A Peptide HPLC Experiment for Biochemistry Laboratory Although it is generally agreed that biochemistry students should become familiar with high-performance liquid chromatography (HPLC), often the only HPLC's available for undergraduate laboratories are isocratic systems with fixed 254-nm wavelength detection. We recently adapted the peptide separation reported by Hrubyl for our undergraduate biochemistry laboratory. Milligram quantities of [Args]-vasotoein,[Args]-vasopressin,and oxytocin can be purchased from BACHEM (Torrance, CA) or Sigma (St. Louis, MO). The separation and quantitation of these three peptide hormones with very similar structures is easily accomplished on an isocratic system using a 254-nm detector. Standard C18 columns (4.6 X 100 mm or 4.6 X 150 mm), 1mL/min flow rate, 2 cmlmin chart speed, and a solvent that was 80%pH 4.0 ammonium acetate and 20% acetonitrile (filtered through a 0.45 pm nylon filter) resulted in baseline separations in less than 6 min per run. Using 1 mg1mL concentrations of the hormones and 25 @Linjections, each milligram of peptide can produce 40 injections (40 chromatography tuns). Preparing standard solutions containing differing amounts of each peptide (by diluting stock solutions), as well as giving each student 100 @Lof hormone mixture to analyze, sets up the identify and quantitate experiment.
' Blevlns, D. D.: Burke, M. F.: Hruby,V. J. Anal Chem. 1980,52,420-424. Martha M. Veotllng Unlverslty of Maryland Baltimore County Baltimore. MD 21228
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Journal of Chemical Education
