The Synthesis Process Of Peptides

The specific synthesis consists of the following cycles:
Remove the protection
FMOC-protected columns and monomers must be protected by an alkaline solvent (piperidine) to remove the protective groups of the amino groups.

Activation and cross-linking
The carboxyl group of the next amino acid is activated by an activator. HBTU/HCTU/HITU/HATU+NMM/DIPEA or HOBT+DIC are commonly used as activators in chemical processes, and the activated monomers react with free amino groups to form peptide bonds. At this step, a large number of super-concentrated reagents are used to drive the reaction to complete. Cycle: The two-step reaction is repeated until the synthesis is complete. After the condensation of amino acids is completed, an appropriate amount of TFA can be used for elution, depending on the acidity and alkalinity, 10% TFA/DCM solution can be selected, to 100% TFA, and so on.

HPLC analysis purification
Analytical HPLC uses a column and pump system that can withstand high transfer pressures, which allows the packing of very fine particles (3-10 μm). As a result, the peptide is highly analyzed within a few minutes. There are two types of HPLC: ion-exchange and reversed-phase. Ion-exchange HPLC relies on direct charge interactions between peptides and solid phases. A COLUMN WITH A SPECIFIC CHARGE IN A CERTAIN PH RANGE EVOLVES INTO AN ION, WHILE A POLYPEPTIDE OR MIXTURE OF PEPTIDES, COMPOSED OF ITS AMINO ACIDS, EXHIBITS AN OPPOSITE CHARGE. Separation is a charge interaction in which a peptide is eluted by variable PH, ionic strength, or both, usually with a solution with low ionic strength, and then gradually strengthened or step-by-step until eluted in the peptide fire. An example of an ion-exchange separation uses a strong cation-exchange column. For example, sulfoethylaspartimide is separated by being positively charged in an acidic pH.

Reversed-phase HPLC conditions are the opposite of normal chromatography, where the peptide is hydrophobic to the column and eluted with reduced ionic strength, e.g., to increase the hydrophobicity of the eluent. Typically, the column consists of a chain of hydrocarbons covalently adsorbed to the silicon, which is G4-G8 carbon atoms in length. As elution is a hydrophobic effect. Long-chain columns are better than short-chain pairs with smaller, high-charged peptides. On the other hand, large hydrophobic peptides are eluted well with short chain columns. However, in general practice, there is little significant difference between the two types of columns, and the other types of carriers are composed of carbohydrates, such as phenyl groups.

A typical operation usually consists of two swasp powders, 0.1% TFA-H2o and 80% acetonitrile 0.1% TFA--H2o dilute acetonitrile. Mix with linear ladders at a rate of 0.5% to 1.0% change per minute. Common columns for analysis and purification are 4.6×250 mm (3-10 μm) and 22×250 mm (10 μm). If the column is packed radially, the sizes are 8×100 (3-10 μm) and 25×250 mm (10 μm).

A large number of various buffers contain many different reagents, such as phosphoric acid, NH4HCO3, sodium acetate, TFA/TEA, sodium phosphate, amylol, etc. Many different combinations can form buffers, but be careful: silicon reversed-phase columns should not be exposed to high pH, or even slightly alkaline pH, for long periods of time, as this can damage the column.

Don't confuse peptide content with purity, the purity of a peptide may be 100%, and the peptide content is related to the amount of anti-ion groups (e.g., Arg, Lys) and the hydrophilicity of the peptide, which is the characteristic of the synthetic peptide itself.