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Reverse Phase LC

 REVERSED-PHASE LIQUID CHROMATOGRAPHY (RP-LC)

Principle

In reversed phase chromatography the stationary phase is non-polar and the mobile phase is polar.  With Silica based materials the non-polar surface is engineered by attaching silanes with a alkyl hydrocarbons tether. With polymer based materials the polymer matrix and thus the particle surface is made by apolar polymers such as Polystyrene or brominated Polystyrene. There is a slight difference in particle and performance properties e between Silica and polymer based materials. This section focusses on Silica based RP-LC,  the section Adsorption-LC on polymer based materials.

Octadecyl (C18) is the most common stationary phase, but octyl (C8) and butyl (C4) are also used in some applications. The designations for the reversed phase materials refers to the length of the hydrocarbon chain.

Elution

In reversed phase 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 like methanol, acetonitrile or THF. Retention increases as the amount of the polar solvent (water) in the mobile phase increases. Reversed-phase chromatography, a partition mechanism, is typically used for separations by non-polar differences.

Reversed Phase (RP) materials are manufactured by bonding alkyl chains to the silica gel base materials. The range covers anything from the very short length C1 to very large C 30 alkyl chain length:

A comparison list of the most important RP materials can be downloaded here

The performance of reversed-phase materials depends on many parameters. Two key properties, hydrophobicity and polarity, are of practical importance and dominate their selection.

CHARACTERISATION OF REVERSED-PHASE MATERIALS

Hydrophobicity

The strength of hydrophobic interaction can be measured by the retention of neutral (non-polar) molecules. The percentage of carbon in the material is a simplistic but useful guide to the retention characteristics of the column. In Figure 1 this loose correlation is demonstrated by the increase in retention observed when alkyl chain length (ie. carbon load) is increased. This results in an increase in hydrophobicity of the stationary phase

RPLC Fig 1

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

Fig 2

RPLC Fig 2

 

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)

The second key property of reversed-phase materials is their of a polar solute (involving neutral solute (involving hydrophobic interaction measured on a relative basis by comparing the retention only).

High Purity Base Deactivated Phases

In recent years a number of new alkyl bonded silicas have been introduced. The cumulative metal ion impurity level within these base silicas has in some cases 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 is produced, which gives significantly improved chromatography for the more basic polar solute molecules. Use of bonded alkyl groups containing hydrophilic substituents (ie. polar embedded) can either 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.

Fig 4

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 (eg. from C18 to cyano or phenyl) is a more powerful tool in altering the selectivity.

Fig. 5

RPLC Fig 5

Hydrophobicity vs. Polarity 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 the resultant phases’ 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. For any separation it is possible to select the most suitable phase from Figure 6. For basic solutes which will interact strongly with surface silanols, lower polarity phases are recommended. If a column of significantly different polar selectivity is required, select phases from a different section of the graph. If only differences in hydrophobicity are desired, simply select phases that are well separated on the hydrophobicity axis

Polarity

Low

Moderate

High

 

ACE CN
Hypersil CPS
Inertsil CN-3
Waters Spherisorb CN
Zorbax TMS
Zorbax SB CN

ACE Phenyl
Hypersil MOS
Inertsil Phenyl-3
NUCLEOSIL 100 C8
Waters Spherisorb C8
Zorbax C8
Zorbax SB Phenyl

Exsil 100 ODS
Exsil 100 ODS1
Hypersil ODS
LiChrospher RP-18
NUCLEOSIL 100 C18
NUCLEOSIL 120 C18
Waters Spherisorb ODS1
Waters Spherisorb ODS2
Zorbax ODS

High

Inertsil C4
SynChropak SCD-100
Vydac SelectaPore 300P
Zorbax SB C3

AquaSep
LiChrospher RP-select B
YMCbasic
Zorbax Rx C8
Zorbax SB C8

Hypersil BDS C18
Inertsil ODS
Inertsil ODS2
NUCLEOSIL 100 C18AB
Zorbax Rx C18
Zorbax SB C18

Moderate

ACE C4
Kromasil C1
Kromasil C4
Vydac SelectaPore 300M
YMC ProC4

ACE C8
Hichrom C8
Kromasil C8
NUCLEODUR Gravity C8
YMC ProC8
Zorbax XDB C8

ACE C18
Develosil ODS-UG
Hichrom C18
Hichrom RPB
HyPURITY C18
Inertsil ODS-3
Kromasil C18
NUCLEODUR Gravity C18
NUCLEOSIL 100 C18HD
Symmetry C18
YMC ProC18
Zorbax XDB C18

Low

 

 

 

 

Hydrophobicity

Fig 6

WIDE PORE (300) REVERSED-PHASE MATERIALS

Introduction

In order 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 60-120 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, due to a reduction in size-exclusion mechanism and enhanced molecular diffusion rates. A pore size of 300 has become the accepted standard for wide pore silicas, and has been found to be suitable for a broad range of molecular weight proteins, peptides and oligonucleotides. In general, peptides exceeding about 50 amino acids and oligonucleotides greater than 25 residues are preferentially analysed on 300 materials. Separations of very large  biomolecules (MW >100,000 Da) may require larger pore size packings (500 to 4000).

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.  The Table below summarises a range of wide pore alkylbonded reversed-phase silica materials. Ion-exchange and size exclusion packings are also available as wider pore materials.

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.

 

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