1. What is the general method for detecting peptide purity?

It is usually determined by high performance liquid chromatography (HPLC).

2. What is the difference between peptide content and peptide purity? 

Peptides contain not only the right peptides, but also impurities such as water and organic salts in production. Peptide purity refers to the correct amount of peptide relative to all impurities (except water). However, peptide content is the percentage of the target peptide relative to all other substances in the sample and is usually determined by N-element analysis and amino acid analysis. Therefore, even if the peptide purity reaches 99%, the content may still be 70%-80%, taking into account water and organic salts.

3. What is the most commonly used technique for peptide isolation and purification? 

The most common technique is reversed phase high performance liquid chromatography (RP-HPLC).

4. Which mobile phase is commonly used for peptide isolation and purification in RP-HPLC?

The most common mobile phases are water and acetonitrile, with trifluoroacetic acid acting as an ion-pair reagent. According to different peptide segments, appropriate gradient elution was used for separation.

5. Why is TFA added to the mobile phase as an ion pair reagent? 

Are there any other mobile phase or ion-pair reagents available for peptide separation and purification? TFA can be used to regulate the pH of the mobile phase as an ion-pair reagent to interact with the peptide, enhance the separation effect, and significantly improve the peak shape. Other mobile phase or ion pair reagents used for the separation and purification of peptides include acetic acid system, phosphoric acid system, hydrochloric acid system, heptafluorobutyric acid, etc., which can achieve a good separation effect by adjusting pH.

6. How to dissolve peptides? 

Most peptides can be dissolved in ultra-pure water. For some insoluble peptides, amino acid sequences need to be analyzed first. Acidic peptides can be dissolved in a small alkaline solution (e.g. 0.1% ammonia) and then diluted to the desired concentration. As a basic peptide, it can be dissolved in a small amount of acidic solutions (such as acetic acid, trifluoroacetic acid) and then diluted to the desired concentration. For hydrophobic peptides, soluble in strong polar organic solvents, such as DMF, methanol, propyl alcohol, isopropyl alcohol, DMSO, etc.

7. What are the commonly used chromatographic fillers in RP-HPLC? 

The most common fillers are C18, C8, and C4 reversed phase columns.

8. How to select chromatographic fillers in purification? 

The physicochemical properties and hydrophobicity of peptides with different sequences are very different. In most cases, the C18 column is the best for the separation of peptides with molecular weights less than 4000 or hydrophilic. C4 columns are suitable for molecular weights over 5000 or hydrophobic ends. The C8 column is between C18 and C4, and its effect is more similar to C18. For some special selective peptides, phenyl and polymer columns can also be selected.

9. What is the principle of selecting polymer fillers in purification?

When higher pH or temperatures are required, polymer HPLC columns with a wide pH range may be suitable for purification. They do not degrade at extreme pH conditions, so they can be separated and purified using strong acids and bases as mobile phases.

10. What will affect the result of peptide purification?

There are many influencing factors, including mobile phase, column type, column temperature, wavelength, chromatograph performance and so on. These differences can lead to errors in the final results

11. How to remove impurities such as TFA and acetonitrile from the mobile phase of the final product after purification by reverse phase chromatography? 

As long as the tolerance range is not exceeded, lyophilization is probably the simplest and most effective way to remove most TFA and acetonitrile. However, this method can not meet the requirements of some peptide drugs with high TFA content. Special salt conversion and desalination should be carried out with them.

12. What are the common salt forms of peptides? How to convert salt form or desalinate? 

Most peptides are purified under the TFA system, so TFA salts are 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.

13. Why does the peptide sample need to be pre-treated before loading the sample column? 

What are the preparation methods? Preparative columns are more likely to be contaminated because they handle more samples than analytical columns. Therefore, samples need to be pre-treated to extend the column's service time. There are five main methods: filtration, centrifugation, pre-column filtration and on-line filtration.

The above are common questions and answers in the purification process of custom peptide synthesis, and hope to help you.