- Why is trifluoroacetic acid used as ion pair reagent in mobile phase? What other mobile phase systems or ion pair reagents can be used for the separation and purification of peptides?
- How to select the chromatographic column packing used in the purification process?
- What are the principles for selecting polymer fillers in the purification process?
- Factors affecting the results of peptide purity test
- After RP-HPLC separation and purification, how to remove TFA, acetonitrile and other impurities in the mobile phase products?
- What kinds of salts do polypeptides generally have? In what way is the salt transferred or desalted?
For those samples that need to be treated before column preparation, the column life can be prolonged.
The main methods of sample pretreatment are extraction, filtration, centrifugation and precolumn.
chromatographic column
Why is trifluoroacetic acid used as ion pair reagent in mobile phase? What other mobile phase systems or ion pair reagents can be used for the separation and purification of peptides?
A: at present, the mobile phase system commonly used by antolex is water plus acetonitrile, with trifluoroacetic acid (TFA) as ion pair reagent. The appropriate gradient elution was selected according to different peptides.
TFA can adjust the pH of eluent and act as ion pair reagent to interact with polypeptide, which can enhance the separation effect and improve the peak shape of the sample.
The mobile phase system or ion pair reagents that can also be used for peptide separation and purification include acetic acid system, phosphoric acid system, hydrochloric acid system, heptafluorobutyric acid, etc. Good separation effect can also be achieved by adjusting pH.
How to select the chromatographic column packing used in the purification process?
A: different sequences have different physicochemical properties and hydrophobicity. In most cases, the separation efficiency of hydrophilic peptides with molecular weight less than 4000 is better than that with C18 column.
For peptides with molecular weight > 5000 and Ji terminal hydrophobicity, C4 column was the best.
C8 column is between C18 and C4, and its application effect is inclined to C18; for some peptides with special selectivity, phenyl column and polymer column can also be selected.
What are the principles for selecting polymer fillers in the purification process?
A: polymer chromatographic column with a wide pH range can be used when the purification needs to be carried out at temperature or higher than normal pH. The advantage of this method is that it will not degrade under the condition of Ji terminal pH. strong acid and strong base can be used as mobile phase for separation and purification.
Factors affecting the results of peptide purity test
A: there are many factors that affect the results of peptide purity test, including mobile phase system, column type, column temperature, wavelength and performance indicators of chromatograph. Each difference may cause errors in the results.
After RP-HPLC separation and purification, how to remove TFA, acetonitrile and other impurities in the mobile phase products?
A: freeze drying method should be able to remove most TFA and acetonitrile. This method is simple and effective as long as it does not exceed the allowable range. The freeze-drying method of some pharmaceutical peptides with higher requirements for TFA content can not meet the requirements completely, so special salt transfer or desalination is needed.
What kinds of salts do polypeptides generally have? In what way is the salt transferred or desalted?
A: most peptides are isolated and purified under the TFA system, so most of the peptides are in the form of TFA salts. Secondly, pharmaceutical peptides are usually in the form of acetate and hydrochloride, and a few Yao peptides have some special salt forms. Ion exchange method and HPLC method are mostly used for salt conversion, while dialysis bag and Chao filter membrane can be used for desalination.
- Does TFA only regulate pH in buffer solution? The higher the TFA concentration is, the more severe the baseline drift is. Does that mean that the lower the TFA concentration is, the better the lower the buffer pH is?
Answer: (1) TFA acts as a kind of ion pair. The concentration of TFA is 0.05-0.1%. If the concentration is too high, the solution will become acid. Long time use may affect the column life.
(2) At the same time, TFA can inhibit the silanol groups on the surface of silica gel and improve the peak shape of alkaline samples. Sometimes 0.1% TFA separation is not ideal. Consider increasing the concentration to 0.2%. However, the chromatographic column should be washed in time after use.
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