase - The highest purity - Mobile Phase eluting power should be adjusted so that solute R f values fall between 0.2 and The overall polarity of the mobile phase determines solute migration rates and hence their R f values. - Polar and ionic solutes are best separated using a blend of a polar organic solvent, such as n-butanol, with water. 2. Thin-layer Chromatography

Solute detection *Visualization - Their spots must be located using a chemical or physical means of visualization. 2. Thin-layer Chromatography

Alternative TLC Procedures 2. Thin-layer Chromatography Figure 2. Two-dimensional TLC of a mixture of 14 amino acids.

Applications of TLC *Advantages of TLC (over GC and HPLC) - The ability to run or more samples simultaneously for immediate and direct comparison with standards. - The basic technique is very cheap, versatile and quick. - All solutes, including those that do not migrate from the origin, are detectable. 2. Thin-layer Chromatography