Standard Purification Protocol for Synthetic peptides
The Standard Purification Protocol for synthetic peptides combines two RPC steps
under conditions providing quite different selectivities and will, in most cases, be
able to render a sufficiently pure target peptide.
Purification Protocol I, for peptides soluble in acidic and neutral solvents
Step one
Step two
Neutral RPC on
Sephasil Peptide C8 or
RESOURCE RPC
at pH 7.0
Acidic RPC on
Sephasil Peptide C8 at
pH ~2.0
Purification Protocol II, for peptides soluble in neutral and alkaline solvents
Step one
Step two
Neutral RPC on
Sephasil Peptide C8 or
RESOURCE RPC
at pH 7.0
Alkaline RPC on
RESOURCE RPC at pH 9.0
Alternative techniques
The alternative techniques may be added to the standard protocols when purity is unsatisfactory even after normal optimisation has been carried out.
Alt I: CIEX at pH 3.0 with 30% ACN added to the eluent. Alt II: SEC in eluents containing ACN.
1. Solubility in RPC eluents
Neutral RPC
Adjust pH to 7.0.
If the sample does not already contain a buffer to control pH in the
range of 7, make up to 10 mM with ammonium phosphate before pH
adjustment.
Filter or spin the sample to remove any formed precipitate.
Determine the content of target peptide in the clarified solutions.
Make up to 60% with acetonitrile.
Filter or spin the sample to remove any formed precipitate.
Determine the content of target peptide in the clarified solutions.
Acidic RPC
Adjust pH to 2 by adding TFA (final concentration around 0.1%)
Filter or spin the sample to remove any formed precipitate.
Determine the content of target peptide in the clarified solutions.
Make up to 60% with acetonitrile.
Filter or spin the sample to remove any formed precipitate.
Determine the content of target peptide in the clarified solutions.
Alkaline RPC
Adjust pH to 9.0.
If the sample does not already contain a buffer to control pH in the
range of 9, make up to 20 mM with b Tris before pH adjustment.
Filter or spin the sample to remove any formed precipitate.
Determine the content of target peptide in the clarified solutions.
Make up to 60% with acetonitrile.
Filter or spin the sample to remove any formed precipitate.
Determine the content of target peptide in the clarified solutions.
Evaluation
Target peptide recoveries < 90% indicate incompatibility with the conditions used.
Standard Purification Protocol for peptides soluble in acidic solvents
Sample preparation
Adjust sample conditions to match those of eluent A.
Filter or spin the sample to remove any particulate matter
Column
Select a Sephasil Peptide C8 to match the amount of sample
Method
Use the recommended values for gradient and flow rate for the column selected
Eluent
Prepare the following eluents:
Eluent A: 0.06% TFA in water.
Eluent B: 0.05% TFA in water containing 60% acetonitrile (final conc.).
Evaluation
If peak symmetry or recovery is unsatisfactory, consult Trouble-shooting
Check purity of collected fractions by:
-Chromatography on µRPC or Mini S.
-Capillary electrophoresis.
- MS.
If purity is unsatisfactory, proceed to OPTIMISATION.
Standard Purification Protocol for peptides soluble in neutral solvents
Sample preparation
Adjust sample conditions to match those of eluent A.
Filter or spin the sample to remove any particulate matter
Column
Select a Sephasil Peptide C8 or a RESOURCE RPC column to match the amount of sample.
Method
Use the recommended values for gradient and flow rate (See column list)
Eluents
Prepare the following eluents:
Eluent A: 10 mM ammonium phosphate pH 7.0 in water.
Eluent B: 10 mM ammonium phosphate in water containing 60% acetonitrile (final conc.).
Evaluation
If peak symmetry or recovery is unsatisfactory, consult Trouble Shooting.
Check purity of collected fractions by:
- Chromatography on µRPC or Mini S.
- Capillary electrophoresis.
- MS
If purity is unsatisfactory, proceed to OPTIMISATION.
Standard Purification Protocol for peptides soluble in alkaline solvents
Sample preparation
Adjust sample conditions to match those of eluent A.
Filter or spin the sample to remove any particulate matter
Column
Select a RESOURCE RPC column to match the amount of sample (See column list)
! Silica-based RPC cannot be used, since silica will dissolve under alkaline conditions.
Method
Use the recommended values for gradient and flow rate (See column list)
Eluents
Prepare the following eluents:
Eluent A: 20 mM Tris-HCl pH 9.0 in water.
Eluent B: 20 mM Tris-HCl pH 9.0 in water containing 60% acetonitrile (final conc.).
Evaluation
If peak symmetry or recovery is unsatisfactory, consult Trouble Shooting.
Check purity of collected fractions by:
- Chromatography on µRPC or Mini S
- Capillary electrophoresis
- MS
If purity is unsatisfactory, proceed to OPTIMISATION.
Alternative Technique I, Cation Exchange
Sample preparation
Adjust sample conditions to match those of eluent A.
Filter or spin the sample to remove any particulate matter
Column
Select a Mono S or a RESOURCE S column to match the amount of sample
Method
Use the recommended values for gradient and flow rate for the column selected
Eluents
Prepare the following eluents:
Eluent A: 5 mM phosphate pH 3.0 in water containing 30% acetonitrile (final conc.).
Eluent B: 5 mM phosphate pH 3.0 in water containing 30% acetonitrile and 1.0 M NaCl (final conc.).
Evaluation
If peak symmetry or recovery is unsatisfactory, consult Trouble Shooting.
Check purity of collected fractions by:
- Chromatography on µRPC.
- Capillary electrophoresis.
- MS.
Alternative Technique II, Size Exclusion Chromatography
Sample preparation
Adjust sample conditions to match those of eluent A.
Filter or spin the sample to remove any particulate matter
Column
Select a Superdex peptide column for target peptides with Mr <3 000.
Select a Superdex 75 column for target peptides with Mr >3 000.
Method
Use the recommended flow rate for the column selected.
Eluents
Depending on the solubility of the target peptide, prepare one of the alternatives below:
50 mM phosphate, pH 7.0 in water containing 30% acetonitrile (final conc.).
0.1% TFA in water containing 30% acetonitrile (final conc.).
70% formic acid in water (applicable to very hydrophobic peptides).
70% acetonitrile in water (applicable to very hydrophobic peptides).
! The resolution of peptides in SEC is affected by the ACN concentration.
ACN concentrations higher or lower than 25 - 30% will cause varying degrees of excessive retardation.
Evaluation
If peak symmetry or recovery is unsatisfactory, consult Trouble Shooting.
Check purity of collected fractions by:
- Chromatography on µRPC.
- Capillary electrophoresis.
- MS.
II. Check solvent delivery
- Run installation test.
Results OK.
Results not OK
Refer to instrument manual.
III. Check the column - Run a known sample.
Results OK
Results not OK. Column malfunction
Refer to trouble-shootingguide below.
Sample incompatible with experimental conditions
Refer to trouble-shooting guide below.
The following table lists symptoms of malfunction.
The actions recommended are intended as "first aids"
Trouble-shooting guide
Symptom
Cause
Action
I. Pressure
- Low
Air in the pump heads.
Worn-out piston seals.
Leaks.
Purge the pump with MeOH.
Replace piston seals.
(Refer to instrument manual)
- High
Clogged tubing or column.
Watch pressure while removing column and tubing in the "upstream" direction until theclogged part is identified.
- Irregular
Solvent delivery malfunction.
Re-run with fresh eluentsPurge the pump with MeOHl.
Replace piston seals.(Refer to instrument manual)
II. Aberrant peak shapes
Always check column first by running a known sample.
-Triangular with sharp front
Overload.
Reduce sample load.
-Excessively broad
Overload.
Column bed irregularities.
Air in the column.
Run column with 2 column volumes of MeOH to remove any trapped air.
Perform recommended CIP.
-Tailing
Column bed irregularities.
Air in the column.
Non-specific adsorption.
Run column with 2 column volumes of MeOH to remove any trapped air.
Perform recommended CIP.
Try additives like ACN or MeOH.
-Fronting
Channels or air in the column bed.
Run column with 2 column volumes of MeOH to remove any trapped air.
-Split peaks
Partially blocked frit.Column bed irregularities.
Air in the column.
Run column "backwards" with 2 columnvolumes of MeOH to remove any particles inthe frit or trapped air.
-Irregular
Solvent delivery malfunction.
Sample incompatible with experimentalconditions.
Run Installation Test.
Run sample stability test.
Try additives like ACN or MeOH,chaotropic salts.
Change conditions, type of column or technique.
- Ghost peaks(Peaks appearing even inblank runs)
Column regeneration incomplete.
Quality of eluent chemicals and /orsolvent too low.
Aged eluents.
Increase eluting component concentration in eluent B.
Use HPLC quality chemicals and solvents only.
Unexpectedly low or missingPeaksLow recovery of activity.
Non-specific adsorption.
Sample incompatible with experimental conditions.
Run sample stability test.
Try additives like ACN or MeOH chaotropic salts.
Try to block non-specific binding sites by injecting e.g. albumin.
Change conditions, type of column or technique.
Base line instability
- Irregular
Air in the detector cell.
Detector problem.
Check constrictor function.Run Installation Test.
- Drifting
Detector problem.
Bleeding of precipitated ornon-specifically adsorbed material.
Faulty or aged eluents.
Run Installation Test.Perform specified CIP procedure.
Re-run with fresh eluents.
Type of base matrix and the ligand fixation chemistry are more important than the choice of ligand type.
2. Scout for optimal gradient slope.
Influences mainly the spacing of peaks
Reducing the gradient slope will
broaden peaks
3. Add a new step. (CIEX or SEC)
Changes overall selectivity.
May require the removal of ACN.
4. Scout for optimal flow rate
Decreases peak width
Only limited effect on final purity
1. Media Screening for RPC Sample preparation
Adjust the sample conditions to match eluent A.
Filter or spin the sample to remove any particulate matter
Method
Use the recommended values for gradient and flow rate for each column to be test
Eluents
Prepare the originally used eluents.
Evaluation
Select the RPC medium providing the highest purity and an acceptable recovery of the target peptide.
If peak symmetry or recovery is unsatisfactory, consult Trouble Shooting.
2. Gradient Optimisation Sample preparation
Adjust sample conditions to match those of Eluent A.
Filter or spin the sample to remove any particulate matter.
Method
Use the chromatography medium and conditions arrived at in the preceding optimisation step(s).
Double the gradient volume for each new experiment.
Evaluation
Select the steepest gradient providing satisfactory purity of the target protein.
3. Adding a New Step Choice of technique I. IEX
CIEX may provide a rather different selectivity even though the pH parameter has been utilised in the standard protocol. Adding 30% normally has a positive effect
on peak symmetry.
Sample preparation
Adjust sample conditions to match those of Eluent A.
Especially reduce any content of ACN to 30% or less.
Refer to Alternative Technique I ,CIEX!
II. SEC
SEC may be one of the few alternatives available for very hydrophobic peptides.
Though to a lesser degree, SEC also contributes to increased overall selectivity.
Varying the ACN content of the eluent may influence the order of elution of peptide
samples and may be used to alter the selectivity even further.
Refer to Alternative Technique II, SEC!
4. Flow Rate Optimisation Sample preparation, column and eluents
See the original method!
Method
Minimum peak widths are obtained at rather low flow rates with peptides.
Thus start with the flow rate recommended column used and then decrease
it in rather large steps.
Evaluation
Select the maximum flow rate providing satisfactory resolution.
