Chromatography

(GC & HPLC)

MED2105
1
2 Introduction

Purposes of chromatography
Definition of chromatography
Basic principles of chromatography
Paper Chromatography (PC)
Thin-Layer Chromatography (TLC)
Column Liquid Chromatography (LC)
Gas Chromatography (GC)
High Performance Liquid Chromatography
(HPLC)
3 Gas Chromatography (GC)
Advantages/disadvantages?

Forensic Significance
Analysis of arson accelerants
Detection of explosives
Pathology /toxicology /clinical analysis:
Drugs
Blood alcohols
Environmental analysis
Vehicle emissions
Trihalomethanes in drinking water
4 Gas Chromatography (GC)
Advantages/disadvantages?
Consumer products
Volatile organics in spices and
fragrances
Trace organics in whiskey
Monomers in latex paint
Purity of solvents
Refinery gas
Composition of gasoline
Pesticides in water, wastewater, and
soil
5 Gas Chromatography (GC)

Samples: Volatile organic chemicals (gas

or liquid)

Limitations:
1. Only for the analysis of small amounts of
compounds that have vapor pressures
high enough to allow them to pass
through a GC column
2. Cannot identify compounds unless
known samples are available
6 Gas Chromatography (GC)

Stationary phase: liquid coating in a long,

narrow column
Mobile phase (carrier gas)
An inert gas (helium or nitrogen)
No interaction with the analytes: simply
carries them down the column when they
are in the vapor state

https://www.uwo.ca/sci/research/biotron/analytical_services/equipment_and_services/gc.html
7 Gas Chromatography (GC)

1) Source of high-pressure pure carrier gas

2) Flow controller
3) Heated injection port
4) Column and column oven
5) Detector
6) Recording device or data station
8 Gas Chromatography (GC)

What is the instrument called?

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9 Gas Chromatography (GC)

1) A small syringe: inject the sample into the

stream of carrier gas in the heated injection
port

2) The sample vaporizes immediately, and the

carrier gas sweeps it into the column that
contains the liquid stationary phase
10 Gas Chromatography (GC)
Differences in separation?

2) Chromatographic Columns
Packed Columns: glass, stainless steel,
copper, or aluminum; 2–6 m in length with
internal diameters of 2–4 mm; packed with
small solid particles with liquid coating
adsorbed

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11 Gas Chromatography (GC)
Differences in separation?

2) Chromatographic Columns
Capillary Columns: constructed from fused
silica and is coated with a liquid coating; 15–
100 m in length with an internal diameter of
approximately 150–300 μm

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12 Gas Chromatography (GC)

2) Chromatographic Columns

Capillary Columns versus Packed Columns

What are the differences in separation?

13 Gas Chromatography (GC)

2) Chromatographic Columns
14 Gas Chromatography (GC)

3) Liquid stationary phase: interacts with the

substances being separated by a number
of intermolecular forces

Examples?
“Like dissolves like”:
Nonpolar liquid coatings separate _______
compounds
Polar liquid coatings separate _______
compounds
15 Gas Chromatography (GC)

Any examples?
3) Liquid stationary phase

What are some criteria?

1. Must not react chemically with the

solutes
2. Must be thermally stable
3. Must have a low volatility
4. Must have a polarity that is appropriate
for the sample’s components.
16 Gas Chromatography (GC)

3) Liquid stationary phase examples

17 Gas Chromatography (GC)

4) Separation
Depends on relative attraction for the
liquid stationary phase and its vapor pressure
The greater a compound’s vapor pressure, the
greater / lower its tendency to go from the
liquid stationary phase into the mobile gas
phase
A more volatile compound spends more/less
time in the gas phase than does a less volatile
compound.
Lower-boiling compounds with higher vapor
pressures travel through a GC column
faster/slower than higher-boiling compounds.
18 Gas Chromatography (GC)

5) Detector
Monitoring the eluent as it comes off the
column
Features:
A low detection limit
A linear response over a wide range of
solute concentrations (which makes
quantitative work easier)
Sensitivity for all solutes or selectivity for a
specific class of solutes
An insensitivity to a change in flow rate or
temperature
19 Gas Chromatography (GC)

5) Recorders and data stations

A computer-based station
The recorded response of the detector’s
electrical signal as the sample passes
through it over time = chromatogram
Each component = one peak
20 Gas Chromatography (GC)

5) Recorders and data stations

a) Retention time (Qualitative)
b) Peak height /peak area (Quantitative)
21 Gas Chromatography (GC)

5) Recorders and data stations

a) Retention time (Qualitative)
The distance from the time of injection to
the time at which the peak maximum
occurs
Under a definite set of experimental
conditions, should be producible
Use standards to identify the components
within a mixture qualitatively

Identity
22 Gas Chromatography (GC)

5) Recorders and data stations

a) Retention time (Qualitative)
How can you confirm the identity of the
unknown sample component?
23 Gas Chromatography (GC)

5) Recorders and data stations

a) Factors changing retention time:
Compound’s structure
The kind and amount of stationary liquid
phase used in the column
Length of the column
The carrier gas flow rate
The column temperature regimen
The solid support
The column diameter
24 Gas Chromatography (GC)

5) Recorders and data stations

b) Peak height /peak area (Quantitative)
Peak height: not a true measure of
concentration (Why?)
25 Gas Chromatography (GC)

5) Recorders and data stations

b) Peak height /peak area (Quantitative)
Area measurement = a more reliable
measure of concentration
E.g., Five pure samples of ethanol and
unknown sample under identical conditions
26 Gas Chromatography (GC)

5) Recorders and data stations

b) Peak height /peak area (Quantitative)

What is this called?

27 Gas Chromatography (GC)

6) Record keeping
Injection port temperature
Column temperature
Detector temperature
Carrier gas flow rate
Injection sample size
Length of column and identity of its
liquid stationary phase
28 High Performance Liquid
Chromatography (HPLC)

Under high pressures of up to 2700 kPa

Use a very much smaller particle size for the
column packing material which gives a
much greater surface area for interactions
between the stationary phase and the
molecules flowing past it
More tightly packed
Allows a much better separation of the
components of the mixture
Samples: both volatile and non-volatile
compounds
29 High Performance Liquid
Chromatography (HPLC)

Normal phase HPLC versus

Reversed phase HPLC

Stationary Phase Mobile Phase

Normal
Phase
Reverse
phase
30 High Performance Liquid
Chromatography (HPLC)

Normal phase HPLC

The column is filled with tiny silica particles, and the
solvent is non-polar, e.g., hexane
_________ compounds in the mixture being passed
through the column will stick longer to the polar silica
than __________ compounds will. The __________ ones
will therefore pass more quickly through the column

Reverse phase HPLC

Silica is modified to make it ____________ by attaching
long hydrocarbon chains
____________ molecules in the mixture will spend most
of their time moving with the solvent; travel through
the column more _______
31 High Performance Liquid
Chromatography (HPLC)

Components:

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32 High Performance Liquid
Chromatography (HPLC)

Components:
33 High Performance Liquid
Chromatography (HPLC)

1) Injection of sample
Sample is introduced
in a flowing mobile
phase that passes
a stationary phase
Mobile phase: a
moving liquid; moved
through the column
by a pump
Stationary phase: in a
column packed with
very small porous
particles
34 High Performance Liquid
Chromatography (HPLC)

2) Chromatogram
Again, look at retention time

Vary depending on:

Pressure used
Nature of the stationary phase the exact
composition of the solvent
Temperature of the column…

Run standards under identical conditions;

analyze retention time and identify the
compounds present
35 High Performance Liquid
Chromatography (HPLC)

2) Chromatogram
Again, look at the area under the peak
Proportional to the amount of X which has
passed the detector
If X was less concentrated, the area under the
peak would be ________ , although the retention
time will still be ___________.
Plot a calibration curve with a set of standards of
known concentrations
36 High Performance Liquid
Chromatography (HPLC)

Coupling HPLC to a mass spectrometer

Give a fragmentation pattern

Compared against a computer
database of known patterns
The identity of a huge range of
compounds can be found without
having to know their retention times
37 High Performance Liquid
Chromatography (HPLC)

Gas Chromatography (GC) Versus High

Performance Liquid Chromatography (HPLC)
Unlike gas chromatography, which is
unsuitable for nonvolatile and thermally
fragile (heat sensitive ) molecules, liquid
chromatography can safely separate a
very wide range of organic compounds,
from small-molecule drug metabolites to
peptides and proteins.
GC: column is heated; HPLC: room
temperature
decomposition
There is a chance of _______________.
38 Question 1 (GC) Exercises

Use the following gas chromatogram of a

mixture of dioxane and cyclohexane
eluted on a non-polar column to answer
the following two questions.
39 Question 1 (GC) Exercises

Which peak is more polar, A or

B?

What is the identity of the

substance labeled peak A?
Explain how you deduced this.
40 Question 2 (GC) Exercises

Which of the following gases is

unsuitable for use as a GC carrier
gas?
a) Nitrogen
b) Helium
c) Argon
d) Oxygen
e) All of the above
41 Question 3 (GC) Exercises

The GC graph obtained after

an experiment is called

a)Chromatograph
b)Chromatogram
c)Chromatophore
d)graph
42 Question 1 (HPLC) Exercises

Which compound (X or Y) is most strongly

adsorbed into the stationary phase?
Which compound (X or Y) has the highest
affinity for the mobile phase?
43 Question 2 (HPLC) Exercises

Which column has greater efficiency?

Which column gives greater resolution
between X and Y?
44 Question 3 (HPLC) Exercises

In normal phase HPLC,

a) the mobile phase is polar and the
stationary phase is nonpolar.
b) the mobile phase is nonpolar and the
stationary phase is polar.
c) eluent strength is increased by adding a
more polar solvent to the mobile phase.
d) eluent strength is increased by adding a
less polar solvent to the mobile phase.
45 Question 4 (HPLC) Exercises

Theoretical plates are used to

a) estimate the efficiency of a column
b) determine the thickness of the
stationary phase
c) measure the distribution of the analyte
between mobile and stationary
phases
d) None of the above
46 Question 5 (HPLC) Exercises
200 mg
Component X

a) How can it be deduced that the sample peak is

also X?
b) By comparing the peak heights, estimate the
amount of X in the sample.
c) Suggest a more accurate way of calculating
the exact amount of X from the peaks.