Removing TFA salt in Peptide Synthesis: Why is it SO Important?

Trifluoroacetic acid (TFA) is a common tool used to cleave peptides from solid phase resins during peptide solid phase synthesis. TFA is also used during peptide purification by reversed phase HPLC. However, due to its potential irritation, special care should be taken when using TFA.

The effects of TFA on cells depend on the context and concentration. In some cases, it can be used as a tool to prepare biologically active compounds; in other cases, it may have direct effects on cells, such as anti-tumor, anti-angiogenic, and induction of apoptosis.

So, If you need to do cell experiments, then removing TFA salt in peptide is an important step in organic synthesis.

Why Do We Need to Remove TFA salts in Peptide R&D?

The effect of trifluoroacetic acid (TFA) on cells depends on its usage scenario and concentration. Under certain conditions, it can be used as a tool to prepare biologically active compounds. In other cases, it may have direct effects on cells, such as anti-tumor, anti-angiogenesis, and induction of apoptosis. However, due to its potential irritation, it needs to be used with caution.

During solid-phase peptide synthesis, trifluoroacetic acid (TFA) is often used to cleave peptides from solid-phase resins. TFA is also used in the process of reversed-phase HPLC purification of peptides.

Therefore, custom peptides synthesis by Omizzur are generally in the form of TFA salts.

If residual TFA affects your R&D experiment, we usually recommend converting to other salts, such as acetate or hydrochloride(HCL).

*** Notice that not all peptides used in peptide experiments need to be desalted from TFA salts (most cell experiments require the removal of TFA salts, which depending on your experimental purpose). Peptide TFA removal will increase the cost of peptide synthesis.

What are the General Salt forms of Peptides? How to Convert Salt Forms or Desalt?

Most peptides are purified under TFA system, so TFA salt is the most common form, followed by acetate and hydrochloride forms, and few peptide drugs are special salt forms.

Ion exchange and HPLC are the most commonly used salt conversion methods, while G25 (Sephadex from GE Healthcare) columns can be used for desalting.

2 Methods for Removing TFA Salt from Peptide:

Method 1: 

High Performance Liquid Chromatography (HPLC). The following is a detailed description of the steps:

I. Basic Principles

TFA (trifluoroacetic acid) is a strong organic acid that is often used in the deprotection step after peptide synthesis, but due to its strong acidity, it may cause unwanted side effects in some applications. Acetate is a weaker organic acid salt that usually has better biocompatibility, so in some pharmaceutical preparations or biological experiments, the acetate form is more suitable.

II. Conversion Process

1. Choose a suitable column: 

For ion exchange, columns filled with ion exchange resins are usually used. These columns can selectively exchange ions based on the charge and size of the ions.

2. Sample Preparation:

Prepare the peptide solution containing TFA salt to ensure that the concentration and purity of the solution meet the experimental requirements.

3. Loading:

Inject the prepared sample into the HPLC system. The HPLC system can accurately control the speed, pressure and temperature of the mobile phase to ensure efficient ion exchange.

4. Mobile Phase Selection:

Use an appropriate buffer as the mobile phase, such as ammonium acetate solution. Ammonium acetate can provide acetate ions to exchange with cations in TFA salts.

5. Elution:

When the sample passes through the column, the TFA salt will undergo an ion exchange reaction with ammonium acetate to form peptides in the form of acetate. During this process, the HPLC system can monitor the composition and concentration of the eluent in real time to ensure the completeness of the conversion.

6. Collection:

Collect the eluted peptides in the form of acetate. If necessary, the collected peptides can be further purified and concentrated.

7. Post-treatment:

According to the experimental requirements, the collected peptides in the form of acetate are subjected to necessary post-treatment, such as removing the solvent, adjusting the pH value, etc.

For Example - Removing TFA salt in Peptide Step by Step:

1. Rinse the C18 reverse phase column with 50% or more acetonitrile solution for 20-30 minutes to remove the residual TFA in the carbon column and pipeline

2. Rinse the carbon column with pure water to prevent the precipitation of ammonium acetate solid when desalting with high concentration ammonium acetate solution

3. Rinse with 95% ammonium acetate solution and 5% acetonitrile for 15-30 minutes for desalting

4. Gradient elution with 0.1% glacial acetic acid to collect compound peaks

5. Freeze-dry the liquid to obtain the compound

Precautions

1. During the ion exchange process, the operator should have certain HPLC operation experience to ensure the success of the experiment.

2. During the conversion process, the experimental conditions, such as temperature, pH value, ionic strength, etc., should be strictly controlled to avoid adverse effects on the activity of the peptide.

3. The collected peptides in acetate form should be fully identified and characterized to ensure that their purity and activity meet the experimental requirements.

4. Through the above steps, the peptide can be successfully converted from trifluoroacetate (TFA salt) to acetate form to meet the needs of different applications.

Method 2: Lyophilization

TFA and acetonitrile can be almost completely removed. It is best to test the residual amount in the lyophilized product before drug experiments. As long as it does not exceed the allowable range, this method is the simplest and most effective.

Lyophilization is usually the preferred sample drying method because it can achieve a high level of drying at a low residual solvent level. In addition, the light, powdery, "fluffy" surface treatment of the sample makes it easy to remove and weigh the sample.

As long as it does not exceed the tolerance range, lyophilization may be the simplest and most effective way to remove most TFA and acetonitrile. However, this method cannot meet the requirements of some peptide drugs that require a higher TFA content.

5 Benefits of Removing TFA Salts From Peptides

1. Improve Purity and Effect

During the peptide synthesis process, especially in solid phase synthesis, some salts (such as trifluoroacetic acid TFA) are often used as cutting agents or purification aids. However, the residues of these salts will affect the purity of the peptide. Removing salts can significantly improve the purity of the peptide, thereby ensuring its effect in subsequent applications.

In the fields of pharmaceuticals and food processing, the purity and activity of peptides are crucial to their effect. Residual salts may interfere with the biological activity or pharmacological effects of peptides, so desalting is an important step to ensure the quality of peptide products.

2. Avoid Toxicity Issues

Some salts (such as TFA) are toxic or potentially toxic in the human body. If these salts remain in the peptide product, it may have adverse effects on human health. Therefore, desalting is to ensure the safety of the peptide product.

3. Meet Application Requirements

For peptides used in preclinical and clinical studies, their purity and safety requirements are extremely high. Residual salts may cause inaccurate or misleading experimental results, so salts must be removed to meet strict clinical requirements.

Regulations in many countries and regions have strict regulations on the salt content of products such as medicines and foods. In order to comply with these regulatory requirements, peptide products must be desalted.

4. Prevent Catalytic Racemization

In solid phase synthesis, if the residual salt is not completely removed, the excess tertiary amine and the salt produced by neutralization will cause catalytic racemization during peptide grafting, thereby reducing the purity and activity of the peptide. Therefore, this problem must be avoided through appropriate desalting and salt transfer steps.

5. Optimize Physicochemical Properties

Certain specific salt forms may affect the solubility, stability and biological activity of peptides. For example, in some cases, acetate has better solubility and stability than TFA salt, so it is necessary to select the appropriate salt form according to specific needs.

 Quote for Amino acids & Peptides:

 1. Click to send inqauiry online:  Make Inquiry Now 

 2. Send mail to us: [email protected]

  * Please mail us product name and quantity, Omizzur services will get back to you within 1 hour.