US EPA

1580073 

Journal Article 

Side Reaction Of Synthetic Peptides During Their Purification By Preparative High-Performance Liquid Chromatography Using Formate Buffers 

Viville, R; Scarso, A; Durieux, JP; Loffet, A 

1983 

1 

Journal of Chromatography
ISSN: 0021-9673 

NIOSH/00139299 

262 

411-414 

A method using high performance liquid chromatography (HPLC) was developed for purifying synthetic peptides. A synthetic polypeptide was prepared on a cross linked esterified chloromethyl polystyrene. Couplings were made with tert-butyloxycarbonyl amino acids and dicyclohexylcarbodiimide. The crude peptide was cleaved from the support by hydrofluoric-acid containing 10 percent anisole. Gel filtration was performed, along with reversed phase liquid chromatography. Peptides were hydrolyzed in hydrochloric-acid at 110 degrees-C for 24 hours; amino acid composition was then determined. Thin layer chromatography and HPLC analysis indicated that the product was transformed into a less polar compound, as indicated by higher retardation factor in n-butanol/pyridine/acetic-acid and a retention time of 4.3 minutes greater than before lyophilization. There was a large excess of concentration formic-acid that suggested than an N-formylation had occurred during the concentration step, the formic-acid present in solution reacted with the free amino acid in the peptide, resulting in N-formylation of the peptide that was converted again to the free peptide through reaction with hydrochloric-acid. The authors conclude that the use of triethylammonium-formate buffer during HPLC is limited for preparative work since concentrations of eluted fractions while purifying synthetic peptides can result in side reactions. 

DCN-127078; Synthetic materials; Proteins; Amino acids; Chemical synthesis; Chemical composition; Chromatographic analysis; Polymers; Peptides; Thin layer chromatography