BINDURA UNIVERSITY OF SCIENCE EDUCATION

CHEMISTRY DEPARTMENT
CH304: ANALYTICAL CHEMISTRY III: ASSGNMENTS: 2014
ASSGNMENT I: DUE DATE 30/09/16
1. Discuss the applications of Ion Chromatography. [7marks]
Answer
Analysis of anions in drinking and process water (chloride, nitrate,
sulphate and hydrogen carbonates)
Nitrates in vegetables
Fluorides in toothpastes.
Bromides, sulphate and thiosulphates in fixing baths
Organic acids in beverages.
Ammonia, potassium and phosphates in soil and fertilizers
Sodium and potassium in clinical samples such as body fluids.
2. Describe the principles of the following chromatographic techniques
i) Size exclusion chromatography
The main purpose of SEC is to provide molecular weight distribution about
polymeric materials. When macromolecules of different molecular weight
pass through a macro-porous gel, smaller molecular weight molecules pass
through the pores of the gel while larger molecular weight molecules by-pass
the pores and are hence eluted faster than smaller molecules
ii) Thin Layer Chromatography
Stationary phase is solid
Mobile phase is liquid
Not retention times but Rf
iii) Affinity Chromatography. [3 4marks]
Analyte molecules are selectively bound to specific sites on the stationary
phase.

The interaction is mostly of biochemical nature

Only the substance with affinity for the ligand are retained on the column
The substance with no affinity to the ligand will elute off
The substances retained in the column can be eluted off by changing the pH
of salt or organic solvent concentration of the eluent

3. Discuss the causes of asymmetry peaks in Liquid chromatography.

[7 marks]
bad column (plugged frit or void)
Contaminated column (buildup of strongly retained sample contaminants)
Column overloads (too large a sample)
A sample solvent that is too strong
Extra-column peak broadening
silanol interactions (including contamination of the column packing by trace
metals inadequate or inappropriate buffering.
use of a higher column temperature with cold (inadequately thermostatted)
mobile phase.
4. Using a block diagram, describe the components of an HPLC system.
[5marks]
Adapted from notes 304 LC slide 21 by Dr M Mupa(2016)

5. Discuss the desirable characteristics of an ideal HPLC detector.

[8 marks]
High sensitivity
Negligible baseline noise
Large linear dynamic range
Non-destructive of the sample
Stable over longer period of time
Convenient and reliable to operate
Inexpensive to purchase and operate

Capable of providing identity of solute/analyte

Response independent of mobile phase composition
Low dead volume
6.Describe the principles of the following modes of detection in HPLC.
i) Fluorescence
Used in molecules that are aromatic, contain multiple double bonds, i.e.,
double bond with high degree of resonance structure
Fluorescence molecules absorbs radiation of one wavelength (from ground
state to excited state) and analytes which are fluorescence will emit
radiation of one wavwlength (from excited state to ground state)
Working Principle
Radiation from a xenon or deuterium lamp passes through an excitation
filter, which provides essentially monochromatic light of desired wavelength
to excite the sample.
This excitation l of light then passes through the column effluent in the flow
cell.
When the sample molecule passes through the column effluent they are
excited and emit light (fluorescence) at a longer l.
A second (emission) filter is positioned at 90 deg. to the first filter to collect
the emitted light. In this way only the light emitted from the sample
fluorescence will pass on PMT for quantitation of the emission signal.

ii) Uv-Vis absorbance [26marks]

Light from the lamp passes through a UV transmitting flow cell (through
which M.P flows and is connected to the column) and falls on a diode which
measures the light intensity I.
Usually, light from the lamp is also directed to the reference diode for
measurement of light intensity I0
The detector electronics then converts the signal from the two diodes into
Absorbance A, which is transmitted to the data system and is measured as
A = log I0/I (ratio of the intensity of absorption between ref and sample
diode)
Analyte concentration (C) in the flow cell is related to the absorbance of the
Analyte (A), molar absorptivity (e), and flow cell length (Lfc) by Beers Law:
A = e Lfc

7. Explain why the development of chiral stationary phases have increased

recently.
[5 Marks]
One enantiomer may be beneficial, e.g. may have therapeutic effect, the
other may have no beneficial effects, e.g. in some instances, it may be toxic
An ever increasing pressure by regulatory authorities against the marketing
of racemic mixtures in foods, drinks, pharmaceuticals and agrochemicals.
Pharmaceutical companies now consider manufacture of racemic drugs as
more risky and hence are investing more effort in accessing chiral
technologies
8. What is the purpose of a pre-column within an HPLC system? [3 Marks]
Method depends on whether the column is for column/preparative or
analytical HPLC.
For column chromatography, a glass column is filled with a lighter solvent
followed by manual filling with stationary phase. The material of large
particulate size > 25.

9. With the help of a reaction scheme describe at least two approaches of

Synthesizing a C18 bonded phase. [8 Marks]

S u r fa c e tr e a tm e n t w ith th io n y l c h lo r id e
OH
HO
Silica gelHO
HO

Cl

OH

nSOCl

OH

-H C l

HO
HO
S i l i c a g eH
lO

OH
Cl

O th e r r e a g e n ts :T iC l4 , S iC l4
G r ig n a r d r e a c tio n
R

Cl
HO

S i l i c a g e Hl O

OH

HO

nRMgCl

HO
S i l i c a g e Hl O
HO

MgCl

Cl

R = -C H
-C H
-C H
-C H

OH

-(C H 2)16-C H 3, C 18 o r O D S
2-(C H 2)10-C H 3, C 12
2-(C H 2)6-C H 3, C 8 o r o c ty l
2 -(C H 2 )2 C H 3 , C 4 o r b u ty l

A n o th e r s y n th e tic r o u te
R

Cl
HO
HO
S i l i c a g eH
lO

OH
Cl

nRLi

HO
HO
S i l i c a g eH
lO

OH
R

nLiCl

Cl
HO
HO
S i l i c a g eH
lO

OH

NH

HO
HO
S i l i c a g eH
lO

H2N
n

Cl

X
n

OH
NH

X
n

X = - C O O H , w e a k c a tio n e x c h a n g e r
- S O 3 H , s tr o n g c a tio n e x c h a n g e r
-C N , -N O 2, n o rm a l p h a s e s
-C H 3, -N H 2
If X is a n a m in o fu r th e r s u r fa c e m o d ific a tio n r e a c tio n c a r r ie d
o u t a s illu s tr a te d in th e r e a c tio n s c h e m e b e lo w .
NH
HO
HO
S i l i c a g eH
lO

NH2
n

OH
NH

+
NH2

NH
Z

HO
HO
S i l i c a g eH
lO

NH

R
X

OH
NH

NH

10. Explain the advantage of a narrow diameter HPLC column over a wider
one.
2

[4 Marks]

11. Why is High pressure needed in HPLC [3marks?]

Due to small size of particles(normally at 450atms)
Also to avoid settling a dense solvent is used.
12. A pump is a major component of an HPLC system. What is the
advantages of?
The following pumps:
Dual piston pump
Dual piston pump are better than single piston pump because they
minimize pulsations
Disadvantage: Expensive

Syringe pump
Advantage: cheaper than the dual piston pump.
Disadvantage: rarely used to deliver mobile phase due to the fact that they
Produce pulse flow
13. Describe the principle behind the following liquid chromatographic
techniques.
a. Size exclusion chromatography and [4 Marks]
The main purpose of SEC is to provide molecular weight distribution about
polymeric materials. When macromolecules of different molecular weight
pass through a macro-porous gel, smaller molecular weight molecules pass
through the pores of the gel while larger molecular weight molecules by-pass
the pores and are hence eluted faster than smaller molecules
b. Ion chromatography. [4 Marks]
Stationary phases have lower exchange capacity.

Ionic strengths of mobile phases can be low (mM)

Since diluted mobile phases can be used, background conductivity is low but
not sufficiently low enough.
Hence ion suppression techniques are also used except for other modes of
detections, e.g. UV detection.
Two types of ion chromatography are currently in use:
suppressor-based and single-column.
They differ in the method used to prevent the conductivity of the
eluting electrolyte from interfering with the measurement of analyte
conductivities.

14. Explain the importance of liquid chromatographic separation of chiral

Compounds. [6 Marks]
15. What are the three common approaches in liquid chromatographic
separations?
Of chiral compounds? [6 Marks]
Conversion of enantiomers into diastereomers and by separation using LC
Enantiomeric resolution by chiral Mobile phase Additives (CMA)
Enantiomeric Resolution by chiral stationary phases.
16. What is the purpose of an interface the hyphenation of a
chromatographic unit to
an MS instrument? [4 Marks]

17. Explain why it is much easier to couple a GC to an MS instrument than LC

THE END
3

ASSIGNMENT II
DUE DATE: 21/10/16
1. Explain the factors that influence choice of ionization technique in MS.
[3marks]

2. Distinguish between the principles of Electron Ionization (EI) and Chemical

Ionization (CI). [8marks]
3.Describe how the Faraday cup detector operates. [4marks]
4. Discuss the advantages and disadvantages of Fast Atom Bombardment
(FAB).
[8 Marks]
5. Describe how the Quadrupole Mass Analyser operates. [5marks]
6. Explain the advantages and disadvantages of Double- Focusing Magnetic
Sector. [7marks]
7. The activity in a 10-ml sample of radioactive waste water containing a
radioactive
isotope with t of 28.1 years was found to be 9.07x106 disintegrations/s.
What is
the molar concentration of radioactive isotope in the sample? [5 Marks]
Since

t =

0.693

and A=N. Then

N=

A t
0.693

Before the number of atoms of can be expressed, it is necessary to express

the activity and half-life in the same units. Converting the half-life for to
seconds gives t as 8.86x108 s (28.1 years).
Therefore
A t (9.097 106 distintegration s1 )(8.86 106 s)
N=
=
0.693
0.693
1.16 106 atoms
Therefore number of moles = 1,16 106/6,023 x 1023
Concentration = 1,16 106/6,023 x 1023 /0,01dm3
=

8. Describe with the help of a diagram the operating principle of the

secondary
electron multiplier (SEM) detector used in mass spectrometry
9.Explain disposal methods of solid radioactive waste. [6 Marks]
Solid waste is transferred to the waste disposal building.
Sludge from the tanks is dried and also transferred to waste disposal building

Waste is characterized to provide information for disposal and shipment

records.
Residual samples and waste disposal
The following disposal options are available:
Contract licensed commercial disposal service to remove all waste.
Return all the sample residues to the client and engage a licenced
commercial disposal service.
Return all samples and associated waste to the client
Waste disposal should be considered as part of the analytical process and
should be budgeted accordingly
10.State possible sample injection approaches in MS. [4 Marks]
11.How are ion sources in MS generally classified? [6 Marks]
12.MALDI-MS is an important analytical instrument for the molecular weight
determination of macromolecules. Explain the operating principle of MALDIMS.
[5 Marks]
4

13.How does a chemical ionization mass spectrum differ from that of an

electron
ionization. Give reasons for your answer? [5 Marks]
14. State any three different ways a sample may be introduced in an MS
instrument. [6 Marks]
15.Explain why an MS instrument is operated at very high vacuum.[4]
16.What are the advantages and disadvantages of Nuclear Activation
Analysis.[7]
17.The half-life of carbon-14 decay is 5730 years. Calculate the decay
constant. [7 Marks]
18.Briefly discuss the advantages of DIDA as an analytical technique.
[10 Marks]
19.Describe distinct regions of the voltammogram for linear sweep
voltammetry with stationary electrode. [8 Marks]
END OF ASSIGNMENT