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 mixture of 14compounds was subjected to a reversed-phase gradient separation going from 5%to 100%acetonitrile with
a gradient time of 60min. The sample was injected at t =time. All peaks were eluted between 22and 50min.
(a) Is the mixture more suitable for isocratic or gradient elution?
(b) If the next run is a gradient, select the starting and ending %acetonitrile
and the gradient time.
Morphine and morphine 3-b-d-glucuronide were separated on two different 50 3 4.6 mm columns with 3-mm particles.61 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,, and time,, 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--d-glucuro-nide emerged at 1.5 min and morphine at 2.8 min. Explain the order of elution.
(c) Find the retention factor k for each solute on column A, using5 0.65 min.
(d) 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-b-d-glucuronide emerged at 2.7 min. Explain the order of elution. Why does the gradient go from high to low acetonitrile volume fraction?
(e) From Equation 25-12 in Box 25-4, estimate k* on Column B assuming S = 4 and with5 0.50 min.
Retention factors for three solutes separated on anon-polar stationary phase are listed in the table. Eluent was a 70 : 30 (vol/vol) mixture of 50 mM citrate buffer (adjusted to pH with) plus methanol. Draw the dominant species of each compound at each pH in the table and explain the behavior of the retention factors.
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 ร pores used?
To which kinds of analytes do these liquid chromatography detectors respond?
(a) ultraviolet
(b) refractive index
(c) evaporative light scattering
(d) charged aerosol
(e) electrochemical
(f) fluorescence
(g) nitrogen chemiluminescence
(h) conductivity
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