CHEM3218 Pharmaceutical Separation Science Revision Questions Dr P.

Behan

Short Questions:

a) Describe the operation of a ‘split’ injection system used in gas chromatography.

b) Outline the operation of a flame ionisation detector.
c) Doubling the length of the column in GC increases the resolution by what factor?
Explain.
d) Write out the Van Deemter equation indicating what each term represents and
describe how it is used in gas chromatography (GC).
e) Briefly explain the difference in polarity between the following:

CH3COCH3 : CH3CH2OH

f) List the following in order of increasing polarity. Explain your reasoning.

CH3CH2OH, CH3CH2Cl, CH3COOH.

g) Calculate the flow rate of carrier gas through a capillary column in cm3/min. given
that the retention time of an unretained species is 1.60 minutes, the column length is
30 m and the internal diameter is 0.25 mm.
h) Define the basic aims of chromatography.
i) The following data was obtained for a two component mixture.

Compound tr (min) Width of peak at base

(min)
A 8.04 0.15

B 8.26 0.15

Calculate the resolution between A and B. Is it suitable for quantitation?

CHEM3218 Revision Question, Dr P. Behan, Page 1 of 8

Long Questions:

Extraction Efficiency & Chromatographic Parameters:

1. Solute A has a partition coefficient of 5 between toluene and water. Suppose that
200ml of 0.02 moldm-3 aqueous solution of A is extracted with toluene What fraction
of A remains in the aqueous phase
a. After 1 extraction with 100ml toluene
b. After 5 extractions with 20ml of toluene

2. The following data were obtained from the chromatogram of a two component
mixture where the retention time of the unretained species was 3.1 minutes.

component Retention time ( min) Width of peak at

base ( min)
A 13.3 1.07
B 14.1 1.16

Calculate
(i) The resolution between A and B.
(ii) The relative retention between A and B.
(iii) The capacity factor for B.
(iv) The number of theoretical plates for B.

3. The following data was obtained from the chromatogram of a three

component mixture. Column length = 10 cm
Compound tR(min) Width of Peak at
base (min)
A 17.8 4.0
B 25.7 5.0
C 29.6 5.5
(i) If the retention time (tR) of an unretained species is 1.7 minutes, determine the
capacity factor for A.
(ii) Calculate the value of N and H (in mm/plate) for peak A, where N is the
number of theoretical plates and H is the height equivalent of a theoretical
plate.
(iii) Calculate the resolution between compounds A and B and between
compounds B and C. Comment on these results in relation to the
quantification of each compound.

4. The following data were obtained from the chromatogram of a two component
mixture separated on a 20 metre capillary column. The retention time of the
unretained species was 1.19 min
component Retention Width of peak
time ( min) at base ( min)
A 8.04 0.15
B 8.26 0.15

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 Unretained 1.19
species
Calculate
(i) The capacity factor for A .
(ii) The relative retention between A and B.
(iii) The height equivalent of a theoretical plate, in millimetres , for B.
(iv) The resolution between A and B.

5. The following data were obtained for a two component mixture separated on a 20
metre capillary column.

Compound Retention Time Width of peak at base

(min) (min)
A 8.26 0.15
B 8.46 0.16
Retention time of unretained species = 1.19 min

Calculate (i) the height equivalent of a theoretical plate in

millimetres for A,
(ii) the selectivity factor between A and B,
(iii) the resolution between A and B and
(iv) the length of column required to increase the resolution
between A and B by a factor of two.

Gas Chromatography:

6. Gas chromatography is used to analyse mixtures of organic compounds. Discuss this

technique under each of the following headings:
a. Basic aims.
b. Advantages.
c. Limitations.

7. Write out the Van Deemter equation, discussing what each term represents and
describe how the equation is used in gas chromatography.

8. List the following in order of increasing polarity: ketones, alcohols, halogenated

hydrocarbons, water, hydrocarbons, ethers, esters

9. List the following in order of increasing polarity: propan-2-ol, methanol, butan-1-ol

ethanol, propan-1-ol.

10. Predict the order of elution of the following compounds:

Propanol (b.p. 97oC), heptane (b.p. 98oC), 2-butanone (b.p. 80oC),
3- pentanone (b.p. 102oC)
(i) From a non-polar column.
(ii) From a column of intermediate polarity and

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 (iii) from a strongly polar column.
In each case explain your reasoning.
11. Discuss the operation of a “split” injection system used in gas chromatography.

12. Outline the use of temperature programming in gas chromatography.

13. Name three types of detectors (not including a mass spectrometer) used in gas
chromatography and briefly compare their use. Outline the operation principles of any
one of them.

14. Hexane and 1-methylethylmethanoate both have boiling points of 68oC. They are
unresolved on an intermediate polarity column. State what the order of elution
would be on a non polar column and a polar column and give reasons for your
answer. Explain why they are unresolved on an intermediate polarity column.

15. Differentiate between packed and capillary columns in gas chromatography.

Liquid Chromatography:

16. “HPLC is a more versatile analytical technique than GC” Discuss this statement.

17. Draw a labelled block diagram of a HPLC. Write a brief note on each component.

18. Describe the differences between normal and reverse phase HPLC.

19. Describe the use of gradient elution in HPLC.

20. Use a diagram explain the fixed loop injection process of HPLC.

21. Describe how you would develop a method for the separation of a mixture of methyl,
ethyl and propylparaben using high performance liquid chromatography (HPLC).

Paraben (R = Methyl, Ethyl or Propyl)

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Mass Spectrometry:

22. Distinguish between electron impact ionisation and chemical ionisation as used in gas
chromatography mass spectrometry (GC/MS).

23. In relation to gas chromatography/mass spectroscopy (GC/MS), write a detailed note

on each of the following:
a. The operation of an ion trap mass spectrometer.
b. Electron impact ionisation (EI) versus chemical ionisation (CI).
c. Total Ion Chromatogram (TIC) versus Selected Ion monitoring (SIM).
d. Continuous dynode electron multiplier detector.

24. Outline the operation of an ion-trap mass spectrometer as used in a hyphenated system
such as GC/MS. Include in your answer a brief description of the advantages of using
such a detector in the hyphenated system compared to a general purpose detector such
as a flame ionisation detector.

25. Below is the GC-MS mass spectrum of benzoic acid (C₇H₆O₂).

(i) From the mass spectrum, identify the m/z for the base peak, the molecular ion
peak and the major fragmentation peaks.
(ii) Suggest and describe ionisation method explaining how the peaks at m/z 122,
105 and 77 in the benzoic acid GC-MS spectrum are formed.

20. The Ethanol electron ionisation mass spectrum shows peaks at m/z 46, 45, 31, 29 &
27
(i) Write the reaction which represents the electron ionisation of ethanol
(ii) What causes the major peaks at m/z = 46, 45, 31 & 29 in the ethanol mass
spectrum?

CHEM3218 Revision Question, Dr P. Behan, Page 5 of 8

21. Below is the mass spectrum of ethyl benzene (C6H5CH2CH3) showing major peaks at
m/z = 106, 911 & 77.

(i) Suggest an ionisation source used in this method and justify your answer.
(ii) Write the reaction for EI for ethylbenzene
(iii) What causes the major peaks at m/z = 106, 91, & 77 in the ethylbenzene mass
spectrum?

26. Discuss the advantages of using a mass spectrometer as a detector with gas
chromatography ( GC/MS).

27. Using the mass spectrum of Neon (Ne) shown below, calculate the average atomic
mass of Ne.

CHEM3218 Revision Question, Dr P. Behan, Page 6 of 8

Quantification:

28. A 0.520 g sample of handcream was shaken vigorously in 20 cm3 of methanol until
well dispersed. 5 cm3 of 100 ppm butyl paraben (internal standard) was added and the
solution made up to 100 cm3 with methanol. Standard solutions of methyl paraben
were made up each containing the same amount of internal standard as the sample. 20
l of each solution were injected into a liquid chromatograph and the following results
obtained:

Standards (ppm) Peak Area (cm2)

Methyl Internal
Paraben Standard
2 0.52 1.04
4 0.95 0.95
6 1.64 1.10
8 2.00 1.01
10 2.80 1.09
Handcream 1.07 0.85

Using this data plot an appropriate graph and use it to determine the % w/w of methyl
paraben in the handcream.

29. The concentration of ethanol in a wine sample was determined by GC using the
internal standard method. A 25 cm3 sample of the wine together with 10 cm3 of
the internal standard, propanol, were made up to 50 cm3 with water.
Standard solutions of ethanol were made up, each containing the same amount
of internal standard as the sample. 1.0 µL samples of each solution were
chromatographed and the following results were obtained.

Ethanol standards Peak height Peak height

( cm) (cm)
%vol/vol Ethanol Propanol
2.0 1.04 2.08
4.0 1.90 1.90
6.0 3.28 2.20
8.0 4.00 2.02
10.0 5.50 2.18
wine 2.11 1.70

Plot an appropriate graph and use it to determine the %vol/vol of ethanol in the
wine. Discuss your result in relation to the label value of 11.5% vol/vol.

30. The concentration of ethanol in a wine sample was determined by GC using the
internal standard method. A 2 cm3 sample of wine together with 5 cm3 of the internal
standard, propanol, were made up to 50 cm3 with water.

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Standard solutions of ethanol were made up each containing the same amount of
internal standard as the sample. 1.0 µL samples of each solution were
chromatographed and the following results obtained:

Ethanol standards Peak area Peak area

(integrator units) (integrator units)
%wt/wt ethanol propanol
0.24 1685 2407
0.48 3483 2488
0.72 4887 2327
0.96 6821 2436
wine 2749 2390

Plot an appropriate graph and use it to determine the % wt/wt of ethanol in the wine.
The density of ethanol is 0.79g cm-3. Compare your result to the label value of 12.5%
vol/vol.

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