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It is usually determined by high performance liquid chromatography (HPLC).
A peptide contains not only a correct peptide but also impurities, such as water and organic salts from production. Peptide purity refers to the number of correct peptides relative to all impurities, except moisture. However, a peptide content is the percentage of a targeted peptide relative to everything else in the sample, and it is usually determined by N elemental analysis and amino acid analysis. Therefore, even if the peptide purity reaches 99%, the content may still be 70%-80% considering water and organic salts.
The most common technique is reversed-phase high performance liquid chromatography (RP-HPLC).
The most common mobile phase is water and acetonitrile, and trifluoroacetic acid is acted as an ion pair reagent. The suitable gradient elution will be used for separation according to different peptides.
TFA can be used to adjust the pH of the mobile phase, and interact with peptides as ion- pair reagent, enhancing the separation effect and significantly improving the peak shape.
Other mobile phases or ion pair reagents for peptide separation and purification include the acetic acid system, phosphoric acid system, hydrochloric acid system, heptafluorobutyric acid, etc., which can be used to achieve great separation effect with pH adjustment.
Most peptides can be dissolved in ultrapure water. For some insoluble peptides, the amino acid sequences need to be analyzed first. Acidic peptides can be dissolved in a small amount of alkaline solution (such as 0.1% ammonia) first, and then they can be diluted to the desired concentrations. As basic peptides, they can be dissolved in a small amount of acidic solution (such as acetic acid, trifluoroacetic acid), and then diluted to the required concentrations. For hydrophobic peptides, they can be dissolved in strongly polar organic solvents, such as DMF, methanol, propanol, isopropanol, DMSO, etc.
The most common fillers are C18, C8 and C4 reversed-phase columns.
Peptides with different sequences show great differences between physiochemical characterizations and hydrophobicity. In most cases, C18 columns will exhibit the best separation effects for peptides with molecular weights less than 4000 or hydrophilicity. C4 columns are suitable for molecular weight more than 5000 or hydrophobic ends. The C8 columns are between C18 and C4, and its effect is more similar to that of C18. For some special selective peptides, phenyl and polymer columns can also be chosen.
The polymeric HPLC columns with a wide range of pH may be suitable for purification when higher pH or temperature is required. They will not be degraded under extreme pH conditions, and thus strong acids and bases can be used as mobile phases for separation and purification.
There are many factors, mainly including mobile phases, column types, column temperatures, wavelengths, and the performance of chromatograph. These differences may cause errors in the final results.
It is the main cause of baseline drift in reversed-phase separation that gradient elution with a fixed TFA concentration will lead to a shift in the absorption baseline at 210-220 nm. It is suggested to make the detection wavelength as close to 215 nm as possible to reduce or eliminate the impact of baseline drift caused by the absorption spectrum changes of TFA. In addition, the baseline drift can be compensated by adding 15% less TFA in solvent A compared with solvent B. For example, if the concentration of TFA in solvent A is 0.1%, 0.085% of that is recommended for solvent B.
The lyophilization might be the simplest and most effective way to remove most of TFA and acetonitrile, as long as it does not exceed the tolerant range. However, this method can not meet the requirements of some peptide drugs which have a high demanding of TFA amounts. Special salt conversion and desalting should be conducted with them.
Most peptides are purified under the TFA systems, so TFA salts are the most common form, followed by the forms of acetate and hydrochloride, and very few peptide drugs are in the special salt forms. Ion change and HPLC are the most common methods for salt conversion, while G25 (a dextran gel from GE Healthcare) column can be used to desalt.
Preparative columns are more likely to be contaminated because they deal with more samples compared with analytical columns. Therefore, it is necessary to pretreat the samples to extend the usage time of the column. There are five main methods: filtration, centrifugation, pre-column, and online filter.
