Appendix VIII Q. Acetic Acid in Synthetic Peptides

Examine by liquid chromatography (2.2.29).

Test solution Prepare as described in the monograph. The concentration of peptide in the solution may be adapted, depending on the expected amount of acetic acid in the sample.

Reference solution Prepare a 0.10 g/L solution of glacial acetic acid R in a mixture of 5 volumes of mobile phase B and 95 volumes of mobile phase A.

The chromatographic procedure may be carried out using:

• — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm),

• — as mobile phase at a flow rate of 1.2 mL/min:

Mobile phase A Dilute 0.7 mL of phosphoric acid R to 1000 mL with water R; adjust the pH to 3.0 with strong sodium hydroxide solution R,

Mobile phase B methanol R2,

• — as detector a spectrophotometer set at 210 nm.

Inject 10 µL of the reference solution and 10 µL of the test solution. In the chromatograms obtained, the peak corresponding to acetic acid has a retention time of 3-4 min. The baseline presents a steep rise after the start of the linear gradient, which corresponds to the elution of the peptide from the column. Determine the content of acetic acid in the peptide.