Chromatographic Methods for Environmental Analysis - Fundamentals of Environmental Sampling and Analysis - page 286

13. Explain the following (a) How column length affects resolution? (b) Why HPLC column
cannot be made very long for a better resolution?
14. The following data apply to a column liquid chromatography: length of pack-
ing¼24.7 cm, flow rate¼0.313 mL/min. A chromatogram of a mixture of chemical
A and B provided the following data:

Retention time, min Width of peak base (w), min

Nonretained solvent 3.1 -

Chemical A 13.3 1.07

Chemical B 14.1 1.16

Calculate the following and make comments regarding the separation:

(a) The number of plates (N) from each peak (chemical A and B)
(b) The plate height (H) for the column on the basis of the average number of plates
(c) The retention factor (k) for each peak
(d) The resolution between chemical A and B
(e) The separation factor (a) between chemical A and B
(f) The length of column necessary to give a resolution of 1.5
15. Refer to Figure 10.2, use a ruler to measure the retention times, and calculate N, H, k,
a for chemical A and B. Assume the time unit is in minutes.
16. Explain the difference between the following: (a) reverse phase HPLC and normal phase
HPLC, (b) isocratic flow and gradient flow.
17. What will be the effect on retention time (i.e., increased or decreased) if the polarity of
mobile phase is (a) decreased in normal phase HPLC, or (b) increased in reverse phase
HPLC?
18. You are to separate three chemicals (A, B, C) on a reverse phase HPLC column with
a UV detector. You first tried isocratic flow with a mobile phase of 10% acetonitrile
and 90% of water at a flow rate of 1 mL/min. The retention times at this condition
are 2.5, 5.5, and 5.9 minutes for chemical A, B, and C, respectively. Which chemical
is most polar? Since the retention times for chemical B and C are so close and peaks
are a little overlapped, what would you do to better separate these two compounds?
Why?
19. Using the properties of solvents given in Table 10.3, answer the following: (a) Why
acetone is not used for HPLC with UV detector? (b) Why isopropanol often produces high
pressure and cannot be used at higher percentage in mobile phase? (c) Which one is most
preferred in RP-HPLC with UV detector: methanol, hexane, isopropanol, or acetone?
20. For a reverse phase chromatography, (a) Which of the following, 1:1 water:methanol;
1:1 water:acetone; 1:1 water:THF, has the highest strength in eluting a hydrophobic
contaminant from a reverse phase column (use solvent properties in Table 10.3)?
(b) Which of the following stationary phase, unbonded silica, cyano, C 3 ,C 8 ,C 18 , has
the strongest retention for non-polar, non-ionic compounds?
21. Define UV-cutoff, refractive index, and polarity index. Why are they important in
HPLC?