25-17.

(a) Nonpolar aromatic compounds were separated by HPLC on an octadecyl (C 18) bonded phase.
The eluent was 65 vol% methanol in water. How would the retention times be affected if 90%
methanol were used instead?

→ Since methanol has a lower polarity than water, the eluent becomes more nonpolar as the

percentage of methanol increases. Therefore, the time for separation of the non-polar

compound (retention time) is shortened.

(b) Octanoic acid and 1-aminooctane were passed through the same column described in part
(a), using an eluent of 20% methanol/80% buffer (pH 3.0). State which compound is expected
to be eluted first and why.

CH3CH2CH2CH2CH2CH2CH2CO2H Octanoic acid

CH3CH2CH2CH2CH2CH2CH2CH2NH2 1-Aminooctane

→ The pKa of a carboxylic acid is 5 and the pKa of an amine is 35. The pKa of an amine refers to
the tendency for an amine to lose an proton, however, in an acidic solution it is more likely the
amine will accept an proton. Therefore the pKa of the conjugate acid must be used. The pKa
of the conjugate acid of a primary amine is 10. The pKa of hydronium is lower than both the
pKa values of octanoic acid and the conjugate acid of 1-aminooctane. This means that in a
solution with a pH = 3.0 both organic compounds will be protonated. The protonated form of
the carboxylic acid is neutral while the protonated form of the amine is positively charged.
Therefore the amine will be more polar than the carboxylic acid and will spend more time in
the polar mobile phase. This will cause 1-aminooctane to elute first.

(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?

→ Since acetonitrile is more polar than methanol, but is still non-polar than water, as the ratio of
acetonitrile in the mixture of water and acetonitrile increases, the total solution becomes non-
polar. Therefore, the retention time increases when polar solutes are eluted.

(d) Polar solutes were separated by normal-phase chromatography on 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?

→ 2-Propanol is more polar than t-butyl ether. Since polar solutes are eluted, the retention time is
shortened.

25-18. In monolithic columns60 the stationary phase is a single porous piece of silica or polymer
filling the entire column and synthesized within the column from liquid precursors. Monolithic
columns offer similar plate height to HPLC particles, but with less resistance to flow. Therefore,
faster flow or longer columns can be used. The figure shows separation of isotopic molecules on
a long monolithic column. Packed columns have too much resistance to flow to be made so long.

Separation of isotopic molecules on a 440-cm-long monolithic C 18-silica column eluted with CH3CN/H2O (30:70
vol/vol) at 30℃.

(a) Unretained thiourea is eluted in 41.7 min. Find the linear velocity ux (mm/s).

10 mm
→ ux = 440cm × ÷(41.7 ×60 seconds) = 1.76 mm/s
1 cm

(b) Find the retention factor k for C6D6.

t r −t m
→ Retention factor (k) =
tm
188.1−41.7
k= min = 3.51 min
41.7

(c) Find the plate number N and plate height for C6D6.
2
5.55t r 5.55× 188.1
2
→ N=¿ 2 = 2 = 192498 plates
w 1/ 2 1.01
L 440 cm
H=¿ = = 2.29×10-3cm = 22.9 μm
N 192498

(d) Assuming that the peak widths for C6H5D and C6H6 are the same as that of C6D6, find the
resolution of C6H5D and C6H6.

→ w : w1/2 = 4 : 2.35

∆ tr 1.01
Resolution (R) = = = 0.59
w av 1.01 × 4 ÷ 2.35
(e) Retention times for C6H5D and C6H6 are 193.3 and 194.3 min, respectively. Find the relative
retention (α ) between C6H5D and C6H6.

t r 2 194.3
→ α) = = = 1.005
t r 1 193.3

(f) If we just increased the column length to increase N, what value of N and what column length
would be required for a resolution of 1.000?

→ Resolution (R) =
4 α ( )
√ N ∙ ( α−1 ) ∙ k 2
1+k 2

1.000 =
4 1.005 ( 1+3.65 )
√ N ∙ ( 1.005−1 ) ∙ 3.65

2 2
1.005 1.+ 3.65
N=¿ 42 × ( ) × ( )
1.005−1 3.65
∴ N =1050000 plates
∴ L=¿ N × H = 22.9 × 10-6 m × 105000 = 24m

(g) Without increasing the length of the column, and without changing the stationary phase, how
might you improve the resolution?

→ Decrease the flow of the mobile phase or decrease the mobile phase strength.

(h) When the solvent was changed from CH 3CN/H2O (30 : 70 vol/vol) to CH 3CN/CH3OH/H2O (10 :
5 : 85), the relative retention for C6H5D and C6H6 increased to 1.0088 and the retention factor
for C6H6 changed to 17.0. If the plate number were unchanged, what would be the resolution?

→ Resolution (R) =
4 α ( )
√ N ∙ ( α−1 ) ∙ k 2
1+k 2

=
4 1.008 ( 1+17.0 )
√192498 ∙ ( 1.008−1 ) ∙ 17.0 = 0.82

25-19. 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)-gimetecan, 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-CNB. Chromatography on the (R,R)-stationary phase gave a
slightly asymmetric peak at tr = 6.10 min with retention factor k = 1.22. Chromatography on the
(S,S)-stationary phase gave a slightly asymmetric peak at tr = 6.96 min with k = 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.
(a) Explain the appearance of the upper chromatograms. Dashed lines are position markers, not
part of the chromatogram. What would the chromatogram of pure (R)-gimatecan look like on
the same two stationary phase?

→ Since the above chromatogram is for almost pure (S)-Gimatecan, (R)-Gimatecan would be
eluted at 6.96 min from (R,R)-stationary phase and 6.10 min from (S,S)-stationary phase.

(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?

→ In the enlarged y-axis of the (S,S)-stationary phase, a slight peak of (R)-Gimatecan is seen.
In (R,R)-stationary phase, (R)-Gimatecan at 6.96 min is lost beneath tail of (S)-gimatecan.

(c) Find the relative retention (α ) for the two enantiomers on the (S,S)-stationary phasd.

t r (S) 6.96
→ Relative retention (α ) = = = 1.14
t r (R ) 6.10

(d) The column provides N = 6800 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?

→ Resolution (R) =
√ N ∙ ( α−1 ) ∙ k 2
4 α ( )
1+k 2

=
√6800 ∙ ( 1.14−1 ) ∙ 1.50
4 1.14 ( 1+1.50 ) = 1.52

25-21. Morphine and morphine 3- β -D-glucuronide were separated on two different 50 × 4.6 mm
columns with 3- μm particles. Column A C18-silica run at 1.4 mL/min and column B was bare silica
run at 2.0 mL/min.

(a) Estimate the volume, Vm, and time, tm, 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.
2
Ld c 50× 4.6 2
→ Vm ≈ ≈ = 0.529 cm3
2 2
2
Ld c 0.529
tmA ≈ ≈ = 0.38min
2F 1.4
2
Ld c 0.529
tmB ≈ ≈ = 0.26min
2F 2.0

(b) Column A was eluted with 2 vol% acetonitrile in water containing 10 mM ammonium formate
at pH 3. Morphine 3- β -D-glucuronide emerged at 1.5 min and morphine at 2.8 min. Explain
the order of elution.

→ In reversed-phase chromatography, its stationary phase is nonpolar or weakly polar and the
solvent is more polar. More polar glucuronide is less retained by reversed-phase column.

(c) Find the retention factor k for each solute on column A, using tm = 0.65 min.

→ Morphine 3- β -D-glucuronide

1.5−0.65
k =¿ = 1.31
0.65

Morphine

2.8−0.65
k =¿ = 3.31
0.65

(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- β -Demerged at 2.7 min. Explain the
order of elution. Why does the gradient go from high to low acetonitrile volume fraction?

→ Polar hydrophilic silica retains glucuronide more than less polar morphine. Gradient goes to
increasing polarity to remove more polar solute. In gradient elution, increasing amounts of
 solvent B are added to solvent A to create a continuous gradient. Typically, solvent A means
a water-soluble solvent, and solvent B means an organic solvent.

(e) From Equation 25-12 in Box 25-4, estimate k* on Column B assuming S = 4 and with t m = 0.50
min.

¿
t G∙ F 5× 2
→ k =¿ = = 11.8
∆ ∅ ∙V m ∙ S 0.4 × 0.529× 4

25-39. The figure shows reversed-phase retention data for three compounds.

(a) Identify whether compounds A, B, and C are weak acids or bases. For each compound, what is
pKa and the retention factor of the fully protonated form?

A : weak acid, pKa = 7.7

B : weak acid, pK a = 3.7

C : weak base, pK a = 4.7

(b) Over what pH range would a method be least rugged with regard to retention of component
C?

→
 About pH 3.0 ~ 6.5

(c) Each different symbol in the plot indicates a different buffer (circle = pH 2.48 phosphate; plus
= pH 5.01 acetate; and so on). Why are different buffers used for this experiment?

→ When separating acids or bases, the aqueous component of the mobile phase must contain a
buffer. HPLC buffer must have adequate buffering capacity at the desired pH, be soluble in the
mobile phase, and be compatible with the detector and instrument hardware. Therefore, different
buffers are required to cover different ranges of pH.