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Liquid-liquid Partition Chromatography

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Principle In liquid-liquid partition chromatography,

sample components are distributed between two immiscible liquid phases, just as they would be if shaken in a separating funnel.

The shaking or equilibrium distribution occurs several thousand times in the column so that a small but highly efficient countercurrent distribution unit results.

One of the liquids involved is obviously the mobile phase. The other is in the form of a thin film both over surface and in the pores of the granular column packing. This is an adsorbent known as a support in view of its main role. The support does not (or al least should not) play any part in chromatographic process.

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Principle, continued Sample molecules are not adsorbed by

stationary phase in liquid-liquid partition chromatography but are simply dissolved in it. The better this solubility, i.e. the worse the solubility in the mobile phase, the longer is the retention time of the components involved, and vice versa.

Liquid-liquid partition system may operate in both the normal- and reversed-phase modes. In the latter case, the stationary phase is non-polar, hence the support must also be hydrophobic so that the liquid film will adhere to it sufficiently well. This is achieved by silanizing the support.

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Phase systems The two liquid phases must not be

mutually miscible so that the sample molecules have a choice between them and can become distributed accordingly.

The solubility of the stationary phase in the mobile phase should be as low as possible, otherwise the eluent will flush the former out the column in a very short time, giving rise to:

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Phase systems, continued

giving rise to: Permanent fluctuation in k’ values

(becoming smaller) as a result of changes in mobile to stationary phase ratio;

An adsorbent surface which is no longer fully covered, making the column useless until it has been re-coated;

Noise and ghost peaks in the detector

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Phase systems, continued

Highly viscous stationary phases are much less efficient than low-viscosity materials as diffusion and hence mass transfer take place more slowly.

Partition systems may be binary or ternary, the two-component type being easier to handle. As the two components must not mutually miscible, their polarities tend to be very different. As a results, the sample molecules have very different degree of solubility in the two phases (whereas it would be better if these were similar).

If a samples dissolves readily in the stationary phase, it is probably sparingly soluble in the mobile phase and is eluted late. The opposite applies for samples which dissolve readily in the mobile phase, the k’ value being too low on elution.

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Phase systems, continued

Three-component system do not have this problem. A component which dissolves the stationary phase is added to the mobile phase, making both similar and producing favorable retention times.

The k’ values can be altered to a certain degree by adjusting the amount of the third component added.

Ternary systems must be very carefully handled (temperature control and stationary phase saturation of the mobile phase being carefully monitored, plus the use of a scavenger column), otherwise the column bleed much more than with carefully selected binary systems.

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Support materialsThe support must be porous to allow

the liquid stationary phase to be taken up. Porous layer beads or fully porous particles are equally suitable.

The maximum potential loading corresponds roughly to the pore volume, i.e. a material with a pore volume of 1 ml/g can be saturated with about 1 g of stationary phase per gram of gel without sticking. This represents 100% loading.

Pores should be between 10 and 50 nm in diameter. If they are too narrow, then mass transfer is impeded; if they are too large, the stationary phase is not retained well enough by capillary forces, and therefore rapidly becomes washed away.

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Column preparation If the column is packed dry, i.e. if the

support (porous layer beads) is large enough, the material can be coated as in gc. A solution of stationary phase in a highly volatile solvent is poured over the support, then stirred or rotated (care is required with dealing with fragile Chromosorb) and the solvent is distilled off slowly.

The granular material is then packed into the column, wet filling is not recommended.

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Column preparation, continued

In situ techniques in which the pre-packed column is coated with stationary phase are more important but far less easy to master

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Liquid-liquid Partition Chromatography in practice

The mobile phase should be saturated with stationary phase, otherwise it will inevitably loosen the film from the support.

The stationary phase must absorb no light at the chosen wavelength when a UV detector is used.

The linear mobile phase flow velocity should not be greater than 1 mm/s as the stationary phase may otherwise be washed out by mechanical erosion.

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Liquid-liquid Partition Chromatography in practice

A heavily loaded pre-column is advisable. The support should preferably be coarse-grained so that the packing does not offer any great flow resistance. This has further advantage of being easily coated and dry packed like GC-column.

The pre-column is changed whenever necessary.

Samples must be dissolved in the saturated mobile phase and not in pure solvent.

A solver stronger than the mobile phase should certainly not be chosen, otherwise the eluent would displace the stationary phase from the support.

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Liquid-liquid Partition Chromatography in practice, continued

A good thermostat system is essential as the solubility of the stationary phase in the mobile phase is a function of temperature. The phase ratio and hence the k’ values can fluctuate under the influence of variations in temperature. Ternary mixtures may become completely miscible at only slightly elevated temperatures.

The sample load ability is about ten times greater than with adsorption system

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The disadvantage of using LLC

The stationary phase which always forms part of the eluent increases the detector noise;

The fraction obtained in preparative work are contaminated with stationary phase, in which case a highly volatile stationary phase is the preferred choice;

Gradient elution is impossible as the stationary phase is washed out by increased elution strength.

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Application

LLPC has multiple uses as the phase system can be freely chosen. Both polar and non-polar compounds can be separated, including those different alkyl groups (homoloques) and those which have different functional groups

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Separation of phenols and acids. Conditions: column, 20cm x 4.6 mm 1.d; support, acid-treated silica gel, 10 µm (gallic acid and 4-hydroxy-3-methoxymandelic acid show strong tailing with untreated silica gel); stationary phase, water; mobile phase, 1 ml/min water-saturated diethyl ether; UV detector, 254 nm.

Peaks: 1 = benzene, 2 = fumaric acid, 3 = gallic acid, 4 = phloroglucinol; 5 = hippuric acid, 6 = 4-hydroxy-3-methoxymandelic acid.

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Separation of sulphonamides. Conditions: column, 25 cm x 3.2 mm i.d, support, LiChrospher Si 100, 10 µm; stationary phase, 0.04 M tetrabutylammonium sulphate and 0.31 M sodium sulphate in 0.25 M borate buffer pH 8.5); mobile phase, butan-1-ol-heptane (25:75), UV detector, 254 nm,

Peaks: 1 = phthalylsulphathiazol; 2 –sulphabenzamide; 3 = sulphisoxazole; 4 = sulphacetamide, 5 = sulphadimethoxine; 6 = sulphachloropyridazine; 7 = sulphadiazine; 8 = sulphaquinoxaline; 9 = sulphamerazine; 10 = sulphamethoxy- pyridazine; sulphathiazol.