Reverse Phased Chromatography (RPC) in Practice

In practice the steps for a typical RPC separation can be summarized as in Figure 1.

Figure 1. Typical RPC separation using gradient elution.

As discussed under the theoretical section, a successful RPC separation is influenced by many parameters and will always need to be optimized to meet the requirements for the application. Steps toward selecting and optimizing media and conditions for an RPC separation of biomolecules are given here in order of priority:

1. Select a medium that provides best resolution under the simplest starting conditions, for example, TFA and acetonitrile in the eluent (proteins and peptides show little difference in selectivity when using silica- or polymer-based media under these conditions).
2. Scout for the pH that provides best resolution. This may be the key factor that defines whether to use a silica- or polymer-based medium.
3. If necessary, scout for a suitable ion-pairing agent to improve selectivity.
4. Optimize the gradient elution to maximize selectivity (the gradient slope only influences the distance between peaks, it will not change their elution order). Select the steepest gradient (lowest gradient volume) that provides acceptable results.
5. Scout for the highest flow rate that gives an acceptable separation.

Media and column selection

Choosing the correct RPC medium and column dimensions is critical for a successful separation and should be based on the goal of the application and the nature of the sample components.

Sample components: hydrophobicity

The selection of an RPC medium in relation to the hydrophobicity of the sample components must be made empirically. Unlike other chromatographic techniques, it is almost impossible to predict the retention of biomolecules in RPC. Important parameters affecting retention of a peptide appear to be a combination of the amino acid sequence of the peptide together with any secondary structure, such as α-helices and β-pleated sheets. The situation for proteins is further complicated by their tertiary structure.

Select less hydrophobic media when separating components that are known to be highly hydrophobic to facilitate elution. Samples that bind strongly to a medium will be more easily eluted from a less hydrophobic medium.

Goal of Separation

Applications involving fractionation of multi-component samples, such as peptide mapping, require extremely high resolution. Preparative reversed phase applications, such as the purification of synthetic peptides, are more concerned with throughput, and resolution may be traded off against speed and capacity. However, if used in the final polishing step, resolution will be crucial.

Resolution in reversed phase chromatography depends on the efficiency of the column and the selectivity. The parameters that can significantly affect selectivity are discussed earlier in this chapter.

Use commercially available prepacked columns whenever possible since maximum efficiency depends on the packing process and the particle size of the medium. The smallest 3 μm particles give the highest efficiency followed by the larger 5 μm media, such as SOURCE™ 5RPC and so on.

Scale of Separation

Use columns packed with 3 μm (μRPC C2/C18) or 5 μm (SOURCE™ 5RPC) particles for micropreparative and analytical separations.

Use 5 μm or 15 μm media for intermediate purification or polishing of laboratory-scale separations. A 15 μm media will be better suited for use with crude samples.

Use 15 μm or 30 μm media for large-scale preparative and process separation, for example, SOURCE™ 15RPC or SOURCE™ 30RPC. These media offer lower pressure requirements at high flow rates and have been optimized to ensure high throughput (amount of sample processed within a defined time) while retaining high performance. SOURCE™ 30 RPC is ideal for the polishing stage of industrial processes.

Note that the nature of RPC separations may cause slight changes in selectivity when changing particle size.

Eluent Conditions

When initial selectivity or sample stability and solubility factors indicate the use of an eluent above pH 7.5, always use a polymer-based medium such as SOURCE™ RPC.

When working with crude protein or peptide samples, it is an advantage to use polymer-based media that can be easily cleaned with alkali (silica-based media dissolve above pH 7.5).

Column Length

Increasing column length may improve resolution when working with large sample volumes.

Longer column lengths may improve resolution of a complex peptide mixture, for example, the resolution of peptides from a peptide digest.

Longer column lengths may improve resolution of closely-related peptides or proteins if a shallow gradient of organic modifier is used.

Eluent Selection and Preparation

Use the highest purity, HPLC grade solvents, acids, bases, salts, ion-pairing agents and water whenever possible. Chemical purity is important since contaminants may produce unwanted extra peaks, ghost peaks or contaminate the final product.

All components must be transparent to UV below 220 nm and soluble under the low polarity conditions used during a separation. Although proteins absorb at 280 nm and synthetic oligonucleotides at 250–260 nm, detection below 220 nm (usually at 215 nm) is necessary when separating short peptides that lack aromatic amino acid residues such as Trp and Tyr. Components recommended in Tables 18 and 19 have been chosen on the basis of providing optimal separation in combination with low background absorbance.

When possible, use volatile components. These can be removed by evaporation from the eluted fractions, along with the organic modifier. Non-volatile salts or acids must be removed by an additional desalting step.

pH and Ion-pairing Agents

Since the net charge of proteins and peptides varies with pH, their net hydrophobicity also varies with pH. Eluent pH is therefore an important influence on elution order and final selectivity.

For samples with unknown properties, start with the most commonly used strong acid that also acts as an ion-pairing agent: 0.1% trifluoroacetic acid as eluent A (reduce to 0.065% if baseline stability needs to be improved).

Add ion-pairing agents at concentrations to enhance binding of hydrophobic components to the medium. Note that other ion-pairing agents are not combined with TFA.

In the case of silica-based media, use ion-pairing agents to minimize mixed-mode retention (page 100), which can impair resolution.

The presence of ion-pairing agents can affect UV absorbance, and changes may be seen as the concentration of organic modifier changes. This may result in ascending or descending baselines during gradient elution. Always run a blank gradient to determine the effect of any additives prior to performing a separation. Adjust the concentration if necessary (refer to eluent balancing, page 112). Note that changing the concentration can change the degree of ionization of sample components and alter their behavior during separation.

Organic Modifiers

For samples with unknown properties, start with the most commonly used organic modifier, acetonitrile.

With a cut-off below 210 nm, acetonitrile has a much lower background absorbance than other common solvents at these low wavelengths, providing better baseline stability as the content of organic modifier is varied during a separation and ensuring optimum detection sensitivity.

Ion-pairing agents may need to be added (see above).

Use 2-propanol when requiring stronger eluting properties or to maintain sample stability. Note that the higher viscosity results in lower column efficiency and increased back pressure.

If the elution profile is still unsatisfactory, refer to Table 19, for a review of other organic modifiers. Note that changing the organic modifier can affect retention time. Changes in the elution order of proteins are likely to be a result of denaturation that significantly alters their hydrophobicity.

Typical Eluent Protocols for Separation of Proteins and Peptides

This section presents some of the more commonly used eluent protocols. Most protocols contain 5% or less of organic modifier in eluent A and 80% or less of organic modifier in eluent B. Note that concentrations above 80% may affect PEEK tubing which is often present in high performance chromatography systems.

Silica-based Media

Eluents A and B should contain at least 0.1% of a strong acid to act as an ion-pairing agent, to maintain a low pH and to minimize mixed-mode retention. Eluent B should contain a significantly higher content of organic modifier. For example:

Eluent A: 0.1% TFA in water, 5% acetonitrile

Eluent B: 0.1% TFA in acetonitrile (maximum 80%)

Polymer-based Media

Since polymer-based media can be used over a wider pH range and without concerns over mixedmode retention and the need to suppress the ionic interactions of silanol groups, a wider range of eluent protocols can be used.

For samples with unknown properties or known to require acidic conditions

Eluent A: 0.065% TFA in 2% acetonitrile

Eluent B: 0.050% TFA in 80% acetonitrile

For samples known to require basic conditions

Eluent A: 0.125% ammonium solution pH 10 in 2% acetonitrile

Eluent B: 80% acetonitrile in eluent A

Other systems

Eluent A: pH 2.1 0.1% formic acid, 2% acetonitrile

pH 2.0 0.1% acetic acid, 2% acetonitrile

pH 2.0 0.1% TFA, 2% acetonitrile

pH 4.5 10 mM sodium acetate, 2% acetonitrile

pH 7.0 10 mM potassium phosphate, 2% acetonitrile

pH 9.0 10 mM Tris-HCl, 2% acetonitrile in buffer

pH 12 10 mM NaOH, 2% acetonitrile in buffer

Eluent B: 70% acetonitrile in eluent A

Although most eluents contain strong acids and organic solvents that give little buffering capacity, adequate buffering capacity should be maintained when working closer to physiological conditions.

Preparation

1. Filter eluents that have had solids added, using a 0.22 μm filter. This prevents particles from clogging the column.
2. Measure volumes of organic solvent and aqueous solutions separately and then mix (this eliminates volume variations that occur when mixing organic and aqueous phases directly).
3. Degas the solutions in a sonication bath (<15 min), under vacuum with magnetic stirring (<5 min) or by purging by helium (<5 min). This prevents bubble formation during elution. Be careful to keep the degassing time as low as possible to prevent evaporation of the organic solvent.
4. Add volatile ion-pairing agents.

Use freshly prepared eluents.

Always use a flow restrictor (compatible with an appropriate pressure range) connected after the detector of a chromatography system to prevent the accumulation of air in the detector.

If eluents have to be stored, the containers must be sealed to avoid changes in composition caused by evaporation and, preferably, kept at 4°C. Do not store aqueous solutions at neutral pH for more than 2–3 days due to the risk of microbial growth. To reduce the risk of bubble formation, allow cold solutions to reach running temperature and degas them before use.

Follow health and safety regulations when using and disposing of the strong acids and organic solvents used in RPC.