Morphine and morphine 3-β-D-glucuronide were separated on two different 4.6-mm diameter × 50-mm-long columns with 3-μm particles.39 Column A was C18-silica run at 1.4 mL/min, and column B was bare silica run at 2.0 mL/min.

(a) Estimate the volume, Vm, and time, tm, at which unretained solute would emerge from each column. The observed times are 0.65 min for column A and 0.50 min for column B.
(b) Column A was eluted with 2 vol% acetonitrile in water containing 10 mM ammonium formate at pH 3. Morphine 3-β-D-glucuronide emerged at 1.5 min and morphine at 2.8 min. Explain the order of elution.
(c) Column B was eluted with a 5.0-min gradient beginning at 90 vol% acetonitrile in water and ending at 50 vol% acetonitrile in water. Both solvents contained 10 mM ammonium formate, pH 3. Morphine emerged at 1.3 min and morphine 3-β-D-glucuronide emerged at 2.7 min. Explain the order of elution. Why does the gradient go from high to low acetonitrile volume fraction?
(d) Find the retention factor k for each solute on column A, using tm = 0.65 min.
(e) From Equation 24-10 in Box 24-4, estimate k*, assuming S = 4 and with tm = 0.50 min.

Transcribed Image Text:

HO-C HO HO NCH, NCH3 HO HO Morphine Morphine 3-B-D-glucuronide