Analytical Science

A course (in 15 Chapters), developed as an Open Educational Resource,

designed for use at 2nd year England & Wales undergraduate level and as a CPD
training resource
https://edocs.hull.ac.uk/muradora/objectView.action?parentId=hull%3A2199&type=1&start=10&pid=hull%3A2351
Author Brian W Woodget
Owner Royal Society of Chemistry
Title Chapter 6 – (1) Introduction to Chromatographic Methods and (2) This Layer
Chromatography
Classification F180, Analytical Chemistry
Keywords ukoer, sfsoer, oer, open educational resources, metadata, analytical science, cpd
training resource, analytical chemistry, measurement science, chromatographic
methods, thin layer chromatography
Description This chapter reviews the variety of separation techniques which come under the
banner of ‘Chromatography’, compares the ‘Plate’ and ‘Rate’ theories of separation
and finally considers in more detail that of ‘Thin Layer Chromatography’. Some of the
images show a degree of animation.
Creative Commons licence http://creativecommons.org/licenses/by-nc-nd/2.0/uk/
Language English
File size 1.4 Mbytes
File format Microsoft PowerPoint (1997 – 2003)

1
© Royal Society of Chemistry 2010
Chapter 6 – Introduction to Chromatographic
Methods. Thin Layer Chromatography
Contents
Introduction to chromatographic analysis
Chromatography is first and foremost a method of separation. However
because chromatographs (sophisticated instruments used for chromatographic
analysis) allow for qualitative identification and quantitative measurement in
addition to performing separations, chromatography is often viewed as a total
method of analysis.

Sample sizes used for analysis are generally very small (often less than
1microlitre (µl), however they are sufficient to provide complex analysis
information.

Once separated the analytes (or solutes) are detected. If mass spectrometry,
infra-red or nuclear magnetic resonance detection is used, the resulting
chromatographic system is referred to as a tandem system. Tandem systems
are very powerful analytical tools allowing samples of nanogram size to be
both separated and characterised.

3
Chromatography relies on having two phases. A phase is a liquid, solid or gas
which can interact either chemically or physically with the analytes (solutes) to be
separated. In chromatography, the two phases are termed:

 A stationary phase, which as the name suggests is in a fixed place;

 A mobile phase which is a gas or liquid, and which moves past (over
or through) the stationary phase.

Analytes are usually introduced into the mobile phase stream. As this stream
moves past the stationary phase, some of the analytes will interact with this fixed
phase, resulting in them being retained. Other analytes remain in the mobile
phase and move on. The retained analytes can then move back into a fresh
portion of mobile phase as it flows past them.

To achieve separation each analyte needs to spend different lengths of time

being retained or held by the stationary phase.

4
5
History of chromatography
The first chromatographic separation was reported in 1903 by
Tswett, who separated plant pigments using a stationary phase
of calcium carbonate and a mobile phase of petroleum ether.
The separation as illustrated in figure (6.1)
shows the two coloured pigments separating
as they progress through the column allowing
them to be collected separately as they emerge
(elute) from the column.

As the pigments formed distinct

coloured bands Tswett coined
the phrase chromatography
(colour writing) from chroma –
colour and graphien - writing

6
Figure 6.1
In 1941 Martin and Synge published a paper describing the principles of partition
chromatography, in which the stationary phase was a high boiling liquid coated
onto a solid support. In 1952 they were jointly awarded the Nobel prize for
chemistry.
In 1952, Martin and James invented gas-liquid chromatography (glc), a
technique which uses a gas as the mobile phase. This is now a widely used
technique for separating volatile or semi-volatile organic substances, for example
herbicide pollutants in water or the components in a petroleum fraction.
Although research into high pressure (now termed high performance) liquid
chromatography (hplc) began in the late 1960’s, the technique did not get
widely used until about a decade later with the advent of instrumental
components that could withstand pressures as high as 6000 psi and pumps that
could deliver these pressures in a smooth rather than a pulsed flow mode. More
recently, further advances have enabled the development of ultra performance
liquid chromatography (uplc) where pressures of 15000 psi are used.
In the 1990s electrochromatography was developed. The mobile phase flow is
driven through a packed capillary column by an electric field rather than a pump.
These developments are summarised in table (6.1) on the next slide.

7
Table 6.1 - summary of historical landmarks in chromatography

8
Principles of chromatography
In column chromatography analytes are injected into the mobile phase stream
and are swept on to the column containing the stationary phase. Those
analytes which do not interact in any way with the stationary phase pass
straight through the column, they are the first to elute (flow out of the column).
Some analytes will interact so strongly with the stationary phase that they
remain on the column, ‘never to be seen again’. In between these two
extremes, are analytes which spend some time interacting with the mobile
phase, during this time they move through the column, and some time bonded
to the stationary phase. Each analyte spends a slightly different length of time
in the two phases and hence by the end of the column, the individual
components of the analyte sample, which had been injected into the mobile
phase stream, are separated and can be detected.

Remember the analogy of the boats on the river (slide 5)

9
As described on the previous slide, analytes distribute themselves between two
phases the stationary and the mobile phase.
As the name suggests the stationary phase does not move. It is:
 A finely divided solid spread as a thin layer onto a sheet of glass or plastic;
 A finely divided solid packed tightly into a glass or stainless steel tube,
referred to as a column;
 A finely divided solid coated with a thin layer of organic liquid and again
packed into a glass or stainless steel column.
 A glass or silica capillary whose inner surface is coated with a low volatile
organic liquid

The mobile phase moves. It can be a gas, a single solvent or a mixture of

solvents.
Modern instrumentation makes use of columns. The sample is injected into
the mobile phase stream at the front of the column. Separation occurs on the
column and the separated analytes then pass out of the column into a suitable
detector placed at the end of the column.
A plot of the detector signal versus time is called a chromatogram, an
example of which can be seen on the next slide.

10
Results aimed for
In any chromatographic separation,

Detector response
the following features are desirable:
 All analytes separated

 Well separated peaks

 Symmetrical, sharp peaks

 Short run times

10 minutes
Retention time
Figure 6.2 - an example of a chromatogram

Ideal results are not always easily achieved, however the following notes provide
the basic information to enable simple chromatographic separations to be carried
out.

11
Column chromatography

Most chromatography is performed on

columns, figure (6.3) on the right is a
schematic of a packed column, the
arrows at the top of the column are
indicative of the mobile phase.

A plot of detector response versus

time is called a chromatogram an
example of which was seen as figure
(6.2) on the previous slide.
Figure 6.3 – schematic diagram
of a packed column

12
Modes of chromatography
The particles which fill a chromatographic column are not all the same
hence the types of interactions between analytes and stationary phases are
not all the same.

In liquid-solid chromatography the stationary phase is a solid such as

calcium carbonate or more likely these days silica or alumina (Al2O3).

In gas-liquid chromatography the stationary phase is a low volatility liquid

coated on to a solid support eg Carbowax 20M - a polyethylene glycol with
a molecular mass of 20,000 g/mole.

In liquid-liquid chromatography, the stationary phase is a liquid, eg a C18

silane, covalently bonded to an inert silica support.

Movement between the stationary and mobile phase can be summarised

by the following equation, where A is an analyte
Amobile Astationary phase

13
The modes of chromatography outlined in the following slides are:

 Adsorption
 Partition
 Bonded phase
 Ion exchange
 Affinity
 Size exclusion

The mode describes the way in which analytes interact with the stationary
phase.

14
Adsorption chromatography
If silica, a silicon-oxygen polymer as illustrated
in figure (6.4) is the stationary phase, the
surface hydroxyl groups interact with the Si OH
analytes, the more the interaction the more the
O
analytes are retained and therefore the longer
they take to pass through the column. Si OH
O Figure 6.4 -
structure of silica

Figure 6.5 – binding of analytes to

silica.
Some analytes (pink) will bind more
than others (blue) and hence
separation is obtained.

15
The mobile phase (solvent) and
analyte molecules compete for the
adsorbent sites of the stationary
phase. The more polar the
molecules the longer they are
retained.

Modification of the adsorbent sites

can make it possible to separate
isomers.

‘Strong solvents’ result in short

retention times as the solvent
molecules successfully compete for
the stationary phase active sites
which means there are fewer
available sites to retain the analyte
molecules.
Figure 6.6 – competition for adsorption
sites
16
Applications of adsorption
chromatography
 Separation of relatively non-polar, water insoluble compounds

 Separation of isomers

 Purification of antibody fragments

17
Partition chromatography
In partition chromatography a solid
support with a high surface area
such as crushed firebrick or
keiselguhr is coated with a high
boiling liquid which acts as the
stationary phase. Separation occurs Solid
because of the differences in support
solubility for the analytes in the
stationary and mobile phases.

The partition coefficient is defined as:

Stationary phase
Analyte

Concn. in stationary phase Figure 6.7 – coated support

K = Equation (6.1) particle
Concn. in mobile phase

18
Applications of partition
chromatography
There are an immense number of possible applications of partition
chromatography. The list below gives just a few examples of where this
technique is routinely applied:

 Determination of water quality;

 Separation of aroma molecules of wine;

 Determination of pesticide residues;

 Quality control of pharmaceutical preparations;

 Identification and measurement on petroleum fractions.

19
Bonded phase chromatography
In bonded phase chromatography, the molecule acting as the stationary
phase is chemically bonded to the solid support.

Si OH + Cl SiR Si O SiR + HCl

R can be a C18 alkane chain or an amine (NH2) or cyano (CN) group or

some other group. The nature of R determines the types of analytes
which can be separated.

The theories of partition and adsorption

chromatography are both used to describe
this mode of chromatography although it is
often classified as a partition technique.
See:
http://en.wikipedia.org/wiki/Reversed-phase_chromatogr
aphy
, for more description of bonded phases
Figure 6.8 – diagram of phase
20
bonded to silica
There are two types of partition chromatography normal phase and reversed phase, they are defined by the relative polarities of the mobile and stationary phases

For this reason, the use of silica (a polar molecule) as the stationary phase (as in adsorption chromatography) is also considered to be a normal phase separation method.

Because of its versatility and wide range of applicability, reversed-phased chromatography is the most frequently used hplc method.

21
Ion exchange (or ion) chromatography
As the name suggests, in ion exchange chromatography, ionic analytes are
exchanged with ionic groups on the stationary phase - usually a polymer with
ionic functional groups.

The mobile phase is an ionic solution, such as a solution of sodium bicarbonate

and carbonate (8 – 12 mmolar) for separating anions, or a sulphonic acid for
separating cations.

Using some form of ion-suppression immediately after the analytical column,

eliminates the high background conductivity of the mobile phase, enabling
conductivity detectors to be used as the end of the column. Conductivity
measurements in the absence of background conductivity, can provide
sensitive detection for a large range of ions.

The next 3 slides explains the mechanism of the suppression effect in ion chromatography

22
 NR3+ OH- Exchanging ion

Polymeric
stationary phase Polymer functional group

Figure 6.9 diagram of the active site on an anion (negative ion)

exchange stationary phase

On the next slide, figure (6.10) shows the analytical and suppressor columns
used in ion exchange chromatography.

23
 Figure 6.10 – Schematic representation of ion exchange separating and
suppressor columns

Cl- Br- -
-
OH
Separating column OH -
-
Cl
Cl - NR3+OH- + NaX NR3+X- + NaOH
OH
Br-- - NaX is the salt of the ion of interest, X is Cl-
OH Br-
-
or Br .
Cl
Cl-OH-

Suppressor column
OH-
H2O SO3-H+ + Na+ + OH- SO3-Na+ + H2O
OH-
OH- represents ions
Conductance detector D of high conductivity Comparatively low
from the mobile conductivity
phase
24
Figure (6.10) on the previous slide, shows that analyte ions literally exchange with
similarly charged ions from the stationary phase. Each analyte ion will interact with
the stationary phase according to its charge, size and to a certain extent shape,
thereby bringing about a separation. As the analytes are ions, a convenient way of
detecting them is to use a conductance detector.

The problem with using a conductance detector is that it will also measure the
conductance of the mobile phase. As the mobile phase is an ionic solvent it will
itself have a high conductance against which the comparatively small analyte signal
needs to be measured.

For a long time the problem described above, meant that ion chromatography was
not sensitive enough to detect low (ppm) levels of ionic analytes. The problem was
solved by a company called Dionex (their name is often synonymous with ion
exchange chromatography), which introduced a suppressor column after the
separating column. The suppressor column also contains an ion exchange resin.
The ions from this resin react with the ions of the mobile phase to produce
molecules which have a lower conductance, e.g. water, than the ions themselves.
The analyte ions will also react with this resin but the molecules formed will be of
equal conductance to those they replace. In the previous slide NaCl (eluting
analyte) would be replaced with HCl.

25
Applications of ion exchange
chromatography
 Separation of vitamins

 Separation of inorganic cations and anions

 Separation low molecular weight organic acids

 Analysis of serum

 Analysis of drugs

26
Affinity chromatography
Affinity chromatography involves the interaction of a ligand (bound to a solid
support with a spacer molecule) with the analyte.

Figure 6.11 - illustration of a ligand bound to the solid support

There are two types of ligand that can be used in affinity chromatography.
Those which are specific and so bind to a specific analyte and those which
are general and which bind to a group of analytes which have similar
properties. A ligand acts in a similar way to an enzyme, a brief description of
which is given on the next slide.
27
 Enzymes in analytical science
An enzyme is typically a protein molecule which enables reactions to proceed under
physiological conditions. Often an enzyme reacts with a very limited number of compounds
referred to as substrates. Binding between an enzyme and a substrate is via weak
covalent bonds the formation of which is strongly influenced by the shapes of the enzyme
and substrates, this has led to the lock and key theory to describe how the shape of the
enzyme allows only those substrates with a complimentary shape to react; this theory also
explains how the ligand in affinity chromatography behaves (seeing binding step in figure
(6.12). Because the bonds between an enzyme and its substrate are weak any
change in the shape of the substrate due to chemical reactions which have taken place
while bound to the enzyme or any change in the conditions under which the reaction is
taking place cause the bonds between enzyme and substrate to further weaken and
ultimately to release the modified substrate. In affinity chromatography it is undesirable to
modify the substrate so one mobile phase is used to give the conditions to enable binding
and a second mobile phase is used to elute the retained analytes, a small change in the
pH or ionic strength of the mobile phase can be sufficient to enable elution.

In affinity chromatography the ligand is bound to the solid support using a spacer molecule
this molecule is present to ensure that the ligand retains its required binding properties as
if it was in close proximity to the solid support the shape of the ligand could be distorted by
additional binding to with the solid support itself.

28
The five steps in affinity chromatography

 activation the ligand is bound to the chromatographic solid

support.

 loading the analytes to be separated are introduced into the

mobile phase stream.

 binding the analytes of interest are retained due to interaction

with the ligand of the stationary phase.

 washing unwanted analytes are eluted from the column.

 elution the analyte(s) of interest are washed from the column

by changing the mobile phase composition.

These five steps are illustrated in figure (6.12) shown on the next slide

29
 activation

load

binding

washing

elution

Figure 6.12 – schematic diagram of affinity chromatography

30
Applications of affinity
chromatography
 Purification of proteins

 Study of drug and hormone interactions with proteins

 Immunoassays

31
Size exclusion chromatography
In size exclusion chromatography (sec) there are no chemical interactions with
the mobile phase. The analytes are separated based on physical interactions -
whether on not they are small enough to fit into the pores of the stationary
phase.

The stationary phases used are wide-pore silica gel, polysaccharides, and
synthetic polymers like polyacrylamide or styrene-divinylbenzene copolymer.
Pore sizes within the solid phases range from 4 – 250 nm.

When an aqueous mobile phase is used, the technique is known as gel

filtration chromatography (gfc sec) and the stationary phase will be
hydrophilic (hydrophilic means water loving). If an organic mobile phase is
used it is known as gel permeation chromatography (gpc sec) and the
stationary phase will be hydrophobic (hydrophobic means water hating).

32
6.13

6.14

Continued on the next slide 33

Figure (6.14) on the previous slide, illustrates how each size exclusion column
is only capable of separating molecules within a limited molecular weight range
(103.5 - 105.6 on the graph shown). All molecules with a mass larger than this,
will pass straight through the column and be eluted together at the start of the
chromatogram ie: they will provide ‘tm‘ the retention time for un-retained
analytes. Molecules which have a mass smaller than the range will be trapped
within the pores of the stationary phase and ‘never seen again’.

In order for molecules to be separated they need mass differences of

approximately 10%.

Note: a fuller description of ‘tm‘ is provided in later slides

34
Application of size exclusion
chromatography
 Determination of the molecular mass distribution of synthetic polymers

 Analysis of sugars

 Analysis and isolation of lipid polymers

 Purification, identification, and quantification of protein mixtures

 Study of polymer reaction kinetics

 Separation and purification of large biomolecules (molecular mass > 10,000

g/mole)

35
Chromatographic terms
Chromatography is a method used for separating a mixture of compounds.

Separation takes place on the column containing the stationary phase. By

washing eluent or mobile phase through the column the sample is separated
into its individual components and is eluted from the column.

A chromatograph is a sophisticated instrument used to separate analytes. The

components of different types of chromatograph will be considered in a later
chapters.

A chromatogram is a recording of the detector response versus time.

36
 Void time or void volume
Figure (6.15) shows a chromatogram
containing two peaks:
 The analyte peak (tr from the point
of injection)
Signal  The unretained peak (tm from the
point of injection
The time tm represents the void time
of the column, which is the time
Point of required for an un-retained solute
Time (those that move through the column
injection
Figure 6.15 column void time at the same rate as the mobile phase)
to move from the point of injection to
the detector. This time can be significant for solutes that elute rapidly from the
column and thus should be allowed for in calculations of relative retention. To
allow for the void time, measurements of retention time are made, not from the
time of injection, but from the retention time of an unretained component ‘tm’
These values are sometimes referred to as ‘Adjusted retention times’ and are
given the symbol ( tr’), see next slide. 37
The chromatogram

Figure 6.16 – a sketch of a chromatogram showing

tm, trA and t’ref
Adjusted retention (t’rA) = trA – tm ( ) Equation (6.2)

adjusted retention of analyte t’rA

Relative retention ratio (R) = = Equation (6.3)
adjusted retention reference t’ref
38
When analytes have been separated, their chromatographic retention time
is indicative of their identity. However it is important to remember that
chromatography is a separation method not an identification method.

In order to identify the analytes giving rise to the peaks on a chromatogram, it

is necessary to compare the results for a sample solution with the results for
standard solutions containing known analytes with known retention times. It is
also very helpful to have prior knowledge of what analytes are expected to be
present. It is also important that standard and sample solutions are
separated using the same chromatographic conditions as retention data
can be greatly affected by any such changes.

On the previous slide the adjusted retention and relative retention ratios
were illustrated. These values are sometimes used instead of the retention
time itself, as they are less influenced by any changes in the experimental
conditions which can occur.

39
The partition coefficient

Once introduced into the chromatographic column, the analytes begin to

distribute themselves between the stationary and the mobile phases in
accordance with their partition coefficients. The partition coefficient (K) is
defined as the ratio of these concentrations at equilibrium. Thus:

Concentration of analyte in the stationary phase (Cs) Equation (6.4)

K =
Concentration of analyte in the mobile phase (Cm)

Separation occurs between mixtures of analytes, when each analyte has a

different ratio of solubility's in the mobile and stationary phases.

40
Resolution -Rs
t r2  t r1
Rs  Equation (6.5)
0.5( w 2  w 1 )

where:
tr1 is the retention time of one analyte
and tr2 is the retention time of the next
analyte to elute
w1 and w2 are the widths of the peaks at
base when approximated to triangles.
Figure 6.17 – a sketch of a Note: both tr measurements are made from
chromatogram showing adjusted that of the unretained peak.
retention times and peak width
Baseline resolution when R = 1.0 for triangles!
Chromatographic peaks are not triangles they are Gaussian in shape; for
Gaussian peaks Rs = 1.5 for baseline resolution (complete separation) as when
Rs = 1.0 only 94% resolution is obtained.

41
The capacity factor
Capacity factor, k’ (kay prime) or more correctly k

The capacity factor is sometimes used instead of the retention time as it is a

comparative method of reporting results and therefore less affected by
experimental variables. Remember the retention ratio, another comparative
method used to report chromatographic results. The capacity factor [equation
(2a.22)] tends to be used most for high performance liquid chromatographic
(hplc) results.

'
t rA
k'  Equation (6.6)
tm
When developing chromatographic methods a k’ value between 5 – 20 is
aimed for, as this usually gives good separation in a reasonable time.

42
Chromatographic plate theory
The plate theory arises from the theories which describe fractional distillation. It
shows why analyte molecules which are injected into the mobile phase at the
same time, give rise to a peak which is Gaussian (bell) shaped and why peaks
have a finite width of many seconds.

According to the theory, an analyte will partition itself between zones (or plates)
of the mobile and stationary phases as they come into contact with each other.

Figure (6.18) on the following slide, illustrates the chromatographic process for a
single analyte with a value of K = 1 and which therefore partitions itself equally
between the mobile and the stationary phases.

43
Figure 6.18 - an animated diagram of a chromatographic separation, illustration
movement of an analyte through a column

The analyte (64 parts) partitions itself between the two phases as they come
into contact.

Mobile phase

64 32 32
64 16 12 16 12 16 12 8
2
8 4 8 4 8 4 8 4 2
4 4
8 2 12 8 12 8 8 2
Cs
16 32 16
K 1
Cm Stationary phase

44
In figure (6.18) the analyte started on one plate of the mobile phase but after 5
equilibrations it was spread across five plates. This demonstrates how dividing the
phases into theoretical plates explains the spreading of analyte molecules during a
chromatographic separation. It also demonstrates why chromatographic peaks
are Gaussian, or bell shaped, in shape rather than being triangular. In reality the
analyte molecules also spread due to
other factors which will be discussed
when the deficiencies or limitations
of the plate theory are considered.

Only five equilibrations were

considered in figure (6.18) , however
if the calculation had been extended
to 10, and the resultant relative
concentrations plotted, a Gaussian Figure 6.19 – Gaussian distribution
distribution plot as illustrated in figure (6.19) plot
would have been obtained. It is important to note that even the shortest
chromatographic column will have many more ‘plates’ than this.

45
 The figure (6.20) below illustrates the effect of the analyte partition
coefficients, the ‘parts’ of the analyte have been plotted as the concentration
of the analyte. Analytes with different partition coefficients separate from
one another as they partition between the theoretical plates.
Concentration of analyte

solvent

1 50 150 250 300 350 400 450

Plate number
Figure 6.20 - Plot showing the separation of three analytes (plus
solvent), each with a different partition coefficient
46
A number of experimental variables affect chromatographic separations:

 The nature of the analyte and the mobile and stationary phases
determines the partition coefficients;
 How sensitive a detector is to a particular analyte - the response factor
changes the size of the peak observed;
 The number of equilibrations which can take place in a column ie the
number of theoretical plates determines how good the final separation of
the analytes will be.

It is important to remember that the plates are purely hypothetical, being a

means of describing the movement of analytes through a column, whereby the
analyte (A) spends time in both phases and equilibrates itself between the two
phases before moving on. The analyte only moves through the column when
it is in the mobile phase. Therefore the rate at which an analyte moves
through the column is proportional to the time spent in the mobile phase.

Differences in the value of K, the partition coefficient, for different analytes

results in them separating into distinct zones or bands.

47
Column efficiency
‘H’ or HETP is the length of column which
corresponds to a theoretical plate. This
needs to be a minimum for efficient
separation

‘N’ is the number of plates per column

‘L’ is the length of the column

From a chromatographic peak, such as

the one shown in figure (6.21), it is
possible to evaluate ‘H’ and ‘N’.

Figure 6.21- chromatographic

peak showing tr and w
Continued on the next slide

48
L  NH Equation (6.7)

Statistically it can be shown that

16t r2 Equation (6.8) From equation (6.9) it is
N 2
w possible to see that for a given
length of column (L) the greater
Thus by combining equations (6.7 & 6.8) the number of plates, the
smaller each individual plate
will be.
L Lw 2
H  Equation (6.9)
N 16t r2
For this reason ‘H’ and ‘N’ are often quoted, e.g. in suppliers catalogues, to
give an indication how good (efficient) a column is at separating particular
analytes. The higher the value of ‘N’ and conversely the lower the value for
‘H’, the more efficient the column. This enables a comparison of similar
chromatographic columns from different suppliers. However it is important
to remember that when selecting a column to perform a particular
separation, ‘like separates like’ i.e. in gas chromatography a polar column
is needed to separate polar analytes. In practise this means that if a
column is efficient at separating one class of analytes (eg alcohols) it will
not necessarily be as efficient as separating those of a different nature (eg
alkanes). 49
Deficiencies of the plate theory
The plate theory is far from perfect and as the following four slides show, it
has a number of limitations:

 The plate theory assumes K is linear and therefore that the plates are
symmetrical;
 It assumes rapid equilibration of the analyte between mobile and
stationary phases;
 All peak broadening is not accounted for;
 The effect of the mobile phase is ignored;
 The dimensions (eg the thickness) of the phases are not taken into
account

50
Eddy diffusion

6.22

The fact that the analyte molecules do not all take the same path through a
packed column means that they do not all reach the detector at the same
time hence peak broadening is observed.

51
Longitudinal broadening
(blotting paper effect)

Narrow at point of
introduction on the
column
Mobile phase
flow
Broadened analyte zone some
time after introduction on to the
column due to the diffusion of
the analyte.

Figure 6.23 - illustration of the effect of longitudinal diffusion

If the velocity of the mobile phase is high then the analyte spends less
time on the column which decreases the effects of longitudinal
broadening.

52
Equilibrium not established

Mobile phase
flow

Time for equilibrium Broadened peak, due to equilibrium being too

to establish so no slow to establish before the mobile phase has
peak broadening. moved on down the column, taking some of the
analyte with it.

If the mobile phase velocity is high the worse the broadening becomes.

Figure 6.24 - illustration of the effect of not establishing an equilibrium of

the analyte between the stationary and mobile phases.

53
Comparison of peak broadening theories
The peak broadening effects on the previous three slides require very different
mobile phase flow rates in order for peak broadening to be minimised.

 Longitudinal broadening requires high flow rates;

 Mass transfer (establishing the equilibrium) requires flow rates to be kept to
a minimum.

As will be shown at the end of the ‘Rate theory’ section of these notes, this
apparent contradiction results in an optimum value for the mobile phase flow
rate.

54
Chromatographic Rate theory
Because of the limitations of the plate theory it has been largely replaced by
the ‘Rate theory’, which is based mathematically upon the Van Deemter
equation. This is shown as equation (6.10) below:
1 B Equation (6.10)
H  Au 3
  Cu
u

Where:
u is the average mobile phase velocity
A takes into account eddy diffusion
B longitudinal diffusion
C mass transfer

For a packed column the Van Deemter equation is directly applicable.

However for capillary columns which are now commonly used in gas-liquid
chromatography, a similar equation applies but without the ‘A’ term which is
now zero, as capillary columns have no column packing.
55
 The effect of the three terms in the van Deemter equation are shown
graphically in figure (6.25) below.

6.25

Remember that we want ‘H’ to be a minimum for efficient separations. So the

above plot illustrates that there is an optimum mobile phase flow rate to achieve
this, the range is shown by the red arrows. The red line shows the
combination of the three broadening terms.

56
Comparison between Plate and
Rate theories
The advantage of the rate theory is that the terms A, B and C are defined in
terms of experimental variables which in practise are seen to affect the
chromatographic separation which is obtained. Typical variables are:

 Thickness of the stationary phase;

 Nature of the analyte;
 Nature of the mobile and stationary phases;
 Column length and diameter.

The plate theory does not take into account all these variables nor does it take
into account the mobile phase flow rate which as has been shown has a
significant effect on the peaks obtained during a separation. However the
plate theory does explain why separation is achieved and the shape of the
peaks.

57
 Types of chromatography

This chapter of this teaching & learning programme has outlined the theoretical
principles of a number of chromatographic techniques. Three of these
techniques will be considered in more detail. These are:

 Thin-layer chromatography (tlc);

 Gas chromatography (gc) – (see Chapter 7 of this teaching and learning

programme)

 High Performance liquid chromatography (hplc) – (see Chapter 7 of this

teaching and learning programme)

58
Thin layer chromatography (tlc)
In thin layer chromatography (tlc) the stationary phase is coated, as a thin
layer, onto a flat sheet of glass, plastic or sometimes metal. The mobile phase
(a single solvent or a mixture of solvents) is poured into the developing tank, a
lid placed on the tank and the atmosphere inside the tank allowed to equilibrate
with the solvent(s). The mixture of analytes is spotted onto the plate at a height
above the level of the solvent. The plate is then placed into the developing
tank, the lid replaced and the mobile phase allowed to rise up the plate by
capillary action. The analytes separate in the usual chromatographic manner
due to their interactions with the mobile and stationary phases. Figure (6.26)
on the next slide, illustrates these procedures diagrammatically.
The line along which the analytes are spotted on to the plate is called the
baseline and the line to which the mobile phase travels is known as the
solvent front.

In tlc, the choice of stationary phase is comparatively limited, silica or alumina

based stationary phases (both are polar) are common, but virtually any
combination of miscible solvents can be used as the mobile phase.

59
6.26

60
tlc mobile phases
Figure (6.27) on the next slide shows the importance of selecting a suitable
mobile phase in order to create the best conditions for separation.
The analytes are of medium ‘polarity’. In this instance the heptane / toluene
mobile phase is not strong (or polar) enough to compete with the polar
sites on stationary phase for the analytes, hence the analytes do not move
far from the baseline where they were spotted initially onto the plate.
Conversely the methanol / toluene mobile phase is too strong (too polar)
and therefore the analytes all move to the solvent front. In this instance the
analytes are unable to compete with the mobile phase for the stationary
phase. When toluene (weakly polar), is used as the mobile phase the
‘polarities’ of the stationary and mobile phases are suitably balanced so that
there is discrimination between the analytes and hence they are separated.

Remember the rule of thumb ‘like separates like’. Here it applies to the
relative polarities of the mobile phase and the analytes. As stated earlier,
there is a limited number of stationary phases for use in tlc, therefore
discrimination between analytes is achieved by altering the mobile phase
composition.
61
Heptane / toluene toluene Methanol / toluene
(non-polar) (polar)

The mobile The mobile phase The mobile

phase is too is sufficiently phase is too
weak compared strong to result in strong compared
to the stationary discrimination to the stationary
phase between the phase
analytes

Figure 6.27 - effect of mobile phase on tlc separations

62
Tlc visualisation
In tlc there are a number of ways of observing or visualising the separated
(and mostly colourless) analytes. These divide into two categories physical
and chemical methods.
Physical Methods
The physical methods are based on the absorbance or emission of radiation,
usually in the ultraviolet (UV) range of the electromagnetic spectrum. Many of
the compounds separated will absorb uv light. Therefore placing a developed
tlc plate under a UV lamp will show up these compounds as darker spots (the
light having been absorbed) against a lighter background. If the separated
analytes are capable of emitting radiation (fluorescing), then placing the plate
under a suitable light source will results in bright, fluorescing spots being
shown.
Some analytes are incapable of absorbing UV light or fluorescing. In this case
a fluorescent compound, such as cadmium or zinc sulphides, is incorporated
into the stationary phase so the whole plate appears bright yellow/green when
held under a uv light source. Once this type of plate is developed the analytes
appear as dark spots as they quench the background fluorescence from the
stationary phase.

63
Chemical Methods
Chemical methods of visualisation can be selective and highlight a few
analytes, or be universal and highlight nearly every analyte. To use a
chemical visualiser the reagent is sprayed on to the plate as a fine mist or the
plate is quickly dipped into a tank of the reagent vapour. Universal visualisers
include iodine and concentrated sulphuric acid.
Iodine forms complexes with organic compounds which are brown in colour
and hence the analytes on the tlc plate appear brown. The advantage of this
method is that the complexes are not very stable and so the spots fade within a
short period of time leaving the analyte unchanged. Sulphuric acid also
produces brown spots but this is because any organic material has been
charred and therefore destroyed.
Selective visualisers include ninhydrin which reacts with amines, peptides and
amino acids to produce purple/pink compounds. Another reagent, fluorescein
can be used to convert hydrocarbons, barbiturates and many other compounds
into fluorescent derivatives which then fluoresce under UV light.

64
Tlc - reporting results
In tlc the retardation factor (Rf) is used to report the results of a separation

Distance travelled by analyte

Rf = Equation (6.11)
Distance travelled by solvent front

Solvent front

Distance to solvent front

Distance travelled
by analyte

Figure 6.28 - diagram of tlc plate to show distance to the solvent front to enable
calculation of the Rf value.

Continued on the next slide 65

The retardation factor (Rf), like many other values in chromatography, is
another ratio value used to report chromatographic results. In tlc, these are the
favoured values, as they are less influenced by changes in the experimental
conditions than absolute measurements of distance travelled by individual
analytes. The following variables all influence how far an analyte moves up the
plate:

 The degree of mobile phase saturation in the atmosphere of the

developing tank;
 The thickness of the stationary phase layer;
 The distance moved by the solvent front;
 The sample size;
 The moisture content of the stationary and mobile phases.

66
Tlc - applications
 Used to check the purity and success of organic syntheses;
 A quick way of checking the purity of reagents;
 Can be used to investigate potential hplc separations as the same
stationary and mobile phases can be used in both techniques;
 Can be useful when a high sample throughput required, as 20+ samples
can be spotted on a single plate.

The main disadvantage of tlc is that it does not readily lend itself to quantitative
analyses, unlike hplc or gc and thus is used mainly for identification purposes
(qualitative analysis)

67
Question 6.1 Sketch and label an idealised chromatogram. How can the resolution between
two peaks be evaluated from such a plot?

Question 6.2 What are the causes of peak broadening and which of the terms in the rate
equation take them into account?

68
Question number 6.1
The answer to this question can be found on slide 41 which shows a
stylised chromatogram and the equation for determining the resolution
between two peaks.

Note that adjusted or absolute retention times can be used, also note
that the width (w) is found by approximating the peak to a triangle this is
why a resolution of 1.5 is required for 100% separation.

69
Question number 6.2
The answer to this question may be found on slides 43 - 57.

There are a number of factors which cause peak broadening these are summarised in the
Van Deemter equation:

 Longitudinal diffusion, the B term;

 Eddy diffusion in packed columns only, the A term;
 Mass transfer, the C term.

The A, B and C terms may be defined in terms of a number of variables which the
chromatographer can control; such as column length, diameter and thickness of the
stationary phase and the nature of both the stationary and mobile phases.
In addition the mobile phase flow rate, as shown by the rate equation, also affects the
peak broadening.

70