Chapter 4

AN INTRODUCTION TO CHROMATOGRPHIC
SEPARATION

Lesson Outcomes:
Identify and explain the different types of chromatographic

analysis
Explain the principles of gas chromatography, high
performance liquid chromatography, ion exchange and size
exclusion and etc.
Interpret analytical information from chromatographic data
Recognize problems arising from resultant spectrum and
chromatograms and suggest probable solutions
2

4.1 A General Description of

Chromatography

 Is a technique used to separate and identify the components

of a mixture
Works by allowing the molecules present in the mixture to
distribute themselves between a mobile and a stationary
phase

mobile phase = solvent or gas

stationary phase = column packing material

How separation occur?

Chromatography used to separate and identify the

components of complex mixtures

Works by allowing the molecules in the mixture to distribute
themselves between a stationary and a mobile phase
Components that are strongly retained by the stationary
phase move slowly with the flow of mobile phase
In contrast, components that are weakly held by the
stationary phase travel rapidly (fast)
As a consequence of these differences in mobility, sample
components separate into discrete bands that can be
analyzed qualitatively and/or quantitatively

Classification of chromatographic methods

1.

Based on physical means

- The way stationary and mobile phases are brought
into contact.

2.

Based on the types of mobile phase

- Either gas, liquid or supercritical fluid.

3.

Based on the kinds of equilibria involved in the in

solute transfer between the phases
-Interaction of analyte between stationary and
mobile phases.

Chromatographic Methods based on

physical means
Column Chromatography
stationary phase is held
in narrow tube; mobile
phase moves by
pressure or gravity
Example:
Gas chromatography
(GC), Supercritical-fluid
chromatography (SFC)
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Planar Chromatography
stationary phase is
supported on a flat plate or
in the interstices of a paper;
mobile phase moves
through capillary action or
gravity
Example:
Thin-layer chromatography
(TLC), Paper
chromatography (PC)

Column Chromatography
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Planar Chromatography

Chromatography based on types of mobile

phase
Column chromatography can further be differentiated
based on the types of mobile phase;
Gas
eg: gas Chromatography
Liquid
eg: liquid chromatography

Supercritical fluid
eg: supercritical fluid chromatography

Gas Chromatography

10

Liquid Chromatography

Supercritical Fluid Chromatography

Classification of Chromatographic Methods

Chromatography

Partition

Liquidliquid

11

Gasliquid

Adsorption

Liquid-solid

Gas-solid

Ion-exchange

Liquid-solid

Size-exclusion

Liquid-solid

Types of chromatography on the basis of

interaction of the analyte with stationary
phase
Adsorption for polar non-ionic compounds
Size Exclusion stationary phase is a porous matrix sieving

Ion Exchange for ionic compounds

- Anion analyte is anion; bonded phase has positive charge

- Cation analyte is cation; bonded phase has negative charge
Partition based on the relative solubility of analyte in
mobile and stationary phases
- Normal stationary phase polar, the mobile phase nonpolar
- Reverse stationary phase nonpolar, the mobile phase polar
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Chromatography based on interaction of

the analyte with stationary phase

4.

13

Size Exclusion separate molecules by size;

sieving- stationary phase is a porous matrix

Adsorption Chromatography

14

Adsorption - of solute on surface of stationary

phase; for polar non-ionic compounds
Components of the mixture selectively adsorb (stick)
on the surface of a finely divided solid stationary
phase.
As mobile phase (gas/liquid) carries the mixture
through the stationary phase, the components of the
mixture stick to its surface with varying degrees of
strength and thus separate.
Stationary phase: solid
Mobile phase: gas/liquid

TLC Plates

15

Partition Chromatography
Partitioning is a distribution (by dissolving) of the

components between 2 immiscible phases.

Separations of the components will be based on
relative solubility of the components in the mobile
and stationary phase
Partition - based on the relative solubility of
analyte in mobile and stationary phases
a. Normal phase stationary phase polar, the
mobile phase nonpolar
b. Reverse phase stationary phase nonpolar, the
mobile phase polar
16

 Example of partitioning using polar stationary phase.

i. Polar components will retain longer than the non-

polar components
ii. Non-polar components will move quickly through
stationary phase and will elute first before the
polar components and vice-versa
The stationary phase actually consists of a thin film
adsorbed (stuck) on or chemically bonded to the surface
of a finely divided solid particles
If the mobile phase is gas, the volatility (vapor pressure)
and solubility in stationary phase plays an important role

17

18

Ion Exchange Chromatography

Method for separating mixture of ions
Ion Exchange - attraction of ions of opposite charges; for

ionic compounds
i.
Anion - analyte is anion; bonded phase has positive
charge
ii. Cation analyte is cation; bonded phase has negative
charge
Sample is aqueous solution of inorganic ions or organic
ions
Stationary phase are small polymer resin beads usually
packed in a glass tube.
i.
These beads have ionic bonding sites on their surfaces
which selectively exchange ions with certain mobile
phase compositions as the mobile phase penetrates
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through it.

 Ions that bond to the charged site on the resin bead

20

are separated from organic or inorganic ions

aqueous solution.
The process is repeated several times by changing of
the mobile phase composition
The process begin with initially running the analysis
using a mobile phase with all the ions in the mixture.
The mobile phase is then change for several times in
a stepwise fashion so that one kind of ion at a time is
removed.
The process is repeated until complete separation
achieved.

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22

Size Exclusion Chromatography

Size Exclusion separate molecules by size;

sieving- stationary phase is a porous matrix

Also called gel permeation chromatography.
Separation technique of dissolved species is based
on the size of the components.
Stationary phase: porous polymer resin particles
(molecular sieves).
The components to be separated enter the pores
of these particles and are slowed down from
progressing through this stationary phase.
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 Separation depends on the sizes of the pores

relative to the sizes of the molecules to be

separated.
Small particles are retarded to a greater extent
than large particles (some of which may not
enter the pores at all) and separation occurs.
Particles with size bigger than the pore size will
be eluted first from the column

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25

Terminologies in Chromatography
Elution: A process in which species are washed through

a chromatographic column by addition of fresh solvent

Mobile phase: Is one that moves over or through an
immobilized phase that is fixed in place in a column or
on the surface of flat plate
Stationary phase: A solid or liquid that is fixed in place.
A mobile phase then passes over or through the
stationary phase
Retention time: Time required for the sample to travel
from the injection port through the column to the
26 detector

4.2 Migration Rates of Solutes

27

4.2.1 Distribution Constant

An analyte is in equilibrium between 2 phases;

Amobile Astationary
The equilibrium constant, K (partition constant) is
the molar concentration of analyte in the stationary
phase divided by the molar concentration of the
analyte in the mobile phase
[ A]stationary
K
[ A]mobile
4.2.2 Retention Factor
Retention factor, k (capacity factor) is used to

describe the migration rate of an analyte on a

tR tM
column
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k' A

tM

4.2.3 Retention Time

Retention time (tR) is the time for the analyte to pass
through the column of the time between the sample
injection and an anlyte peak reaching a detector at
the end of the column

29

4.2.4 Dead Time

Dead time (tM)is the time a non-retained compound

spends in the mobile phase (the amount of time the

non-retained compound spend in the column.
tM can be expressed as the time required for an
average molecule of the mobile phase to pass
through the column

4.2.5 Adjusted Retention Time

The adjusted retention time (tR) is the time a
compound spends in the stationary phase
tR is the difference between the retention time (tR)
and the dead time (tM) for a compound
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tR ' tR tM

4.2.6 Selectivity Factor

Selectivity factor ) is the ratio of the capacity

factors of two peaks which describes the separation

of two species (A and B on the column)
The selectivity is always greater than one (>1)
If the selectivity equals to one (=1), the
compounds cannot be seperated
The higher the selectivity, the more separation
between two compound or peaks
k' B

k' A
*Note that species A elutes faster than species B
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4.3 The Efficiency of

Chromatographic Column

32

Description of Column Efficiency

The efficiency is related to the number of

compounds that can be separated by the column

The efficiency is expressed as the number of
theoretical plates (N, unitless) or as the height
equivalent to a theoretical plate (HETP, milimeters)
The efficiency increases as the height of
theoretical plate decreases, thus more
compounds can be separated by the column and
vice versa
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The Theoretical Plate Model of

Chromatography
The plate model proposes that the chromatographic

column contains a large number of separate layer

(theoretical plates)
The analyte moves down the column by transfer of
equlibrated mobile phase from one plate to the next
The column

Theoretical plates

34

*Note that the plates do not really exist, theyre just figment
of imagination in order to understand the separation
process in the column

Where,
H = height of the plate
L= length of the column
N= number of theoretical plate

L
H
N

The number of theoretical plates that a real column

can be found by examining a chromatographic peak

after elution
2

tR
N 16
W

or

35

5.5tR
N 2
w1 2

Where,
W = width of the peak

Where,
w = w1/2 is the peak width at
half-height

4.4 Column Resolution

36

4.4.1 The Effect of Retention and

Selectivity Factors on Resolution
Resolution is the measure of how well species

have been separated

2[(tR )B ( tR )A]
WA WB

Where,
Z = separation btwn peak A and B
WA and WB = widths at the base of peaks A and B

37

 Acceptable resolution is Rs=1.0 and the best resolution

between two peaks requires Rs>1.5

To relate with the number of plates (N), the selectivity
factor () and the retention factors (k) of the two
solutes;

N
R
4

1 k ' B

1 k ' B

Simplified: Rs = N

38

Rs values of less than 1.0 are considered unresolved

peaks

39

4.4.2The Effect of Resolution on

Retention Time
Relationship btw the resolution of a column and

retention time

16 R H (1 k ' B )3
(tR ) B

1 (k ' B ) 2
2
S

Where,
= velocity of the mobile phase

Simplified: tR = Rs2
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Example:
17.63 min

16.40 min
1.30 min

Length of column: 30 cm
Peak widths:
A = 1.11 min
B = 1.21 min

Calculate:
i) column resolution, Rs
ii) the average number of plates, N
iii) the average plate height, H
iV) length of column to achieve Rs 1.5
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Answer:
i)

2[(tR )B (tR )A]

Rs
WA WB

ii)

tR
N 16
W

iii)

L
H
N

iv) (Rs)1 = N1
(Rs)2
N2
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= 1.06

= 3.44 x 103

= 8.7 x 10-3 cm

= 60 cm

Variables Affecting Column Efficiency

1.
2.

3.
4.

43

Mobile phase flow rate

Particle size
Diameter of column
Film thickness

Effect of Mobile Phase Flow

From both the plots for LC and GC, we can see that

both show a minimum in H at low linear flow rates

H increases as the mobile phase flow rate
increases
44

Effect of Particle Size

Effect of particle size on plate height for a packed GC column

The numbers to the right is the packing particle

45

diameters
The smaller the particle size, the more pressure is
needed to push the mobile phase through the
column
H increases as the particle size increases

Effect of Diameter of The Column

For packed column, the most important variable

that affect column efficiency is the diameter of the

particles that making up the packing
The increase in column diameter will increase the
plate height

46

Effect of Film Thickness

With thick films makes the analyte molecule travel

slower. As a result, thick films will increase in plate

height

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4.5 Applications of Chromatography

48

Qualitative and Quantitative Analysis

25.6 min

Retention time tell as about

compound identity =
qualitative
Peak Area or height tell us
how much of compound is
there = quantitative

49

Qualitative Analysis
Based on retention time

- Provided the sample produce the peak at the

same retention time as a standard under
identical conditions

50

Same compound
due to the same tR

51

Quantitative Analysis
1. Analysis based on Peak Height

52

The height of chromatographic peak is obtained

by connecting the base lines on either side of
the peak by a straight line and measuring the
perpendicular distance from this line to the
peak.
Suitable if the peak is very narrow and have
uniform shapes or when peak area values are
not available.

2. Analysis based on Peak Area

Peak areas are usually the preferred method of

quantitation since peak areas are independent of
broadening effects.
Most modern chromatographic instruments are
equipped with computer or digital electronic integrator
that permit precise estimation of peak areas.

A = peak height (h) x w1/2

w1/2 = width at height

53

3. Calibration method (also known as external method)

54

Involve preparation of series of standard solutions

that approximate the composition of the unknown.
The peak heights or areas are plotted as a function
of concentration.
The concentration of the component(s) to be
analysed is determined by comparing the
response(s) (peak(s)) obtained with the standard
solutions.

4. Internal Standard Method

Internal standards are widely used in chromatography

because the small quantity of sample solution injected

into the chromatograph is not very reproducible in
some experiments.
By knowing the concentration of the standard
compound and its relative response, the
concentration of analyte can be determined.
Internal standards are also desirable when sample loss
can occur during sample preparation steps prior to
analysis.

55

Calibration involves plotting the ratio of the analyte

signal to the internal-standard signal (Area ratio or
height ratio) as a function of the analyte
concentration of the standards
Signal from analyte is compared with signal from the
internal standard to find out how much analyte is
present
Area Ratio =

Peak Area/Height Analyte

Peak Area/Height internal standard

Area or
height ratio
Concentration of analyte in sample
56

Concentration of analyte in standard solution

5.

Analysis based on Peak Area using Response Factor

Method
A measure of relative mass spectral respond of an
analyte compare to each internal standard
The response factor for compound X can be
calculated using the following equation:
Response factor =

57

Peak Area of Compound X

Concentration of Compound X

 Example:

58

An injection containing benzene at a concentration of

2000 g/mL is made and results in a peak area of
100,000. An injection of the sample with the unknown
concentration of benzene has a peak area of 57,000.
Calculate the concentration of benzene present in the
sample.
Answer:
Step 1: Calculate the Response Factor
Response Factor for benzene = 100000 = 50
2000
Step 2:
Concentration of benzene in sample = 57000 = 1140 g/mL
50

6.

Area Normalization Method

In the normalization method, the areas of all
eluted peaks is normalized
The percentage content of one or more
components of the substance to be examined is
calculated by determining the area of the peak(s)
as a percentage of the total area of all the peaks,
excluding those due to solvents or any added
reagents and those below the disregard limit
Content of a component in the sample, C:
Ci = Ai x 100
AT
Ai = Area of component peak in the chromatogram
AT = Total area of the peaks in the chromatogram

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 Example:

A mixture contains only three compounds, X, Y and Z. An

injection of 1 L of the mixture resulted a
chromatogram with peak areas of 232, 650 and 984
arbitrary units. Calculate the percentage of each
compound.
Answer :
By assuming the response factor of the three
compounds is identical, the total peak area and the
percentage of each compound can be calculated.
Sample calculation:
Compound
Peak
Composition
Area
(%)
232 x 100 = 12.43%
X
232
12.43
1866
Y
650
34.83
60

984

52.73

TOTAL

1866

100

Tailing and Fronting

A common cause of tailing and fronting is a

distribution constant that varies with

concentration.
Fronting also arises when the amount of sample
introduced onto a column is too large.

61

 Band broadening can be reduced by using:

1. Smaller particle
2. Narrower columns

3. Lower temperature (GC)

4. Thinner liquid stationary phase

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