Post Cleavage Purification and Analysis HEADING_TITLE

Precipitation and Isolation

Transfer the product from the cleavage block to centrifuge tubes. Fill each tube only ~1/10. Add 9 parts cold diethyl ether (pre-chilled in a dry ice/acetone bath or otherwise reduced to ~ -70°C). At this point the product will crash out of solution forming a fluffy white flocculent. Centrifuge for five

minutes at 3300 rpm; a well-formed pellet should result. Decant the supernatant, and resuspend the product in cold ether again. Repeat this process at least 3-4 times, or until the odor of TFA is not detectable.  Allow the ether to evaporate slowly overnight from the open centrifuge tube. The resulting dry product is suitable for HPLC analysis or MS.

Washing in this manner will remove TFA as well as deprotection byproducts and excess scavengers. This process tends to remove lower molecular weight impurities such as residual scavengers, and may improve %purity by a couple percentage points. Alternatively, batch scale HPLC purification can be utilized for higher purity.

Analytical HPLC

Most peptides are analyzed on C18 columns such as a 25 cm Spirit™ C-18 Peptide Column for routine analysis. Typical sample concentrations are ~ 1 mg/mL in water or buffer A (0.10% TFA in water). Purity is determined by calculating %purity from comparing peak areas to the total area.

Typical gradient reverse phase HPLC buffers are:

A = 0.10% TFA in water, and

B = 0.10% TFA in CH3CN

Peptides are typically analyzed using a shallow gradient.  A gradient that may be used for general peptide analysis is 0%B to 60%B in 20 minutes. 

Mass Spectroscopy

MW of the product is verified via mass spectroscopy. One common method used lab is direct injection on a single quad instrument with an electrospray interface. Other widely used methods include LC/MS and MALDI techniques.  Some ion trapping instruments are also available on which one may obtain sequence data.

Yield Calculation

Calculate % yield by comparing the dry mass of the product obtained above to the theoretical yield calculated from the following equation:

Theoretical Yield (mg) = sresin * mresin * MWproduct


sresin = resin substitution in mmol/g

mresin = resin dry mass in g

MWproduct = MW of the product in mg/mmol

HPLC Purification of Peptides

Depending on how the synthesized peptide will be used, the crude peptide cleaved from the resin and isolated may be sufficiently pure.  The table below indicates typical applications and purity levels.



Tissue culture; ligand for affinity purification; non-quantitative antibody blocking experiments

80% or greater

In vivo studies; bioassays; markers for electrophoresis; monoclonal antibodies

90% or greater

ELISA; RIA; enzyme substrate

95% or greater

NMR; chromatography standards


If the synthesized peptide requires HPLC purification, then a 30-minute gradient from 0% to 70% acetonitrile on a Spirit™ C-18 Peptide Column will usually provide peptide with satisfactory purity.  The HPLC solvents should contain 0.1 % trifluoroacetic acid (TFA) which acts as an ion-pairing reagent and improves the shape of the peptide peaks.  Long peptides or relatively hydrophobic peptides should be purified on a Spirit™ C-4 or C-8 column.  If the crude peptide has impurities that elute close to the product, a shallower gradient, such as 0%-30% acetonitrile or 10%-40% acetonitrile can provide better separation.   Hundreds of milligrams of crude peptide were purified at one time using a gradient of 0.1%B per minute.[1]

The crude peptide should be dissolved in a minimal volume of 0.1% aqueous TFA.  If the peptide is not soluble in dilute TFA,  it may dissolve in  6M guanidine hydrochloride containing 0.1% TFA.  (6M guanidine hydrochloride solution can be prepared by dissolving 1 gram of guanidine in 1 ml of water).  The guanidine salts elute in the void volume of the column while the peptide elutes later.  Inject the peptide solution onto the HPLC column and monitor the eluant from the column at 220 nm.  Collect fractions as the peptide elutes. 

Test the fractions and combine all fractions that contain only the pure peptide.  The combined fractions can be lyophilized to isolate the purified peptide.

Removing Trifluoroacetic Acid (TFA) From Peptides

Trifluoroacetic acid is toxic.  Depending on how the peptide will be used, it may be necessary to exchange TFA for a more biologically benign acid, such as HCl or acetic acid.  Recently several common methods of exchanging or removing trifluoroacetate were evaluated.[2]  The following procedures can be utilized to efficiently exchange acid anions.  The trifluoroacetate, fluoride ,chloride and acetate content of the peptide can be accurately measured by ion chromatography.[3] 

TFA/HCl Exchange

  1. Dissolve the peptide in 100 mM HCl.
  2. Allow the solution to stand at room temperature for 1 minute.
  3. Freeze the solution in liquid nitrogen.
  4. Lyophilize the frozen solution to obtain the peptide hydrochloride salt.

TFA/Acetate Exchange

  1. Prepare a small column (10-fold to 50-fold excess of anion sites in the column relative to anion sites in the peptide) of strong anion exchange resin.
  2. Elute the column with a 1M solution of sodium acetate.
  3. Wash the column with distilled water to remove the excess sodium acetate.
  4. Dissolve the peptide in distilled water and apply it to the column.
  5. Elute the column with distilled water and collect the fractions containing the peptide.
  6. Lyophilize the combined peptide containing fractions to obtain the peptide acetate salt.


[1] Harris, PWR; Lee, DJ; Brimble, MA. J. Pept. Sci. 2012, 18, 549-55.

[2] Roux, S.; Zékri, E.; Rousseau, B.; Paternostre, M.; Cintrat, J.-C.;Fay, N. J. Pept. Sci. 2008, 14, 354-359.

[3] For a sample procedure, see Dionex Application Note 115, “Determination of Trifluoroacetic Acid (TFA) in Peptides”, Dionex Corporation, 2002.