CHM260

CHAPTER 4:
AN INTRODUCTION TO
CHROMATOGRAPHIC
SEPARATIONS
 SUBTOPICS
 A general description of chromatography
- Principles of chromatographic separations
- Classification of chromatographic methods
 Terminologies in chromatography
 Migration rates of solutes
 Band broadening and column efficiency
- tailing and fronting of chromatographic peaks
- quantitative description of column efficiency
- variables that affect column efficiency
 Column resolution, Rs
 Applications of chromatography
- qualitative
- quantitative
 A GENERAL DESCRIPTION OF CHROMATOGRAPHY

 Definition of chromatography: A physical method of

separation in which the components to be separated
are distributed between two phase, one of which is
stationary phase, while the other (the mobile phase)
moves in a definite direction.
 This method is used to separate and identify the
components of complex mixtures.
 The sample is dissolved in mobile phase such as gas,
liquid or supercritical fluid.
 This mobile phase is then forced through an
immiscible stationary phase, which is fixed in place in
a column or an solid surface.
 Those 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.

 As a consequence of these differences in mobility,

sample components separate into discrete bands or
zones that can be analyzed qualitatively and or
quantitatively.

 Plot: Chromatogram
 Principles of chromatographic separations
Sample Mobile phase

Component A
Stationary
Component B
phase
Component C

 A small volume of sample is placed at the top of the column (filled with
stationary phase and solvent).
 Sample is eluted with mobile phase.
 The individual components interact with the stationary and mobile
phase to different degrees.
 Component A interact more strongly to the stationary phase than
component B and component C.
 Then, as the mixture travels down the column, component A will be
retarded with respect to the component B and C.
 In time, they will separate into bands, and be eluted at different times.
a) Diagram showing the
separation of a
mixture of
components A and B
by column elution
chromatography

b) The output of the

signal detector at the
various stages of
elution
 Classification of Chromatographic Methods
 Can be categorized based on the followings:
1. Based on physical means
 The way stationary and mobile phase 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 solute transfer between the phases
 Interaction of analyte between stationary and
mobile phases
 Based on physical means
Column chromatography
 Stationary phases is held in narrow tube.
 Mobile phase moves by pressure or gravity.
 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.
 Examples: thin-layer chromatography (TLC), paper
chromatography (PC)
 Based on the types of mobile phase

Mobile Phase

Gas
Example: Gas Supercritical fluid
Chromatography Liquid Example:
Example: Liquid Supercritical-
Chromatography fluid
Chromatography
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
 Chromatography

Partition Adsorption Ion-exchange Size-exclusion

Liquid- Liquid-solid Liquid-solid Liquid-solid

liquid Gas-liquid
Gas-solid
Classification of Column Chromatographic Methods
 Partition Chromatography
 The stationary phase is a liquid supported on an inert
solid.
 Mobile phase: liquid (liquid-liquid partition
chromatography) or gas (gas-liquid chromatography-
GLC).
 In normal-phase chromatography of liquid-liquid
chromatography: A polar stationary phase (e.g., methanol
on silica gel) is used with a nonpolar mobile phase (e.g.,
hexane). Favors retention of polar compounds and elution
of nonpolar compounds.
 In reversed-phase chromatography of liquid-liquid
chromatography: A nonpolar stationary phase is used with a
polar mobile phase. Nonpolar solutes are retained more
and polar solutes more readily eluted.
 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.
 Adsorption Chromatography
 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 the surface of it with varying
degrees of strength and thus separate.
 Example: Thin-layer chromatography (TLC),
stationary phase is plane (a solid supported on an
inert plate), mobile phase is liquid.
TLC PLATES
 Ion-exchange Chromatography
 Method for separating mixture of ions.
 Sample: aqueous solution of inorganic ions /
organic ions.
 Mechanism of separation: based on ion exchange
equilibria.
 Stationary phase – small polymer resin “beads”
usually packed in a glass tube
- These beads have ionic bonding sites on their
surfaces which selectively exchange ions with
certain mobile phase compositions as the mobile
phase penetrates through it.
 Size-exclusion Chromatography
 Also called molecular or gel chromatography.
 Technique for separating dissolved species on the
basis of their size.
 Stationary phase: porous polymer resin particles
(molecular sieves).
 Small particles are retarded to a greater extent than
large particles (some of which may not enter the
pores at all) and separation occurs.
 Types of size exclusion chromatography
• Gel filtration chromatography:
- mobile phase (eluent) is water, the packing is
hydrophilic.
- used to separate high molecular weight polar
(water-soluble) species such as protein.

• Gel permeation chromatography:

- mobile phase is on organic solvent, the packing is
hydrophobic.
- used to separate high molecular weight non-polar
(water-insoluble) species such as polymer.
 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:
- is the time interval between its injection onto a column
and the appearance of its peak at the other end of the
column.
 Migration rates of solutes

 Distribution constant, K

 Retention time, tR

 Capacity factor, k’

 Selectivity factor, 
 Distribution constant, K
 When the elusion process occured, analyte can be in the
stationary phase (adsorb) when it do not move or in the mobile
phase when it eluted through the column.
 These two condition can achieve equilibrium.
 Let say, we have analyte A. The distribution equilibrium is
written as:
A mobile  A stationary
 Therefore, the equilibrium constant K is called distribution
constant and is defined as:
c stationary
K = c mobile
c = Molar concentration
 K is also called partition coefficient or partition ratio.
 Retention Time, tR
 Time required for the sample to travel from the injection
part through the column to the detector.

• A typical chromatogram for a two-component mixture. The

small peak on the left represents a species that is not
retained on the column and so reaches the detector almost
immediately after elution is started.
 tM
- Time taken for the unretained species to reach the
detector.
- Sometimes called dead time.
- Rate of migration of the unretained species is SAME
as the average rate of motion of mobile phase
molecules.
- tM can be expressed as the time required for an
average molecule of the mobile phase to pass through
the column.
 Retention Factor (Capacity factor), k’
 Term used to measure the migration rates of analytes on columns.
 A measure of how long a sample is retained with respect to the
dead volume.

k’A = KA VS
VM) [unitless] for analyte A

Vs = volume stationary phase

VM= volume mobile phase

 How is k’A related to tR and tM?

k’A =
tR – tM
tM
When k’A is  1.0, separation is poor
When k’A is > 30, separation is slow
When k’A is 2-10, separation is optimum
 Selectivity Factor, 

 is defined as:  = KB distribution constants

KA
= k’B capacity factors
k’A
= tR(B) – tM
tR(A) – tM retention times

 A measure of the relative migration rates of species A

and B with a stationary phase material in
chromatography
Response
tR

tR

tM

1 3 6
Retention time , min

tR – tM tR(B) – tM
k’ =  =
tM tR(A) – tM
 Band Broadening and Column Efficiency

 Band broadening reflects a loss of column efficiency.

 The slower the rate of mass-transfer processes
occuring while a solute migrates through a column,
the broader the band at the column exit.
 Some of the variables that affect mass-transfer rates
are controllable and can be exploited to improve
separations.
  TAILING AND FRONTING OF CHROMATOGRAPHIC PEAKS
 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.
 Poor separations and less reproducible elution times.
 QUANTITATIVE DESCRIPTION OF COLUMN EFFICIENCY
• Column efficiency is expressed in terms of plate height (H)
and plate count/ the number of theoretical plates (N).
• The relationship between H and N is:
Column length
N = L
Number of H Plate height
theoretical
plates

 The efficiency of chromatographic columns increases as

the number of plates (N) becomes greater and plate height
(H) become smaller.

** Efficient column has small plate height (H)

 The Theoretical Plate Model of Chromatography

• The plate model: the chromatographic column is contains a

large number of separate layers, called theoretical plates.
• Separate equilibrations of the sample between the stationary
and mobile phase occur in these "plates".
• The analyte moves down the column by transfer of equilibrated
mobile phase from one plate to the next.
• The plates DO NOT really exist.
- The plates are a figment of the imagination to understand the
processes in the column.
- A way of measuring column efficiency.
 Experimentally, H and N can be approximated
calculated from the width of the base (W or Wb) of
the chromatographic peak.

The equation:

tR 2
N = 16 W

 N can be calculated using tR

and W
 To obtain H, the length of
the column must be known
 Another method for approximating N is to
determine W½, the width of the peak at half its
maximum height.

N = 5.54 tR 2
W½
 VARIABLES THAT AFFECT COLUMN EFFICIENCY

 The efficiency of a chromatographic column is

affected by the amount band broadening that occurs
as a compound passes through the column.
 VARIABLES AFFECTING COLUMN EFFICIENCY

* Increases as temperature increases and viscosity decreases.

** In liquid, DM values for small and medium-size molecules are on the
order of 10-5 cm2s-1; for gases, DM values are ~105 times larger
 VARIABLES AFFECTING
COLUMN EFFICIENCY

Mobile phase Diameter of Film

Particle size
flow rate column thickness
 EFFECT OF MOBILE PHASE FLOW RATE ON
PLATE HEIGHT

 From both the plots for LC and GC, we can see that
both show a minimum in H at low linear flow rates.
*** Flow rate of mobile phase directly proportional to the
plate height (H)
 EFFECT OF PARTICLE SIZE ON PLATE HEIGHT
 The smaller the particle size, the more uniform the column
packing, then the more tolerant to the change in mobile-phase
velocity.
Plate height H, cm

Linear, velocity, cm/s

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

The numbers to the right of each curve are particle diameters

*** Particle size (dp) directly proportional to the plate height (H)
EFFECT OF DIAMETER OF THE COLUMN ON PLATE HEIGHT

 For packed column, the most important variables

that affect column efficiency is the diameter of the
particles that making up the packing.
EFFECT OF DIAMETER OF THE COLUMN ON PLATE HEIGHT
 While for open tubular column, the diameter of the
column itself is an important variables.

 Refer to table 26-3, the mobile phase mass-transfer

coefficient CM is known to be inversely proportional
to the diffusion coefficient of the analyte in the mobile
phase, DM.
 CM is proportional to the square of the particle
diameter of the packing material, d2p (packed
column).

 CM is proportional to the square of the column

diameter, d2c (open tubular column).
 As a conclusion, the bigger the column diameter, the
smaller the diffusion coefficient DM. Therefore, we can
say that increase in column diameter will increase the
plate height (column is not efficient).
** Efficient column:
small plate height (H), DM, CM, dP , dc
 EFFECT OF FILM THICKNESS ON PLATE HEIGHT
 When stationary phase is an immobilized liquid, the mass-
transfer coefficient Cs is directly proportional to the square of
the thickness of the film on the support particles , d2f and
inversely proportional to the diffusion coefficient, Ds of the
solute in the film.

 With thick films, molecules must on average travel farther to

reach surface, and with smaller diffusion coefficient (Ds),
analyte molecule travel slower. As a result, slower rate of mass-
transfer and an increase in plate height (column is less
efficient).
*** thickness of film (df) directly proportional to the H
 SUMMARY OF EFFECTS OF VARIABLES TO
COLUMN EFFICIENCY
 Flow rate of mobile phase directly proportional to plate
height (H).
 For packed column, particle size on plate height (dp)
directly proportional to plate height (H).
 For open tubular column, diameter of column (dc)
directly proportional to plate height (H).
 Thickness of film (df) directly proportional to plate height
(H).

*** COLUMN IS EFFICIENT IF PLATE HEIGHT (H) IS

SMALL
 METHODS FOR REDUCING BAND BROADENING OR TO
IMPROVE THE EFFICIENCY OF A CHROMATOGRAPHIC
COLUMN
 For packed column, the diameter of the particles (dp)
must be as small as possible.
 For open tubular column, the diameter of column should
be small and narrow.
 Liquid stationary phase, the thickness of the layer of
adsorbed liquid should be minimized because the mass-
transfer coefficient Cs is directly proportional to the
square of the thickness of the film on the support
particles , d2f
 Increase the length of column (for GC).
 Column resolution, Rs

 A measure of the separation of two chromatographic

peaks.

 Baseline resolution is achieved when Rs = 1.5

Rs = 2[tR(B) – tR(A)]
WA + WB
 Effect of Capacity Factor & Selectivity Factor on
Resolution

 Relationship between the resolution of a column and

the capacity factor k’, selectivity factor  and the
number of plates N is given by this equation:

Rs = √N  - 1 k’
4  1 + k’

Simplified: Rs = √N
 Effect Resolution on Retention Time
 Relationship between the resolution of a column and
retention time:

2
tR = 16Rs2H  ( 1 + k’)3
u -1 (k’)2

Simplified: tR = Rs2
 The general elution problems
• A single set of conditions is often unsatisfactory for
good separation of all components in a complex
mixture in an acceptable time (wide range of k values).

The general
elution problem in
chromatography
 Based on the chromatograms:
i) Chromatogram (a):
- all components separated but time is long and peak
widths are broad at long times
ii) Chromatogram (b):
- a faster separation and good peak shapes for 5, 6 but 1, 2
and 3,4 are not resolved
iii) Chromatogram (c):
- intermediate case where peaks 1, 2 still not resolved

 Common solution: change separation conditions

dynamically – e.g. temperature gradient, solvent gradient
 Methods to improve resolution
1. Optimizing k (capacity factor)
i) change the temperature (in Gas Chromatography)
ii) change the composition of the mobile phase (in Liquid
Chromatography)

100 °C

150 °C

200 °C
 Effect of solvent variation on chromatograms.
Analytes: (1) 9,10-anthraquinone; (2) 2-methyl-9,10-anthraquinone; (3) 2-
ethyl-9,10-anthraquinone; (4) 1,4-dimethyl-9,10-anthraquinone;
(5) 2-t-butyl-9,10-anthraquinone
2. Increase  (selective factor) while maintaining k in the range of
1 to 10.
- k is optimised first, and then  is increased by one of the
following procedures:

a) Changing mobile phase composition

- Example: separation of anisole and benzene.
i) Mobile phase a 50% mixture of water and methanol,
k=4.5 for anisole, k=4.7 for benzene,  = 4.7/4.5 = 1.04.
- peaks were overlap
ii) Substitution of an aqueous mobile phase
containing 37% tetrahydrofuran, k=3.9 for anisole,
k=4.7 for benzene,  = 4.7/3.9 = 1.20
b) Changing column temperature
c) Changing composition of stationary phase
d) Use special chemical effects
- incorporate into the stationary phase a species
that complexes or interacts with one or more
components in the sample.
- Example: an adsorbent impregnated with a
silver salt to improve the separation of olefins.
Complexes between the silver ions and
unsaturated organic compounds was formed
thus improve the results.
 Example

 Length of column: 30 cm
 Peak widths (at base) for A & B were 1.11 & 1.21
min respectively.
 Calculate:
i) column resolution, Rs
ii) the average number of plates, N
iii) the plate height, H
iv) length of column to achieve Rs 1.5
 17.63 min
Response
tR

16.40 min
tR

1.30 min
tM

1 3 6
Retention time , min
i) Rs = 2[tR(B) – tR(A)]
WA + WB
Rs = 2(17.63 min – 16.40 min)
(1.11 min + 1.21 min)
= 1.06

ii) 2
N = 16 tR
W

NA = 16 16.40 min 2
1.11 min
= 3.49 x 103
NB = 16 17.63 min 2
1.21 min
= 3.40 x 103
 Therefore, calculate the N average
N average = NA + NB
2
Naverage = 3.44 x 103

iii) H = L / N
= 30 cm / 3.44 x 103 = 8.7 x 10-3 cm
iv) (Rs)1 = √N1
(Rs)2 √N2

1.06 = √ 3.44 x 103

1.5 √N2
N2 = 6.9 x 103

L = N x H
= 6.9 x 103 x 8.7 x 10-3
= 60 cm
 Applications of Chromatography

Qualitative Analysis
 Qualitative Analysis
 Based on retention time
Identification of analyte can be determined by
comparing its retention time, tR with the retention
time of standard under identical conditions (using
same column and same separation method).
 Same compound
due to the same tR
Quantitative Analysis
 GC chromatogram

Abundance

Time

 Area of the each peak in the chromatogram

corresponds to the amount/concentration of the
compound
 Quantitative Analysis
 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.

Height
 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.
w1/2

A = peak height (h) x w1/2

w1/2 = width at ½ height
 Area Normalization Method
 Determine the relative composition of a sample.
 In the normalization method, the areas of all eluted peaks
are computed.
 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

 Detector response is assumed to be the same for different

components.
 Components of the sample injected are assumed to be all
detected and to produce peaks.
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.

Solution:
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 Area Composition
232 x 100 = 12.43% (%)
X 232 12.43
1866
Y 650 34.83
Z 984 52.73
TOTAL 1866 100
 Calibration Method (also known as external method)
- The standard sample and sample are analyzed separately
and the resulting peaks are compared.
- Both analyses must be performed under identical
conditions.
- The peak heights or areas are plotted as a function of
concentration.
- The concentration of the component(s) to be analyzed is
determined by comparing the response(s) peak(s)
obtained with the standard solutions.

Area or
height
Concentration of analyte in sample

Concentration of analyte in standard solution

 Internal Standard Method
- Equal amounts of an internal standard substance is
introduced into each standard and sample.
- Determine concentrations of sample components
using the concentrations of the internal standard and
its response relative to the target compounds.
- Useful for analyses in which the quantity of sample
analyzed or the instrument response varies slightly
from one run to another due to factors that are
difficult to control.
For example, gas or liquid flow rates that vary by a
few percent in a chromatography experiment could
change the detector response.
 Internal Standard Method
- A calibration curve is only accurate for the one set of
conditions under which it is obtained.
- 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.
 Internal Standard Method

- Criteria for the internal standard:

i) not react with the substance to be examined
ii) it must be stable
iii) must not contain impurities with a retention
time similar to that of the substance to be
examined
iv) well separated from the peaks of all other
components in the sample (Rs >1.25).
 Internal Standard Method
- 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.
Area ratio = Peak area/height analyte
Peak area/height internal standard

- This ratio for the samples is then used to obtain analyte

concentrations from a calibration curve.

Area or
height ratio
Concentration of analyte in sample

Concentration of analyte in standard solution

 Example:
 GC analysis was done to determine the concentration of compound A
in sample solution. An internal standard (IS) compound was added
into each standard solutions A and sample.
Standard solutions A

Standard 1 Standard 2 Standard 3 Standard 4

Determining an Unknown Concentration of A in sample solution

Concentration
of A in sample
Sample solution
 Example:
A 5 mL blood sample from a suspect is spiked with 0.5 mL of
aqueous 1% propanol internal standard. A 10 µL portion of
the mixture is injected into the GC and the peak areas are
recorded. Standards are treated in the same way. The
following results were obtained.
% Ethanol (wt/vol) Volume of 1% Peak area ethanol Peak area
propanol (mL) propanol
0.020 0.5 114 457
0.050 0.5 278 449
0.100 0.5 561 471
0.150 0.5 845 453
0.200 0.5 1070 447
Unknown (Blood) 0.5 782 455
Determine concentration of ethanol present in the suspect’s
blood.
Step 1: Calculate ratio peak area EtOH/PrOH
% EtOH Peak area EtOH Peak area PrOH Ratio peak area
EtOH/PrOH
0.02 114 457 0.249452954
0.05 278 449 0.619153675
0.1 561 471 1.191082803
0.15 845 453 1.865342163
0.2 1070 447 2.393736018
Unknown (Blood) 782 455 1.718681319

Step 2: Plot ratio EtOH/PrOH versus EtOH concentration

3
2.5 y = 12.029x + 0.0128
R² = 0.9988
Peak area ratio

2 Concentration of EtOH, x :
1.5 From y = 12.029x + 0.0128
1 when y = 1.718681319,
0.5 x = 0.14182022 ~ 0.142 %
0.142
0
-0.05 0 0.05 0.1 0.15 0.2 0.25
% Ethanol (wt/vol)
 Analysis based on Peak Area using Response Factor Method

• Definition: A measure of relative mass spectral respond of an

analyte compare to each internal standard.
• If the detector were equally sensitive to each component in a
mixture, the peak areas could be used directly to give the
percentage composition of the mixture by dividing the area of
each peak by the total area under all of the peaks.
• However, the detector generally has a different response to
each component.
• For examples:
i) Flame Ionization Detector (FID) in GC give response
according to the number of carbon atom in compound.
ii) Thermal Conductivity Detector (TCD) give response to
the changes in the thermal conductivity of solute.
• The response factor for compound X can be calculated using
the following equation:

Response Factor, RF = Peak area compound X

Concentration / Amount of Compound X

• Determination amount of analyte in sample.

Step 1: Analyze a sample containing a known amount of analyte
and record the peak area. Calculate the response factor.

Response Factor, RF = Peak area analyte

Concentration / Amount of analyte
Step 2: Inject the sample with unknown analyte concentration and
record the peak area. Calculate the concentration of analyte in
sample.
Concentration/Amount of analyte = Peak Area Analyte in Sample
Response Factor
 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.

Step 1: Calculate the Response Factor

Response Factor for benzene = 100000 = 50
2000
Step 2:
Concentration of benzene in sample = 57000 = 1140 µg/mL
50