An HPLC method was developed for the separation of the R(+) and S(-) enantiomers
ID: 1053722 • Letter: A
Question
An HPLC method was developed for the separation of the R(+) and S(-) enantiomers of Ibuprofen (Advil) a chiral non-steroidal anti-inflammatory drug. The HPLC system was equipped with a 5 cm silica guard column and a 35 cm x 4.6 mm silica analytical column. The mobile phase consisted of a mixture of Methanol, H2O and acetonitrile at a ratio of 50:40:10. The analysis showed 1,3,5-Tri-t-butylbenzene was un-retained in the column and eluted in 6.5 minutes while the R(+) enantiomer and the S(-) enantiomer of Ibuprofen eluted in 11.45 minutes and 12.05 minutes respectively.
1) Calculate the capacity factor of the S(-) enantiomer in the analytical column.
2) If the volume of the stationary phase is 1/6th of the volume of the mobile phase in the column (Vs = 0.34 Vm). Find the partition coefficient, K, for the system.
3) If the width of the peak at the base of the R(+) and the S(-) enantiomers peaks are 36 sec and 45 sec respectively. For the R(+) and the S(-) enantiomers peaks of Ibuprofen calculate:
The relative retention (a).
The separation factor (g).
The resolution and state if the peaks are baseline separated.
The number of theoretical plates for R(+) enantiomer peak.
The plate height for R(+) enantiomer peak.
Explanation / Answer
1)capacity factor ,K=(tR-to)-1
tR=retention time of the peak ,to=dead time of the column
'k'for S=(12.05/6.5)-1=0.85
2)partition coefficient,K=molar concentration of analyte in the stationary phase/molar concentration of analyte in the mobile phase=Vs/Vm=0.34
3)The relative retention (a) for S(-)=tR of S(-)/tR of std=12.5/6.5=1.85
The relative retention (a) for R(+)=11.45/6.5=1.76
3)separation factor (g)=ratio of capacity factors of both enantiomers=S/S'=(12.05/6.5)-1/S=(11.45/6.5)-1=0.85/0.76=1.12
4)resolution ,R=[tR2-tR1/(W0.5 h1-W0.5 h2)]*1.18=
W0.5 h=peak width at half maximum height
R=[(12.05-11.45)*60 seconds/45-36]*1.18=4.7
5)Number of theoretical plates for R(+) enantiomer peak=N=5.54(tR/W0.5h)^2=(11.45*60/36)^2*5.54=364
6) plate heigh=H=L/N
L=length of column(mm)=5 *10 mm
H=50 mm/364=0.14mm
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