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Chapter 25: Q3P (page 707)
In hydrophilic interaction chromatography , why is eluent strength increased by increasing the fraction of water in the mobile phase?
The mobile phase is the aqueous phase. So, increasing the fraction of water in the mobile phase increases its polarity, which increases the elute strength in HILIC.
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Question: Literature search problem: Human serum albumin (HSA) is an important protein ingredient in cryopreservation media used in procedures such as in vitro fertilization. Search the literature for a high-performance liquid chromatography method for the determination of human serum albumin and the stabilizer N-acetyl tryptophan in medical devices.
(a) Give the citation (authors, title, journal name, year, volume, pages) for the research paper that fits the criteria of this analysis.
(b) What alternative methods could be used for analysis of human serum albumin?
(c) What type of analytical column is used for the separation?
(d) How long was the gradient? How long were the additional wash and equilibration steps within the gradient method?
(e) What parameters were assessed in the method validation?
(f) Why were particles with 300 Å pores used?
Question: How do additives such as trienthylamine reduce tailing of certain solutes?
If along HPCL column has a place height of 5.0 what will be the half-width (in seconds) of a peak eluted at 10.0min? if plate height,what will be?
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?
a. Why are HPLC particles porous?
b. Why are the particles withpores used for small molecules but wide porestationary phases used to separate polypeptides and proteins?s
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