CHROMATOGRAPHY 1

 Chromatography is a technique
for separating complex mixtures of
chemicals.

 Used primarily for organic analysis.

 Chromatography is derived
from Greek words chroma, which
means "color", and graphein,
which means "to write".
  Technique – a general way of
measuring the target analyte(s),
using a particular technology,
Some  e.g., Gas Chromatography
Attributes of  Method – a specific set of
instructions, written as a Standard
Analytical Operating Procedure (SOP)
Techniques  e.g., a laboratory’s SOP entitled
“The Determination of
Organochlorine Pesticides and
PCBs in Water, Soil and Fish Tissue
by GC/ECD”
 A method is a way of applying a
technique.
2
  Sensitivity - : the capability of a
method or instrument to
discriminate between
measurement responses
Some representing different levels of a
Attributes of variable of interest.

Analytical  Sensitivity is quantified as the

slope of the calibration curve (for
Techniques linear calibrations).
 For non-linear calibrations, it is the
slope at the concentration of
interest (tangent).

3
  Selectivity – the capability of a
method or instrument to respond
to a group or class of analytes
Some (and no others).

Attributes of
Specificity – responsiveness to the
Analytical 
analyte of interest and only the
Techniques analyte of interest. This is often
desirable in a method or
instrument. Not many instruments
or techniques are specific.

4
Container with liquid

 Some molecules of
compound A exist
as a vapour in the
gas above the
A
A liquid.
A AA

A
 Some molecules of
A are also dissolved
in the liquid.

5
Container with liquid

 Molecules of A go
from the gas
phase into the
liquid.
A
A  Molecules go from
A
A A the liquid into the
gas.
A
 There is a dynamic
equilibrium.

6
Container with liquid

Partition Coefficient

K = Cliquid/Cgas

A The ratio of the

A concentration of A in
A A
A A the liquid phase to that
A in the gas phase
A A
K depends on what A is,
what the liquid is, and
the temperature.

7
Container with liquid

Partition Coefficient

K = Cliquid/Cgas

A
A A As the temperature is
A A raised, more molecules
A A
of A would go from the
liquid phase to the gas
A A
phase.
As temperature
increases, K decreases.

8
Different container, 2
compounds
B
B

 Most of A is in gas phase, a little of it in liquid.

 Most of B is dissolved in the liquid, little of it in
the gas phase.
 KA = CA,liquid < KB = CB,liquid
CA,gas CB,gas
9
A container with openings 10
at each end

 Consider the container to be a long,

narrow tube containing a liquid.
 The gas above the liquid flows from one
end of the tube to the other.
A container with openings
at each end

 A mixture of A and B are introduced at

one end of the tube (the end at which
the gas is coming in).
 A and B are pushed down the tube by
the “carrier gas” or mobile phase.
11
A container with openings
at each end

 A and B each partially dissolve in the

liquid (stationary) phase.
 If B has a greater partition coefficient
than A, more of B than A goes into the
stationary phase.
12
A container with openings
at each end

 Because more of B goes into the

stationary phase, it is retained (held back)
on the column more than A is.

13
A container with openings
at each end

 Compounds A and B are physically

separated from each other because they
have different partition coefficients.

14
A container with openings
at each end

 After enough time, A reaches the far end

of the column.
 Some time later, B also reaches the far
end of the column.
15
  The example in the previous slides
is the basis for gas
chromatography.
 A gas chromatograph is an
instrument for separating a
Gas complex mixture and detecting
Chromatography each compound in turn.

 How GC Columns Work - YouTube

 Gas Chromatography GC –
YouTube

16
A Gas Chromatograph 17
Carrier Gas for GC 18

Properties of a Carrier Gas

1. High purity.
2. Inert – should not react
with the stationary phase,
mobile phase or
analyte(s).
3. Compatible with the
detector.
4. Cheap and readily
available.
  Rotameters – measurement of flow
before the column by position of a
ball floating in a calibrated glass
tube
 Bubble meters – after the column
or detector. Inexpensive and
relatively accurate.
Flow
Measurement  Electronic flow sensor
 Modified thermal conductivity
detector
 Large range
 Must be calibrated
 Response will vary depending on
carrier gas

19
A Gas Chromatograph 20
Sample injection into the GC

 Purpose of the port –

flash evaporate
sample and
introduce it into
column
 Tinj at least 50°C hotter
than column
 Septum must be
stable at Tinj
 Septum must be
replaced regularly to
maintain seal

21
  A syringe is used to inject a
known volume of liquid or
Sample gas sample onto the
column.
injection
into the  Typical volume injected

GC  Gases – 0.5 to 25 mL
 Liquids – 0.1 to 10 µL

22
  Manual and automatic
(autosampler) injection.

Sample  Precision of injection ± 1%

injection  If manual injection is used, the

into the injection technique itself is a major
source of error in chromatography
GC  Syringe must be filled accurately
 Injection must be rapid, so that
sample enters column as a narrow
plug

23
A Gas Chromatograph 24
  2 Major types – packed columns
and capillary columns

Columns  Typical packed column

 1/8” to ¼” OD, stainless steel or

used in 
glass tube
6 to 20 feet long
GC
 Typical capillary tube
 0.1 to 0.5 mm ID
 10 to 100 m long

25
Packed column 26
Support Particles in a 27

Packed Column
Multipath in a Packed
Column

 In a packed column, a molecule may follow

a relatively straight route down the column
(red).
 It may follow a more tortuous route that
takes longer to reach the end of the column
(blue).

28
  Specifications from the
manufacturer:

Example  Stationary Phase: DB-VRX

Capillary 


Length: 60 m
ID: 0.32 mm
Column  Thickness of liquid coating on wall:
1.80 µm
 Usable Temperature Range: -10 to
260°C

29
Example Capillary Column 30

 Columns are available with different phases, diameters and

film thickness to aid in separation.
Example Capillary Column 31
  Capillary columns provide
greater separation
efficiency over packed
columns.
Example
Capillary  Since separation is based on
the interaction of the solute
Column with the stationary phase
therefore the selection of
the phase is important.

32
 33
Example Capillary Column
 The stationary phase must not
react with the solute, it must be
thermally stable, it must have
low volatility and its polarity
must match that of the solute.

 The “backbone” of the

stationary phase is a polymer of
poly-dimethyl siloxane.
Example Capillary 34
Column

 Replacing the “R” or methyl

groups with other functional
groups will change the polarity
of the stationary phase.

 Replacing 50% of the methyl

groups with phenyl produces a
slightly more polar phase.

 Adding trifluoropropyl or
cyanopropyl groups will further
increase the polarity.
Example Capillary Column 35

 Rule of Thumb is;

In non-polar columns,
separation is based on
MW (boiling point).

In polar columns,
separation is based on
polarity.
A Gas Chromatograph 36
 Detectors can be classified as
destructive or non-destructive.

Detectors for Gas Detectors can be selective

Chromatography (responding only to a certain
class of compounds) or non-
selective (responding to almost
all compounds).

37
  High sensitivity – possible selectivity

 Rapid response to concentration

Properties changes

of a good  Large linear dynamic range

GC
Electronic stability (low drift, low
Detector

noise)

 Low sensitivity to variations in flow,

pressure and temperature

38
Thermal 39
Conductivity
Detector
 General Purpose (not
selective)
 Nondestructive
 Limit of detection ~ 10-8
g/mL carrier (others
more sensitive by 104 to
107)
 Linear range ~ 106
 Change in resistance of
a wire based on
variations in the thermal
conductivity of the gas
flowing through it.
  Very widely used

 Selective – responds to most

organic compounds

Flame  Destructive

Ionization  Limit of detection ~ 5 pg

Detector carbon/sec

 Linear range ~ 107

 Production of ions in a flame result

in a measurable current

40
Flame Ionization Detector 41
Flame Ionization 42
Detector
 Compounds with little or no
response on FID:

 Noble gases, NH3, CS2, NOx,

CO, O2,
 H2O, CO2, N2
 Formic acid, formaldehyde

 Compounds for FID detectors

require C-H bonds.
FID 43
Response of FID and TCD 44
Electron 45
Capture
Detector
 Selective – responds only to
gas phase molecules that
absorb electrons

 Non-destructive

 Limit of detection ~ 0.1 pg

Cl/sec

 Linear Range ~ 104

 Absorption of ß particles by
species containing halogens,
nitrates, organometallics
ECD 46
  Selective – compounds ionized by
UV

 Limit of detection ~ 2 pg
carbon/sec

 Linear range ~ 107

Photoionization
Detector  UV light is used to directly ionize
sample components. The resulting
current is measured.

 Hand-held, battery-operated
versions are widely used in military,
industrial, and confined working
facilities for health and safety.

47
 48
Photoionization Detector
Detectors

49
Chromatograms 50

 Each peak has a retention volume, V.

 The retention volume is the volume of
carrier gas that passes through the
column before the peak elutes (comes
out the far end of the column).
Chromatograms 51

 Each peak has a retention time, t.

 The retention time of a peak is the time from
injection until the peak reaches the detector.
 As the retention time gets longer, the peak
gets wider and shorter.
Chromatograms 52

 The peaks are separated from one another based on the

differences in partition coefficient between different
compounds.
 Ideally, each peak is due to one compound, but this isn’t
always true.
 If two compounds have the same partition coefficient,
they will co-elute, i.e., come out at the same time.
Chromatograms
53

ttmm

 The small peak near the start is due to a species

that has K = 0, i.e., it has no retention at all.
 tm is the “dead time”
 Dead time represents the average rate of
motion of the mobile phase.
Chromatograms
54

ttmm

 The retention time for a compound depends

on the column and chromatographic
conditions. It is found experimentally.
 The area under the peak is proportional to
the amount of the compound.
 55
Peak Overlap

 The purpose of
chromatography is to separate
the mixture into discrete peaks,
ideally one peak, completely
separated from its neighbors,
for each compound. Each
compound could then be
measured by the detector, in
turn.

 This ideal is often achieved, but

not always.
Peak Resolution

Resolution is defined
as the ratio of peak
separation to
average peak
width.

R = d
0.5( w1 + w2)

56
Peak Resolution

 First pair of peaks, R = 0.5

 Second pair, R = 0.75
 Third pair, R = 1.0

57
What happens as compounds
move down the column?

 They separate from each other.

 Their bands “spread.”
 Their bands are lower in height.
58
The “General Elution
Problem”
Good Separation – but
takes too long

Practical time – but

poor separation

Still not quite there

59
The “General Elution
Problem”
 There are two
ways to deal
with the
“General
Elution
Problem”
 Increase band
separation
 Decrease band
spread

60
  The chromatographic column sits
in an oven.

 The temperature may be held

Temperature constant during the analysis –
of the isothermal.

column
 If the temperature is varied during
the analysis, it is done through
temperature programming.

61
Temperature Program

 A – Initial
temperature, hold
for a time
 B – temperature
Ramp, specified in
degrees per
minute from initial
temp to final temp
 C – Final
Temperature, hold
for a time.
62
  It is a way to solve the general
elution problem in gas
chromatography.
 With homologues, tR increases
exponentially with the number of
carbons.
Why do  As tR increases, the width of the
temperature peak increases, and height
programming? decreases, making quantification
more difficult.
 Since the solubility of a gas in a
liquid decreases as the
temperature goes up, we can
reduce the retention of a material
by increasing the temperature.

63
Effect of Temperature 64
programming

 With temperature
programming, the peaks
that are more highly
retained, come out
sooner.
 The more highly retained
peaks remain sharper –
they are not broadened
as much.
Partition Coefficient 65

K = Cliq
Cgas

K = Cstat
Cmobile

The higher the value of K, the more

the analyte ‘sticks’ to the stationary
phase and the longer it takes to elute
from the column.
Retention Time (tr) 66

The Average Rate of Analyte Migration can be calculated

by:
v = L / tr
where L = Length of Column
tr = Retention Time of Analyte

The Average Rate of Mobile Phase movement can be

calculated by:
u = L / tm
where L = Length of Column
tm = Dead Time
Retention Time (tr)& 67

Partition Coefficient (K)

 It is helpful to be able to relate the rate of analyte
migration to it’s Partition Coefficient and to the volumes of
the stationary and mobile phases:

v = u * 1/(1 + KVs/Vm)

 where u = average rate of movement of mobile phase

 K = partition coefficient of analyte
 Vs = volume of stationary phase
 Vm = volume of mobile phase
  Another way to express the rate of
analyte migration is the Capacity
Factor:
k’A = KAVs/Vm

Capacity
The main reason for the usefulness
Factor

of this factor is that the above
equation can be stated in terms of
experimentally determined times
from the chromatogram:
k’A = (tr - tm)/tm

68
 Capacity Factor 69

 Capacity Factor = k’A = (tr - tm)/tm

Capacity Factors greater than 20 - 30 result in VERY long analysis

times. Values between 1 & 5 are ideal.
  In order to compare the
chromatographic characteristics
of one molecule with another the
ratio of partition coefficients is
often used...

Selectivity  This ratio is called the Selectivity

Factor Factor:
 α = KA / KB (The Ratio of Capacity
Factors is Also known as the
Selectivity Factor)
 NB! the value is ALWAYS +ve... the
partition coefficient with the
largest value is always the
numerator

70
Selectivity Factor 71

 As with the Capacity Factor (one

compound), the Selectivity Factor (two
compounds) can be expressed in terms of
experimental chromatogram measurements:

(Tr ) B − Tm
Selectivity Factor = α=
(Tr ) A − Tm

Capacity Factor = k’A = (Tr - Tm)/Tm

  Partition Coefficients, Retention
Times, Capacity Factors and
Selectivity Factors tell us about
Band how long it takes the average
molecule to get down the
Broadening column...
&
Column  BUT recall... there are two
problems associated with the
Efficiency ‘General Elution Problem’:
 migration time
 band broadening...

72
 Band  The theoretical shape of a
Broadening chromatographic peak is
Gaussian (i.e., ‘Normal’)...
&
 Why?...
Column  because the path of each
Efficiency individual molecule down the
column is determined by how
much time it spends in each
phase
 there is a lot of randomness in this
process because thermal (kinetic)
energy plays an important role,
and each molecule will possess
more or less Ek

73
 Band
The result of the random individual
Broadening

processes is a symmetrical spread
& of velocities around the mean
value, which represents the
Column behavior of the average particle.
Efficiency
 The breadth of the band increases
as it moves down the column
because more time is allowed for
the random processes to occur...
The zone breadth is directly
related to residence time and
inversely related to velocity.

74
Column Efficiency is a measure of
how well a column can prevent
band broadening.
Two ways to express...
Plate Height
Number of Theoretical Plates
N=L/H
where
N = Number of Theoretical
Plates
L = Column Length
H = Plate Height

75
 Column
Resolution  Resolution: The ability of a column
to separate two analytes
 a value of 1.5 = complete
separation
 improved by lengthening the
column (but this increases analysis
time)
 Resolution is dependent on:
 Number of plates
 Selectivity Factor
 Capacity Factor

76
  The Selectivity Factor (α) and the
Capacity Factor (K) can be
adjusted independently by
varying the temperature or by
Column changing the composition of the
mobile phase (or if you’ve got lots
Resolution of time... the composition of the
stationary phase.
&
Retention  N can be changed by changing
the length of the column
Time
 H can be changed by altering
flow rate, or stationary phase
particle size

77
 Triangle of Chromatography 78

60 m
0.25 mm OD
0.1 µm

15 m 0.53 mm OD
0.25 mm OD 1.5 µm
0.1 µm
Applications of 79
Chromatography
 Note that a chromatogram
provides only one piece of
information about each species.

 Also note that for qualitative

analysis, you can never be sure
that more than one species
exhibits the same
chromatographic properties i.e.,
they may co-elute.

 Thus, although many methods are

based on chromatography alone,
it is often used in combination with
other techniques like IR, and Mass
Spectrometry
  Quantitative Analysis
 Calibration curves are based on
the peak height vs concentration
or the peak area vs concentration

 Because variables are often

Applications of difficult to control frequent re-
Chromatography standardization is required for
most methods.

 Because injection volumes are

small (~0.1 - 10 µL) errors can be
large if injection is not
reproducible and accurate.

80
 81

Applications
 82

Applications
 83

Applications
 84

Applications
Applications of Chromatography 85

 A GC system with a single

column and single detector does
have limitations.
 The separation potentially works
well for samples with a small
number of analytes.
 As stated earlier, complex
mixtures are subject to co-elution
of the analyte peaks.
 Commonly referred to as 1-
dimensional chromatography.
 Limited information to “positively”
identify an analyte is available.
 How can we add dimensionality?

i.e., How can we increase the

likelihood of a “positive”
identification from a complex
Applications of mixture of analytes?
Chromatography

Multiple detectors….?
Multiple columns….?
Both…..?

86
Applications of 87
Chromatography

 Multiple detectors…..

 Two different detectors can be

utilized by splitting the sample
between 2 or more detectors
using a glass press fit “y-splitter”
just prior to the detectors.

 Uses the differing chemical &

physical properties of analytes to
“confirm” detection.
Applications of Chromatography 88

 Multiple detectors…..
Applications of Chromatography 89
 90
Applications of
Chromatography
 Multiple columns…..

 Two dis-similar phase columns may be

used in parallel by splitting the injected
sample using a “y-splitter” just after the
injection point.

 Detectors are usually the same.

 Allows for a second confirmation of the

analyte's identity.
Applications of 91
Chromatography
Applications of 92
Chromatography
Applications of 93
Chromatography
Applications of 94
Chromatography
 How about both….?

A classical concept;
Applications of
Chromatography
Two-way paper chromatography
involves using two solvents and
rotating the paper 90° in between.
This is useful for separating complex
mixtures of similar compounds.

95
Applications of 96
Chromatography
 Two-dimensional gas
chromatography, or GC x GC was
created by Professor Phillips in 1991.

Applications of
Chromatography It has extensively been applied to
many kinds of applications: fuels
analysis, forensics, food and flavours,
environmental, metabolomics,
biomarkers, and clinical.

97
  Each fraction collected after the
first separation will be further
separated in a column involving a
different separation mechanism.

 The transfer of the fractions is done

through a modulation device
Applications of (modulator) which traps, focuses
Chromatography and reinjects them onto the
secondary column.

 This leads to a 2-Dimensional

chromatogram offering higher
peak capacity and more
separation power.

98
Applications of 99
Chromatography
  The figure on the next page shows
an example of a peak, which
resulted from three coeluting
compounds.

 After modulation, the peak is

divided into 8 small fractions, of
Applications of which each are subjected to a
Chromatography very fast separation in the second
dimension (column).

 The second-dimension
chromatograms are then stacked
side-by-side and software offers a
2D contour plot visualisation and
often a 3D visualization.

100
Applications of Chromatography 101
  GC x GC Modulation

 Cryogenic liquid nitrogen is used

to trap the molecules eluting from
the first dimension. The first stage
Applications of immobilizes the compounds
Chromatography during the modulation period, the
molecules previously immobilized
in the second stage are released
and can flow into the second
column and be separated.

102
  GC x GC Columns Set

 Typically, a non-polar column for

the first dimension followed by a
more polar column in the second
dimension.
Applications of
Chromatography  The GC x GC set-up includes a
long first dimension column
(typically 30 m – 60 m) that is
connected to a short second
dimension column (typically 0.5 m
– 1.3 m)

103
 GC x GC Detectors

Considering the very fast secondary

separation using a very short column,
eluting peaks are very narrow thus
requiring an exceptionally fast data
acquisition.
Applications of
The following can be used:
Chromatography
Flame ionization detector (FID),

(micro) electron capture detector

(µECD)

Mass spectrometry detectors such as

fast time of flight (TOF/MS).

104
Applications of 105

Chromatography
Applications of 106

Chromatography
 Peak Identification

For a peak to be considered as a “real &

confirmed” value there are three identification
criteria that must be met;

1 – the retention time must be within -1 to +3

seconds of the corresponding isotopically
labelled internal standard.

Here the difference is 20.36 – 20.34 = 0.02

minutes

0.02 minutes = 1.2 seconds.

107
 Peak Identification

2 – All ion current intensities must be >2.5 times the

background noise.

108
 Peak Identification

3 – the integrated ion currents for the two quantitation ions

must have a ratio to each other that is between the upper and
lower established limits.

© Copyright 2022 | ALS Limited 109

 Peak Identification
Let’s look at a real-life example;

110
 Peak Identification
2,2’,5,5’-Tetrachlorobipenyl

PCB 52 is a tetra-chlorinated biphenyl.

Retention time of 16.97 minutes and the

corresponding isotopically labelled analog has
a retention time of 16.96. A difference of 0.01
minutes.

0.01 minutes is 0.6 seconds

Therefore criteria #1 has been met;

The retention time must be within -1 to +3

seconds of the corresponding isotopically
labelled internal standard.

111
 Peak Identification
The average background noise is about 5% (depending on where you draw
the baseline – subjective).
As we can see the PCB 52 peak is more than 2.5 times the background
noise.

Therefore criteria #2 has been met;

All ion current intensities must be >2.5 times the background noise.
 Peak Identification 113

The ion current of the primary ion is 975.2 and the

secondary ion is 1135.7.

A ratio of 975.2 / 1135.7 = 0.86

The theoretical ratio for a tetra substituted PCB is 0.77

(same as for the tetra substituted TCDD/F) and must
be between 0.65 & 0.89.

This satisfies criteria #3;

The integrated ion currents for the two quantitation

ions must have a ratio to each other that is between
the upper and lower established limits.

113
Peak Identification 114

Rule #1 – Passed (RT) Rule #2 - Passed (S/N) Rule #3 - Passed (Ratio)