Instrumental Analytical

Techniques

An Overview
of
Chromatography and Spectroscopy
1
 •Chromatographic Techniques
-Thin layer and column chromatography
-Gas Chromatography (GC)

-High Performance Liquid Chromatography

(HPLC)
•Spectroscopic Techniques
- Atomic Absorption Spectroscopy(AAS)
- Colorimetry
- UV-Visible Spectroscopy (UV-Vis)
2
 Chromatography
A technique exploiting the interaction of the
components of a mixture with a stationary phase and a
mobile phase (solvent) in order to separate the
components.

Components are separated by different levels of

adsorption to the stationary phase and solubility in the
the mobile phase.

3
 Types of Chromatography
Paper Chromatography and Thin Layer Chromatography (TLC)

Column Chromatography

Gas
GasLiquid
LiquidChromatography
Chromatography(GLC)
(GLC)

High
HighPerformance
PerformanceLiquid
LiquidChromatography
Chromatography(HPLC)
(HPLC)

4
 Thin Layer (and Paper) Chromatography

TLC plates are inert supports (glass, plastic, aluminium) with a

thin veneer of chromatographic media (silica,etc…)

• Apply a concentrated drop of sample (•) with a capillary or dropping tube to bottom of plate (origin pencil line)

• Stand plate in a sealed vessel.

• carefully add solvent
(keep solvent level below sample).

•
• Allow solvent to adsorb up the plate,
drawing the sample with it.

5
 Thin Layer (and Paper) Chromatography

The ratio of distance travelled by the component (from origin) compared with
the distance travelled by the solvent front (from origin) is called the R f value.

x
Solvent
Solventfront
front a Rf of = a/x
b
c Rf of = b/x

Rf of = c/x
•

6
 Thin Layer and Paper Chromatography
A solution of a mixture is applied as a spot/band at the bottom of
the plate and allowed to travel with the solvent up the plate.

Mixed Unknown +
standards standards standards

• • • • • •

A B C A+B+C A+B+C ??
7
 Column Chromatography
A mixture is applied to a solid support in a chromatography
column, and eluted by a solvent.

Elute with solvent

1 2 3 4

Absorbent
medium

tap
Cotton wool
plug

8
 Gas Liquid Chromatography

9
 Gas Liquid Chromatography
A mixture is injected into a very thin“steel-jacketed”
chromatography column. Inject sample as gas or liquid. A solid
component can be dissolved in solvent but a solvent peak will
also be seen.
Inject sample
Gas mobile phase dense liquid (on solid) SP

Column in oven up to approx. 300 C.

Substance must be able to vaporise and not decompose
Extremely
sensitive

Elute with inert gas

FID detector

10
 Gas Chromatogram of High Grade Petrol

11
 Qualitative known Rf values under standard conditions

mixture of hydrocarbons eg petrol

mixture of alcohols

air

Must be able to be vaporised up to about 300oC

But
Must not decompose

Calibration graph of a series of standards of

Quantitative known concentration plotting area under peak
vs concentration

Eg. How much ethanol is in the blood?

12
 A Use a series of
r standards of ethanol
e to determine area
a under peak.

u Construct
n calibration graph
d Area (or height at
e first approx.) is
r proportional to
concentration.
p
Find area of unknown
e and read off
a concentration
k 0
0 0.10 0.20 0.30 0.40 0.50
13 Concentration of alcohol in grams/Litre
 High Performance Liquid Chromatography
(HPLC)

A mixture is injected into a “steel-jacketed” chromatography column

and eluted with solvent at high pressure (4000psi or approx 130
atm).

Inject sample as gas or liquid. A solid component can be dissolved in solvent but a solvent
peak will also be seen.

Elute with solvent UV detector

14
 STATIONARY PHASES

The surface of the stationary phase can be altered to create a surface wirh different
bonding properties in TLC, column chromatography, GLC and HPLC.

Normal Polarity

Reverse Polarity

Ion Exchange

Size Exclusion

15
 STATIONARY PHASES
(NORMAL POLARITY)

Silica or alumina possess polar sites that

interact with polar molecules.
silica
O
Polar Group HO Si
O

Components
Componentselute
eluteininincreasing
increasing
order
orderof
ofpolarity.
polarity.

Most polar…….Least polar

16
 STATIONARY PHASES
(REVERSE POLARITY)

If the polar sites on silica or alumina are capped with non-polar

groups, they interact strongly with non-polar molecules.
silica
C18 phase Me O
Si O Si
Me O

Components
Componentselute
eluteinindecreasing
decreasing
order
orderof
ofpolarity.
polarity.

Most non-polar…….Least non-polar

17
 STATIONARY PHASES
(CATION EXCHANGE)

Silica is substituted with anionic residues that interact

strongly with cationic species (+ve charged)
Cations exchange Na+ silica
O
Na O S
O

+ve
+vecharged
chargedspecies
speciesadhere
adheretotothe
thesupport
support
and
andare
arelater
latereluted
elutedwith
withacid
acid(H
(H+))
+

Most +ve…….Least +ve

18
 STATIONARY PHASES
(ANION EXCHANGE)

Silica is substituted with cationic residues that interact

strongly with anionic species (-ve charged)
Anions exchange Cl- silica
Me
Cl Me N CH2
Me

-ve
-vecharged
chargedspecies
speciesadhere
adheretotothe
thesupport
support
and
andare
arelater
latereluted
elutedwith
withacid
acid(H
(H+))
+

Most -ve…….Least -ve

19
 STATIONARY PHASES
(SIZE EXCLUSION)

Size exclusion gels separate on the basis of molecular size.

Individual gel beads have pores of set size, that restrict
entry to molecules of a minium size.

Large
Largemolecules
moleculeselute
elutefast
fast(restricted
(restrictedpath),
path),
while
whilesmall
smallmolecules
moleculeselute
eluteslowly
slowly(long
(longpath
pathlength)
length)

Larger molecules…….Smaller molecules

20
 Regions of the Electromagnetic Spectrum

Light waves all travel at the same speed through a vacuum but
differ in frequency and, therefore, in wavelength.
21
 Spectroscopy
Utilises the Absorption and Emission
of electromagnetic radiation by atoms

Absorption:
Low energy electrons absorb energy to move to higher energy level

Emission:
Excited electrons return to lower energy states

22
 Absorption v. Emission
Energy is emitted by
electrons returningto
lower energy levels
3rd
Excited 2nd
States
1st

Energy is absorbed as
electrons jump to
higher energy levels
Ground State

23
 Emission Spectra of Elements
Continuous

Sodium

Hydrogen

24 Calcium
 Absorption Spectra

Sodium

For other Spectra, click on the hyperlink below:

http://www.achilles.net/~jtalbot/data/elements/index.html

25
 The Spectroscopic Techniques are based on the fact that

Light absorbed (Absorption)

is directly proportional to the

Concentration of the absorbing component.

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 An introduction to Colorimetry
Colorimetry is a quantitative technique which makes use of the
intensity in colour of a solution is directly related to the
concentration of the coloured species in it.

Colorimetry can be used if the substance to be analysed is coloured,

or if it can be made coloured by a chemical reaction.

The concentration of the unknown solution can be estimated

by the naked eye by comparing its colour to those of a series of
standard solutions prepared by successive dilution. However at
low concentrations, colour may not be detected.

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A more accurate quantitative analysis can be made
using an instrument called a Colourimeter. The light
source of a kind that will be absorbed by the solution, ie if the
solution is blue then light of a colour other than blue will be
absorbed by it.

Simple
colourimeters
allow a choice
of three
wavelengths
using blue,
green and red
Light Emitting
Diodes (LEDs)
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29
 Red Detector
LED
measures
Green red light
LED absorbed

Blue
LED

In this example, the blue solution would absorb red (or green) and
reflect blue. The chosen red LED is passed through the a transparent
plastic or glass cell (cuvet) of fixed pathlength (1cm) containing the
blue solution to be investigated and a Detector measures the amount of
light absorbed measured.
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 Collect data Absorbance Concentration

0.0
0.00
• A set zero adjustment 0.125
enables the instrument to 0.20
0.40 0.250
factor out any absorbance of
the solvent and the material 0.380
0.59
the cuvet is made from. 0.78 0.50
0.35 unknown

• concentration of a species in solution

is proportional to the light absorbed

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Absorbance
1.00

0.80
Note that graphs
may not be linear
0.60 over a wide range
of concentrations

0,40

0.20

0
0 0.10 0.20 0.30 0.40 0.50
32 Concentration in mol/Litre
 1.00

0.80
Note that graphs
may not be linear
0.60 over a wide range
of concentrations

0.40

0.20

0
0 0.10 0.20 0.30 0.40 0.50
33 Concentration in mol / Litre
• The concentration of an unknown solution of a food
colouring can be determined by measuring its absorbance
and reading the concentration from the calibration graph.

Using the data in the graph above, if a sample of this food

colouring was found to have an absorbance of 0.35, then
its concentration would be ______ M.

Questions

• What would happen to absorbance if the path length of the

cuvet was doubled?

• What would happen if the cuvet was handled on the

transparent outer surface?
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35
 Atomic
Absorption
Spectroscopy

36
 Absorption Wavelengths of Iron

37
 Atomic Absorption Spectrophotometer (AAS)

38
 AAS Operation

Hollow
Cathode
Lamp

Gas Mixture Adjustment Display Monochromator

39 Controls Flame
 Atomic Absorption Spectrometer

Atomised
sample in Detector
flame

Monochromator
Lens Lens
Hollow
Cathode Flame
Lamp Solution Amplifier

Display
40
 Close-up view of AAS

Electrons return to ground

Less
Ions energy
absorb is
energy,
state,and photons emitted in
transmitted
jumpall to detector
to directions
excited state

Hollow Cathode Lamp

Ions in Flame
emits several unique
wavelengths of light

Transmittance
41
 Atomic Absorption Spectrometry

• measures small concentrations of metal ions in solution

– Al, As, Au, B, Ca, Cd, Co, Cr, Cs, Cu, Fe, Ge, K, Li, Mg, Mn,
Mo, Na, Ni, Pb, Si, Sr, Ti, V, W and Zn

• used by industry
− analysis of ores for metal content
− quality control of metals in steel
− testing water for metals ions
− analysing food and pharmaceuticals for metal ions

42
 Advantages of using AAS

• very sensitive:

can detect concentrations as small as a few parts

to µg / Litre (parts per billion)

• generally very specific:

set wavelength is strongly absorbed by the

particular metal ion being analysed (and not by
other components)

43
 A Source of Error

• Another species may be absorbing at the same wavelength.

44
 UV-Visible Spectroscopy

A UV-visible spectrophotometer measures the amount

of energy absorbed by a sample.

45
 The optics of the light source in UV-visible spectroscopy
allow either visible [approx. 400nm (blue end) to 750nm
(red end) ] or ultraviolet (below 400nm) to be directed at
the sample under analysis.
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47
Why are carrots orange? Carrots contain the pigment
carotene which absorbs blue light strongly and reflects
orange red and so the carrot appears orange.

400nm 500nm 600nm 700nm

Y O
E R
BLUE GREEN A RED
L
LO N
W G
E

420nm 520 nm 600nm

48
Carotene
• beta-Carotene forms orange to red crystals and occurs in the chromoplasts of plants and in the
fatty tissues of plant-eating animals.
• Molecular formula: C40H56
• Molar Mass 537
• Melting point 178 - 179 °C

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 Absorbance is set to 0% or light transmitted using a solvent
blank in a cuvet. This compensates for absorbance by the cell
container and solvent and ensures that any absorbance
registered is solely due to the component under analysis.

The sample to be analysed is placed in a cuvet (as for

colorimetry).

Qualitative analysis is achieved by determining the radiation

absorbed by a sample over a range of wavelengths. The results are
plotted as a graph of absorbance/transmittance against wavelength,
which is called a UV/visible spectrum.

50
The UV- Visible absorption spectrum for carotene
in the non-polar solvent, hexane
I
N
T
E
N
S
I
T
Y 400nm 700 nm

O
F

A ultra- violet visible infrared

B
S
O
R
P
T
I
O 320nm 460nm 540nm
N

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 Although the light absorbed is dependent on pathlength
through the cell, a usual standard 1cm pathlength is used so
that pathlength can effectively be ignored.

Quantitative analysis is achieved in a manner similar to

colorimetry. The absorption of a sample at a particular
wavelength (chosen by adjusting a monochromator) is
measured and compared to a calibration graph of the
absorptions of a series of standard solutions.

What can be analysed?

In its quantitative form, UV-visible spectroscopy can be
used to detect coloured species in solution eg. bromine ,
iodine and organic compounds or metal ions that are
coloured, or can be converted into a coloured compound.

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