Introduction

Partition or liquid-liquid chromatography (LLC) is a powerful separation technique which has been successfully used for the separation and analysis of acids and proteins. The basis of LLC is the distribution of sample molecules between two immiscible liquid phases, a stationary phase and a mobile phase (Figure 1). In conventional LLC, the stationary phase is mechanically held to a support by adsorption. In liquid chromatography (LC), the most widely used supports are those with chemically bonded organic stationary phases. This may be described as a separate LC method: bonded-phase chromatography (BPC).

LLC offers unique selectivity for various samples since a wide range of liquid phases can be used (Table 1). In normal-phase LLC, the support is coated with a polar stationary phase, whereas a relatively nonpolar solvent is used as mobile phase. In reversed-phase LLC, the two LLC phases are interchanged so that the less polar liquid is now the stationary phase and the polar liquid is the mobile phase. LLC systems composed of aqueous/aqueous, aqueous/organic or organic/organic solvents can be used, depending on the particular separation problem. A variety of supports have been used (Table 2), but silica-based matrices appear to be best suited for LLC.

The sample molecules are distributed between the stationary and mobile phases depending on their physicochemical properties and the properties of the

Table 1 Liquid-liquid chromatographic systems commonly used for LLC and LLPC of compounds, nucleic acids and proteins

Sample

Chromatographic system

Stationary phasea

Mobile phase

Compounds

Normal-phase LLC

^'-Oxydipropionitrile

Pentane, cyclopentane, hexane,

heptane or isooctane. Same, but

with 10-20% of chloroform, di-

chloromethane tetrahydrofuran,

acetonitrile, dioxane, etc.

1,2,3-Tris(2-cyanoethoxy)propane

See above

Triethylene glycol

See above

Trimethylene glycol

See above

Ethylene glycol

Di-n-butyl ether or nitromethane

Dimethylsulfoxide

Isooctane

Water/ethylene glycol

Hexane/CCl4

Water

n-Butanol

Nitromethane

CCl4/hexane

Reversed-phase LLC

Cyanoethylsilicone

Methanol/water

Dimethylpolysiloxane

Acetonitrile/water

Hydrocarbon polymer

Methanol/water

Nucleic acids

LLPC

4.4% PEG 8000/6.2% dextran 500a

Proteins

LLPC

4.4% PEG 8000/5.2% dextran 500a

4.4% PEG 8000/6.2% dextran 500a

5.2% PEG 8000/10.0% dextran 40a

2.7% PEG 20000/4.5% dextran 500a

LLPC

10% PVP 30/10% dextran 40a

LLPC

PVA 10/dextran 40a

LLPC

11.25% PEG 1500/12.75% potassium phosphatea

16.75% PEG 1500/16.0% sodium citratea

LLC, liquid-liquid chromatography; LLPC, liquid-liquid partition chromatography; PEG, polyethylene glycol; PVP, polyvinylpyrrolidone; PVA, polyvinyl alcohol.

aOr complete system.

LLC, liquid-liquid chromatography; LLPC, liquid-liquid partition chromatography; PEG, polyethylene glycol; PVP, polyvinylpyrrolidone; PVA, polyvinyl alcohol.

Figure 1 Schematic illustration of LLC. IU, international units; VR, retention volume.

a wide variety of sample types, including water-soluble and oil-soluble compounds, ionic and nonionic compounds, as well as biopolymers such as nucleic

Table 2 Supports commonly used for LLC of compounds, nucleic acids and proteins

Sample Support type Name Particle size (¡m) Pore size

Compounds

LLC Porous

Pellicular

Diatomaceous earth Silica, spherical

Silica, irregular

'Inactive' silica, spherical

'Active' silica, spherical

Corasil modified with octadecylsilane Perisorb A modified with dimethylsilane

Zipax modified with octadecylsilane Vydac modified with ethylnitrile

Nucleic acids/proteins Polyvinyl modified with polyacrylamide Silica modified with polyacrylamide

Agarose modified with dextran

Chromosorb LC-1 37-44

Spherosil XOA 600 5-8

Zorbax 6

Porasil 10

LiChrosorb Si-60 5,10

Zipax 25-37

Liqua-Chrom 44-53

Corasil I 37-50

Perisorb A 30-40

Vydac 30-44

Bondapak C18/Corasil 37-50

Perisorb-RP-2 30-40

Permaphase-ODS 25-37

Vydac SC-polar 30-44

LiParGel 650 25-40

LiParGel 750 25-40

LiChrospher-Diol 100 10

LiChrospher-Diol 1000 10

LiChrospher-Diol 4000 10

Superdex 200 prep. 24-44 grade

a Data not available. Exclusion limit for globular proteins of 5 x 106 Da and for dextrans of 106 Da. b Data not available. Exclusion limit for globular proteins of 5 x 107 Da and for dextrans of 107 Da. cData not available. Exclusion limit for globular proteins of 106 Da and for dextran of 105 Da. LLC, liquid-liquid chromatography; BPC, bonded-phase chromatography.

phases (and support). In the case of compounds, the separation is generally based on the type and number of substituent groups and by differences in molecular weight (up to about 2000 Da). Hence, LLC is very useful for the separation of homologues and mixtures of compounds of different functionality. Biopolymers (nucleic acid fragments) can be separated mainly owing to differences in molecular weight (up to about 4000 base pairs). In the case of proteins, conditions can be designed in such a way that differences in their overall exposed surface properties are detected. In fact, LLC has proved to be a unique tool for protein analysis, that is for purification and fractionation, detection and separation of conformational isomeric forms, examination of surface properties related to biological specificities and for providing information about the events upon binding of ligand and about possible ligand-induced conformational changes. In this article, we describe the use of LLC for the separation and analysis of compounds, nucleic acids and proteins, focusing on the latter group.

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