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Purification related issues

1. What is the difference between the polypeptide content and the purity of the peptide? In addition

to the polypeptide itself, a polypeptide product includes impurities such as water and organic salts

introduced during the production process. The purity of the peptide refers only to the content of the

peptide product contained in the polypeptide itself and the content of impurities. Excluding impurities

such as moisture; and the content of the polypeptide refers to the net content of the target polypeptide

in the product, which is generally detected by N-element analysis or amino acid molecule method;

therefore,even if the purity of a polypeptide reaches 99%, the product also contains Moisture and

organic salt, etc., its content may be only 70-80%.

2. How to dissolve the peptide? Why do the peptide samples need to be pre-treated before the column 

is prepared? What pretreatment methods are available? Most of the peptides can be dissolved in 

ultrapure water. For some insoluble peptides, the amino acid sequence should be analyzed first. The 

acidic peptide can be first dissolved in a small amount of alkaline (such as 0.1% ammonia) solution, 

and then diluted to the desired concentration. For the basic polypeptide, it can be dissolved in a 

small amount of acidic (such as acetic acid, trifluoroacetic acid) solution, and then diluted. To the 

desired concentration, for the hydrophobic polypeptide, it can be dissolved by a strong polar organic 

solvent such as DMF, methanol, propanol, isopropanol, DMSO or the like. Preparation of chromatographic 

columns Because of the large number of samples processed, they are more susceptible to contamination

than analytical columns, so it is necessary to pre-treat the samples to effectively extend the life of the 

prepared columns. There are five main methods: extraction, filtration, centrifugation, pre-column, and 

online filter.

3. Why do you add trifluoroacetic acid as an ion pair reagent in the mobile phase, and which mobile 

phase systems or ion pair reagents can be used for the separation and purification of peptides? 

The current mobile phase system is water plus acetonitrile and trifluoroacetic acid (TFA). For ion pairing 

reagents, the polypeptides are separated by elution according to different polypeptides in a suitable 

gradient. The addition of trifluoroacetic acid can adjust the pH of the eluent and interact with the peptide 

as an ion-pairing reagent to enhance the separation effect and significantly improve the peak shape. 

Other mobile phase systems or ion-pairing reagents which can be used for peptide separation 

and purification include acetic acid system, phosphoric acid system, hydrochloric acid system, 

heptafluorobutyric acid, etc., and can achieve good separation effect by appropriately adjusting pH.

4. How to choose the column packing used in the purification process? The physicochemical properties 

and hydrophobicity of different sequences of peptides are very different. In most cases, the molecular 

weight is less than 4000 and the hydrophilic peptide is best separated by C18 column. The 

molecular weight is greater than 5000 and The extremely hydrophobic peptide is best separated by 

C4 column, while the C8 column is between C18 and C4 columns. Its application effect is more similar to 

that of C18 column. For some special selective peptides, phenyl column and polymerization can also be 

selected. Column.

5. What are the principles for selecting a polymer filler during the purification process? When purifying 

it above normal pH or temperature conditions, a polymer column with a very wide pH range can be 

used. The advantage is that it is not under extreme pH conditions. It will degrade and can be separated 

and purified using strong acid and strong base as mobile phase.

6. Factors affecting the purity test results of peptides There are many factors affecting the purity test 

results of peptides, including: mobile phase system, column model, column temperature, wavelength 

and chromatograph performance indicators. Each difference may cause errors in the results. .

7. What are the reasons for the frequent baseline drift during peptide testing? How to solve it? 

Gradient elution of fixed trifluoroacetic acid concentration sometimes causes absorption baseline drift 

at 210-220 nm detection, which is the baseline for many reversed phase separations. The cause 

of the drift. Reducing or eliminating baseline drift due to spectral absorption changes of trifluoroacetic 

acid requires as close as possible to the detection wavelength near 215 nm and compensates for baseline 

drift in solvent B with 15% less trifluoroacetic acid in solvent A. For example, when trifluoroacetic acid 

in the solvent A is 0.1%, 0.085% can be used in the solvent B. 8. After separation and purification by 

reversed-phase chromatography, how to remove TFA, acetonitrile and other impurities in the mobile 

phase product of the product? Freeze-drying method should remove most TFA and acetonitrile. As 

long as it does not exceed the allowable range, this method is the simplest. Effective. Some methods 

for lyophilization of some drug peptides that require higher levels of TFA are generally not fully met and 

require special salt or desalting.

8. What kinds of salts are there in the form of peptides? By what way to transfer salt or desalting? 

Most of the peptides are isolated and purified under the TFA system, so the polypeptides are most in 

the form of TFA salts, and the second drug peptides are generally acetate. In the form of the hydrochloride 

salt, very few drug peptides have some special salt forms. Most of the salt transfer methods are ion exchange 

and HPLC, while desalting can be carried out with dialysis bags and ultrafiltration membranes.

9. Does TFA only play a role in regulating pH in the buffer? The higher the concentration of TFA, the more 

the baseline drift is. Does it mean that the concentration of TFA is as low as the pH of the buffer is allowed?

(1) TFA acts like an ion pair. Generally, the concentration is between 0.05 and 0.1%. Too high a concentration 

will make the solution acidic. Long-term use may affect the life of the column.

(2) At the same time, TFA can inhibit the silanol group on the surface of the silica gel and improve the 

peak shape of the basic compound. Sometimes 0.1% TFA is not well separated. Consider increasing the 

concentration to 0.2%. However, be careful to flush the column in time after use. (3) When the gradient is 

taken, it drifts into the baseline, but has little effect on the preparation.

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