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Reverse Phase Liquid Chromatography

Reversed Phase Liquid Chromatography (RP-LC)

In Reverse Phase Liquid Chromatography the stationary phase is non-polar and the mobile phase is polar. With Silica based materials the non-polar surface is altered by means of attaching silanes with a alkyl hydrocarbons tethers.

With polymer-based materials the polymer matrix that is to say the particle surface is made by apolar polymers such as Polystyrene or brominated Polystyrene.

There is a slight difference in particle and performance properties between Silica and polymer-based materials. As a result of that we focus here on Silica based RP-LC. The polymer based RPLC materials are discussed under Adsorption-LC.

Silica based Octadecyl (C18) material is the most common stationary phase. Octyl (C8) and butyl (C4) are popular but not the best choice in biochromatography. The designations for the reversed phase materials refers to the length of the hydrocarbon chain. . The range covers anything from the very short length C1 to very large C 30 alkyl chain length:

Elution in Reverse Phase Liquid Chromatography

The partition-mechanism in Reverse Phase Liquid Chromatography, is typically used for separations by non-polar differences

In Reversed Phase Liquid Chromatography the most polar compounds elute first with the most non-polar compounds eluting last.

The mobile phase is generally a binary mixture of water and a miscible polar organic solvent. Typical examples are methanol, acetonitrile or THF. Retention increases as the amount of the polar solvent (water) in the mobile phase increases..

A comparison list of the most important RP materials can requested through Contact Form

The performance of reversed-phase materials depends on very many parameters. However, hydrophobicity and polarity are of practical importance and dominate selection.

Characteristics In Reversed Phase Liquid Chromatography

Hydrophobicity

The strength of hydrophobic interaction can be measured by the retention of neutral (non-polar) molecules. Percentage of carbon in the material is also a simplistic but useful guide to the retention characteristics of the column.

In Figure 1 the loose correlation is demonstrated. An  increase of retention is observed when alkyl chain length (i.e. carbon load) is increased. This results in an increase in hydrophobicity of the stationary phase RPLC Fig 1

Reverse Phase Liquid Chromatography Column Fig-1
Increasing alkyl chain length (ie. carbon load) increases hydrophobicity and retention

Figures 2 and 3 compare the retention obtained for a selection of non-polar solutes with a range of commercially available C8 and C18 columns.

Fig 2

Reverse Phase Liquid Chromatography Fig.2
Separation behavior on C8 bonded phases

RPLC Fig 3

Fig 3 C18 Hydrophobicity Comparison, Separation of dimethyl phthalate, toluene, biphenyl and phenanthrene on C18 bonded phases using a methanol-water (90:10) eluent
Polarity (Silanol Activity)

High Purity Base Deactivated Phases

About the year 2000 new alkyl bonded silicas have been introduced. The cumulative metal ion impurity level within these base silicas has been reduced to <10ppm.  As a result the number of isolated silanol groups and hence the polarity of the silica surface is also reduced.

When coupled with the use of more effective and reproducible bonding processes, a new generation of reversed-phase materials was produced. The resultant is significantly improved chromatography for the more basic polar solute molecules. Use of bonded alkyl groups containing hydrophilic substituents (i.e. polar embedded) did enhance the above effect and/or offer alternative selectivity.

Figure 4 demonstrates the reduced polarity of high purity base deactivated materials compared to lower purity products. Toluene is used as a hydrophobic reference. High purity (low polarity) materials generally give better peak shape with strongly basic compounds. However, low purity (high polarity) materials may offer a unique selectivity.

RPLC Fig 4

Optimising Selectivity

Figure 5 illustrates the change in polarity and hydrophobicity for Kromasil C18, C8 and C4 materials. As discussed previously, a decrease in hydrophobicity on reducing alkyl chain length is observed. Greater ligand density and hence lower polarity is also seen as the length of the alkyl chain is reduced from C18 to C4. Such variations offer the possibility of reduced analysis time and improvements in peak shape, but no major change in selectivity. Changing the chemistry of the bonded phase (e.g. from C18 to cyano or phenyl) is a more powerful tool in altering the selectivity.

Fig. 5

RPLC Fig 5

Hydrophobicity vs. Polarity in Reverse Phase Liquid Chromatography a comparison

Traditional C18 (ODS) phases are hydrophobic and have a high polarity due to the lower purity silicas on which they are based. Use of the new high purity silicas reduces  silanol activity and improves reproducibility.

Employing a polar embedded functionality may also result in a reduced polarity material. Shorter alkyl chain phases are found at the lower hydrophobicity area of the graph. Alternative bonded phases (including phenyl and cyano) based on high purity silicas are best considered to effect changes in polarity.

WIDE PORE (300Å) REVERSED-PHASE MATERIALS

Introduction

Indeed, for a sample molecule to freely access the interior of the pores of the packing material, its diameter must be smaller than the average pore diameter.

For high molecular weight solutes, the use of lower pore size materials of 60-120Å may result in frictional drag within the pore leading to restricted diffusion and reduced column efficiency.

 The use of larger pore silica-based bonded phases leads to improvements in resolution, capacity and recovery of proteins and other biomolecules.  There is a reduction in size-exclusion mechanism and enhanced molecular diffusion rates. A pore size of 300Å has become the accepted standard for wide pore silicas. That is to say it has been found to be suitable for a broad range of molecular weight proteins, peptides and oligonucleotides. In general, peptides exceeding 50 amino acids and oligonucleotides greater than 25 residues are preferentially analyzed on 300 Å materials. Separations of very large biomolecules (MW >100,000 Da) may require larger pore size packings (500 to 4000Å).

Bonded phases

Alkyl bonded silica phases are the most commonly used materials for the reversed-phase separation of biomolecules. The shorter C4 matrices are generally recommended for large hydrophobic peptides and most proteins. Peptide maps, natural and synthetic peptides and small hydrophilic proteins are best chromatographed on C8 columns. C18 columns are often chosen for the analysis of small peptides. Other bonded wide pore phases, including cyano and phenyl, are available in some brands.

Column Dimensions

Wide pore silica phases are available in a range of column dimensions from rapid analysis to preparative and process scale. Increased column capacity favours these wide pore materials for preparative separations of samples with molecular weight >5000 Da.