Learning Objectives

1 Apply the principles of chromatography

Describe the methods used

2
in the analysis of compounds

Explain the chemistry behind the

3
methods of analysis of compounds

4 Identify which method is more effective

for the analysis of compounds
1
Analysis of Compounds

Analysis of

1 Fibres Cotton, wool, silk, polyester, nylon?

2 Dyes

3 Athletes’ blood / urine Unauthorised

substances?
4 Suspected illegal drugs
2
Analysis of Compounds

Complication

1 Complex mixtures

2 A few or one compound(s) of interest

3
Analysis of Compounds

1 How to separate out mixtures

Find compound(s) of interest

2 Identify the compound

What is it?

4
Chromatography

Separate a mixture into its components

Stationary phase Inert absorbent material

Mobile phase Liquid or gas

Longer experiment Greater separation

5
Types of Chromatography

1 Thin Layer Chromatography (TLC)

6
Thin Layer Chromatography

Blank Thin layer of

plate absorbent
material

Inert
backing
7
Thin Layer Chromatography

Coloured compounds Inspection

Colourless compounds

UV light
TLC plate
under
Chemical staining UV light
8
Thin Layer Chromatography

Unknown mixture C

Standards A & B

TLC to identify compounds?

Presumptive test
9
Thin Layer Chromatography

Too many organic compounds

TLC plate too small

10
Thin Layer Chromatography

Unknown spot
corresponding
to standard

11
Thin Layer Chromatography

Too many organic compounds

TLC plate too small

Unknown spot corresponding to standard

Compound is present

Compound may be present

12
Thin Layer Chromatography

Standards

13
Thin Layer Chromatography

Unknown

14
Thin Layer Chromatography

Solvent
front

Run TLC

15
Thin Layer Chromatography
Solvent front
Compounds present

1 Green

2 Black

16
Thin Layer Chromatography
Solvent front
Compounds present

1 Green

2 Black

3 Yellow
Unknown
4 Red

17
Thin Layer Chromatography

Retention factor (Rf)

Numerical method to
describe position of spots

18
Thin Layer Chromatography
Solvent front y

x1 Rf = x1 / y

x2 Rf = x2 / y

x3 Rf = x3 / y

x4 Rf = x4 / y

19
Gas Chromatography

Mobile phase Carrier gas

Uses a column to contain the stationary phase

Coil of packed metal tube

20
Gas Chromatography

In Out

To the detector
and computer

GC column
21
GC vs TLC

TLC Measure distance moved by

Rf
components in a fixed time

GC Measure time taken by components

to move to the end of the column

Retention time

22
GC Results

Unknown sample Known standards

23
GC Results
Solvent peak

Can be ignored

Area under peak

Proportional to the
Unknown sample amount of material
24
Gas Chromatography

1 Separate mixture into components

2 Measure amount of components present

Quantification

25
Types of Chromatography

1 Thin Layer Chromatography (TLC)

2 Gas Chromatography (GC)

3
High Performance Liquid Chromatography or
High Pressure Liquid Chromatography (HPLC)

26
HPLC

Mobile phase Liquid

Organic solvents or
aqueous solutions

Steel columns High pressures

27
Why Use High Pressures?

Stationary phase densely packed in the column

High pressures

Get good flow rates

Steel columns to withstand pressures

28
HPLC

Similar to GC

Measure time taken by components

to move to the end of the column

Measuring retention time

29
HPLC

HPLC chromatogram GC chromatogram

30
HPLC

Area under peak

Proportional to the
amount of material

HPLC chromatogram
31
HPLC vs GC

1 Efficient, highly selective, widely applicable

2 Small amounts of sample required

3 Non-destructive of the sample

4 Quantitative

5 High resolution
32
HPLC vs GC

GC HPLC

1 Simple 1 Non-volatile samples

2 Inexpensive Samples that are

2
thermally unstable
3 Rapid
33
What is it?

1 Chromatography

Separate mixture into components

Comparison to authentic samples

2 Spectroscopy
Identify components
3 Spectrometry
34
Infrared (IR) Spectroscopy

Identification of compounds

Based on molecular vibrations

Stretching and bending of chemical bonds

Energy change match that of IR light

35
Infrared (IR) Spectroscopy

Irradiate a molecule with IR

light of the correct frequency

Molecule starts to vibrate

36
Infrared (IR) Spectroscopy

Stretching frequency depends on

1 Masses of atoms involved

2 Stiffness of the bond

Single Double Triple

bonds bonds bonds

37
Infrared (IR) Spectroscopy

Double
bonds (stiff)

bonds to
H (light)
Triple bonds
(very stiff)

38
Infrared (IR) Spectroscopy
1500 cm-1

Characteristic of the
class of compounds

39
Infrared (IR) Spectroscopy
1500 cm-1

Characteristic of the
individual compound

40
Infrared (IR) Spectroscopy
1500 cm-1

Molecular
fingerprint

41
IR spectrum of diamorphine

Diamorphine

Diagnostic “Fingerprint
region”

42
Mass Spectrometry (MS)

Measure mass of molecule

How?

43
Mass Spectrometry (MS)

Light molecule Large deflection

Heavy molecule Small deflection

Measure mass of molecule

Measure the degree of deflection

44
Cocaine

Molecular formula

C17H21NO4

45
Cocaine

Molecular weight

Carbon 12 x 17 = 204

Hydrogen 1 x 21 = 21

Nitrogen 14 x 1 = 14
C17H21NO4
Oxygen 16 x 4 = 64

Total = 303
46
Cocaine

Molecular weight = 303

Cocaine? No

Same atoms can be

arranged in different ways C17H21NO4

Isomers
47
C17H21NO4 Isomers

Cocaine

48
Mass Spectrum

Pattern of fragments at detector

49
Mass Spectrum

Fragments
Molecular
ion (M+)

mass
amount of deflection
50
Mass Spectrum

Pattern of fragments at detector

Highest weight Molecular ion

Lower weights Fragment ions

Always the same pattern

51
Mass Spectrum

Cocaine?

Authentic sample of cocaine

Compare the 2 fragmentation patterns

Same Cocaine
52
Mass Spectrum
Cocaine

Molecular
ion (M+)
Diamorphine

53
Techniques

1 Chromatography

Separation of mixtures

2 Spectroscopy and spectrometry

Identification of components
54
Techniques

Combining separation and identification

Gas chromatography -
Mass Spectrometry (GC-MS)

Hyphenated technique

55
GC-MS

Auto- GC output
sampler

Goes to mass
spectrometer

GC
MS MS Detector

56
GC-MS

GC output Retention time of components

Quantify amount

MS output Identification

Combines separation and identification

57
Summary

1 Chromatography

TLC, GC and HPLC

Separation of mixtures

58
Summary

1 Chromatography

2 Infrared spectroscopy

Identification by molecular vibrations

Molecular stretching and bending

59
Summary

1 Chromatography

2 Infrared spectroscopy

3 Mass spectrometry

Identification by fragmentation

60