CHM 510

TOPIC 1

CHROMATOGRAPHIC
SEPARATIONS

1
 Course Learning Outcomes
Students should be able to:
1. Understand the general concepts and principles of
separation in chromatographic technique.
2. Understand the efficiency of separation:
a) Resolution
b) Plate Theory
c) Rate Theory (van Deemter Theory)
3. Apply chromatographic equations in solving problems.
4. Plot & interpret chromatogram.
2
 Methods of separation in
chemical analysis
Method Basis of method
Distillation Differences in volatility of compound
Extraction Differences in solubility in 2 immissible
liquids
Chromatography Samples that are multicomponent &
Electrophoresis complex

3
Definition of chromatography

‰ Chromatography is a separation method in which

the components in a mixture to be separated are
distributed between two phases,
- a stationary phase
- a mobile phase,

moves in a definite direction.

4
 The chromatographic process

COLUMN
Tubing

Mobile Phase Carrier Gas

Sample
Stationary Phase
(Liquid Phase)
‰ Stationary phase – the one that stays in place
inside the column or on a solid surface (flat sheet);
phase that is stationary in chromatography.

‰ Mobile phase – the solvent moving through,

carrying with it the component mixture
9 GC - carrier gas (He)
9 HPLC - liquid (H2O, organic solvent)
9 SFC - supercritical fluid (CO2)

6
‰ Any chromatography system is composed of
three components :

1. Stationary phase
2. Mobile phase
3. Mixture to be separated

7
Stationary phase in Stationary phase fixed on a
a column (HPLC) solid surface (thin layer
chromatography)
8
 Types of chromatography

‰ Gas chromatography (GC)

‰ High performance liquid chromatography (HPLC)
‰ Thin layer chromatography (TLC)
‰ Supercritical fluid chromatography (SFC)

9
 General principle of
chromatography

‰ Components of a mixture are carried through the

stationary phase by the flow of a mobile phase.

‰ Separation occur because of differing affinities of

components with stationary phase & mobile phase.

10
‰ Those components that are strongly retained by the
stationary phase move slowly with the flow of mobile
phase.

‰ Components that are weakly held by the stationary

phase travel rapidly.

‰ These differences in mobility will cause the sample

components separate.

11
 The separation process
Flow of Mobile Phase
Injector Detector
T=0

T=10’

T=20’

Most interaction with stationary phase Least 12

 Chromatographic results
Plot of Component
Concentration
High Concentration
Component Bands

Low Concentration

Component Molecule
Concentration
Page 13
 Chromatogram

‰ If a detector that responds to solute concentration is

placed at the end of the column during elution & its
signal is plotted as a function of time (or volume of
added mobile phase) a series of peak is obtained.

‰ Such plot is called chromatogram.

14
‰ Peak positions used to identify components;
peak areas to determine amounts of each
component/analyte.

15
 Some useful terms
‰ Process in which components are washed through a
stationary phase by the movement of a mobile phase :
ELUTION

‰ Mobile phase described as ELUENT

‰ Component leaving column is ELUATE

‰ Solid that is dissolved in liquid : SOLUTE

‰ The chemical /substance that is determined in an

analytical procedure : ANALYTE
16
 Retention time, tR
‰ tR : time for the analyte to pass through the column or the
time from sample injection to detection.

‰ Each analyte will have a different tR.

tR = retention time
tM = void time
tS = adjusted retention time

17
‰ A compound not-retained by the stationary phase will
elute out of the column at time tM, called the void time
or the dead time (sometimes designated by to ).

tM :
9 the time a non-retained compound spends in the
mobile phase, or
9 the amount of time the non-retained compound
spends in the column, or
9 the time taken for the mobile phase to pass through
the column.
18
 Adjusted retention time, tRʹ

‰ tʹR or tS is the time a compound spends in the

stationary phase.

‰ tʹR is the difference between the tR and tM for a

compound.

tʹR = tR - tM
19
 Retention/capacity factor, kʹ

‰ kʹ used to described the migration rate of solutes

on column.

‰ The kʹ for analyte A is defined as :

(t R ) A − t M K AVS
kA =
' or k =
'
A
tM VM

20
9 kʹ can be derived from chromatogram.

9 A large kʹ (> 20-30) favors good separation, but

increased elution time.

9 Good separation, 2< kʹ >10.

21
 Selectivity factor, α
‰ α described the separation of 2 species (A & B)
on the column.

k B'
α = ' (Retention factor)
kA
(t R ) A − t M (t R ) B − t M
kA =
'
and k B =
'

tM tM
(t R ) B − t M
α = (Retention time)
(t R ) A − t M
22
‰ α always greater than 1.

‰ If α = 1, the 2 compounds cannot be separated.

‰ The higher the α, the more separation between 2

compounds or peaks.

‰ When calculating the α, species A elutes faster than B.

23
 Efficiency of separation
‰ Two factors affect how well two components are
separated :
9 difference in retention time between peaks
(farther apart, better separation)
9 peak widths
(an efficient separation will produce narrow peaks)

‰ Solutes in a column spread into a Gaussian profile.

24
 Efficiency of separation
Gaussian peak shape :
Detector response

σ σ
w1/2=2.35σ
h

1/2h
w=4σ

t0 tr
time 25
 Resolution, Rs

‰ The degree (how well) of separation of 2 peaks: Rs

‰ Although α, describes the separation of peak

centres, it does not take into account peak widths.

26
 tR2
Detector response

∆t
tR1

wb1 wb2
Time

2∆t 2[tR2 - tR1]

Rs = w =
b1 + wb2 wb1 + wb2
Δt = Difference between retention times of two peaks
= tR2 - tR1
 Overlap of 2 peaks with different degrees of Rs

Rs=0.50 Rs=0.75

t0 2σ time t0 3σ time

Rs=1.00 Rs=1.50
Rs≥1
is good

t0 4σ time t0 6σ time
Higher Rs, better separation
28
‰ Rs =1.5 represents baseline resolution, or complete
separation of two neighboring solutes Æ ideal case.

‰ Rs =1.0 considered adequate for most separations.

29
 Sample problem 1

Ethanol & methanol are separated in a GC column

with tR of 370 & 385 s and a base widths of 16.0 &
17.0 s, respectively? Calculate the Rs.

30
 Sample problem 2
The separation of 4 compounds gave Rs values of
0.5, 1.8 & 10.5. Sketch the chromatogram &
comment on the efficiency of separation.

31
 Column efficiency

‰ The column efficiency is expressed as a number of :

1. Theoretical plates, N
2. Plate height, H or Height Equivalent to a
Theoretical Plate, HETP.

‰ H & N provide useful measures to compare the

performance of different columns for a given analyte.

32
 Theoretical plates, N
‰ View column as divided into a number of adjacent
imaginary segments called theoretical plates.

‰ N is a number indicating how good a column is for a

separation.

Plate theory :
Treats separation in discrete stages, more
stages = more plates

33
 Plate theory

Column divided into theoretical

plates

34
‰ Equilibration of the solute between the stationary
& mobile phase occur in these “plates”.

‰ The analyte moves down the column by transfer

of equilibrated mobile phase from 1 plate to the
next.

35
‰ Plates do not really exist; they are a figment of the
imagination that helps to understand the processes
at work in the column.

‰ The larger the value of N is for a column, the

better the column will be able to separate two
compounds.

‰ N ~ few hundred to several hundred thousand.

36
‰ Columns with high N

9 More efficient (i.e., higher column efficiency).

9 Have a narrower peak at a given tR

than a column with a lower N.

37
 tR
Start

wh

wb

N = 16 (tR/Wb)2 = 5.54 (tR/Wh)2

‰ N is specific for each solute on a given column.

‰ Increasing tR increases N.
‰ N depends upon the length of the column.

L
N =
HETP

39
‰ N’s relation to Rs :

N ⎛ α − 1 ⎞⎛⎜ k 2′ ⎞
⎟
Rs = ⎜ ⎟⎜
4 ⎝ α ⎠⎝ 1 + k avg ′ ⎟
⎠
t’R2 k’2 K2
α = relative retention = = =
t’R1 k’1 K1
‰ N required to obtain a certain Rs :

′
2
⎛ α ⎞ ⎛ 1 + k avg ⎞
2

N = 1 6 Rs ⎜ 2
⎟ ⎜⎜ ⎟⎟
⎝ α −1 ⎠ ⎝ k 2′ ⎠

N1 N2 N2>N1

t0 time t0 time
‰ To improve Rs :

9 By lengthening the column, thus

increasing the N.

42
 Plate height (H) or Height Equivalent to a
Theoretical Plate (HETP)

‰ H or HETP is approximately the length of column

required for one equilibration of solute between
mobile & stationary phase.

‰ Compare efficiencies of columns with different

lengths:

H or HETP = L / N
‰ re: L = column length 43

‰
 2
L Lw
HETP = = 2
N 16tR
The smaller the H, the narrower the peak width

As HETP ↓, Rs increases (N ↑)
‰ Greater separation occurs with :
9 greater number of N
9 H or HETP becomes smaller
Chromatographic relationships
 Sample problem 3a
Substances A and B were found to have tR of 6.4 &14.4 min,
respectively, on a 22.6 cm column. An unretained sample of
air passed through the column in 1.30 min. The widths of the
peak bases were 0.45 and 1.07 min. Calculate the:
(a) column resolution
 Sample problem 3b

Substances A and B were found to have tR of 6.4 & 14.4

min, respectively, on a 22.6 cm column. An unretained
sample of air passed through the column in 1.30 min. The
widths of the peak bases were 0.45 and 1.07 min.
Calculate the:

(b) the av. no. of plates in the column

 Sample problem 3c
Substances A and B were found to have tR of 6.4 & 14.4 min,
respectively, on a 22.6 cm column. An unretained sample of air
passed through the column in 1.30 min. The widths of the peak
bases were 0.45 and 1.07 min. Calculate the:
(c) the plate height for B
 Sample problem 4
A separation of 3 compounds on a column (25 mm x 4.6 mm x 10
µm) produced the following data.

Peak tR (min) Peak width (min)

Unretained 1.2 -
1 3.4 0.11
2 3.8 0.13
3 4.7 0.17

a) The average number of plates from the above data.

b) The average plate height for the column.
49
 Column Performance

Why do bands spread out?

Page 50
 Why bands spread ???

A band of solute spreads as it travel through a

chromatographic column
 Zone/band broadening

‰ Band broadening reflects a loss of column efficiency.

‰ The slower the rate of mass-transfer processes

occurring while a solute migrate through a column, the
broader the band.
52
‰ Mechanisms which contribute to band broadening :

9 Multiple paths / Eddy diffusion, A

9 Longitudinal diffusion, B

9 Mass transfer between phases / equilibration time

between phases, C

53
If we consider the various mechanisms which
contribute to band broadening, we arrive at the
van Deemter equation for plate height (H) :

H = A + B/v + Cv

54
 Total band broadening

‰ Each term introduces its part in the total band

broadening, therefore the sum of all of them
will give the total column plate height.

H = HA + H B + H C

55
 Van Deemter equation
‰ Tell how the column & flow rate affect the H
(H must minimum!!!).

H = A + B/v + Cv
v = flow rate or average velocity of the mobile phase.
A, B & C = coefficients related to multiple paths,
longitudinal diffusion & mass transfer between phase,
respectively.
56
 H = A + B/v + Cv
van Deemter equation says :

“ There are band broadening mechanisms that are

proportional to v, inversely proportional to
v & independent to v “

57
 A term - Multiple Paths
Each molecule takes a different path
through column.

‰ The mobile phase moves through the column which

is packed with stationary phase.

‰ Solute molecules will take different paths of different

lengths through the stationary phase at random.

‰ This will cause broadening of the solute band.

 A term - Multiple Paths

time

Some paths are longer than others, molecules

entering the column at the same time (left) but
eluted out at different times (right).
 A term- Multiple paths
A = 2λdp
‰ A is directly proportional to the packing particle
diameter, dp.
‰ λ is a function of packing uniformity and column
geometry (same shape).
‰ In packed columns, A ≠ 0, in open tubular column,
A =0.
‰ A is small when using smaller particles size &
uniform size.
 B term - Longitudinal diffusion
Diffusion is a process in which analyte migrate from
a more concentrated to a more dilute region

Longitudinal diffusion in column chromatography

is a band broadening process in which solute
diffuse from the concentrated center of zone to
more dilute region (towards & opposed to the
direction of the flow of mobile phase
 B term - Longitudinal diffusion
Low High Low
Conc. Conc. Conc.

‰ Solute diffuses out from the center to the edges.

‰ Solute [ ] is lower at the edges of a band.
‰ This causes band broadening.
B term - Longitudinal diffusion
Start

Time # 1 Time # 2 Time # 3

 B term - Longitudinal diffusion

B/v = 2γDM/v

‰ The longitudinal diffusion effect on H is inversely

proportional to v because the solute spends less
time in the column at high v & less diffusional
broadening occurs.
 B term - Longitudinal diffusion

B/v = 2γDM/v
‰ B is directly proportional to the solute diffusion
coefficient in the mobile phase, DM.

‰ DM is smaller in liquids than in gases. B term is

common source of band broadening in GC, where the
rate at which molecules diffuse is high, whereas in
HPLC, diffusion rates are much smaller. So, B/v is less
pronounced in LC than in GC.
65
 B term - Longitudinal diffusion

B/v = 2γDM/v
‰ The obstructive factor, γ shows that longitudinal
diffusion is hindered by the packing.

‰ γ is lower for a packed column (ie γ = 0.6) than an

unpacked (capillary) column (ie γ = 1).

66
C term-Mass transfer between phases

The term Cv comes from the finite (or a certain

amount ) time required for solute to equilibrate
between mobile & stationary phase.

67
 C term-Mass transfer between phases

‰ If the velocity of the mobile phase is high & the

molecule has a strong affinity for the stationary
phase, then the molecule in the mobile phase will
move ahead of the molecule in the stationary phase.

‰ The band of analyte is broadened.

‰ The higher the velocity of mobile phase, the worse

the broadening becomes.
 C term-Mass transfer between phases
Mobile
phase slow equil.
Stationary
phase
bandwidth bandwidth

Analyte molecules at the front of a band are swept

ahead without equilibrating with the stationary
phase & those at the trailing edge are left behind
for a longer time in the stationary phase
 Mass transfer coefficient (Cs & CM)

Hmass transfer = Cv = (Cs + CM)v

‰ CMv is the mobile phase mass transfer term.

‰ CSv is the stationary phase mass transfer term.

70
• The mass-transfer effect on H is directly
proportional to v because

9 the solute residence time is longer at low v,

the deviation from equilibrium is less & zone
broadening or H is smaller.

71
 Carrier Packed Column
Gas
Liquid Phase

Capillary Column
Carrier
Gas
Liquid Phase Column
Wall
The stationary phase mass transfer (Csv)

Csv = (fS(k’)df2/DS)v
df = stationary phase film thickness (most important
factor)
DS = diffusion coefficient of the solute in the film
A complex function of fs(k’) of the retention factor k’

Csv is less if the df is smaller, or the

solute DS is larger
73
‰ Decreasing df, reduces H & increase efficiency
because solute can diffuse faster from the farthest
depth of the stationary phase into the mobile phase.

‰ E.g.,
9 With thick films, molecules must on the average
travel farther to reach the surface; with smaller Ds,
they travel more slowly.

9 The consequence of both factors lower rate of

mass transfer & an increase in H.

74
 The mobile phase mass transfer (CMv)

CMv = (fM(k’)dp2/DM)v
dp = particle diameter of packing
DM = mobile phase diffusion coefficient

CMv is less if dp is smaller (hence greater surface

area) or the solute diffusion coefficient in the
mobile phase, DM is larger

Small particles reduce the distance solute must diffuse

in the mobile phase
75
76
 Note:
Both longitudinal & mass transfer broadening depend
upon the rate of diffusion of analyte molecules but the
direction of diffusion in the 2 cases is different.

‰ Longitudinal broadening arises from the tendency of

molecules to move in directions that tend to parallel
the flow.

‰ Mass transfer broadening occurs from diffusion that

tends to be at right angles to the flow.

77
As a consequence,

9 The extend of longitudinal broadening is

inversely related to flow rate.

9 For mass transfer, the faster the mobile phase

moves, the less time there is for equilibrium to
be approached.

78
 van Deemter Plot
‰ Determine the optimum mobile phase flow rate to
obtain the minimum H.

H H = A + B/v + Cv
B Cv
v
Hmin
A

v
vopt
Zone broadening can be reduced by using :
1. Smaller particles size
2. Narrower column
3. Thinner liquid stationary phase

80
Why optimization of flow rate is one of the important steps
in chromatographic analysis?

‰ For B/v, high flow rate will decrease the term & thus
reduces band broadening.

‰ But for Cv, low flow rate is required to give the

molecules enough time to reach equilibrium.

‰ Thus, an optimum flow rate is high enough to reduce

diffusion & low enough to give enough time for
achieving equilibrium.

81
Decrease in flow rate will increase
diffusion, H will increase

Increase in flow rate –

not enough time for
equilibrium – inefficient
mass transfer, H
increases

82
 Sample problem 5

Which term in van Deemter equation is most important

at low flow rates? Why?

83
 Sample problem 6a
Specify what would be the effect (increase, decrease or no change)
of the change on the H. Explain.
a) Change the particle size from 3µm to 5 µm (HPLC).

84
 Sample problem 6b

b) Increase thickness of liquid stationary phase (film

thickness).

85
 Effect of film thickness

5 µm Thick Film
H

0.25 µm Thin Film

Average Linear Velocity (ū)

 Sample problem 7
Van Deemter curves below show the effect of particle size on H.
Explain why the curve for the smallest particles (0.1-0.15mm) is
flat at high flow rate?

87
 Sample problem 8

How does a narrow bore column with small particle

size of packing achieve its advantages over a wide
bore column with bigger particle size of packing?

88
 Effect of column ID
ID (mm)
0.6 0.53
H (mm)

0.32
0.4
0.25
0.2

20 40 60
Average Linear Velocity (ū cm/s)
THE END

90