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The mobile phase
The eluent
Organic solvents are used to increase the eluting strength by decreasing the polar properties of the eluent in RPC. This solvent is referred to as the organic modifier and should be completely miscible with water, the other solvent component of the eluent.
Lower alcohols or acetonitrile (ACN) are often used for organic molecules, while peptides and proteins (when stable enough) are eluted by ACN or isopropanol-containing eluents.
ACN has a very good UV transparency and contributes rather little to the eluent viscosity and thus to the back pressure over the column.
For peptide and protein separations ACN is the by far most commonly used organic modifier and iso-propanol is turned to only when required by the sample stability.
Eluent components to modify eluting strength
Organic
modifier | Suitable for: | UV cut-off | Viscosity | Comments |
Methanol |
| 210 | Medium- low | May destabilise protein structure |
Ethanol |
| 205 | Medium- low | May destabilise protein structure |
Isopropanol |
| 210 | High | Least effect on protein structure |
Acetonitrile
(ACN) |
- Org. molecules
- Proteins
- Peptides
| 190 | Low | Less effect on protein structure |
Fig 4.1. Properties of frequently used eluents.
The three experiments shown in Figure 4.2 all use the same sample and identical gradient slopes. As seen, methanol provides the best resolution. However, ACN is normally preferred due to its better optical properties and the lower viscosity encountered.
Fig 4.2 The effect of changing eluent. A seen, the eluents shown
differ in eluting strength rather than in their influence on selectivity.
Eluent pH
Peptides and proteins are ampholytes i.e. their net charges vary with pH (see Basic principles in IEC) and their net hydrophobicities vary accordingly.
Eluent pH is therefore an important parameter influencing elution order and spacing of the peaks.
Fig 4.3. Protein and peptide net charges vary with ambient pH, a fact that influences their hydrophobic properties and thus their chromatographic behavior in RPC.
Fig 4.4. Peptide net hydrophobicity is influenced by pH and when run at different pH values, peptide elution positions may change considerably.
The pH change from 2 to 6.5 actually rearranges the elution order of the angiotensin derivatives of figure 4.4 quite considerably.
Eluent pH
Ionic molecules tend to interact by a process called ion pairing. The process has much in common with IEX only that the interaction takes place in free solution (Fig 4.5). The ion pairing tendency increases with decreasing polar properties of the solvent.
Fig 4.5. Ion pairing agents associate with sample
molecules giving altered net hydrophobicities.
Ion pairing agents
Ion pairing agents that are frequently used in RPC to block charges thereby rendering a charged sample molecule less hydrophobic.
Commonly used ion pairing agents are listed in Figure 4.6.
Fig 4.6. Frequently used ion pairing agents.
Ion pairing agents have a pronounced influence on selectivity in RPC and as seen in Figure 4.7 both peak spacing and elution order is affected.
Fig 4.7. Ion pairing agents have a pronounced effect on selectivity.
Note, however, that part of the effect seen in the
experiments above depend on the running pH used. |
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