L3 Chromatography in Forensic Science

Chromatography in
Forensic Science
MED2105
1
2 Introduction
 Purposes of chromatography
 Definition of chromatography
 Basic principles of chromatography
 Paper Chromatography (PC)
 Thin-Layer Chromatography (TLC)
 Liquid Chromatography (LC)
 High Performance Liquid Chromatography
(HPLC)
 Gas Chromatography (GC)
3 Introduction
Limitations of Liquid–Liquid Extractions

1. If we have several analytes in a mixture,
we may need to complete a separate
extraction for each analyte.
2. The extent of a separation depends on
the distribution ratio of each species in
the sample.
4 Introduction
Purposes:
1) Purify organic compounds
2) Separate complex mixtures
3) and/or identifying the components in a
mixture
 Can it separate colourless substances?
 Separate almost everything: proteins, nuclei

acids, carbohydrates, viruses
5 Introduction
Definition of chromatography
 A physical method of separation in which the
components of a mixture are distributed
between two phases
 A stationary phase
 A mobile phase
 Different molecules (analytes) have different

degrees of attraction to the stationary phase
(sorbent)
 Different rates of travel
6 Introduction
 Separation based on the distribution of

individual components of the mixture
between stationary and mobile phases
 Effectively establish an equilibrium for
each component
 Differences in equilibrium allow

separation
7 Introduction
Imagine you
have some
proteins, how
would you
separate them?
Based on what
principles?
https://www.sciencebuddies.org/science-fair-projects/project-ideas/biotechnology
8 Intro - 4 Basic principles
1) Adsorption
 A solid stationary phase and a liquid or
gaseous mobile phase
 Each solute has its own equilibrium
between adsorption onto the surface of
the solid and solubility in the solvent, the
best adsorbed ones travel more slowly.
 E.g. Liquid chromatography using a
column containing silica gel or alumina
1) Adsorption
 E.g. silica gel in TLC
 Intermolecular forces
 Examples?
 Polar versus
nonpolar
compounds?
 Stronger forces with
the stationary phase;
migrate slowly
http://www.expertsmind.com/topic/packing-material-or-stationary-phase/adsorption-chromatography-913002.aspx
2) Partition
 Stationary phase: a non-volatile liquid
which is held as a thin layer (or film) on
the surface of an inert solid
 Mobile phase: a gas or a liquid
 Solutes distribute themselves between
the moving and the stationary phases,
with the more soluble component in the
mobile phase reaching the end
 E.g. Paper chromatography
2) Partition
3) Ion exchange
 Stationary phase:
coated solid with ions;
ions of the opposite
charge are
electrostatically bound
 Mobile phase: a liquid
 Charged molecules:
large proteins, small
nucleotides and amino
acids
https://www.biochemden.com/ion-exchange-chromatography/
4) Molecular exclusion
 Mixture passes as a gas
or a liquid through a
porous gel
 Large solute particles:
pass through
unchanged
 Small solute particles:
permeate the gel and
slow down
 Separation based on
particle size
https://liveapi.authorcafe.com/preview.php?assetid=27950
4) Molecular exclusion
16 Introduction
Summary:
17 Introduction
1) Paper Chromatography (PC)

2) Thin-Layer Chromatography (TLC)
3) Liquid Chromatography (LC)
4) High Performance Liquid
Chromatography (HPLC)
5) Gas Chromatography (GC)
How do you choose the method?

18 Introduction
19 Paper Chromatography (PC)
 Partition of solutes between water in the

paper fibres (stationary phase) and the
solvent (mobile phase)
 Separating amino acids, hormones, drugs,
salts and complexes
https://microbenotes.com/paper-chromatography/
In the field of Forensic science

 Separate inks from fountain and fibre-
tipped pens,
 Separate food colourings and dyes
Same principle:
 Partition of solutes between water in the
paper fibres (stationary phase) and the
solvent (mobile phase)
 Separation is most efficient if the atmosphere is

saturated in the solvent vapour
 Chromatographic
chamber
 Chromatogram
 Solvent front
 Retention factor
https://microbenotes.com/paper-chromatography/
Choice of the solvent:

 should not react chemically with any of the
components of the sample mixture.
 composition of solvent mixture should not change
with time. It means that none of its components
should be volatile.
 should not interfere with the detection of spots.
 The distribution ratio should be independent of
solute concentration
 The minimum difference between the Rf values of
any two components should be 0.05 or 0.1 so that
they may be separated easily.
Two-dimensional
Using two solvents

to separate a multi
component mixture
Based on retention factor,

 Can identify the known solutes
 But
 Need to use the correct solvent and type
of chromatography paper (same
chemical and physical properties of the 2
phases); same experimental conditions
(temperature and pressure) Standard Unknown
conc.
• • • • • • •
Factors affecting Rf values:
 Absorbent
 Solvent
 Chromatography paper/plate
 Application technique
 Temperature of the solvent and plate
 Presence of other cations or anions as
impurities
Exercises
28 Thin-Layer Chromatography
(TLC)
 Simple, inexpensive, fast, efficient

 Require only milligram quantities of
material
 Determine the number of compounds in
a mixture; establish whether or not two
compounds are identical
 Samples: nonvolatile organic

compounds
 What about liquid compounds?
(TLC)
 Forensic science: tentative identification
of dyes, pigments and screening for drugs
(e.g. opiates, cocaine, amphetamines,
barbiturates, and cannabis)
 Summary: mixture is separated as it

moves through a thin layer of stationary
phase (silica gel or aluminium oxide on
glass, aluminium or plastic sheets) by the
migrating solvent (mobile phase)
 The polarity of the compound determines

the relative positions of the components
(TLC)
 Mobile phase: a pure solvent or a mixture

of solvents (developing solvent)
 Stationary phase: glass, metal, or plastic
plates coated with a thin layer of
adsorbent
Silica gel (SiO2∙ xH2O)
Aluminium oxide (Al2O3 ∙ xH2O)
 Are they polar or non-polar?
 What do they bind? Intermolecular
forces?
(TLC)
 Stationary phase: glass, metal, or plastic

plates coated with a thin layer of
adsorbent
Modified Silica gel by replacing the
hydroxyl groups normally attached to
silicon atoms with alkoxy groups and
with long-chain alkyl groups, such as
-(CH2)17CH3
What do they bind? Intermolecular
forces?
This is called reversed-phase TLC.
(TLC)
 Dissolve sample in a volatile organic solvent

 What are some examples?
 Commercial micropipets
1.0 cm from the bottom
How do we mark? Pen?
Only 1-5uL of sample
No more than 2mm diameter
Why?
Lightly and gently
Use known standards
(TLC)
 In a close developing chamber

containing a developing solvent
 Saturated with solvent vapors
Insert a piece of filter paper
three-quarters
Add a shallow layer of
developing solvent (3-4mm)
Why shallow?
(TLC)
Developing the chromatogram

 Solvent front: 1-1.5cm from top of the
plate
 Mark the solvent front before evaporation
 Evaporate the solvent in a fumehood
(TLC)
Visualization techniques
1) Use adsorbents with a fluorescent indicator
 Short-wavelength ultraviolet lamp (254 nm)
 Fluoresces in UV light
 Outline each spot with a pencil
(TLC)
Visualization techniques
2) Dip briefly in visualizing solutions to form

colored compounds upon heating
 E.g. p-anisaldehyde, vanillin, and
phosphomolybdic acid
3) Use iodine (I2) vapor
 Produce yellow-brown colored
spots
(TLC)
Analysis of a Thin-layer Chromatogram
 determine the ratio of the distance each
compound has traveled on the plate
relative to the distance the solvent has
traveled
 depends on structure as well as the

adsorbent and mobile phase used
(TLC) Exercises
 What are the Rf of compound 1 and 2?
Can Rf bigger than 1?

(TLC)
(TLC)
Exercises
Finding a Suitable Developing Solvent
 Chromatographic behavior is the result of
competition by the stationary phase (________)
and the mobile-phase (________) for the
compounds being separated.
(TLC)
Finding a Suitable Developing Solvent
(TLC)
Ideal Rf values should be in the
range 0.30–0.60
Standard Unknown
conc.
• • • • • • •
(TLC)
Accurate record keeping:
 Brand, type of backing, and adsorbent

on the TLC plate
 Developing solvent
 Method used to visualize the
compounds
 Rf value for each substance
44
Exam questions
Exercises
 State two reasons for using chromatography. [2]

 State the stationary phase in paper
chromatography and thin-layer
chromatography. [2]
 The diagram shows the results of a
paper chromatography experiment.
Determine the Rf value for substance A.
Distances measured on this diagram are
61 mm to the solvent front and 43 mm
to spot A.
45 Exam questions Answers
Reasons of using chromatography: low cost, fast, simple

etc
Paper chromatography: Cellulose fiber

TLC: Silica gel, aluminum
46
Multiple choice
Exercises
Chromatography is based on the

A) Different rate of movement of the solute in a
column
B) Separation of one solute from other constituents
by being captured on the adsorbent
C) Different rate of movement of the solvent in the
column
D) None of the above
In reverse phase chromatography, the stationary

phase is made
A) Non-polar
B) Polar
47
Multiple choice
Exercises
Thin layer chromatography can be used to

distinguish between different amino acids. If a
particular amino acid has low solubility in the
mobile phase used, then the other amino acid ...
A. will spend more time dissolved in the mobile

phase than attached to the stationary phase.
B. will have a low Rf value.
C. must have a high molecular mass.
D. will move at a speed close to that of the solvent.
48
Multiple choice
Exercises
In the TLC lab, ink from the t shirts was spotted on silica
coated plates. The plates were then placed in a beaker that
had a small amount of ethyl acetate in it.
A. The stationary phase in the TLC lab was

a. the blue ink b. the silica coating
c. the ethyl acetate d. the pyrex beaker
B. The mobile phase in the TLC lab was

a. the blue ink b. the silica coating
c. the ethyl acetate d. the pyrex beaker
C. An ink sample that travels farther up the TLC plate has a

greater attraction to
a. the mobile phase b. the stationary phase
c. the pyrex beaker d. the plastic in the TLC plate
49 Comparison
TLC versus LC
 The stationary phase is contained inside a
column, rather than applied as a coating on a
plate.
 The column is commonly made of glass, but
some are made of metal or other materials.
 The sample to be separated is loaded from the
top. The eluting solvent, or mobile phase, is also
added from the top.
 The solvent flows down the column by gravity,
carrying with it the components of the sample.
 Same principle: components travel at different
rates effecting the separation
50 Comparison
TLC versus LC
1. Thin Layer Chromatography (TLC)
 stationary phase: spread over glass or plastic
sheet
 mobile phase: liquid; drawn up plate by
capillary action
2. Column Chromatography (LC)

 stationary phase: contained in a column
 mobile phase: liquid; passes through column
(gravity or pressure)
51 Liquid Chromatography (LC)
 Column chromatography
 Samples: compounds of low volatility
 Quantities: 1 few micrograms to 10g for
HPLC
 Mobile phase: an elution solvent, liquid
 Stationary phase: a solid adsorbent with
a liquid coating packed into a column
(silica gel, aluminum oxide)
 What is the difference between 2
phases?
If silica gel is the adsorbent, is compound

A more or less polar than compound B?
 Retention time, tr, is the elapsed time from the

introduction of the solute to the peak maximum
 Time of mobile phase required to elute non-

retained components is called the column’s void
time
 Baseline width
 Resolution is a quantitative measure of the

degree of separation between two
chromatographic peaks, A and B, and is defined
as
 Higher R, better separation

Exercises
 In a chromatographic analysis of lemon
oil a peak for limonene has a retention
time of 8.36 min with a baseline width of
0.96 min. γ-Terpinene elutes at 9.54 min,
with a baseline width of 0.64 min. What is
the resolution between the two peaks?
 How to improve Resolution?

Theoretical plates
 A quantitative means of evaluating column
efficiency that treats the column as though it
consists of a series of small zones, or plates, in
which partitioning between the mobile and
stationary phases occurs.
 A column’s efficiency improves with an increase
in the number of theoretical plates or a decrease
in the height of a theoretical plate.
Absorbent particles size:

63-210 um (gravity LC)
Smaller than 63um (HPLC)
Smaller particle size; more

effective separations but slower
eluent flow rates; use smaller
columns or with greater
atmospheric pressure
Similar to TLC:
 The more strongly a compound adsorbs
to stationary phase, the slower it will
travel - adsorption is based on
interactions of functional groups in the
molecule with the stationary phase
(silica or alumina)
 Hydrogen bonding?
 Van der Waals interactions?
 Dipole dipole interactions?
?
Differences in normal phase and reverse phase?
Make sure
 Stationary phase is saturated with solvent.
Why?
 An inert material is at the bottom of the
column
Example?
Why
 Solutes can be extracted from the liquid
collected
What is it called?
 Evaporate off the solvent
65 Questions Exercises

L3 Chromatography in Forensic Science

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Chromatography in

Limitations of Liquid–Liquid Extractions

 Can it separate colourless substances?

 Separate almost everything: proteins, nuclei

 Different molecules (analytes) have different

 Separation based on the distribution of

 Differences in equilibrium allow

1) Paper Chromatography (PC)

How do you choose the method?

 Partition of solutes between water in the

In the field of Forensic science

 Separation is most efficient if the atmosphere is

Choice of the solvent:

Using two solvents

Based on retention factor,

Factors affecting Rf values:

 Simple, inexpensive, fast, efficient

 Samples: nonvolatile organic

 Summary: mixture is separated as it

 The polarity of the compound determines

 Mobile phase: a pure solvent or a mixture

 Stationary phase: glass, metal, or plastic

 Dissolve sample in a volatile organic solvent

 In a close developing chamber

Developing the chromatogram

2) Dip briefly in visualizing solutions to form

 depends on structure as well as the

 What are the Rf of compound 1 and 2?

Can Rf bigger than 1?

 Brand, type of backing, and adsorbent

 State two reasons for using chromatography. [2]

Reasons of using chromatography: low cost, fast, simple

Paper chromatography: Cellulose fiber

Chromatography is based on the

In reverse phase chromatography, the stationary

Thin layer chromatography can be used to

A. will spend more time dissolved in the mobile

A. The stationary phase in the TLC lab was

B. The mobile phase in the TLC lab was

C. An ink sample that travels farther up the TLC plate has a

2. Column Chromatography (LC)

If silica gel is the adsorbent, is compound

 Retention time, tr, is the elapsed time from the

 Time of mobile phase required to elute non-

 Resolution is a quantitative measure of the

 Higher R, better separation

 How to improve Resolution?

Absorbent particles size:

Smaller particle size; more

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