TOPIC: INTRODUCTION TO CHROMOTOGRAPHY

COLUMN RESOLUTION
The ability of a column to separate a sample into a series of
chromatographic peaks each representing a single component
of the sample is of prime interest in chromatography.
Resolution in chromatography means the degree of separation
of components of similar character.The resolution is expressed
in terms of retention time (tr ) and peak width (W).
If two peaks have peak width W1 and W2 at the base and
respective retention times are tr1 and tr2 then resolution R of
the two peaks is

∆𝒛 (𝒕𝒓𝟐 − 𝒕𝒓𝟏)
𝑹= =
𝒘𝟏 + 𝒘𝟐 𝒘𝟏 + 𝒘𝟐
𝟐 𝟐
. .

𝟐(𝐭𝐫𝟐 − 𝐭𝐫𝟏)
=
𝐰𝟏 + 𝐰𝟐
Thus column resolution is the difference in retention times
between the two peaks divided by the combined average width
of the elution peaks.The resolution of an elution is a
quantitative measure of how well two elution peaks can be
differentiated in a chromatographic separation.When two
components of a sample are poorly resolved the peaks overlap
to an extent that makes identification and determination of the
components impossible.
Figure given below shows chromatogram for species 1 and 2 on
four columns having different resolving powers of 0.50 0.75,
1.00 and 1.50
 FIGURE
Figure11

Tm is un retained peak
The resolution (R) for each of these columns is defined as:
𝟐(𝐭𝐫𝟐 − 𝐭𝐫𝟏)
𝐑=
𝐰𝟏 + 𝐰𝟐
It is evident from the figure that a resolution of 1.5 gives an
essentially complete separation of components 1 and 2 as there
are two distant peaks where as a resolution of 0.75 does not.
In the figure 1 R equals to 0.50. Peaks are merging with each
other hence a bad resolution.
Hence if resolution R > 1, separation is good.
If R < 1, separation is not good
If R> 1.5 then resolution is very good.

Question 1: Ethanol and Methanol are separated in a capillary

GC column with retention time of 370 and 385 seconds
respectively and base width W of 16.0 and 17.0 seconds. An un
retained air peak occurs at 10.0 Seconds
Calculate the resolution.
Solution:
tr1 = 370S. W1= 16.0S
tr2= 385S. W2= 17.0S
2(tr2−tr1)
We know R= =
w1+w2
 = 2(385-370)S / (16.0+ 17.0S)
= 2(15) / 33
= 30/33 = 0.91

QUESTION 2: A solute with a retention time of 5 min has a

width of 12S at the base. A neighbouring peak is eluted at 5.4
minutes with a width of 16S what is the resolution of these two
components ?
2(tr2−tr1)
Solution We know R= = w1+w2

tr1= 5 minutes= 300 seconds.

tr2= 5.4 min= 324 seconds
W1= 12 S
W2= 16 S
R= 2(324-300)S /(12+16)S
= 2×24/ 28 = 1.7
Introduction to chromatography.

Retention time (tr) and adjusted retention time (tr')

The retention time of a solute is the time taken usually in minutes by the solute to elute out
of the column of specified length at a constant flow rate of the mobile phase.
Or
It is the time elapsed between sample introduction( beginning of chromatogram) and the
maximum signal of the given compound at the detector. It is measured from the time at
which the sample is injected, to the point at which a display shows a maximum peak for that
compound.
Or
The retention time(tr) is the sum of the time the compound interacts with the stationary
phase as well as the time it takes for a component to get from the start to the end of the
system without any interaction.
If there is no retention that is no interaction of the component with the stationary phase,
the component elutes at the (tm) time.
If tr = 5 min.
And tm= 1 min.
Then t'r = tr-tm
=(5-1) min
= 4 min
Where t'r is the adjusted retention time that is the time which sample spends in the column
and not with the stationary phase and tm is the time required for the mobile phase to
transverse the column and is the time it would take an unretained solute to appear. In gas
chromatography an air peak often appears from unretained air injected into the sample and
the time for this to appear is tm.
If a sample contains several components then each will have a different retention time. The
retention time is more important in chromatography as this time is used to identify the
nature of the solutes by comparison with retention times of standard solutes
Firstly, we inject a known standard and identify its retention time in the chromatogram .
Then we compare this time to a peak in the sample chromatogram. If the times match then
there is a high probability of identifying the sample peak.
Factors influencing retention time
1 Boiling point of a compound.
2 Polarity of the compound relative to the polarity of stationary phase in the column
3 Column temperature
4 Flow rate of the carrier gas
5 Column length

In the diagram
tm it is the time taken by mobile phase to pass through the column.
tr1 retention time of solute 1
tr2 retention time of solute 2
w½ is the peak width at half the height.
W= Peak width at the base in same units as retention time.

W is not the base width of the peak but the width obtained from the intersection of the
baseline with tangents drawn through the inflection of the Gaussian curve at each side of
the peaks and then extended if necessary to form a triangle with the abscissa ( x Axis). The
area of the triangle corresponds to approximately 96% of the area under the curve.
 Capacity Factor (K')
It is an important constant and is related to the nature of the solute, the
stationary phase, the mobile phase and the temperature.
It is an indication of how long a compound can be retained by the
stationary phase.
Or
It is a measure of the efficiency of a separation process occurring in the
column.

𝐊𝐕𝐬
Capacity factor 𝐊′ =
𝐕𝐦
Where K= Partition co efficient
Vs= Total volume of stationary phase in the column.
Vm= Total volume of mobile phase in the column.
Capacity factor (K') can also be calculated by the expression
𝐭𝐫 − 𝐭𝐦
𝐊′ =
𝐭𝐦

𝐭𝐫 𝐭𝐦
= −
𝐭𝐦 𝐭𝐦

𝒕𝒓
= −𝟏
𝒕𝒎
Where tr= retention time of the target solute that is retained by column
packing.
tm= Retention time of solute that is not retained or time required for
mobile phase to traverse the column.
If tr=tm then the sample is not retained by the stationary phase.
The capacity factor for a solute can frequently be varied over a
considerable range by altering the composition of the mobile or the
stationary phase, usually the composition of mobile phase is varied to
optimise a given separation.
𝐕𝐬
We know 𝐊 ′ = 𝐤
𝐕𝐦

Thus if the size of solute particle decreases then surface area

increases and volume of stationary phase (Vs) increases, accordingly
capacity factor increases.
Retention volume (Vr)
The retention volume of a solute is the volume of a mobile phase required to carry the solute through
the column to elution.

Or

It is also defined as the measure of the fraction of time spent by the solute in the mobile phase.

Or

It is the product of retention time and eluent flow rate.

Retention volume of a component could be split into two parts

1 Reduced retention volume.

2 Dead volume.

Reduced Retention Volume: It is the volume of the eluent that passed through the column while the
component was sitting on the surface.

Dead Volume: It is the volume of the eluent that passes through the column while the component was
moving with the liquid phase.

The second part(dead volume) is equal to the total volume of the liquid phase in the chromatographic
column.

Retention volume is independent of the flow parameters for the particular run, but it depends on the
geometrical parameters of the column.

Retention volume will be different for the same compound eluted on the different columns packed
with the same type of adsorbent.

Retention volume Vr= Vm+Vs×K

Vm= Volume of mobile phase.

Vs=Volume of stationary phase.

K= Partition coefficient.
Q) A chromatogram shows two similar sized peaks, one with the retention time of 22.5 minutes and
peak width of 0.82 cm and the second with a retention time of 24 minutes and a peak width of 0.94
centimetres. The void time is 0.32 minutes. Calculate the resolution of the peaks and capacity factor
of the second peak. The chart speed is 0.5 cm/min

Solution
We know

𝟐(𝐭𝐫𝟐 − 𝐭𝐫𝟏)
𝐑=
𝒘𝟏 + 𝒘𝟐
tr1= 22.5 minutes.

tm= 0.32 minutes

tr2= 24.0 minutes

W= Retention Time/Chart Speed

𝟎. 𝟖𝟐 𝐜𝐦
𝐖𝟏 =
𝟎. 𝟓𝐜𝐦
𝐦𝐢𝐧
.
=1.64 min
𝟎. 𝟗𝟒𝐜𝐦
𝐖𝟐 =
𝟓𝐜𝐦
𝟎. 𝐦𝐢𝐧
.
=1.88 min.

We know

𝟐(𝐭𝐫𝟐 − 𝐭𝐫𝟏)
𝐑=
𝒘𝟏 + 𝒘𝟐

𝟐(𝟐𝟒. 𝟎 − 𝟐𝟐. 𝟓)𝐦𝐢𝐧

=
(𝟏. 𝟔𝟒 + 𝟏. 𝟖𝟖)𝐦𝐢𝐧
 𝟐. 𝟎 × 𝟏. 𝟓
=
𝟑. 𝟓𝟐

𝟑. 𝟎
=
𝟑. 𝟓𝟐

= 0.85

𝐭𝐫−𝐭𝐦
We know capacity factor 𝐊 ′ = 𝐭𝐦
For second peak tr2= 24.0 min

Capacity factor
(𝟐𝟒. 𝟎 − 𝟎. 𝟑𝟐 )
𝐊′ =
𝟎. 𝟑𝟐
𝟐𝟑.𝟔𝟖
= 𝟎.𝟑𝟐

= 74

Chart speed refers to the days when the signal from the
instrument was outputted to a chart recorder
SELECTIVITY FACTOR OR SEPARATION FACTOR ( α )

The ability of a column to resolve two solutes is of prime interest in

chromatography. The resolution can be described in thermodynamic terms
without regard to peak width.
The selective factor is a measure of different retentions of two components in the
mixture. Two components will move through the mobile phase at the same speed
but have different degrees of interaction with stationary phase. Selectivity factor
is a major of this .Thus selectivity factor(α) is the ability of the chromatographic
system to chemically distinguish between sample components
Selectivity factor is related to the partition coefficient for the two species and is
given by
α = K2/K1……….(i)
Where K2 is the partition coefficient of more strongly adsorbed component.
and K1 is the partition coefficient of less strongly adsorbed component.
Thus α must always be greater than unity.
𝐕𝐬
We know K' (capacity factor) = 𝐊 𝐕𝐦 … …. (𝒊𝒊)

Where K = partition coefficient.

Vs = total volume of stationary phase in the column.
Vm = total volume of mobile phase in the column.
Hence K2' = K2 Vs/Vm and K1' = K1 Vs/Vm
Therefore K2 = K2' Vm/Vs and K1 = K1' Vm/Vs
Substituting K2 and K1 in equation 1 we get
K2
α = K1
𝐊𝟐′ 𝐕𝐦
× 𝐕𝐬
𝟏
α= 𝐊𝟏′ 𝐕𝐦
× 𝐕𝐬
𝟏
.
 K2′
α= … … … (𝐢𝐢𝐢)
K1′

Here K2 is the capacity factor for component 2 and K1 is a capacity

factor for component 1.
This is the relation between selectivity factor and capacity factor
(Retention Factor)

We also know K' (capacity factor) = (tr-tm)/tm

tr= retention time of peak solute that is retained by column packing.
tm= retention time of solute that is not retained.
Hence
(𝐭𝐫)𝟏 − 𝐭𝐦
𝐊𝟏′ =
𝐭𝐦

(𝐭𝐫)𝟐 − 𝐭𝐦
𝐊𝟐′ =
𝐭𝐦

Substituting values of K2' And K1' in equation (iii) we get

𝐊𝟐′
. α= 𝐊𝟏′
(𝐭𝐫)𝟐−𝐭𝐦 𝐭𝐦
. α= × (𝐭𝐫)𝟏− 𝐭𝐦
𝐭𝐦

(𝐭𝐫)𝟐−𝐭𝐦
. α=(𝐭𝐫)𝟏−𝐭𝐦

This is an equation that permits the determination of α from an

experimental chromatogram, that is how well the peaks are separated
without taking peak width into consideration.
Question 1: Ethanol and Methanol are separated in a capillary GC column with retention time of 370
and 385 seconds respectively and base width W of 16.0 and 17.0 seconds. An un retained air peak
occurs at 10.0 Seconds Calculate the resolution and also separation factor.

Solution:

tr1 = 370S. W1= 16.0 S tr2= 385 S.

W2= 17.0 S.

𝟐(𝐭𝐫𝟐−𝐭𝐫𝟏)
We know 𝐑 = 𝐰𝟏+𝐰𝟐

= 2(385-370)S / (16.0+ 17.0S)

= 2(15) / 33

= 30/33 = 0.91
(𝐭𝐫)𝟐−𝐭𝐦
We know . α=(𝐭𝐫)𝟏−𝐭𝐦

. α= (385-10)seconds/ (370-10) seconds.

= 375/360
= 1.04
Theoretical plates and efficiency.
The efficiency of separation of a chromatographic column can be expressed in
terms of the number of theoretical plates in the column. The concept of
theoretical plate has been derived from distillation theory.
According to Plate theory of chromatography developed by Martin and Synge,
chromatographic column consists of a series of discrete yet continuous identical
layers.
There occurs an equilibration of the solute between the stationary and mobile
phase in these layers.
A theoretical plate represents a single equilibrium step in other words these
continuous layers are termed as theoretical plates.
The migration of the solute is assumed to occur by a series of stepwise transfers
between one plate to the other immediately below.Thus the number of
equilibrations taking place in a column is equal to the number of plates that the
column possesses.
In all forms of column chromatography the efficiency of a column is usually
expressed in terms of plate count (N) of the column. The more theoretical plates
available within a column the more equilibration between the stationary and
mobile phase and the better the quality of the separation (the sharper the peak)
that is higher the efficiency of a column.
If two solute components have very small difference in their partition
coefficient they will need a large number of equilibrations before they can be
completely resolved from each other
Thus the column which has a large number of plates will be better suited for
resolving these components where as a column which has small number of
plates will not be efficient.
The number of plates contained in a column of length (L).
N= L/H ………..(i)
Where L= length of column
H = Plate height (HETP: height equivalent to theoretical plate)
To avoid a long column (H) should be as short (thin or small) as possible. These
concepts apply to all forms of column chromatography but the parameters are
easy to determine in gas chromatography.
It has been found experimentally that

H= σ²/L ……………..(ii)
Where σ² = variance of the chromatographic band.
L=Distance which the band has travelled through the column
σ=standard deviation of the Gaussian chromatographic peak and
is equal to the width at the steepest portion of the curve ( the
inflection point)

From (i) and( ii )we get

𝑳
𝑵=
𝑯
𝐿
𝑁= σ2
L

𝐿2
𝑁=
σ2

If (N) is greater, then column efficiency is greater.

It has been found that width at half height Wh= W1/2= 2.35 σ and peak base
width w= 4 σ

We know number of plates

𝐋
𝐍=
𝐇
𝐋𝟐
𝐍= 𝟐
𝛔
But W= 4 𝛔 or W= 4tr
𝒘
𝛔 = 𝟒
 𝐋𝟐
𝐍= 𝟐
𝛔
𝑳𝟐
𝑵=
𝑾 𝟐
(𝟒)

𝟏𝟔
𝑵 = 𝑳𝟐 × ( )
𝑾𝟐
𝑳𝟐
𝑵 = 𝟏𝟔( )
𝑾𝟐
𝐭𝐫 𝟐
Or N= 𝟏𝟔 × (𝐖 𝟐 ) where tr= retention time
.
N=number of plates of a column
W= Peak width measured at the base in same units as tr

Relation between number of plates and width of the peak measured at

a height of one half of the peak height Wh = W1/2

We know Wh = 2.35 σ
𝒘𝒉
Or σ=𝟐.𝟑𝟓

We know W= 4 σ

W=4(wh/2.35)

𝐰𝐡
𝐖 𝟐 = 𝟏𝟔 × ( )²
𝟐. 𝟑𝟓
 𝑾𝒉𝟐
Or 𝒘𝟐 = 𝟏𝟔 ( 𝟓.𝟓𝟐 )

𝑾𝟐 = 𝟐. 𝟖𝟗 𝑾𝒉𝟐
We know
𝒕𝒓𝟐
𝑵 = 𝟏𝟔( 𝟐 )
𝒘
𝒕𝒓𝟐
𝑵 = 𝟏𝟔( )
𝟐. 𝟖𝟗𝒘𝒉𝟐
𝟏𝟔𝒕𝒓𝟐
Or 𝑵 = 𝟐.𝟖𝟗 𝒘𝒉𝟐
𝒕𝒓𝟐
Or 𝑵 = 𝟓. 𝟓𝟒 𝒘𝒉𝟐
𝒕𝒓𝟐
𝑵 = 𝟓. 𝟓𝟒 𝟏
𝒘(𝟐)²

As wh= W1/2
Van Deemter Equation
Number of equations have been developed that relate the efficiency
of chromatographic columns in other words the height of a
chromatographic plate with three kinetically controlled processes.
a) Eddy diffusion or multipath effect
b) Longitudinal diffusion or molecular diffusion
c) Non equilibrium mass transfer.
The earliest and simplest of these is Van Deemter equation which was
derived for gas chromatography. It provides an approximate
relationship between the flow rate (u) of the mobile phase and the
plate height (H).
𝐁
𝐇=𝐀+ + 𝐂𝐮
𝐮
Where A, B, and C are constants for the given system.The quantity (A)
is associated with Eddy diffusion (B) with longitudinal diffusion (C)
with non equilibrium mass transfer.The magnitude of these effects
are determined by controllable variables such as particle size of
packing, diffusion rates, thickness of stationary phase.
Van Deemter equation was developed for gas chromatography but it
also holds good for liquid chromatography.
The three kinetically controlled mechanisms which need to be
considered for the efficiency of chromatographic columns are
1.Eddy diffusion or multiple path effect or flow dispersion: It is any
diffusion process by which substances are mixed in any fluid system
due to eddy motion.In Eddy diffusion there are multiple paths in the
column or in the plates which differ in length. The solute molecules
passing down the chromatographic column travel separate
paths.Hence solute molecules elute at different times. Thus
broadening of the elution band results (Band Broadening)
Eddy diffusion is independent of the gas or mobile phase velocity. The
Eddy diffusion is related to particle size and geometry of packing by
A= 2 λ dp
Where dp is average particle diameter and λ is empirical
constant with values ranging from 0.8 to 1.0 for a well packed
column. Its value is minimised by using small and uniform
particles.
Eddy diffusion does not happen in gas chromatography
because maximum times we are using capillary columns
either wall coated open tubular (WCOT) columns having
stationary phase coated directly into the inner wall of the
tubing or support coated open tubular (SCOT) columns
having a finely divided layer of solid support material
deposited on the inner wall on which stationary phase is
coated.
In open tubular capillaries the (A) term will be zero because
multiple path effect does not occur.
Hence in Gas Chromatography Van Deemter equation is
𝐁
𝐇= + 𝐂𝐮 [𝐀 = 𝟎]
𝐮
In packed columns however multiple distinct routes
(channels) exist through the column packing which results in
band spreading and hence (A) term will not be zero.

Longitudinal diffusion or axial diffusion or molecular

diffusion
It refers to the diffusion of individual solute molecules in the
mobile phase along the longitudinal directions of the
column.It is the diffusion broadening of the band while the
band is transported along the column by the flow of the
solvent.It occurs along the axis of the column.
When mobile phase runs through the column even with zero
velocity it is longitudinally diffused due to which there is the
turbulence effect that is there is the higher concentration of
the solute in the middle part and lower concentration at both
sides.Hence the molecules migrate from the concentrated
centre parts of the band towards the forwards and rear edges
of the band that is towards more dilute regions thus band
broadening.
Longitudinal diffusion (B) is given by
B= 2 γ Dm
Where γ = Constant called
obstruction factor = 0.6 to 0.8
Dm = Diffusion Co Efficient of the
solute in the mobile phase
As the sample components are fixed in a given analysis, the
only way to change (B) is by varying the type, pressure and
flow rate of the carrier gas. This effect will be more
pronounced,the longer the solute remains in the column ,so
it is reduced by high flow rate and short columns
Denser gases also reduce molecular diffusion.Thus
molecular diffusion in case of CO2 and N2 will be less
compared to He or H2.
Longitudinal or molecular diffusion is most important where
the mobile phase is a gas because diffusion rates in gas are
several orders of magnitude greater than those in the liquid.
In LC molecular diffusion in the stationary phase is very
small compared to that in the gases.
Hence in column chromatography
H[HETP] = A+ Cn [B=0]
Non equilibrium mass transfer.
Mass transfer means the net movement of mass from one
phase or component to another during adsorption. Diffusion
is a mass transfer phenomenon that causes the distribution
of chemical species to become more uniform in space as
time passes.
The interphase mass transfer is due to the finite time
required for equilibrium of the solute to be established
between stationary and mobile phase. Chromatographic
separation occurs because solute moves between stationary
and mobile phase. Sample components (analytes) are
retained in chromatography columns due to their interaction
with the stationary phase.Analyte molecules present in the
flowing mobile phase diffuse towards the mobile/stationary
phase interface and enter into the stationary phase. To
maintain partition equilibrium some molecules will return to
the mobile phase.
After some time there will be a reverse process as the
analytes move from the stationary phase into the mobile
phase. This results in continuous mass transfer between the
flowing mobile phase and stationary phase during the course
of separation.
Resistance to mass transfer is dependant on the speed with
which the partition equilibrium between mobile phase and
stationary phase is obtained. Since the resistance to mass
transfer in the mobile phase is not the same for all molecules
of one type of analyte, this will also result in peak broadening
of that analyte in the column .The term C×U is decreased by
decreasing the flow rate as more time is available for
equilibrium to be approached. In Addition it is minimised if
the channels through which the mobile phase flows are
narrow so that solute molecules do not have far to diffuse in
order to reach the stationary phase. In liquid
chromatography this term dominates due to slow diffusion in
the liquid mobile phase.
It is minimised by using small particles,thin stationary phase
films, low viscosity mobile phases.
The larger the particle size of the stationary phase and the
more viscous the mobile phase the greater this effect
becomes.The greater the mobile phase velocity the greater
the band broadening due to this effect.
Figure shows the contribution of each term in the Van
deemter equation as a function of mobile phase velocity as
well as their net effect.