Basic Chromatography

Chromatography
Chromatogram - Detector signal vs. retention

time
Detector Signal 1 2
time
Textbook: Skoog et al.

Chapter 31-33
CHROMATOGRAPHY
• It is a separation technique based on the different
interactions of compounds with two phases, a mobile
phase and a stationary phase, as the compounds
travel through a column.
Mobile phase: a phase that flows through the stationery

phase, carrying the analyte with it.
Stationary phase: a phase that is fixed in place in a

column that interacts with the analytes.
Column: a solid surface on which the stationary phase is

bound or coated
How does it work
In all chromatographic separation the sample is transported in a
mobile phase. The mobile phase can be a gas, a liquid or a
supercritical fluid.
The mobile phase is then forced through a stationary phase

held in a column or on a solid surface. The stationary phase
needs to be something that does not react with the mobile phase
or the sample.
The analytes then have the opportunity to interact with the

stationary phase as they move past it. Analytes that interact
greatly, then appear to move more slowly. Analytes that
interact weakly, then appear to move more quickly. Because
of this difference in rates, the analytes can then be separated
into their components.
Chromatography
Types of chromatography
1. Liquid chromatography (LC)
i) High Performance Liquid Chromatography (HPLC)
ii) Ion-exchange chromatography (IEC)
iii) Gel permeation/ filtration chromatography (GPC)
iv) Partition or paper chromatography
v) Size exclusion chromatography (SEC)
2. Gas chromatography (GC)
i) Gas liquid chromatography (GLC)
ii) Gas-solid chromatography (GSL)
3. Supercritical fluid chromatography (SFC)
Classification of chromatographic
methods
Column chromatography
Principle of separation: it is based on differential
adsorption of substance by the stationery phase.
Stationary phase: adsorbent filled in a column.
Mobile phase: various solvents.

Thin layer chromatography
Principle of separation: it is based on different
adsorption of the components on the surface of an
adsorbent.
Stationary phase: adsorbent, mostly silica or aluminum

oxide, forming a thin layer on a glass, plastic or aluminum
plate.
Mobile phase: various organic solvents or mixture of

solvents.
Partition or paper chromatography
Principle of separation: it is based on differences in the
partition coefficients of components of a mixture
between two liquids.
Stationary phase: thin layer of water adsorbed on the

cellulose filaments of a filter paper.
Mobile phase: solvent, partly miscible or immiscible with

water.
Ion exchange chromatography
Ion-exchange: RH + K+A- RK + H+A- :
Ions of an electrolyte solution will be replaced with an
equivalent amount of ions of the same sign from an ion-
exchanger.
Principle of separation: it is based on different affinity of
the ions of the mixture to the ion-exchanger (resin).
Stationary phase: cation or anion exchangers.
Mobile phase: aqueous solution of ionic compounds.

Gel permeation or size exclusion
chromatography
Principle of separation: the small particles of a
mixture can penetrate easier into the pores of the
packing material than the larger molecules thus
elute later.
Stationary phase: grains of a swelling three-

dimensional polymer with definite pore size.
Mobile phase: solution of components to be

separated
1: the solution being studied (black particles)
is poured into the column
2: after washing of the column with a solvent
Gas chromatography
Principle of separation: different adsorption of the
components of the mixture
Stationary phase: adsorbent or wetted carrier

material
Mobile phase: inert gas (nitrogen, hydrogen, helium)

High performance liquid
chromatography
Principle of separation: different partition or
solubility of the components of the mixture
Stationary phase: liquid
Mobile phase: liquid

Theoretical Plate Model
• Based on distillation theory
• Actual plates exist where
vapour passes through a liquid
phase.
• Equilibrium between the
phases is assumed.
• Number of plates affect
separation efficiency.
• The height of a plate can be
measured.
• Chromatography column -
plates cannot be observed i.e.
theoretical plates.
Theoretical Plate Model
Fundamentals of chromatography
t0 or tM – death time = retention of compound with no

interaction with stationery phase
tR(1) – total retention time of component 1
tR(2) – total retention time of component 2
t’R(1) – net retention time of component 1 (= tR1-t0)
t’R(2) – net retention time of component 2 (= tR2-t0)
w1 – base peak of component 1
w2 – base peak of component 2
Factors affecting the retention time
i) Length of column
ii) Packing material
iii) Type of carrier gas
iv) Flow rate of carrier gas
v) Temperature of column
Equations in chromatography
Column efficiency
N – plate number or number of theoretical plates
Large N indicates good column efficiency (should be ≈ 10000

for HPLC
N is increased by:
- increased temperature, column length
- decreased stationery phase particle size, flow rate, mobile
phase viscosity
Chromatographic Efficiency
Plate Height (H): Used to compare columns of
different lengths
H=L/N
L = column length, N = number of plates
The smaller / lower H, the better column efficiency

Variables affecting column efficiency
1) Linear velocity of mobile phase
2) Diffusion coefficient in mobile phase
3) Diffusion coefficient in stationery phase
4) Retention factor
5) Diameter of packing particles
6) Thickness of liquid coating

Example
An analyte eluted from a 15.0m column in 5 min. The width at the
base of the peak was measured to be 0.5 min.
Calculate the number of plates and plate heights.
N = 16( 5 min/0.5 min) 2 = 1600
H = 15.0 m/ 1600 = 0.0094 m = 9.4 mm

Capacity or retention factor (k or k’)
✓ k’- is the rate at which the analyte migrates through the column
✓ Estimates the ratio of the retained to unretained compounds
Kc = distribution constant
β = phase ratio (only applicable to liquid
stationary phase)
✓ k’ = 1– 5: Acceptable
✓ k’ < 1: analyte elutes too quickly. Difficult to quantify accurately
✓ k’ > 5: compounds will take longer to elute - long run
To manipulate k’ – change temperature, stationary phase,

mobile phase
Selectivity factor (α)
✓ α - is the ratio of the distribution constant of the more
strongly retained solute (B) to the distribution constant of
the less strongly held solute (A)
✓ Larger α means better separation
✓ The separation of two components is only possible if α >1
✓ To manipulate α - change mobile phase and stationery

phase
Example
Given the following chromatogram. Calculate the capacity factors for
benzene and toluene and the selectivity factor for the separation.
What are the implications of ?
Resolution (Rs)
✓ Rs - is the ratio of the distance between two peak
maxima to the average of the peak width at the base line
✓ How well two peaks that elute next to each other are
separated.
2(t RB − t RA ) N   −   kB 
RS = RS =    
w b1 + w b2 4     1+ kB 
Resolution (Rs)
Rs = 1.5 full separation, <1.5 incomplete separation
To manipulate Rs: change column temperature, mobile phase,

stationery phase
Example
The retention time for compound A and B is 14.10 and
15.30 min, respectively in a 30.0cm column. The peak
width is 1.09 min (A) and 1.12 min (B). Calculate the
resolution between the two compounds.
2(t RB − t RA )
RS =
w b1 + w b2
2(15.30 − 14.10)
=
1.09 + 1.12
= 1.08
Effect of retention factor and selectivity factor on
resolution
2
   1+ kB 
2
N   −   kB 
RS =     N = 16 Rs 
2
  
4     1+ kB    −1   kB 
Effect of resolution on retention time

2
16 Rs H    1+ kB 
2 2
(t R )B =    
u   −1   kB 
Example
Using the previous example. The resolution is calculated to be
1.08, average plate number is 4000 and plate height is 5.0x10-3
cm. If the resolution required to separate the compounds is 1.5,
calculate i) the number of plates required and ii) the retention time
for compound B to elute.
(Rs )1 =
N1
(t R )1 (Rs1 )2
(Rs )2 =
N2
(t R )2 (Rs2 )2
1.08
=
4000 (15.30)1 = (1.08)2
1.5 N2 (t R )2 (1.5)2
N 2 = 7.7 x103 (t R )2 = 29.5 min
Rate theory
✓Plate theory assumptions:
• there is a rapid equilibrium of the analyte between the
stationary and mobile phases.
• the partition coefficient (K) is linear.
✓Plate theory does not take into account:

• kinetics of separation i.e. elution rates, solute diffusion
✓Rate theory takes into account

• effect of mobile phase flow rate
• different paths that solute molecules travel through stationary
phase
Rate Theory – Van Deemter
•
Eddy Diffusion (A)
✓ Solute molecules take random, different paths through
the stationary phase.
• May cause broadening of
solute band.
✓ Minimise eddy diffusion by :
• Selecting well packed columns
• Using smaller stationary phase particles
• Using particles with a narrow size
distribution
Longitudinal Diffusion (B)
✓ Molecules disperse in all directions because of concentration
gradient (less at the edges of the band than at the center).
• Analyte diffuses out from the center to the edges
• Bigger problem at low flow rate
• BUT high flow rate may result in
no equilibrium.
✓ Minimise by:
• Using higher mobile phase flow rates
• Keep system tubing short and as a narrow as possible

Mass Transfer (C)
✓ Equilibrium of the analyte between the phases takes time.
✓ The time “allowed” for this interaction depends on the
velocity of the mobile phase.
✓ Too high flow rate – analyte in mobile phase will move
ahead of analyte in the stationary phase.
✓ This results in band broadening.
Mass Transfer (C)
✓ Minimise mass transfer effects:
• Using smaller (diameter) stationary phase particles
• Using lower mobile phase flow rates
• Heating the column (at higher temperatures the diffusion
processes are speeded up and the differences in elution time

from the particle pore are reduced)
** For an open tubular column A = 0
• Widely used to calculate mobile flow rate
Effect of particle size
Smaller particle sizes yield higher overall peak efficiencies

and a much wider range of usable flow rates
Effect of particle size
✓The A term of the van Deemter equation is smaller with
smaller particles, because the differences in path lengths is

less dramatic.
• This results in the curve being shifted downward (to lower H).
✓The C term is smaller because the equilibrium between the
mobile and stationary phases is more efficient.
• This results in a shallower slope for the curve at higher values
of u.
Effect of carrier gas
✓ Nitrogen has the lowest minimum but its
minimum value has steep rise on both sides.
✓ Helium shows of flatter minimum, more resistant

to resolution changes over a wider flow velocity
range.
✓ Hydrogen has the flatest minimum spread over

a range of velocities, thus its resolution is highly
stable over a wide range of flow velocities.
Nitrogen has the lowest minimum but is accompanied
with steep rise on both sides of the minimum value. It
has better separation efficiency at its minimum but the
efficiency of separation gets reduced significantly on
either side with changes in flow velocities. 12cm /s
Helium shows of flatter minimum and an improved

resistance to changes in resolution over a broader
range than nitrogen. However, its use is limited by its
availability and higher cost in comparison to
nitrogen.20cm /s minimum flowrate
Hydrogen has the flatest minimum spread over a range

of velocities, ie, its resolution is stable over a range of
flow velocities around the optimum value. 40cm /s

Basic Chromatography

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Chromatography

Chromatogram - Detector signal vs. retention

Textbook: Skoog et al.

Mobile phase: a phase that flows through the stationery

Stationary phase: a phase that is fixed in place in a

Column: a solid surface on which the stationary phase is

The mobile phase is then forced through a stationary phase

The analytes then have the opportunity to interact with the

Stationary phase: adsorbent filled in a column.

Mobile phase: various solvents.

Stationary phase: adsorbent, mostly silica or aluminum

Mobile phase: various organic solvents or mixture of

Stationary phase: thin layer of water adsorbed on the

Mobile phase: solvent, partly miscible or immiscible with

Stationary phase: cation or anion exchangers.

Mobile phase: aqueous solution of ionic compounds.

Stationary phase: grains of a swelling three-

Mobile phase: solution of components to be

Stationary phase: adsorbent or wetted carrier

Mobile phase: inert gas (nitrogen, hydrogen, helium)

Stationary phase: liquid

Mobile phase: liquid

t0 or tM – death time = retention of compound with no

Large N indicates good column efficiency (should be ≈ 10000

L = column length, N = number of plates

The smaller / lower H, the better column efficiency

2) Diffusion coefficient in mobile phase

3) Diffusion coefficient in stationery phase

5) Diameter of packing particles

6) Thickness of liquid coating

Calculate the number of plates and plate heights.

N = 16( 5 min/0.5 min) 2 = 1600

H = 15.0 m/ 1600 = 0.0094 m = 9.4 mm

✓ Estimates the ratio of the retained to unretained compounds

✓ k’ < 1: analyte elutes too quickly. Difficult to quantify accurately

✓ k’ > 5: compounds will take longer to elute - long run

To manipulate k’ – change temperature, stationary phase,

✓ Larger α means better separation

✓ The separation of two components is only possible if α >1

✓ To manipulate α - change mobile phase and stationery

maxima to the average of the peak width at the base line

Rs = 1.5 full separation, <1.5 incomplete separation

To manipulate Rs: change column temperature, mobile phase,

Effect of resolution on retention time

✓Plate theory does not take into account:

✓Rate theory takes into account

the stationary phase.

• May cause broadening of

✓ Minimise eddy diffusion by :

• Selecting well packed columns

• Using smaller stationary phase particles

• Using particles with a narrow size

gradient (less at the edges of the band than at the center).

• Analyte diffuses out from the center to the edges

• Bigger problem at low flow rate

• BUT high flow rate may result in

• Using higher mobile phase flow rates

• Keep system tubing short and as a narrow as possible