3. 3
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.
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4. 4
Column Chromatography
Gas Liquid Chromatography (GLC)Gas Liquid Chromatography (GLC)
High Performance Liquid Chromatography (HPLC)High Performance Liquid Chromatography (HPLC)
Paper Chromatography and Thin Layer Chromatography (TLC)
Types of Chromatography
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5. 5
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.
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6. 6
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 Rf value.
Solvent frontSolvent front
x
a
b
c
Rf of = a/x
Rf of = b/x
Rf of = c/x
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7. 7
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.
A B C A+B+C
standards
Mixed
standards
Unknown +
standards
•• •• •• •• •• ••
A+B+C ??www.PharmInfopedia.com
8. 8
Column Chromatography
A mixture is applied to a solid support in a chromatography
column, and eluted by a solvent.
Elute with solvent
21 3 4
Absorbent
medium
Cotton wool
plug
tap
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10. 10
Elute with inert gas
Column in oven up to approx. 300 C.
Substance must be able to vaporise and not decompose
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.
FID detector
Gas mobile phase dense liquid (on solid) SP
Inject sample
Extremely
sensitive
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12. 12
mixture of hydrocarbons eg petrol
air
mixture of alcohols
Qualitative
Must be able to be vaporised up to about 300o
C
Must not decompose
Quantitative
Eg. How much ethanol is in the blood?
known Rf values under standard conditions
Calibration graph of a series of standards of
known concentration plotting area under peak
vs concentration
But
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13. 13
A
r
e
a
u
n
d
e
r
p
e
a
k
0.30 0.40 0.500.10 0.20
Concentration of alcohol in grams/Litre
0
0
Use a series of
standards of ethanol
to determine area
under peak.
Find area of unknown
and read off
concentration
Construct
calibration graph
Area (or height at
first approx.) is
proportional to
concentration.
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14. 14
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).
Elute with solvent UV detector
Inject sample as gas or liquid. A solid component can be dissolved in solvent but a solvent
peak will also be seen.
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15. 15
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
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16. 16
HO Si
O
O
STATIONARY PHASES
(NORMAL POLARITY)
Silica or alumina possess polar sites that
interact with polar molecules.
Most polar…….Least polar
Components elute in increasing
order of polarity.
Components elute in increasing
order of polarity.
Polar Group
silica
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17. 17
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.
Most non-polar…….Least non-polar
Components elute in decreasing
order of polarity.
Components elute in decreasing
order of polarity.
C18 phase
silica
Si
Me
Me
O Si
O
O
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18. 18
STATIONARY PHASES
(CATION EXCHANGE)
Silica is substituted with anionic residues that interact
strongly with cationic species (+ve charged)
Most +ve…….Least +ve
+ve charged species adhere to the support
and are later eluted with acid (H+
)
+ve charged species adhere to the support
and are later eluted with acid (H+
)
Cations exchange Na+
silica
S
O
O
ONa
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19. 19
STATIONARY PHASES
(ANION EXCHANGE)
Silica is substituted with cationic residues that interact
strongly with anionic species (-ve charged)
Most -ve…….Least -ve
-ve charged species adhere to the support
and are later eluted with acid (H+
)
-ve charged species adhere to the support
and are later eluted with acid (H+
)
Anions exchange Cl-
silica
Me N
Me
Me
CH2
Cl
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20. 20
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.
Larger molecules…….Smaller molecules
Large molecules elute fast (restricted path),
while small molecules elute slowly (long path length)
Large molecules elute fast (restricted path),
while small molecules elute slowly (long path length)
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21. 21
Regions of the Electromagnetic Spectrum
Light waves all travel at the same speed through a vacuum but
differ in frequency and, therefore, in wavelength.
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22. 22
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
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23. 23
Absorption v. Emission
Ground State
1st
2nd
3rd
Energy is absorbed as
electrons jump to
higher energy levels
Energy is emitted by
electrons returningto
lower energy levels
Excited
States
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24. 24
Emission Spectra of Elements
Calcium
Hydrogen
Continuous
Sodium
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26. 26
The Spectroscopic Techniques are based on the fact that
Light absorbed
is directly proportional to the
Concentration of the absorbing component.
(Absorption)
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27. 27
An introduction to Colorimetry
Colorimetry can be used if the substance to be analysed is coloured,
or if it can be made coloured by a chemical reaction.
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.
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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28. 28
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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30. 30
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.
Red
LED
Green
LED
Blue
LED
Detector
measures
red light
absorbed
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31. 31
• concentration of a species in solution
is proportional to the light absorbed
Absorbance Concentration
0.0
0.125
0.250
0.380
0.50
unknown
• A set zero adjustment
enables the instrument to
factor out any absorbance of
the solvent and the material
the cuvet is made from.
0.00
0.20
0.40
0.59
0.35
0.78
Collect data
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34. 34
• 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?www.PharmInfopedia.com
41. 41
Close-up view of AAS
Ions absorb energy,
jump to excited state
Electrons return to ground
state,and photons emitted in
all directions
Less energy is
transmitted to detector
TransmittanceTransmittance
Ions in Flame
Hollow Cathode Lamp
emits several unique
wavelengths of light
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42. 42
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
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43. 43
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)
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44. 44
A Source of Error
• Another species may be absorbing at the same wavelength.
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46. 46
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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48. 48
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
BLUE GREEN RED
O
R
A
N
G
E
Y
E
L
LO
W
420nm 600nm520 nm
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49. 49
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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50. 50
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.
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).
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51. 51
540nm320nm 460nm
ultra- violet visible infrared
700 nm400nm
I
N
T
E
N
S
I
T
Y
O
F
A
B
S
O
R
P
T
I
O
N
The UV- Visible absorption spectrum for carotene
in the non-polar solvent, hexane
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52. 52
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.
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?
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