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

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 — 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

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

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Supercritical Fluid Chromatography
 Classification of Chromatographic
Methods

Chromatography

Partition Adsorption Ion-exchange Size-exclusion

Liquid- Gas- Liquid-solid Gas-solid Liquid-solid Liquid-solid

liquid liquid

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 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. Size Exclusion – separate molecules by size;

sieving- stationary phase is a porous matrix

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 Adsorption Chromatography
— 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
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 TLC Plates

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 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

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 — 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
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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
19 penetrates through it.
 — Ions that bond to the charged site on the resin
bead 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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21
22
 Size Exclusion Chromatography
— Size Exclusion – separate molecules by size;
sieving- stationary phase is a porous matrix
— Example: Gel filtration and 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
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to the detector
 4.2 Migration Rates of Solutes

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 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
column tR - tM
k' A =
28
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
30 tR ' = tR - tM
 4.2.6 Selectivity Factor
— Selectivity factor α) 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 (α>1)
— If the selectivity equals to one (α=1), the
compounds cannot be seperated
— The higher the selectivity, the more separation
between two compound or peaks
k' B
a=
k' A
*Note that species A elutes faster than species B
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 4.3 The Efficiency of
Chromatographic Column

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 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
*Note that the plates do not really exist, they’re just figment
of imagination in order to understand the separation
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process in the column
 Where,
L H = height of the plate
H= L= length of the column
N N= number of theoretical plate

— The number of theoretical plates that a real column

can be found by examining a chromatographic peak
after elution 2
æ tR ö Where,
N = 16ç ÷ W = width of the peak
èW ø
or 2
5.5tR Where,
N= 2 w = w1/2 is the peak width at
w1 2 half-height
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 4.4 Column Resolution

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 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 ] ∆Z
Rs =
WA + WB
Where,
ΔZ = separation btwn peak A and B
WA and WB = widths at the base of peaks A and B

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 — 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 (σ) and the retention factors (k’) of
the two solutes;
N æ a - 1 öæ k ' B ö
R= ç ÷ç ÷
4 è a øè 1 + k ' B ø

Simplified: Rs = √ N

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 Rs values of less than 1.0 are considered unresolved
peaks

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 4.4.2The Effect of Resolution on
Retention Time
— Relationship btw the resolution of a column and
retention time
2
16 R H æ a ö (1 + k ' B ) 3
2
(tR ) B = S
ç ÷
u è a - 1 ø (k ' B ) 2

Where,
Ʋ = velocity of the mobile phase

Simplified: tR = Rs2

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 Example:
17.63 min

16.40 min
Length of column: 30 cm
Peak widths:
1.30 min 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 ] = 1.06
Rs =
WA + WB
2
ii) æ tR ö
N = 16ç ÷ = 3.44 x 103
èW ø

iii) L = 8.7 x 10-3 cm

H=
N

iv) (Rs)1 = √N1 = 60 cm

(Rs)2 √N2
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 Efficeiency….
— Determines the number of compounds that can
be separated by the column
— The narrower the peak, the more efficient it is
— The more broad the band, the less efficient it is
— The more plates in the column, the more efficient
it is

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 Variables Affecting Column
Efficiency

1. Mobile phase flow rate

2. Particle size
3. Diameter of column
4. Film thickness

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 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
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 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

diameters
— The smaller the particle size, the more pressure is
needed to push the mobile phase through the
column
46 — 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

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 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

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 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

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 Qualitative Analysis
— Based on retention time
- Provided the sample produce the peak at the
same retention time as a standard under
identical conditions

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 Same compound
due to the same tR

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 Quantitative Analysis
1. Analysis based on Peak Height
— 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.

53
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 ½ height

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 3. Calibration method (also known as external method)
— 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.

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 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.

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 — 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

57 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 = Peak Area of Compound X

Concentration of Compound X

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 — Example:
An injection containing benzene at a concentration of
2000 µg/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 µg/mL
59 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 µL 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
232 x 100 = 12.43% Area (%)
X 232 12.43
1866 Y 650 34.83
Z 984 52.73
61 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.

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 — 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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