What is reversed phase chromatography?

Reversed phase chromatography (RPC) is a separation technique that separates molecules according to hydrophobicity. Unlike in hydrophobic interaction chromatography (HIC) the RPC medium is highly substituted with hydrocarbon chains making it very hydrophobic. Proteins, peptides and oligonucleotides adsorb to the hydrocarbon chains even in pure water. In HIC, hydrophobic interaction has to be promoted by salts to force the sample molecules to adsorb while RPC requires organic solvents to desorb them. Elution uses gradients of increasing concentrations an organic solvent like acetonitrile in water. RPC provides very high resolution and is used to separate molecules with only minor structural differences such as peptides produced by enzymatic digestion or failures after peptide synthesis. In fact, RPC is the preferred separation technique for peptides. Unlike peptides, proteins contain a substantial degree of tertiary structure important for its biological function. The strong adsorption in RPC requires rather harsh eluents, a fact that often comes in conflict with protein stability and makes RPC less suitable for protein separations where recovery of biological activity is essential. This picture shows hydrophobic and hydrophilic parts on the surface of lysozyme. The most hydrophobic parts are dark red, the less hydrophobic lighter red. The most hydrophilic parts are shown in dark blue, while the less hydrophilic parts are lighter blue.

The separation mechanism

Reversed Phase Chromatography (RPC) utilises solubility properties of the sample in much the same way as the organic chemist does when he purifies a crude sample by partitioning it between two different liquid phases in a separation funnel (Fig 1.1). Fig 1.1 Purification by partitioning the sample between two liquid phases. The distribution is controlled by the difference in polar properties of the respective phases. The distribution of sample components between the two phases will depend on their respective solubility characteristics and the polar properties of the phases. The solubility "rule of thumb" says: "equal dissolves equal" i. e. non-polar substances dissolve preferably in non-polar solvents, while polar ones dissolve in polar solvents. By varying the polar properties of one of the phases (the lower phase in Fig 1.1) one can change the distribution of the components to be purified as well as the contaminants. A more efficient way to use the very same principle is to turn to column chromatography (figure 1.2) in which the funnel experiment is repeated a very large number of times per run. Fig 1.2 Reversed Phase Chromatography utilises solubility differences between the sample components by a continuous re-partitioning mechanism. In the column the sample is continuously re-partitioned between a stationary non-polar phase (the beads in the column) and a mobile phase (the liquid pumped through the column). The distribution between the stationary and the mobile phase is controlled by the polar properties of the mobile phase. The more of a sample component that dissolves in (adsorbs to) the stationary phase, the more the zone will be retarded while carried down the column by the mobile phase or eluent. The somewhat odd name reversed phase chromatography deserves an explanation. The most commonly used solubility-dependent chromatography techniques are: The name reversed phase chromatography was coined in relation to the older technique, normal phase chromatography. Solubility properties The ability of a solvent to dissolve a substance depends on its ability to interact with the substance. Non-polar substances are held together by Van der Waals interactions (Fig 1.3) which polar solvents do not break, since dipole-dipole interaction between the solvent molecules is too strong. Fig 1.3 Hydrocarbon chains attract each other mainly by Van der Waals interactions. Polar substances are held together by the attraction between dipoles (Fig 1.4). It takes polar solvents to break these bond in order to bring about solubilisation. Fig 1.4 Dipoles interact in a head-to-tail manner between the polar centres. Hydrogen bonding is typical for water and makes water molecules appear in clusters rather than as "free" single molecules (Fig 1.5). The HX- group is also a dipole, which makes water an excellent solvent for polar substances especially those containing HX-groups ( X= O; N; F; Cl). Fig 1.5. Hydrogen bonding occurs betweenmolecules containing HX-groups. Each water molecule can bind four other water molecules. This fact constitutes the very special properties of water and makes it an excellent solvent for polar substances. The difference in hydrophobicity forms the basis for separation in RPC. Fig 1.6 Because of their solubility in water, polar substances are called hydrophilic. Non-polar substances, on the other hand, are called hydrophobic because they do not dissolve in water. There is no distinct limit between hydrophilic and hydrophobic behaviour, but rather a continuum. Adsorption mechanisms RPC was first applied to relatively small organic molecules which more or less dissolved in the hydrocarbon phase, in other words the distribution between the stationary and the mobile phases worked in close analogy to the separation funnel. Fig 1.7 Organic molecules are "embraced" by the carbon chains of the stationary phase. With peptides and proteins, however, the mechanism is a bit different. First of all both peptides and proteins carry a mix of hydrophilic and hydrophobic amino acids accessible for interaction with the RPC ligands. Moreover, they are rather large at least in comparison to the traditional organic target molecule. They therefore cannot be completely "embraced" by the hydrocarbon phase. Instead there is a high probability for multi-point attachment. Hydrophobic surfaces are known to combine by a mechanism called hydrophobic interaction (see below!). Together this leads to adsorption rather than to dissolution and the properties of the interaction for peptides and proteins deviates distinctly from that of the typical organic molecule. Fig 1.8 Unlike the typical organic target molecule peptides and proteins adsorb to the stationary phase often by multi-point attachment. Hydrophobic interaction Hydrophobic surfaces are surrounded with layers of highly organized water molecules, when exposed to aqueous solvents. A decrease in the amount of this organized water would lead to a thermodynamically more favourable situation by an increase in entropy (DS). Hydrophobic surfaces therefore combine to minimise the total area exposed to the aqueous solvent and thereby the amount of organized water. RPC media carry ligands consisting of hydrocarbon chains, which can combine with hydrophobic surfaces of peptides and proteins in this way. In fact, in RPC the hydrophobic interaction is strong enough to adsorb these molecules in pure water. To bring about desorption, eluents consist of mixtures of water and organic solvents like acetonitrile. Fig 1.9. In contrast to bulk water, hydrophobic surfaces are covered by a shell of highly ordered water molecules. The carbon chains of the stationary phase combine with the hydrophobic areas of peptide and proteins to minimise this shell and so gain in entropy. Fig 1.10. A decrease in the polar properties of the mobile phase will weaken the hydrophobic interaction. Fig 1.11. Protein tertiary and quaternary structures depend to a large extent on hydrophobic interaction as a stabilising force. RPC eluents are designed to weaken hydrophobic interactions and are thus potential denaturants. Peptides and proteins are made to be biologically active under "physiological" conditions i.e. contact with quite polar solvents. Hydrophobic interaction is one of the important forces stabilizing the tertiary and quaternary structures of proteins and eluents that weaken hydrophobic interaction are also potentially denaturants. If a protein becomes partially unfolded at an eluting strength below or near that needed to desorb the protein, more hydrophobic areas may be uncovered resulting in increased co-operative binding. This in turn requires an even higher eluting strength for desorption leading to further unfolding and so on. Such a chain of events may lead to complete denaturation and/or irreversible adsorption. Fig 1.12 Peptide secondary structure is stabilized mainly by hydrogen bonding and is less sensitive to RPC eluents. Protein tertiary structure depends on hydrophobic interaction as one of the stabilizing forces and is thus sensitive to RPC eluents. Peptides contain a low (if any) degree of tertiary structure and are readily renaturated. They are therefore less likely to be harmed by RPC eluents. Proteins, on the other hand, depend on their tertiary and quaternary structures for their biological function and are much more difficult to renaturate. RPC of proteins is therefore a delicate balance between desorption and denaturation and care must be taken to satisfy this balance or the protein may be irreversibly destroyed. Desorption curves The adsorption reaction is a dynamic equilibrium between free and adsorbed molecules and is controlled by the content of an organic solvent in the eluent. It can be described in terms of a desorption curve obtained by plotting the relative amount of free sample molecules as a function of the organic solvent concentration as shown in Figure 1.13. (Desorption curves have little practical value and are used here only to demonstrate the working principles of RPC.) In a column experiment all transport of a sample down the column is carried out by the mobile phase (the eluent) and acts only on the molecules present in the mobile phase. When a sample travels down the column, its velocity is proportional to the portion of sample molecules present in the mobile phase. The desorption curve thus represents the velocity of a sample zone as a function of the organic solvent concentration. The concentration interval corresponding to the desorption curve will be referred to as the partition zone. Fig 1.13 The desorption curve reflects the distribution of the sample between the mobile and the stationary phase. Within the partition zone this distribution varies as a function of the salt concentration and the elution velocity varies accordingly.

		Explanation of symbols
Symbolic representation of a section of an RPC bead.	The least hydrophobic sample component.	Sample component with intermediate hydrophobicity.	The most hydrophobic sample component.	Very hydrophobic contaminant.

1. Equilibration Buffer A is pumped through the column until conditions matches those intended.	2. Sample application and wash Sample is added and adsorbed and non-adsorbed components are washed out.	3. Gradient elution Gradient is started and adsorbed sample components are eluted in order of their respective Hydrophobicities. Elution order:			4. Regeneration Remaining hydrophobic contaminants are washed out.

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 Org. molecules 210 Medium- low May destabilise protein structure Ethanol Org. molecules 205 Medium- low May destabilise protein structure Isopropanol Proteins Peptides 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.

There are primarily two types of RPC media, one based on silica beads covered with a bonded non-polar phase of carbon chains and one based on a naked non-polar polymer matrix (Fig 5.1). Fig 5.1. Properties of RPC media. There is no big difference in selectivity between polymer-based and silica-based RPC in the commonly used pH range (2 - 7.5), at least not with ACN as the organic modifier. Above pH 7.5 , however, silica dissolves leaving polymer based RPC as the only alternative. It is interesting to note that the amino acids cysteine, tyrosine, and lysine all have pKa values in the pH range 9.1 - 10.4. RPC in this pH interval would consequently provide new selectivities for peptides and proteins containing these amino acids. Polymer-based media can be cleaned by alkali, a great advantage when running crude protein and peptide samples. Small organic molecules behave as if they were dissolved in the hydrocarbon phase, while peptides and proteins behave as if they were adsorbed to it. As a consequence, organic molecules are sensitive to the chain length (the depth) of the bonded phase. Proteins and peptides, on the other hand, are much less sensitive in this respect (Fig 5.2). Proteins and many peptides carry both hydrophilic and hydrophobic areas on their surfaces and the net hydrophobicity varies depending on the ratio between the two. Fig 5.2. "Classical" hydrophobic organic molecules are sensitive to the carbon chain length, while more or less identical results are obtained for proteins and larger peptides, regardless of the carbon chain length. Generally, most components interact with RPC media in a way characterised partly by dissolution and partly by adsorption.

Reversed phase chromatography in practice

Use of Reversed phase chromatography

High resolution mode (gradient elution)	Group separation mode (step elution)
Separates peptides, proteins and oligonucleotides according to net hydrophobicity.	Concentrates dilute oligonucleotide and peptide samples.
Suitable for intermediate steps and polishing in multi-step purification protocols.	Suitable for so called solid phase extraction.
Main technique for the purification of synthetic peptides.	Suitable for desalting of peptide and oligonucleotide samples.
Main technique for the analysis of peptides and for peptide mapping.

Experimental

	High resolution mode (gradient elution)	Group separation mode (step elution)
Reversed phase chromatography medium	Use 10 - 30 mm media for purification work. Use 5 - 12 mm media for analytical work. Use polymer-based media for protein separations (allows cleaning under alkaline conditions).	Use media with 30 mm beads or larger to allow higher flow rates.
Column	Typical column lenghts are 1 - 10 cm.	Column length is less important. Short and "fat" columns, however, will allow higher flow rates.
Eluents	Oligonucleotides are separated under alkaline conditions Standard conditions for peptides are ACN; TFA at pH ~ 2.	Eluent optimisation is not necessary for desalting and concentrating the sample. Sample salts will elute during sample application.
Sample volume	Sample volume restricted to 0.5 - 5% of column volume in isocratic mode. No sample volume restriction in gradient mode.	The sample volume is not important since the sample will bind to the column.
Sample amount	5 - 10 % of the total loading capacity of the column used can be applied without loss of resolution.	Around 40 % of the total loading capacity of the column used can be applied.

• Adjust pH of buffer B before adding the organic solvent.
• Adjust buffer pH at the temperature intended for the experiment.
• Always test that target protein/ peptide is soluble and stable in buffer A and B.
• Use HPLC grade chemicals for analytical experiments.

• Filter or spin the sample to remove any particulate matter.
• Especially with large sample volumes, adjust sample pH and salt content to match those of "buffer A".
• Buffer exchange on Sephadex G-25 is a rapid and mild way to adjust sample conditions.
• Especially with large sample volumes, adjust sample temperature to match that of "buffer A".

Reversed phase chromatography profile

Reversed Phase Chromatography

Handbooks

Hydrophobic Interaction and Reversed Phase Chromatography pdf
Protein Purification Handbook pdf