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SUGGESTED ANSER FOR TUTORIAL 4

QUESTION 1

Gas chromatography (GC), liquid chromatography (LC), and supercritical fluid chromatography
(SFC) are three types of separation methods with different mobile phases.

a) Compare and discuss how each mobile phase affects

i) the B term of van Deemter in each of the above separation method.


(6 marks)

Compared with liquids and gases, the diffusion rates of solutes in supercritical
fluids is about ten times greater than that in liquids ( and about three times less
than in gases). Diffusion coefficient of solutes is highest in gas, followed by
supercritical fluid and liquid. Therefore, also able to dissolve large, non volatile
molecules. Gas with the lowest density can only elute volatile solutes with
small molecular weight.

ii) the types of sample that are suitable for each of the above separation method.
(6 marks)

Liquid has the highest density, therefore able to dissolve and elute large and
non volatile solutes as well small and volatile solutes. Compared with gas,
supercritical fluid has higher density (liquid-like), therefore also able to dissolve
large, non volatile molecules. Gas with the lowest density can only elute volatile
solutes with small molecular weight.

b) For each of the above separation methods, discuss one main parameter (exclude flow
rate) that can be manipulated to improve the separation of analytes.
(6 marks)
In GC, temperature: low T for volatile, high T for less volatile analytes.

In LC, the composition of mobile phase affects the elution of analytes. The higher
the eluent strength, the faster the elution.

In SFC, pressure is more significant than temperature to affect the elution of


analytes. As the pressure in the system is increased, the supercritical fluid
density increases and correspondingly its solvating power increases. Therefore,
components most retained in the column can be eluted faster.

(Q2 OCT 2010)

QUESTION 2

For each of the following, give a reason to the mentioned problem and suggest an approach to
overcome the problem without changing the extraction method used.

Alkanes can be extracted from urban air particulates using supercritical fluid extraction (SFE)
with CO2 at 75 bar (45C), whereas the polyaromatic hydrocarbons (PAHs) remain unextracted.
(3 marks)

CO2 at 75 bar (45C) has low density therefore could not dissolve higher molecular
weight molecules. Therefore, increase the pressure. As the pressure in the system is
increased, density of with CO2 increases and able to dissolve and extract the PAHs.

Or

CO2 is non polar therefore not able to extract PAHs which are slightly more polar than
alkanes. Add a small amount of polar modifier into with CO2 to increase its polarity and
enhance the solvating power. Thus PAHs can be extracted.

(Q6b OCT 2010)

QUESTION 3

a) What is supercritical fluid?


(2 marks)

Supercritical fluid is a substance above Tc and Pc (critical point). It exists in a


single phase which is neither liquid nor gas.

b) Explain the differences in properties of the mobile phase in GC, HPLC and supercritical
fluid chromatography (SFC).
(6 marks)

Compared with gas, supercritical fluid has high density (liquid-like)(higher than
gas, lower than liquid), therefore able to dissolve large, non volatile molecules and
increase column loading.

Compared with liquid, supercritical fluid has low viscosities, therefore able to use
higher flow rate for faster analysis.

The diffusion rates of solutes in supercritical fluids is about 10x greater than that
in liquids ( and about 3x less than in gases). This results in a decrease in
resistance to mass transfer in the column and allows for fast separations (faster
than HPLC).

c) Discuss the kind of gradient used in SFC.


(4 marks)
Pressure programming.

As pressure in the system is increased, the supercritical fluid density increase


and correspondingly its solvating power increases. Therefore, as the density of
the supercritical fluid mobile phase is increased, components retained in the
column can be made to elute. (This is similar to temperature programming in GC
or using a solvent gradient in HPLC.)

d) Discuss the advantages of supercritical fluid extraction (SFE) over solvent extraction.
(4 marks)
Faster extraction: due to greater diffusion rate and lower viscosity.
Solvent strength: can be varied with pressure and temperature.
Recovery of analytes: By releasing pressure of CO2 (environmental friendly).
Less organic solvent (organic modifier).
(Q4 JAN 2012)

QUESTION 4

a) Compare the diffusion coefficient for gas, liquid and supercritical fluid and relate this
property to band broadening in gas chromatography, liquid chromatography and
supercritical fluid chromatography (SFC).
(3 marks)
Diffusion rate: liquid <SF< gas (1)
Therefore band broadening is greater in SFC than LC but less than in GC (2)

b) Explain the function of a modifier used in SFC.


(3 marks)

A modifier is required when CO2 is used to separate polar compounds (1). Since
CO2 is non polar (1), a polar modifier is added to enhance the solvating power
(making it polar) and enhance the separation (1).

c) Supercritical fluid extraction is faster with easy analyte recovery method compared to
liquid-liquid extraction. Explain.
(4 marks)

Faster due to the higher diffusion coefficient and lower viscocity (1) for fluid
compared to liquid causing greater mass transfer (1).

Many supercritical fluids are gasses at ambient condition (1). Thus, recovery of
analyte is simple compared to organic liquids, as no vaporization step is required
(1).

d) You are given a water sample contaminated with several relatively non polar pesticides.
Outline the steps required for an efficient a solid phase extraction technique. Please
suggest specific sorbent and solvent in each step.
(6 marks)

1. Column (non polar such C18) () must be conditioned (1) using methanol or
and water ();
2. Sample loading (1)
3. Inteference elution (1) using polar solvent such as water ()
4. Elution of analytes (1) using non polar solvent such as hexane ().

e) In the analysis of herbicides in soil sample, a pressurized liquid extraction (PLE)


technique is proposed prior to LC analysis. PLE technique is able to achieve comparable
efficiency as Soxhlet technique but at a shorter analysis time. Explain.
(5 marks)
High temperature: (1)
- can disrupt the strong solute-matrix interactions (1)
- decreases the viscosity of organic solvents allowing improved penetration of
the matrix, and hence improved extraction (1).

High pressure: (1)


- solvent remains liquefied above their boiling point and allows the solvent to
penetrate the sample matrix.
(Q5 JUNE 2012)

QUESTION 5

a) Give two characteristics of a supercritical fluid which can enhanced the separation of
analytes compared to gas.
(2 marks)
Higher viscosity
Higher density

b) The efficiency of separation in supercritical fluid chromatography (SFC) is better than


GC and HPLC. Explain.
(4 marks)

Diffusion rate for supercritical fluid is 3x less than gas (1), thus less band
broadening due to B term compared to GC (1).

Viscosity of supercritical fluid is less than liquid (1), therefore faster analysis
compared to HPLC (1).

c) Carbon dioxide has a limitation to extract polar compounds. Explain.


(3 marks)

CO2 is non polar (1), therefore have ability to extract non-polar compounds only
(1). Need to add a strongly polar modifier to enhance the polarity of CO 2 and
improve the extraction (1).

d) SFC can separate a mixture of volatile and non-volatile compounds efficiently.


Explain.
(4 marks)

Use pressure programming (1). Start with low pressure to separate volatile
compounds (1), then increase pressure to increase solvating power (1), therefore,
non volatile compounds can be eluted faster (1).

e) Discuss two advantages of supercritical fluid extraction (SFE) compare to solvent


extraction.
(4 marks)
i) SFE elimination of organic solvents
ii) CO2 use in SFE is low cost
iii) Nonflammable compounds
(Q3 JAN 2013)
QUESTION 6

a) The analysis of eight pesticides mixture (of various polarities and volatilities) was
conducted using supercritical fluid chromatography (SFC). The separation was done
using carbon dioxide as the mobile phase, isobaric condition of 300 bar and isothermal
condition of 100 C.

i) Only 6 peaks were observed. The polar pesticides were not detected. Explain
and suggest an approach to rectify the problem.
(4 marks)

Non polar carbon dioxide (1) is not suitable for polar analytes (1).
Add polar modifier (methanol) to make the mobile phase more polar for the
polar analytes (2).

ii) In the above analysis, it was found that the non volatile pesticides were retained
too long resulting in a very broad peaks. Explain the use of pressure
programming approach in solving this problem.
(4 marks)

Use lower pressure for volatile analytes (1). Increase pressure will increase
the density of mobile phase and correspondingly its solvating power
increases (1), thus able to solvate the non volatile analytes (1). Faster
elution (1).

b) Supercritical fluid extraction (SPE) is considered as a fast and clean extraction technique
compared to liquid-liquid extraction. Explain.
(4 marks)

Use (1) (no/less organic solvent), hence produced clean extracts (1). Fast
extraction due to greater diffusion rate (1) and lower viscosity (1) of supercritical
fluid.

(Q3 DEC 2013)

QUESTION 7

a) Compare the density and viscosity of supercritical fluid with liquid and gas. Discuss how
these properties are considered an advantage for supercritical fluid chromatography
(SFC) compared to HPLC and GC.
(6 marks)

Viscosity of SF is lower than liquid but higher than gas (1). Density of SF is lower
than liquid but higher than gas (1).

Lower viscosity of SF will contribute to faster flow rate of analysis compared to


liquid, (1) hence faster analysis time than HPLC (1).

Compared with gas, supercritical fluid has high density (liquid-like)(higher than
gas, lower than liquid), therefore able to dissolve large non volatile molecules (2).
b) T
he separation of a mixture of polychlorinated biphenyl (PCBs) using SFC with carbon
dioxide as the mobile phase resulted in long analysis time. Discuss an approach to
reduce the analysis time of the separation.
(4 marks)

SFC with CO2 limted to separation of non polar compounds due to non polar
character of CO2. (1)

To reduce the analysis time, the mobile phase must be made more polar (1) by
adding polar modifier (1), e.g. methanol (1).

(Q3 JUNE 2014)

QUESTION 8

Describe the fundamental difference between ion exchange chromatography (IEC) and size
exclusion chromatography (SEC) in term of its principle of separation.
(4 marks)

Ion exchange chromatography separates analytes based on exchange equilibria


between ions in solution and ions of same charge on the surface of a solid exchanger
site (2)

Or

IEC relies on charge-charge interactions between the charges in your sample and the
charges immobilized on the resin of your choice (1). Separations based upon exchange
of ions (cations or anions) on an ion exchange stationary phase (resin) column (1).

Size exclusion chromatography is based on the average residence time of large


molecular weight analytes in a silica or polymer packing with a network of uniform
pores (1). Separation mechanism is based on sieving not partitioning (1).

(Q2b JUNE 2014)