An Introduction to

Chromatographic Separations

1
Liquid chromatography :
 Was invented and named by Mikhail Tswett, a
Russian botanist, in 1903 who first invented
and named.

 He used glass column filled with finely

divided chalk (calcium carbonate) to
separate plant pigments.

 Hence name chromatography, where Greek

CHROMA means COLOUR and GRAPHE in
means WRITE.
2
3
4
5
 Different components of a sample are transported in
a mobile phase (a gas, a liquid, or a supercritical
fluid). The mobile phase (also called eluent)
penetrates or passes through a solid or immiscible
stationary phase.

 Solutes (eluates) in the sample usually have

differential partitioning or interactions with the
mobile and stationary phases.

 Since the stationary phase is the fixed one then

those solutes which have stronger interactions with
the stationary phase will tend to move slower (have
higher retention times) than others which have lower
or no interactions with the stationary phase will tend
to move faster.
6
 Interactions between a solute and a stationary
phase take place when both have similar
characteristics, for example in terms of polarity.

 But when properties are so different, a solute

will not tend to stay and interact with the
stationary phase and will thus prefer to stay in
the mobile phase and move faster; a polar
solvent and a non polar stationary phase is a
good example.

7
 Do you want
Chocolate

List the major classification of chromatography

and their mobile phase

8
 Based on the nature of the mobile phase,
chromatographic techniques can be
classified into three classes:

1. Liquid chromatography (LC)

2. Gas chromatography (GC)
3. Supercritical fluid chromatography (SFC)

 Other classifications are also available

where the term column chromatography
where chromatographic separations take
place inside a column, and planar
chromatography, where the stationary phase
is supported on a planar flat plate, are also
used.
9
 Elution
 Elution involves passing a mobile phase
inside the column whereby solutes are
carried down the stream but on a differential
scale due to interactions with the stationary
phase.
 As the mobile phase continues to flow,
solutes continue to move downward the
column.
Distances between solute bands become
greater with time and as solutes start to
leave the column they are sequentially
10
detected.
11
 The time a solute spends in a column is called
retention time and this depends on the fraction
of time that solute spends in the mobile phase.

 As solutes move inside the column, their

concentration zone continues to spread and the
extent of spreading (band broadening) depends
on the time a solute spends in the columns.

The dark colors at the center of the solute zones

in the above figure represent higher
concentrations than are concentrations at the
sides. This can be represented schematically as:

12
13
 Chromatogram
This is the plot of detector signal (absorbance,
fluorescence, refractive index, etc..) versus
retention time of solutes in a chromatographic
column.

The areas under the peaks in a chromatogram

are usually related to solute concentration and
are thus very helpful for quantitative analysis.

14
 Distribution Constants
 Solutes traveling inside a column will interact with
both the stationary and mobile phases. If, as is our
case, the two phases are immiscible, partitioning of
solutes takes place and a distribution constant, K,
can be written:
K = CS/CM (1)
Where; CS and CM are the concentrations of solute in
the stationary and mobile phases, respectively. If a
chromatographic separation obeys equation 1, the
separation is called linear chromatography. In
such separations peaks are Gaussian and
independent of the amount of injected sample

15
Retention time: The time required for an
analyte to travel through the column after
injection till the analyte peak reaches the
detector is termed the.
Dead or Void time tM : is the time spent by
that species to exit the column.

Solutes will move towards the detector in

different speeds, according to each
solute’s nature.

16
Chromatography Retention Times

tM = retention time of mobile phase (dead time)

tR = retention time of analyte (solute)
ts’ = time spent in stationary phase (adjusted retention time)
L = length of the column
17
 Chromatography: Velocities
Linear rate of solute migration!

Velocity = distance/time  length of column/ retention times

L
Velocity of solute: v 
tR
L
Velocity of mobile phase:  
18 tM
 average linear velocity of a solute, v, can be
written as

v = L/tR (2)

Where, L is the column length and tR is the

retention time of the solute. The mobile phase
linear velocity, u, can be written as:
u = L/tM (3)

The linear velocity of solutes is a fraction of the

linear velocity of the mobile phase. This can be
written as:
v = u * moles of solute in mp
total moles of solute
 Chromatography
Velocity/Retention time and Kc

v    fraction of time in mobile phase

moles of solute in mobile phase
v   
total moles of solute
cMVM
v   
20
cMVM  cSVS
 This can be further expanded by substitution
for the moles of solute in mp, CMVM, and the
total number of moles of solute, CMVM + CSVS.
v = u * CMVM / (CMVM + CSVS)
Dividing both nominator and denominator by
CMVM we get

v = u * 1/ (1 + CSVS/ CMVM) (4)

Now, let us define a new distribution constant,

called the capacity or retention factor, k’, as:

k’ = CSVS/ CMVM = K VS/VM (5)

21
Substitution of 1,2 and 5 in equation 4 we
get:
L/tR = L/tM * {1/(1 + k’)}
Rearrangement gives:
tR = tM (1+k’) (6)
This equation can also be written as:
k’ = (tR – tM)/tM

NOTE: The retention factor is also called the capacity factor, the capacity ratio, and the
partition ratio, and is sometimes given the symbol k′. Keep this in mind if you are using other
resources. Retention factor is the approved name from the IUPAC Gold Book

22
Q1. In a chromatographic analysis of low molecular weight acids, butyric acid elutes

with a retention time of 7.63 min. The column’s void time is 0.31 min.

Calculate the retention factor for butyric acid.

Q2.

Figure above is the chromatogram for a two-component mixture.

A. Determine the retention factor for each solute assuming the
sample was injected at time t = 0.
B. Determine the selectivity factor?

23
 The Selectivity Factor
 For two solutes to be separated, they should have
different migration rates. This is referred to as
having different selectivity factors with regard to a
specific solute. The selectivity factor, a, can be
defined as:
a = kB’/kA’ (7)
Therefore, a can be defined also as:
a = (tR,B – tM)/ (tR,A – tM) (8)

For the separation of A and B from their mixture, the

selectivity factor must be more than unity.

24
The Shapes of Chromatographic Peaks
Can be:
 symmetrical normal error peaks (Gaussian
peaks). This assumption is necessary in
order to continue developing equations
governing chromatographic performance.

 However, in many cases tailing or fronting

peaks are observed.

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 Gaussian peaks (normal error curves) are easier to deal
with since statistical equations for such curves are well
established and will be used for derivation of some basic
chromatographic relations.

 It should also be indicated that as solutes move inside a

column, their concentration zones are spread more and
more where the zone breadth is related to the residence
time of a solute in a chromatographic column.

27
 Plate Theory
 Multiple partitions take place while a solute is moving
towards the end of the column.

 The number of partitions a solute experiences inside a

column very much resembles performing multiple
extractions.

 It may be possible to denote each partitioning step as an

individual extraction and the column can thus be regarded
as a system having a number of segments or plates, where
each plate represents a single extraction or partition
process.
28
Therefore, a chromatographic column
can be divided to a number of theoretical
plates where eventually the efficiency of
a separation increases as the number of
theoretical plates (N) increases.
 In other words, efficiency of a
chromatographic separation will be
increased as the height of the theoretical
plate (H) is decreased.

29
 Column Efficiency and the Plate Theory

If the column length is referred to as L,

the efficiency of that column can be
defined as the number of theoretical
plates that can fit in that column length.
This can be described by the relation:

N = L/H

30
 Definition of Plate Height
From statistics, the breadth of a Gaussian
curve is related to the variance s2. Therefore,
the plate height can be defined as the
variance per unit length of the column:
H = s2/L (9)
In other words, the plate height can be defined
as column length in cm which contains 34%
of the solute at the end of the column (as the
solute elutes). This can be graphically shown
as:

31
32
The peak width can also be represented
in terms of time, t, where:
t = s/v (10)
t = s/(L/tR) (11)
The width of the peak at the baseline, W,
is related to t by the relation:
W = 4t where 96% of the solute is
contained under the peak.
s = L t/tR
s = LW/4tR (12)
33
s2 = L2W2/16tR2
s2 = HL
H = LW2/16tR2

N = 16(tR/W)2 (13)

Also, from statistics we have:

W1/2 = 2.354 t (14)
s2 = LH

34
s = L t/tR
s = L (W1/2/2.354)/tR
s2 = L2 (W1/2/2.354)2 /tR2 (15)
Substitution in equation 9 gives:
LH = L2 (W1/2/2.354)2 /tR2
H = L (W1/2/2.354 tR)2
N = L/H
N = 5.54 (tR/W1/2)2 (16)

35
s = L t/tR
s = L (W1/2/2.354)/tR
s2 = L2 (W1/2/2.354)2 /tR2 (15)
Substitution in equation 9 gives:
LH = L2 (W1/2/2.354)2 /tR2
H = L (W1/2/2.354 tR)2
N = L/H
N = 5.54 (tR/W1/2)2 (16)

36
Asymmetric
Peaks
The efficiency,
N, can be
estimated for
an
asymmetric
chromatograp
hic peak using
the relation:

37
N = 41.7 (tR/W0.1)2 / (A/B + 1.25) (17)

Draw a horizontal line across the peak at

a height equal to 1/10 of the maximum
height.

Where W0.1 = peak width at 1/10 height =

A+B

38
 Band Broadening
 Apart from specific characteristics of
solutes that cause differential
migration, average migration rates for
molecules of the same solute are not
identical.

 Three main factors contribute to this

behavior:

39
Longitudinal Diffusion
Molecules tend to diffuse in all directions because
these are always present in a concentration zone
as compared to the other parts of the column.

This contributes to H as follows:

HL = K1DM/V

Where, DM is the diffusion of solute in the mobile

phase. This factor is not very important in liquid
chromatography except at low flow rates.

40
This contributes to H as
follows:

HL = K1DM/V

Where, DM is the diffusion of

solute in the mobile phase.
This factor is not very
important in liquid
chromatography except at
low flow rates.

41
Asymmetric
Peaks
The efficiency,
N, can be
estimated for
an
asymmetric
chromatograp
hic peak using
the relation:

42
N = 41.7 (tR/W0.1)2 / (A/B + 1.25) (17)

Draw a horizontal line across the peak at

a height equal to 1/10 of the maximum
height.

Where W0.1 = peak width at 1/10 height =

A+B

43
Band Broadening

Apart from specific characteristics of

solutes that cause differential
migration, average migration rates for
molecules of the same solute are not
identical. Three main factors
contribute to this behavior:

44
Longitudinal Diffusion
Molecules tend to diffuse in all directions because
these are always present in a concentration zone
as compared to the other parts of the column.

This contributes to H as follows:

HL = K1DM/V

Where, DM is the diffusion of solute in the mobile

phase. This factor is not very important in liquid
chromatography except at low flow rates.

45
This contributes to H as
follows:

HL = K1DM/V

Where, DM is the diffusion of

solute in the mobile phase.
This factor is not very
important in liquid
chromatography except at
low flow rates.

46
Resistance to Mass Transfer
Mass transfer through mobile and stationary
phases contributes to this type of band
broadening.

1. Stationary Phase Mass Transfer

This contribution can be simply attributed to

the fact that not all molecules penetrate to
the same extent into the stationary phase.
Therefore, some molecules of the same
solute tend to stay longer in the stationary
phase than other molecules
47
Quantitatively, this
behavior can be
represented by the
equation:

Hs = K2 ds2 V/Ds

Where ds is the
thickness of
stationary phase and
Ds is the diffusion
coefficient of solute
in the stationary
phase.

48
 Mobile Phase Mass Transfer
Solute molecules which happen to pass
through some stagnant mobile phase
regions spend longer times before they can
leave. Molecules which do not encounter
such stagnant mobile phase regions move
faster. Other solute molecules which are
located close to column tubing surface will
also move slower than others located at the
center. Some solutes which encounter a
channel through the packing material will
move much faster than others.
49
HM = K3dp2V/DM

Where dp is the
particle size
of the
packing.

50
Multiple Path Effects

Multiple paths which

can be followed by
different molecules
contribute to band
broadening.

Such effects can be

represented by the
equation:

HE = K4 dp

51
The overall contributions to band broadening are then,

Ht = HL + HS + HM + HE

Where; Ht is the overall height equivalent to a

theoretical plate resulting from the contributions of
the different factors contributing to band
broadening.

Ht = k1dp + k2DM/V + K3 ds2 V/Ds + K4 dp2 V/DM

Ht = A + B/V + CSV + CMV

•

52
H = A + B/V+ CV

It turned out that resistance to mass transfer

terms (K3 ds2 V/Ds and K4 dp2 V/DM) are most
important in liquid chromatography and
thus should be particularly minimized. This
can be done by:
1. Decreasing particle size
2. Decreasing the thickness of stationary
phase
3. Working at low flow rates
4. Increase DM by using mobile phases of low
viscosities.

53
On the other hand, the longitudinal
diffusion term (k2DM/V) is the most
important one in gas chromatography.
Reducing this term involves:

1. Working at higher flow rates

2. Decreasing DM by using carrier gases
of higher viscosities

54
 Van - Deemter Equation
From the abovementioned contributions to band
broadening, the following equation was suggested to
describe band broadening in liquid chromatography
(LC)

H = A + B/V+ CV

Where; A represents multiple path effects, B/V

accounts for longitudinal diffusion, and CV accounts
for resistance to mass transfer. The figure below
shows a plot of H against the different factors in the
equation

55
56
The optimum flow rate can be found by taking the first
derivative of equation 23.

dH /dV = O - B/V2 + C

V is optimum when dH /dV= O , therefore,

C = B/V2optimum

Voptimum = {B/C}1/2 (24)

This theoretically calculated velocity is always small

and in practice almost twice as much as its value is
used in order to save time.

57
 Particle size and flow rate
The relation of H and the flow rate of the
mobile phase is highly dependent on
particle size. H will become almost
independent on flow rate at very small
particle size. In this case, faster
separations can be achieved, using
higher flow rates, without affecting H,
and thus band broadening. The figure
below shows such an effect:
58
59
 Resolution
One of the basic and most important
characteristic of a chromatographic
separation is undoubtedly the resolution
term. Resolution between two
chromatographic peaks is a measure of how
well these peaks are separated from each
other, which is the essence of the separation
process. Resolution of the two peaks in the
figure below are different and one finds no
trouble identifying that the lower
chromatogram has the best resolution while
the top one has the worst resolution:

60
 Chromatography: Peak Resolution

Poor resolution

More separation

Less band spread

61
62
 Chromatography
Determining the Number
of Theoretical Plates

N  number of pates
2
 tR 
N  16  
W 
W1/2 2
 tR 
N  5.54  
 W1/ 2 

63
Resolution can be defined from the following
figure as:

64
R = DZ/(WA/2 + WB/2) = 2 DZ/(WA + WB) (18)

R = 2(tR,B – tR,A)/(WA + WB) (19)

For a separation where WA = WB = W, we can

write:

R = (tR,B – tR,A)/W

However, we have the equation:

N = 16 (tR/W)2, or for peak B we have:
W = 4 tR,B /N1/2

65
R = (N1/2/4)(tR,B – tR,A)/tR,B
We can now substitute for the retention
time using the equation derived earlier:
tR = tM (1+k’)
Thus we have:

R = (N1/2/4){(tM(1+kB’) - tM(1+kA’))
/tM(1+kB’)}
Rearrangement gives:
R = (N1/2/4)(kB’ – kA’)/(1+kB’)
66
Dividing both nominator and
denominator by kB’:
R = (N1/2/4)(1 – kA’/kB’)/{(1+kB’)/kB’}

However, a = kB’/kA’

R = (N1/2/4)(1 – 1/a) (kB’)/(1+kB’)

R = (N1/2/4){(kB’)/(1+kB’)}{(a – 1)/a)} (21)

67
Therefore, resolution can be viewed as a
composite contribution of three terms:

a. Efficiency term where R is proportional to

N1/2
b. Retention term where R is proportional to
k’/(1+k’) which suggests that the retention
parameter should be optimized. A value for k’ in
the range from 5-10 is preferred as smaller
values (low retention) results in bad resolution
while a very high k’ value means very long
retention with exceedingly small improvements
in resolution:

68
69
These data can be better viewed as a plot where
as k’ was increased, almost a plateau was
realized.

70
The overlap of two Gaussian peaks of equal area and
amplitude, at various values of resolution (R) is
presented below:

(a) R = 0.50
Overlap of two peaks = 16%

(c) R = 1.00
Overlap of two peaks = 2.3%

(d) R = 1.50
Overlap of two peaks = 0.1%

c. Resolution is dependent on a selectivity term {(a

– 1)/a)}. As the selectivity is increased, resolution
increases as well. When a = 1, resolution is zero.
71
 Effect of Other Parameters on
Resolution
a. The resolution of a column is proportional to
the square root of its length since N = L /
HETP.

R = (L1/2/4H1/2){(kB’)/(1+kB’)}{(a – 1)/a)} (22)

b. Retention time as related to resolution can
be obtained by the following treatment:
N = L/H
tM = NH/u
72
tR,B = tM (1+kB’)
tR,B = (NH/u) (1+kB’)

N = 16R2{(1 + kB’)/kB’}2{a /(a - 1)}2

Substitution gives:
tR,B = (16R2H/u){(1 + kB’)3 /kB’2}{a /(a - 1)}2

Therefore, one can also write:

tR,A/tR,B = RA2/RB2
73
To obtain a high resolution, the three terms
must be maximised.
An increase in N, the number of theoretical
plates can simply be done by lengthening the
column. This leads to two opposing effects
where resolution is increased but at the
same time this causes an increase in
retention time and thus increased band
broadening. In addition, a longer column may
not always be available. An alternative is to
increase the number of plates, the height
equivalent to a theoretical plate by adjusting
elution variables (mobile phase
composition), and other factors affecting
selectivity.
74
 Chromatographic Relationships

75
It is often found that by controlling the
capacity factor, k', separations can be
greatly improved. This can be achieved
by changing the temperature (in Gas
Chromatography) or the composition of
the mobile phase (in Liquid
Chromatography).

76
When a is close to unity, optimising k' and
increasing N is not sufficient to give good
separation in a reasonable time. In these
cases, a is increased by one of the following
procedures:
1. Changing mobile phase composition
2. Changing column temperature
3. Changing composition of stationary
phase
4. Using special chemical effects (such as
incorporating a species which complexes
with one of the solutes into the stationary
phase or use of surfactants)

77
 The General Elution Problem
Look at the chromatogram below in which six
components are to be separated by an elution
process:

78
It is clear from the figure that the separation is
optimized for the elution of the first two
components. However, the last two
components have very long retention and
appear as broad peaks. Using a mobile
phase composition that can optimize the
elution of the last two compounds will,
unfortunately, result in bad resolution of the
earlier eluting compounds as shown in the
figure below where the first two components
are coeluted while the resolution of the
second two components becomes too bad:
79
80
One can also optimize the separation of the
middle too components by adjusting the
mobile phase composition. In this case, a
chromatogram like the one below can be
obtained:

81
However, in chromatographic separations, we
are interested in fully separating all
components in an acceptable resolution.
Therefore, it is not acceptable to optimize the
separation for a single component while
disregarding the others. The solution of this
problem can be achieved by consecutive
optimization of individual components as the
separation proceeds. In this case, the mobile
phase composition should be changed
during the separation process.

82
First, a mobile phase composition suitable for the
separation of the first eluting component is
selected, and then the mobile phase
composition is changed so that the second
component is separated and so on. The change
in mobile phase composition can be linear,
parabolic, step, or any other formula. The
chromatographic separation where the mobile
phase composition is changed during the elution
process is called gradient elution. A separation
like the one below can be obtained:

83
84
 Qualitative Analysis
Usually, the retention time of a solute is the
qualitative indicator of a specific analyte.

The retention time of an analyte is thus

compared to that of a standard. If both have
the same retention time, this may be a good
indication that the identity of the analyte is
most probably that of the standard.

85
Development in qualitative analysis in chromatography
involves use of detectors that can give structural
details of solutes, like
diode array,
Fourier transform infrared,
mass spectrometers, etc.
In such cases, qualitative analysis with high degree of
certainty can be accomplished.
 It should therefore be clear that a similar retention
time of a component and standard does not imply a
100% identification but rather a good possibility.
 However, if the retention time of a compound in
question does not match that of the standard, we are
100% sure that the anticipated compound is either
absent or present at a concentration below the
detection limit of the instrument.

86
 Quantitative Analysis
Chromatographic separations provide very good
and reliable information about quantitative
analysis of sample constituents. Either the
peak height or peak area can be used for
quantitative analysis.
Peak heights are easier and faster to use and
usually result in good precision, especially
when reproducible sample injections are made.
However, late eluting peaks may have small
peak heights but large width which may cause
large errors.
Peak areas are better for quantitative analysis as
the area under the peak is integrated which is
an accurate measure of concentration.
87
The Internal Standard Method
Uncertainties in sample injection can be
overcome by use of an internal
standard. In this method, a measured
quantity of an internal standard is
added to both standard and sample,
and the ratio of analyte signal to
internal standard is recorded. Any
inconsistency in injection of the sample
will affect both the analyte and internal
standard.
88
Properties of the internal standard
should include:
The retention times of internal standard
and analyte should be different and the
two peaks must be well separated, R
>1.25
The detector response factor for the
analyte and the internal standard
should be the same.
Using internal standards can significantly
improve precision to better than 1%.
89
 TUTORIAL QUESTIONS
1. Complete the sentence
a. Running a column is called …….; the
solvent is the ……; the …….. comes out .
b.The efficiency of separation is •
determined by two major factors namely
…………… and ……..
c. There are ……., …… and ……. Peaks •
in chromatography

90
2.Define the following terms?
a. Retention time
b. Distribution constant
c. Void time
3. List
a. Three major classification of
Chromatography
b. Three differences between LC and GC

91
4. Described two major factors that
determines the efficiency of a column

5.Explains the Van-Deemter Equation.

6. State three factors on which resolution of

chromatography depends.

92
7. Consider a chromatography experiment in which two
components with capacity factors k1’ = 4.00 and k2’= 5.00 are
injected into a column with N = 1.00 × 103 theoretical plates.
The retention time for the less-retained component is

tr1 =10.00 min.

(a) Calculate tm and tr2. Find w1/2 and w for each peak.

(b) Sketch the chromatogram, supposing the two peaks have

the same height.

(c) Calculate the resolution of the two peaks.

93
8. (a) Find the capacity factors for octane and nonane in Figure (below).
(b) Find the ratio: (time octane in stat. phase)/(time octane on column). (c)
Find the relative retention for octane and nonane.
(d) Find the partition coefficient for octane by assuming that the volume of
the stationary phase equals half the volume of the mobile phase.

94
9. A gas chromatogram of a mixture of toluene and ethyl acetate is shown
below.
(a) Use the width of each peak (measured at the base) to calculate the
number of theoretical plates in the column.
(b) (b) Use the width of the toluene peak at base to calculate the w1/2.
Compare to the measured value.

95
10. The relative retention for two compounds in gas chromatography is
1.068 on a column with a plate height of 0.520 mm. The capacity factor for
compound 1 is 5.16.
(a) Find the separation factor (𝛾) for the two compounds.
(b) (b) What length of column will separate the compounds with a
resolution of 1.00?
(c) (c) The retention time for air (tm) is 2.00 min. If the number of plates is
the same for both compounds, find tr and w1/2 for each peak.
(d) (d) If the ratio of stationary phase to mobile phase is 0.30, find the
partition coefficient for compound 1.

96
10. A gas chromatogram of a mixture of toluene and ethyl acetate is
shown below.
(a) Use the width of each peak (measured at the base) to calculate the
number of theoretical plates in the column.
(b) (b) Use the width of the toluene peak at base to calculate the w1/2.
Compare to the measured value.

97