Analytical Chemistry Section D Separation Technique.

Analytical Chemistry

Section D

Separation Technique

<Instant Notes, D. Kealey & P.J. Haines>

Contents

1. Principles of Chromatography 2. Thin-layer Chromatography

1. Principles of Chromatography

Chromatographic Separation

Chromatography is the process of separating the components of mix-tures(solutes) that are distributed between a stationary phase and a flowing mobile phase according to the rate at which they are trans-ported 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 de-fined 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 mecha-nisms by which this occurs give rise to different modes of chromatog-raphy.

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.


Chromatographic Separations(2)


Table1. A classification of the principal chromatographic tech-niques

Solute Migration and Retention(1)

*Distribution Ratio, D

⇒ D = CS / CM

(CS : the total solute concentration in the stationary phase, CM : 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.


Solute Migration and Retention(2)

*Planar Separations(PC and TLC)

- Retardation Factor, Rf (0≤ Rf≤1)

⇒ Rf = 1 / (1+k)

(The maximum value of Rf is 1 → solute migrating at the

same velocity as the mobile phase.

The minimum value for Rf 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)


⇒ Rf =

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


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.)


Sorption Processes


Table 2. Adsorbents for chromatographic separations (listed in order of decreasing po-larity.)

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, cellu-lose powder, ion-exchange resins, restricted pore-size mate-rials, and chiral selectors.

Mobile Phase

Single solvents or blends of two or more solvents having the ap-propriate overall polarity necessary to achieve the required separa-tion are used as mobile phases. They range from nonpolar hydrocar-bons 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 chro-matographic performance have been introduced, and new stationary phases are becoming available.

Applications of TLC

Thin-layer chromatography is used primarily as a qualitative analyt-ical technique for the identification of organic and inorganic solutes by comparisons of samples with standards chromatographed simul-taneously. Quantitative analysis is possible but precision is relative poor.


Principles and Procedures(1)

*Distribution Ration, D

⇒ D = CS / CM

(Those with the largest values moving the least, whilst those with the smallest values moving with the advancing mobile phase, or solvent front.)


Figure 1. TLC plates


*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 min-utes to allow the atmosphere in the tank to become satu-rated with the solvent vapor.

② Small volumes of liquid samples and standard, or solu-tions, 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.



*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 rela-tive to the solvent front, i.e. its Rf value, which will lie between 0 and 1, and unknowns are identified by comparisons with stan-dards run simultaneously.


Stationary Phase(1)

*Microparticulate sorbents - particle diameters of between 10 and 30 μm.

- The smaller the mean particle size and the nar-rower the size

range, the better the chromatographic performance in terms

of band spreading (efficiency) and resolution.


Stationary Phase(2)


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 Rf values fall between 0.2 and 0.8.

- The overall polarity of the mobile phase determines solute

migration rates and hence their Rf values.

- Polar and ionic solutes are best separated using a blend of

a polar organic solvent, such as n-butanol, with water.


Solute detection

*Visualization

- Their spots must be located using a chemical or physical

means of visualization.


Alternative TLC Procedures


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 10-20 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.


Analytical Chemistry Section D Separation Technique.

Documents

Transcript of Analytical Chemistry Section D Separation Technique.