Higson: Analytical Chemistry

Solutions to Exercises – chapter 8

8.1) Why should you always use pencil to mark the starting line within

paper chromatography?

A pencil should be used since inks contain pigments that will themselves be

soluble and hence separate when the chromatogram is run.

8.2) What is the Rf value of a chromatogram and what are the largest

and smallest Rf values possible?

The Rf values for a component is the ratio of the distance travelled by a spot

and the distance travelled by the solvent front from the starting line, ie:

Distance travelled by centre of Spot

Rf =
Distance travelled by Solvent Front

The smallest Rf value that can be obtained is 0 and the largest 1.

8.3) What is meant by a stationary phase and a mobile phase?

All forms of chromatography rely on a mixture coming in contact with two

phases and then moving one phase relative to the other. The two phases are

known as the stationary phase and the mobile phase. The components of the

sample distribute themselves between the two phases as dictated by their

relative solubility within the two phases. One of the phases (the stationary

phase) is static (or immobilised). The stationary phase may be either solid or

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liquid. The mobile phase passes through or along the stationary phase and

may be either liquid or gaseous.

8.4) What is the basis of electrophoretic separations?

Electrophoretic separations are those performed via electrophoresis.

Electrophoresis separates components within mixtures via electrical migration

so that ionised or highly polar components move under the influence of an

electric field. The rate of movement of a substance is a function of both its

molecular weight and its charge due to the resistance to movement through

the medium through which it has to travel.

8.5) What phenomenon gives rises to band broadening within

chromatography and why does band broadening increase with longer

columns?

The broadening of chromatographic peaks is a phenomenon normally referred

to as band broadening. Band broadening is caused by a number of effects. A

major cause of band broadening originates from flow distribution effects of the

mobile phase within columns. A second cause is the diffusion of molecules

within the mobile phase as it passes along the stationary phase. A third effect

is Eddy diffusion of solutes. Band broadening increases as the column length

increases since a greater time is given for all of the effects to occur.

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8.6) Discuss the relative advantages of using paper and thin-layer

chromatography.

Paper chromatography offers simplicity and inexpensiveness. Thin layer

chromatography offers better separations than are often obtainable with paper

chromatography and also tends to offer more reproducible behaviour.

8.7) Describe the principles of gas chromatography.

Gas chromatography utilises a carrier gas as the mobile phase together with a

stationary phase within a packed or open tubular capillary column.

If a liquid stationary phase is used, the technique is known as gas-liquid

chromatography or GLC. A schematic for a gas chromatography is shown

within Fig 8.9 of the book.

Separation occurs by partitioning gaseous samples between a carrier gas and

the stationary phase. The sample must either be in the gaseous phase (or be

transferred into the gaseous phase by heating), so that it can be passed into

the carrier gas stream.

8.8) What samples can be separated by HPLC and not GLC.

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GLC requires samples to be volatile. HPLC however allows the separation of

solutes that may not be readably be evaporated into the gaseous form.

8.9) Why might gas chromatography be coupled to mass spectrometry

and how can this be achieved?

Mass spectrometry can be used as a powerful and selective form of detector

for HLPC since the mass of each molecular species can be identified as it

elutes from the HPLC column.

There are a number of ways in which mass spectrometry may be coupled to

HPLC. One approach, for example is via the use of a thermospray interface.

In this device, the HPLC effluent passes through a heated stainless steel

capillary tube to evaporate much of the solvent to form an analyte / solvent

aerosol. This aerosol may then be passed into the mass spectrometer.

8.10) A solute has a distribution coefficient of 9.5 between hexane :

water. 10g of the solute is dissolved in 50ml of water. Calculate the

concentration of the solute remaining in the water sample, following

extraction with 1, 2 and 3 extractions with 10ml aliquots of hexane.

Distribution coefficient KD water : hexane of 9.5

10g of solute dissolved in 50ml water.

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[S ]
Water
KD =
[S ]Hexane ]

⎛ 50 ⎞
4

[solute]Water = ⎜⎜ ⎟⎟ x10
⎝ (10 x 9 . 5) ⎠

⎛ 50 ⎞
4

=⎜ ⎟ x10
⎝ 145 ⎠

= (0.3488)4 x 10

= 0.014

= 0.14g solute remaining within the original water sample.

8.11) How might the number of theoretical plates in a chromatographic

column be determined?

The number of theoretical plates, N, may be obtained by analysing a

chromatogram since:

N = 16 (tR / w)2

Where tR is the retention time and w is the width of the base of the

chromatographic peak.

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8.12) If the width of a solute peak is 7.5mm at one half its height, what

will be its base width by extrapolation?

If we assume that a chromatographic peak to be Guassian in shape, then the

width at half height will be equal to +/- 1σ = 2σ. The width of the base of the

peak can be taken by extrapolation of the tangents to give a peak base width

equal to +/- 2σ = 4σ.

If 2σ = 7.5mm then 4σ = 2 x 7.5mm = 15mm.

8.13) If the base width of a chromatographic peak is equivalent to 2.4s

duration calculate the half height peak width for this peak.

In a similar manner to the calculation of problem within problem 8.12, the half

width half height peak width will be equivalent to 2.4/2 s = 1.2s.

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8.14) A chromatographic peak is found to have a retention time of 72s.

The base width of the peak is determined to be 6.5 seconds by

intersection of the sides of the peak with the baseline. If the column is

1m in length, calculate the HETP in terms of cm/plate.

N = 16 (tR/w)2

∴= 16(72/6.5)2

= 16 x (11.077)2

= 16 x 122.69

∴N = 1963.17

HETP = L/N = 1/1963.17 = 5.0x10-4m or 0.05cm

8.15) A gas chromatogram of a mixture of ortho and para isomers of

cresol gives peaks with integrated areas of 35.7 and 10.5 respectively.

Assuming the detector responds equally to both isomers calculate the

percentage of each isomer within the mixture.

The ratio of the isomers = 35.7:10.5 = 35.7/10.5

35.7 + 10.5 = 46.2

100 / 46.2 = 2.165

therefore in percentage terms the ratio of the isomers within the mixture =

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(35.7x2.165) : (10.5x2.165)

= 77.27 : 24.73

8.16) If the retention time for a chromatographic peak, tR, is 85s and tmob

is 40s, calculate the capacity factor, k’.

t −t
R mob
k’=
t mob

85 − 40 45
It follows k’ = = = 1.125
40 40

8.17) Calculate the capacity factor, k’, for a chromatographic peak if its

retention time, tR, is 95s and tmob is 45s.

tR − t mob
k’ =
t mob

95 − 45 50
It follows k’ = = = 1.11
45 45

8.18) If the HETP of a column is 0.01cm per plate and the number of

theoretical plates is 5000, calculate the length of the column.

L
HETP =
N
∴L = HETP x N

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∴L = 0.01 x 5000

∴L = 50cms

8.19) Suggest approaches for helping to improve the resolution

between different substances within a chromatographic separation.

There are a number of approaches that can be adapted including:

• Careful choice of packing and also of packing material

• Lengthening the column
• Careful choice of mobile phase (and also solvent mixtures when
used)

8.20) Define what is meant by (a) the selectivity factor and (b) the

capacity factor.

(a) The selectivity factor, α, for two solutes is defined as the ratio of the larger

(l) partition coefficient, kl’ and the smaller (s) partition coefficient ks’: ie:

k'
α= l

k's

t −tR mob
(b) The capacity factor is equivalent to k’ = , where tR = the retention
t mob

time for a solute to elute from a column and tmob is the time taken for solutes

to travel the length of the chromatographic column.

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8.21) If two chromatographic peaks have capacity factors of 1.4 s-1 and

3.4 s-1, calculate the selectivity factor.

t −t
R mob
The capacity factor k’ =
t mob

For the first capacity factor:

If we take tmob = 1 then for the first chromatographic peak:

t −1
R
1.4 =
1
1.4 = tR-1, and

tR = 1.4 + 1 = 2.4

For the second capacity factor:

t −t
R mob
k’ =
t mob

t −1 R
k’ 3.4 =
1
∴3.4 = tR-1

∴tR = 3.4+1

tR = 3.4=1 = 4.4

(t ) − 1
The selectivity factor α = R l

(tR ) − 1 s

4. 4 − 1 3 . 4
∴α = = = 2.43
2. 4 − 1 1 . 4

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