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Chapter 25: Q30P (page 710)
what are criteria for an adequate isocratic chromatographic separation?
Criteria for an adequate isocratic chromatographic separation
If k is too small. The first peak must be distorted by the solvent front. If k is too great, the separation takes too long.
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(a) When you try separating an unknown mixture byreversed-phase chromatography with 48%acetonitrile 50%water,the peaks are too close together and are eluted in the range k = 2- 6Should you use a higher or lower concentration of acetonitrile in thenext run?
(b) When you try separating an unknown mixture by normal-phasechromatography with 50%hexane50% methyl t-butyl ether, thepeaks are too close together and are eluted in the range k = 2 - 6Should you use a higher or lower concentration of hexane in thenext run?
The antitumor drug gimatecan is available as nearly pure (S)-enantiomer. Neither pure (R)-enantiomer nor a racemic (equal) mixture of the two enantiomers is available. To measure small quantities of (R)-enantiomer in nearly pure (S)-gimatecan, a preparation was subjected to normal-phase chromatography on each of the enantiomers of a commercial, chiral stationary phase designated (S,S)- and (R,R)-DACH-DNB. Chromatography on the (R,R)-stationary phase gave a slightly asymmetric peak at tr 5 6.10 min with retention factor k 5 1.22. Chromatography on the (S,S)- stationary phase gave a slightly asymmetric peak at tr 5 6.96 min with k 5 1.50. With the (S,S) stationary phase, a small peak with 0.03% of the area of the main peak was observed at 6.10 min.
Chromatography of gimatecan on each enantiomer of a chiral stationary phase. Lower traces have enlarged vertical scale. [Data from E. Badaloni, W. Cabri, A. Ciogli, R. Deias, F. Gasparrini, F. Giorgi, A. Vigevani, and C. Villani, “Combination of HPLC ‘Inverted Chirality Columns Approach’ and MS/MS Detection for Extreme Enantiomeric Excess Determination Even in Absence of Reference Samples.” Anal. Chem. 2007, 79, 6013.]
(a) Explain the appearance of the upper chromatograms. Dashed lines are position markers, not part of the chromatogram. What Problems 709 would the chromatogram of pure (R)-gimatecan look like on the same two stationary phases?
(b) Explain the appearance of the two lower chromatograms and why it can be concluded that the gimatecan contained 0.03% of the (R)-enantiomer. Why is the (R)-enantiomer not observed with the (R,R)-stationary phase?
(c) Find the relative retention (a) for the two enantiomers on the (S,S)-stationary phase.
(d) The column provides N 5 6 800 plates. What would be the resolution between the two equal peaks in a racemic (equal) mixture of (R)- and (S)-gimatecan? If the peaks were symmetric, does this resolution provide baseline separation in which signal returns to baseline before the next peak begins?
Question: Explain how to use a gradient for the first run to decide whether isocratic or gradient elution would be more appropriate.
(a) You wish to use a wide gradient from 5 vol % to 95 vol %B for the first separation of a mixture of small molecules to decide whether to use gradient or isocratic elution. What should be the gradient time, ,for a column containing particles with a flow of
You optimized the gradient separation going from 20vol%to 34 vol% Bin 11.5min at 1.0mLFindfor this optimized
separation. To scale up to a column, what should be the gradient time and the volume flow rate? If the sample load on
the small column was 1mgwhat sample load can be applied to the large column? Verify that is unchanged.
a) UHPLC can provide exquisite resolution when run slowly on long columns or rapid separation with reasonaple resolution if short columns are run fast. the drug acetaminophern run on a UHPLC column has a retention time of min and a width at half-height of .Find the plate number and plate height. How many diameter particles places placed dide-by are equal to one theoretical plate?
b) From figure,we expect an optimum plate height of how many particles placed side-by- side are equal to one theroretical plate? Do you think the column in (a) is being run for maximum resolution or maximum speed?
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