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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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(a) Nonpolar aromatic compounds were separated by HPLC on an octadecylbonded phase. The eluent was 65 vol% methanol in water. How would the retention times be affected if 90% methanol were used instead?
(b) Octanoic acid and 1-aminooctane were passed through the same column described in (a), using an eluent of 20% methanol/80% buffer (pH 3.0). State which compound is expected to be eluted first and why.
role="math" localid="1656416023291"
(c) Polar solutes were separated by hydrophilic interaction chromatography (HILIC) with a strongly polar bonded phase. How would retention times be affected if eluent were changed from 80 vol% to 90 vol% acetonitrile in water?
(d) Polar solutes were separated by normal-phase chromatographyon bare silica using methyl t-butyl ether and 2-propanol solvent. How would retention times be affected if eluent were changed from 40 vol% to 60 vol% 2-propanol? (Hint: See Table 25-4.)
The figure shows the separation of two enantiomers on a chiral stationary phase.
Sepation of enantiomers of Ritalin by HPLC with a chiral stationary phase.[Data from R.Bakhitar,L.Ramos,and F.L.S.Tse, 鈥淨uantification of methlylphenidate in plasma using chiral Liquid-chromatography/Tandem mass spectrometry: Application to Toxicokinetric studies,鈥滱nal Chim Acta 2002,469,261.]
(a)From and find N for each peak.
(b) Fromandfind the resolution.
(c)Givenmin, use Equationwith the average N to predict the resolution.
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, 鈥淐ombination of HPLC 鈥業nverted Chirality Columns Approach鈥� and MS/MS Detection for Extreme Enantiomeric Excess Determination Even in Absence of Reference Samples.鈥� 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: 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 脜 pores used?
a. Why is high pressure needed in HPLC?
b. For a given column length , why do smaller particles give a higher plate number?
c. What is bonded phase in liquid chromatography?
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