School of Science

Hamilton Campus

Session 2013-14

Trimester 1

Module Code: BIOL08006

ANALYTICAL TECHNIQUES

Date: 14 January 2014

Time: 1400 - 1600

Duration: 2 Hours

Instructions to Candidates:
1. Answer ALL multiple choice questions from section A.
Answer ONE question from section B.
Answer FOUR questions from section C.
2. Ensure you enter your student number on any
Graph paper used
Multi choice answer sheet
and attach it to the answer booklet.
3. Calculators must not be used to store text and/or formulae nor be
capable of communication. Invigilators may require calculators to be
reset.

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Section A
(Multiple Choice Questions)
Answer ALL Questions using the multi choice answer sheet provided

1.

Which of the following pieces of information cannot be obtained from an

infra-red spectrum?
(a) The molecular mass
(b) The presence of C=O bonds
(c) The presence of O-H bonds
(d) The identity of a compound through comparison with other
spectra
(e) The functional groups present in a compound

2.

What is a chromophore?
(a) A group of atoms in a compound responsible for the
emission of electromagnetic radiation
(b) A group of atoms in a coloured compound
(c) A group of atoms in a compound responsible for the
absorption of electromagnetic radiation
(d) A coloured compound
(e) A fluorescent label attached to a non-coloured compound

3.

Which of the following statements about tetramethylsilane is incorrect?

(a) It is inert.
(b) It is volatile and can be easily distilled off and used again.
(c) It is used to provide a reference against which other peaks
are measured.
(d) It has 12 equivalent hydrogens
(e) It produces a single peak at =10.

4.

The path of ions after deflection in a mass spectrometer depends on:

(a) Only the mass of the ion
(b) Only the charge of the ion
(c) Both the charge and the mass of the ion
(d) Neither the charge nor the mass of the ion
(e) The abundance of each ion

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5.

To check that a secondary alcohol has been completely oxidised to a

ketone, you can:
(a) Check that the IR spectrum has absorptions at 3500 cm-1
and 1650 cm-1
(b) Check that the IR spectrum has no absorptions around
3500 cm-1
(c) Check that the IR spectrum has no absorptions around
1650 cm-1
(d) Check that the IR spectrum has no absorptions at 3500
cm-1 and 1650 cm-1
(e) Check that the IR spectrum has weaker absorptions at
3500 cm-1 compared to 1650 cm-1

6.

A diode array detector (DAD) coupled

advantageous because:

with

UV

detection

is

(a) It allows lower concentrations of analyte to be detected

(b) It allows a single wavelength of detection to be more
precisely chosen
(c) It speeds up the detection at a single wavelength
(d) It covers a range of wavelengths
(e) It allows automatic background correction

7.

The advantage of the ICP-AES technique over conventional AES

analysis is that:
(a) Multi-element analysis can be performed in sequential runs
(b) Analysis of a single metal is faster
(c) Several atomic absorption spectra can be recorded
simultaneously
(d) Multi-element analysis can be performed in one run
(e) Emission and absorption spectra can be recorded
simultaneously

8.

Which one of the following statements about the mass spectrum of

CH3Br is correct?
(a) The last two peaks are of equal size and can occur at m/z
values of 94 and 96
(b) The last two peaks have abundances in the ratio 3:1 and
can occur at m/z values of 94 and 96
(c) There is just one peak for the molecular ion with an m/z
value of 95
(d) There is just one peak for the molecular ion with an m/z
value of 44
(e) There is just one peak for the molecular ion with an m/z
value of 96
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9.

The proton nmr spectrum of propane will consist of:

(a) A triplet and a singlet
(b) A triplet and a septet
(c) A doublet and a sextet
(d) A doublet and two triplets
(e) A triplet and a quartet

10. Lead levels in drinking water could be determined by using?

(a) UV spectroscopy
(b) GC-MS
(c) NMR
(d) FTIR
(e) AAS

11. High performance liquid chromatography (HPLC) cannot be used to:

(a) Determine the caffeine content of coffee samples
(b) Identify the various pigments from a leaf extract
(c) Determine the mercury content of a fish sample
(d) Identify the active compounds present in Anadin Extra
(e) Separate types of organic pesticides

12. A characteristic feature of any form of chromatography is:

(a) Use of an inert carrier gas
(b) Use of molecules that are soluble in water
(c) Calculation of an Rf value for molecules separated
(d) Use of a mobile and stationary phase.
(e) Calculation of peak area ratios

13. The isoelectric point, or pI, of an amino acid or protein is:

(a) The pH at which the amino acid or protein has no net
charge
(b) Zero at pH 7.0
(c) The pH at which the amino acid or protein is neither
hydrophobic nor hydrophilic
(d) The measure of the hydropathy of an amino acid or protein
(e) The voltage at which the amino acid or protein stops
moving in an electrophoresis gel

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14. Which functional group is regularly employed in the stationary phase of

cation-exchange chromatography?
(a) Trimethyl (ammonium), -N(CH3)3+
(b) Peroxide, -O2(c) Magnesium, -Mg+
(d) Carbonate, -CO32(e) Sulphonic acid, -SO315. Which of the following statements about paper and gas chromatography
is correct?
(a) The Rf and Rt values of a substance are solely determined
by the interaction of the substance with the stationary
phase
(b) A low Rf value is indicative of a substance that absorbs
strongly onto the stationary phase
(c) A substance with a long retention time in gas
chromatography is likely to have a high Rf value in paper
chromatography
(d) A long retention time in gas chromatography is indicative
of a substance with a strong adsorption on to the
stationary phase
(e) A non-polar stationary phase can be used in both paper
and gas chromatography

16. Which of the following is commonly used as the mobile phase in GCMS?
(a)
(b)
(c)
(d)
(e)

H2O
He
CH3CN
Air
Acetylene

17. In an electrophoresis system, using an agarose gel, proteins are

separated due to differences in total charge. What decides the proteins
total charge?
(a) Voltage
(b) Proteins size
(c) Support medium
(d) Isoelectric point
(e) Buffers pH
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18. Which type of liquid chromatography separation depends on interactions

between solute, mobile phase, and immobilised liquid stationary phase?
(a) Partition
(b) Ion-exchange.
(c) Size exclusion
(d) Tlc
(e) Adsorption
19. Tomatoes contain lycopene (max = 444, 470 and 502 nm) and carotene (max = 442 and 472 nm).
Which technique could be used for the analysis of lycopene and carotene in tomatoes, after suitable sample preparation?
(a) AAS.
(b) GC with flame ionisation detection.
(c) HPLC with UV-Vis detection.
(d) GC-MS.
(e) HPLC with UV detection

20. In a mixture of the five proteins listed below, which should elute second
in size-exclusion (gel-filtration) chromatography?
(a) Cytochrome C, Mr = 13,000
(b) RNA polymerase, Mr = 450,000
(c) Serum albumin, Mr = 68,500
(d) Immunoglobulin G, Mr = 145,000
(e) Transferrin, Mr = 80,000

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Section B
Answer ONE Question

1.

2.

a) Outline the principles of Fourier Transform infrared (FTIR)

spectroscopy and explain the advantages of a FTIR spectrometer
over Dispersive infrared spectrometers.

(10)

b) Explain how the use of size exclusion chromatography (SEC) brings

about the separation of protein mixtures. Compare the linear
relationship between mass and relative mobility of proteins in SEC vs
SDS-PAGE.

(10)

a) Draw and label a block diagram of a gas chromatography (gc)

system using capillary columns. Describe the function and essential
characteristics required of the main components in the system.
Reference should be made to the mechanism of the separation
process and, choosing the stationary phase and operating
temperature.

(10)

b) Explain what is meant by validation of an analytical procedure and

outline the basic guidelines involved. Briefly describe the meaning of

(10)

(i)
(ii)

accuracy
precision

within the process of validation.

3.

a) Describe the structure of the stationary phase, choice of mobile

phase, mechanism of separation and a typical application of the
following hplc mechanisms:

(10)

(i) C 18 reversed phase

(ii) Ion exchange
b) Write an account of mass spectrometry. Reference should be made
to:
The instrumentation
The processes involved in producing the spectrum
A sketch of a typical spectrum
How the results are used

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Section C
0B

1B

1.

Answer FOUR questions

With the aid of the infrared and nuclear magnetic resonance correlation
charts provided, fully interpret the spectra Worksheet Q1 IR, Worksheet
Q1 NMR and Worksheet Q1 MS and hence deduce which of the
following structures A, B or C correlates to the spectra given.

A.

B.

C.

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Worksheet Q1 IR

3H
Worksheet Q1 NMR
Integration is
numeric
3H
2H

2H

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Worksheet Q1 MS
Q1 Worksheet MS

2.

Phosphate ions can be determined by reaction with ammonium

molybdate followed by the addition of a few drops of stannous chloride
to produce a blue colour.
This is then followed by spectrophotometry at = 650 nm.
A phosphate stock solution of concentration 100 mg dm-3 is used to
prepare standards of concentration 1, 2, 3, 4 and 5 mg dm-3. The
absorbance is measured in a 1 cm pathlength cell.
a) Describe how you would prepare the standard solutions.

(2)

b) Which type of cell would you use for this experiment, and why?

(1)

c) Using the results below and the graph paper provided plot a
calibration graph for the phosphate standards.

(4)

Tube Number
1
2
3
4
5
Phosphate (mg dm-3)
1
2
3
4
5
Absorbance values 0.15 0.32 0.50 0.59 0.72
d) How can you tell if your calibration graph conforms to the
Beer-Lambert law?

(2)

e) A sample was analysed under the same conditions and shown to

have an absorbance of 0.55. Use the calibration graph to
calculate the concentration of phosphate in the sample.

(1)

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3.

4.

In flame atomic absorption spectrophotometry, using an air-acetylene

flame, levels of copper in water are measured at 324.75 nm and the limit
of detection is quoted as 4 ppb or 4 g L-1.
a) Explain what is meant by the Limit of Detection and outline in
general terms how its value would be determined.

(4)

b) Explain how the energy equivalent to 324.75 nm is generated and

how it is then absorbed by the sample to produce the data required
to determine the level of copper.

(6)

a)

(2)

(i)

Explain why the particles used as stationary phase in

high performance liquid chromatography (hplc) have
very small diameters, typically between 3 and 10 m.

(ii)
(iii)

Explain why a high pressure pump is required in hplc.

Explain why mobile phases are degassed prior to use in
hplc and state one method for doing this.

(2)

(2)
b) Explain what is meant by gradient elution in hplc and comment on
the advantages it brings to the process.

5.

(4)

a) The carcinogen benzene is often measured in air by adsorbing

onto a charcoal tube followed by solvent extraction from the
adsorbent and then gas chromatography of the resultant solution.
An internal standard is added to the extract before gc and the
results are compared to a calibration graph of benzene standards
containing the same amount of internal standard.
(i) Describe how benzene is identified in the sample.
(ii) Explain the purpose of the internal standard.

(2)
(2)

b) Briefly highlight the advantages and disadvantages of using

nitrogen (N2), helium (He) or hydrogen (H2) as the carrier gas in
gas chromatography.

(3)

c) Explain why a temperature programme is often used in gas

chromatography.

(3)

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6.

Two proteins have the following characteristics:

Protein A
20,100
4.5

Molecular Weight
Isoelectric Point

Protein B
68,000
5.8

State and explain their relative mobilities when using:

a) SDS-PAGE (sodium dodecylsulphate-polyacrylamide gel
electrophoresis)
b) Isoelectric focusing (IEF)
Reference should be made to the media used and the mechanism
of separation.

END OF QUESTION PAPER

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