Performing an RPC Separation with SOURCE™ Media

Performing a separation

Use the instructions given here as a basis from which to optimize a separation.

For samples with unknown properties or known to require acidic conditions

Eluent A: 0.1% TFA in 2% acetonitrile

Eluent B: 0.1% 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

First-time use or after long-term storage

  1. Flush out the storage solution with at least 5 column volumes of eluent A
  2. Wash the column using a gradient of 5 column volumes from 0–100% eluent B (or continue at 100% eluent B until UV signal is stable).
  3. Wash the column with a gradient of 2 column volumes from 100–0% eluent B.
Flow: 0.2 ml/min (SOURCE™ 5RPC ST 2.1/150)
  1 ml/min, (SOURCE™ 5RPC ST 4.6/150)
  2 ml/min, (SOURCE™ 15RPC ST 4.6/100)
  1–5 ml/min, (RESOURCE™ 1 ml)
  1–5 ml/min, (RESOURCE™ 3 ml) or, for larger columns, 200 cm/h (SOURCE™ 15RPC) and
  100–1000 cm/h, (SOURCE™ 30RPC)
  Collect fractions throughout the separation.
  1. Equilibrate the column with at least 10 column volumes of eluent A until the UV signal is stable.
  2. Dissolve the sample in a small volume of eluent A. Filter or centrifuge to remove particulate matter if necessary. Apply to the column.
  3. When the UV signal is stable, that is, when all unbound material has washed through the column, elute using a gradient of 10–20 column volumes from 0–100% eluent B.
  4. Wash the column with at least 5 column volumes of 100% eluent B (or until UV signal is stable) to elute any remaining material.
  5. Wash with a gradient of 2–3 column volumes from 100%–0% eluent B.
  6. Re-equilibrate with 10 column volumes of eluent A or until UV signal is stable.

Do not exceed the maximum recommended flow for the medium.

Check column performance regularly by determining column efficiency and peak symmetry. See Appendix 2.

Cleaning

Correct preparation of samples and eluents, including filtration, the removal of any particulate matter and a final wash step in 100% eluent B, should keep most columns in good condition. However, reduced performance, reduced flow, increasing back pressure or complete blockage are all indications that the medium needs to be cleaned using more stringent procedures in order to remove tightly bound, precipitated or denatured substances.

It is recommended to reverse the direction of flow during cleaning so that contaminants do not need to pass through the entire column length. The number of column volumes and contact time required for each cleaning step may vary according to the degree of contamination. If the cleaning procedure does not restore column performance, change the top filter before trying alternative cleaning methods. Take care when changing a filter as this may affect the column packing and interfere with performance.

Contact time, organic solvent and pH are significant parameters for successful cleaning, and different protocols may have to be developed and used in combination according to the nature of the contaminants. Examples of cleaning protocols are as follows:

Eluent A: 0.1% TFA

Eluent B: 0.1% TFA in 80% acetonitrile

Note: acetonitrile cannot be used for bioprocess applications, 2-propanol is an accepted alternative.

Flow: 0.5 ml/min, (SOURCE™ 5RPC ST 4.6/150);
  0.5 ml/min, (SOURCE™ 15RPC ST 4.6/100)
  1.0 ml/min, (RESOURCE™ 1 ml)
  1.0 ml/min, (RESOURCE™ 3 ml) or, for larger columns, 100 cm/h (SOURCE™ 15RPC) and 100 cm/h, (SOURCE™ 30RPC)
  1. Equilibrate the column with at least 10 column volumes of eluent A until the UV signal is stable.
  2. Wash using a gradient of 20–30 column volumes from 0–100% eluent B.
  3. Wash the column with at least 10 column volumes of 100% eluent B.
  4. Wash using a gradient of 20–30 column volumes from 100–0% eluent B.
  5. Wash the column with at least 10 column volumes of eluent A.
  6. Equilibrate the column in at least 10 column volumes in the eluent A that will be used for the separation if different the eluent used in step 5. Transfer between the two eluents should be performed using a 2–3 column volume gradient if the two eluents are significantly different.

Change to 0.1%TFA in 2-propanol for eluent B if column performance is not restored. Note that 2-propanol will increase back-pressure, and flow rates may need to be reduced.

For removal of contaminants known to be acid- or alkali-soluble the following eluents can be used, following the same procedure as outlined above:

Removal of acid-soluble contaminants

Eluent A: 90% acetic acid

Eluent B: 80% acetonitrile or 50% 2-propanol

Removal of alkali-soluble contaminants

Eluent A: 0.5 M NaOH

Eluent B: 50% acetonitrile or 50% 2-propanol

If neither of the protocols for acid- or alkali-soluble contaminants is successful, wash the column in 5–10 column volumes of 6 M guanidine hydrochloride.

SOURCE™ RPC media can be cleaned using aggressive chemical agents since the polystyrenebased matrix is extremely stable. Sodium hydroxide is a very effective cleaning agent and SOURCE™ RPC can be equilibrated with several column volumes of 0.5–1 M NaOH for cleaning.

The ability to use such a strong cleaning agent is a major advantage of using SOURCE™ RPC for large-scale separations. At production scale, other cleaning protocols may be applied in order to fulfill regulatory requirements.

Media characteristics

Composition: rigid, monodisperse, polystyrene/divinyl benzene particles (5 μm, 15 μm or 30 μm) with an optimized pore size distribution.

Table 25. Media characteristics for SOURCE™ RPC media.

Product Temperature stability for regular use pH stability* Mean particle size
SOURCE™ 5RPC 4°C to 60°C Long term: 1–12
Short term: 1–14
5 μm
SOURCE™ 15RPC 4°C to 40°C Long term: 1–12
Short term: 1–14
15 μm
SOURCE™ 30RPC 4°C to 40°C Long term: 1–12
Short term: 1–14
30 μm

*Long-term pH stability refers to the pH interval where the medium is stable over a long period of time without adverse side effects on the chromatography performance.

Short-term pH stability refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.

All ranges are estimates based on the experience and knowledge within Cytiva.

Chemical stability

For daily use, SOURCE™ RPC media are stable in:

  • all commonly used aqueous buffers: 1 M HCl, 1 M NaOH, 1 M HCl/90% methanol, 90% acetic acid, 0.45 M NaOH/40% 2-propanol, 6 M guanidine hydrochloride, 1-propanol, 20% ethanol, acetone.
  • aqueous solutions pH 1–12 including trifluoroacetic acid (up to 0.3%), pentafluoropropionic acid (up to 0.3%), heptafluorobutyric acid (up to 0.3%), perchloric acid (up to 0.3%), formic acid (up to 0.3%), acetic acid (up to 60%), ammonium acetate (10–50 mM), phosphoric acid (10–50 mM).
  • water-miscible organic solvents including methanol, ethanol, acetonitrile, 1-propanol, 2-propanol.
  • ion-pairing agents such as trifluoroacetic acid, tributylphosphate, triethylammonium phosphate, tetrabutylammonium salts, hexylsulfate.

Note that ethanol, 1-propanol and 2-propanol will increase back-pressure, and flow rates may need to be reduced.

Avoid detergents, oxidizing agents and solutions <pH 1 and >pH 12.

Storage

SOURCE™ 5RPC columns: wash with 70% acetonitrile. SOURCE™ 15RPC and SOURCE™ 30RPC columns: wash with at least 10 column volumes distilled water, equilibrate with at least 10 column volumes 20% ethanol or 70% acetonitrile. Note that columns used for bioprocess applications can only be stored in 20% ethanol.

Store at 4°C to 30°C. Ensure that the column is sealed well to avoid drying out. Store unused media at 4°C to 8°C in 20% ethanol. Do not freeze.

Materials