TUTORIAL 3 CHM510

CHAPTER 3: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

QUESTION 1

a) Octanoic acid and aminooctane were separated by reversed-phase high

performance liquid chromatography (HPLC) on a C18 silica bonded stationary
phase, using an eluent of 20% methanol/80% buffer (pH 3) which was pumped at
high pressure through the column.

i) Why is high pressure needed in HPLC?

(2 marks)

High pressure is needed in HPLC to ensure the gases are soluble

because gases become insoluble at low pressure (ie. at the end of the
column or detector) (1) in which the gas bubbles create difficulties with
pumps (destabilize pressure), columns (bad separation) & detectors (1).

ii) Why is silica stationary phase generally limited to operating in the pH range of
2-8?
(2 marks)

Silica stationary phase generally is limited to operating in the pH range of

2-8 because it dissolves above pH 8 and siloxane bond (Si-O-Si) to the
stationary phase hydrolyses below pH 2. These defects will cause
changes in tR and loss of resolution

iii) State which compound is expected to be eluted first and why.

(3 marks)

iv) Explain briefly how the retention times would be affected if 80% methanol were
used instead.
(2 marks)

b) In a gradient elution normal phase separation, what should be changed in regard

to acetone (starting solvent) and hexane when the following situations develop?

i) The peaks are bunched together near tM

(3 marks)

ii) Peaks continue to elute for a considerable time after completion of the gradient.

(3 marks)

c) Describe how an ion exchange chromatography can be used to purify water or to

prepare deionized water.
(4 marks)
d) For size exclusion chromatography, explain total permeation and exclusion limit.

(4 marks)

Total permeation limit defined as the limit which molecules that are smaller
than the pore size can enter all pores (1) and elute together as the last peak
in the chromatogram (1) ;

Molecules larger than the pore size cannot enter the pores (1) and elute
together as the first peak in the chromatogram (1) is called total exclusion
limit.

(Q3 OCT 2007)

QUESTION 2

a) A mixture of alcohol compounds was analysed on a 25 cm C18 column. The

mobile phase contained a mixture of 30% methanol and 70% water. Explain how
the retention time would be affected if:

i) the composition of the mobile phase is changed to 70% methanol and 30%
water
(3 marks)

ii) the C18 bonded phase contained unreacted silanol groups. Name the process
to reduce the concentration of unreacted silanols.
(3 marks)

iii) the C18 column is changed to a phenyl bonded phase column.

(3 marks)

b) Draw and label completely a chromatogram for the following sugar compounds
separated on a '500 A' column with a fractionation range of 50 - 1000: xylose
(MW150), glucose (MW 180), maltose (MW 342), maltoheptaose (MW 1153) and
starch (MW >1500). What are the exclusion limit and the permeation limit for this
separation?
(5 marks)

c) In ion exchange chromatography, retention is based on the attraction between

solute ions and charged sites bound to the resin.

i) Give a type of resin known as a "strong-acid resin" in cation exchange

chromatography. Explain briefly what will happen to the retention time of
analytes if the pH used is less than 4.
(3 marks)
 ii) How is gradient elution employed in ion exchange chromatography?
(2 marks)

d) Isomer compounds can be separated by adsorption chromatography. Explain

briefly.
(2 marks)

(Q3 OCT 2008)

QUESTION 3

For each of the following, discuss the reason for the mentioned problem and suggest an
approach to overcome the problem.

a) In a separation using a C18 column with a mobile phase of acetonitrile/water

80/20, the first two peaks eluted very close to tM.
(4 marks)

b) 7-chlorophenol and 2-chlorophenol were not separated on a bonded siloxane with

diol functional groups stationary phase.
(4 marks)
7-chlorophenol and 2-chlorophenol are isomers.
The can have very different physiosorption characteristics due to steric
effect in the molecules

Adsorption chromatography

Stationary phase : Silica or alumina particles (polar).

Mobile phase : A non polar solvent (e.g. hexane) modified with a small
amount of polar solvent (e.g. acetone, diethyl ether, methylene chloride).

Detector : Normally UV.

Note:
Mechanisme of the separation :

Analytes compete with mobile phase for the binding sites on the
stationary phase.
Analytes in the liquid sample interacts with adsorption sites on solid
surface.
Polar groups (-OH) on solid surface form dipolar interaction (e.g. H
bonds) with sample.
Analytes are separated based on repeated adsorption-desorption steps
onto the solid support.

c) In the analysis of cations in water sample using single column ion chromatography,
most of the cations could not be detected due to the low sensitivity of the
conductivity detector
(4 marks)

d) The cations in a water sample at pH 2 cannot be separated on a carboxylic acid

resin.
 (4 marks)

e) When a Bio-gel 150 with a molecular mass range of 15,000 - 150,000 is used for
size exclusion chromatography packing, protein A (MW 185,000) and protein B
(MW 158,000) are not retained and eluted together as a single peak.
(4 marks)

(Q4 OCT 2009)

QUESTION 4

For each of the following, give a reason for the mentioned problem and suggest an
approach to overcome the problem.

a) In a normal phase separation of 5 compounds using isocratic mixture of

hexane:acetone (80:20), the retention time for the last two peaks were too long.

(5 marks)

b) For the separation of organic acids using ion exchange chromatography, the
compounds were separated using carboxylic acid COO-H+ resin as the stationary
phase at pH 4. The compounds were not separated due to overlapping of peaks.
(5 marks)

c) You are given the task to separate anions in a water sample. In your laboratory,
the liquid chromatograph available can only run partition chromatography. When
the separation of anions in the sample was carried out using reverse phase
chromatography with methanol:water (50:50) as the mobile phase, no peak was
observed.
(5 marks)
Ion-pair chromatography (IPC)

Mobile phase : aqueous buffer containing organic solvent (MeOH/ACN) &

IPcontaining a counter ion of opposite charge to the analyte. Typically :
Alkyl amines or tetra alkyl amines salt are added to ion pair with acids.
Alkyl sulfates, sulfonates or phosphates sodium salt are used to ion pair
with bases.

Mechanism of separation :
The counter ion combines with the analyte ion to form an ion-pair(neutral
species/uncharged) that is retained by the RP packing.

Elution of the ion-pairs is then accomplished with an aqueous solution of

methanol or other water soluble organic solvent.

Note:
IPC is a method for improving the separation of charged/ionized (cation &
anion) & very polar analytes on a reversed phase column.

The use of IP reagents can enhance peak shape & tR when common
remedies such as modifying eluent ratios or changing stationary phase fail.
d) When a Bio-gel 150 with a molecular mass range of 22,000 - 250,000 was used
for size exclusion chromatography packing, the first three compounds were
separated well. However, the last two compounds were eluted as a single peak.

(5 marks)

(Q4 OCT 2010)