ATOMIC SPECTROSCOPY

BASED ON FLAME
ATOMIZATION (CHAPTER 3)
CHM 260
 Lesson Outcomes
2

¨ Explain the principles of Flame Atomic Spectroscopy

(absorption and emission)
¨ Draw and label the schematic diagram of AAS and
AES
¨ Able to explain the functions of each component in
AAS and AES
¨ Discuss the difference in terms of parts and functions
of AAS and AES
3.1 Fundamental Principle
Atomic Spectroscopy
 Atomic Spectroscopy
4

¨ A class of spectroscopic methods in which the species

examined in the spectrometer are in the form of ATOMS
(not molecules or ions)
¨ Spectral transitions are observed to occur between
electronic energy levels in the visible, UV and x-ray
regions
¨ The wavelengths of the observed absorption/emission
lines are characteristic of a particular ELEMENT
¨ The intensity of the spectral line is proportional to the
number of atoms undergoing the corresponding
transition
 En
Emission
Absorption

E1

E1

5
 Atomic Spectra
6

¨ Each element has a characteristic spectrum.

Example: Na gives a characteristic line at 589 nm
¨ Atomic spectra feature sharp bands
 Applications of Atomic Spectroscopy
7

¨ Wide application in industry & research

¨ High sensitivity for qualitative and quantitative
determination of metallic elements
eg:
- Clinical studies : Ca & Mg
- Environmental pollutants : Pb, Hg, Cu, Cd etc
- Forensic studies
- Toxicological studies : Heavy metals (As, Cr, Hg
etc)
 Elements detectable by atomic absorption

8
 ¨ Three techniques (methods) included in atomic
spectroscopy
1. Atomic absorption spectroscopy (AAS)
2. Atomic emission spectroscopy (AES)

3. Atomic fluorescence spectroscopy

(Focus on 1 & 2 only)

9
3.2 Flame AAS
Flame Atomic Absorption Spectroscopy
 AAS Principle
11

¨ AAS is based on the same principle as the flame test

used in qualitative analysis
¨ When an alkali metal salt or a calcium, strontium or
barium salt is heated strongly in the Bunsen flame, a
characteristic flame colour is observed:
Na →yellow
Li →  crimson
Ca → brick red
Sr →  crimson
Ba →green
 ¨ In the flame, the ions are reduced to gaseous metal
atoms
heat
compound atom
¨ The high temperature of the flame excites a valence
electron to a higher-energy orbital. The atom then
emits energy in the form of (visible) light as the
electron falls back into the lower energy orbital
(ground state)

12
 ¨ The ground state atom absorbs light of the same
characteristic wavelengths as it emits when returning
from the excited state to the ground state
¨ The intensity of the absorbed light is proportional to
the concentration of the element in the flame
(quantitative analysis)
¨ Absorbance or emission of atomic vapour is
measured (Oxidation states (e.g. Fe2+, Fe3+) cannot
be distinguished)

13
 3.2.1 Atomization Process (Sample
14
Introduction)
¨ In order to perform atomic spectroscopy, atoms of the
analyte must first be formed, usually in the form of an
atomic vapor
¨ Atomization is the process by which a sample is
converted to an atomic vapor
- Solution of the analyte is evaporated rapidly at an
elevated temperature to yield a finely divided solid
¨ Atomizer is a device used to convert a sample to an
atomic vapor
 Type of Atomizer Typical Atomization
Temp, °C
Flame 1700-3150
Electrothermal vaporization (ETV) 1200-3000
Inductively coupled argon plasma 4000-6000
(ICP)
Direct current argon plasma (DCP) 4000-6000
Microwave-induced argon plasma 2000-3000
(MIP)
Glow-discharge plasma (GD) Nonthermal
Electric arc 4000-5000
Electric spark 40 000 (?)
15
 3.2.2 Flame Atomization
16

Process occurring during atomization process:

1. Nebulization

Conversion of the liquid sample to a fine spray

2. Desolvation

Solid atoms are mixed with the gaseous fuel

3. Volatilization

Solid atoms are converted to a vapor in the flame

(molecules/atoms/ions.)
 What are the process involve to change the analyte
from one state to another?

There are three types of particles that exist in the flame:

1. Atoms 2. Molecules 3. Ions

17
 3.2.3 Properties of Flame
18

Types of flame:
Fuel Oxidant Temperature °C Max burning
velocity cms-1
Natural gas Air 1700-1900 39-43
Natural gas Oxygen 2700-2800 370-390
Hydrogen Air 2000-2100 300-400
Hydrogen Oxygen 2550-2700 900-1400
Acetylene Air 2100-2400 158-266
Acetylene Oxygen 3050-3150 1100-2480
Acetylene Nitrous oxide 2600-2800 285

*Selection of flame type depends on the volatilization

temperature of the atom of interest. Most common is
Acetylene/air.
 Flame Structure
19

¨ Primary combustion zone is

characterized by existence of some
non atomized species & presence of
fuel species that emit in the blue
region of the electromagnetic
spectrum
¨ Interzonal region is the hottest part of
the flame and best for atomic
absorption
¨ Oxidation of the atoms occurs in the
secondary combustion zone where the
atoms will form molecular oxides and
are dispersed into the surroundings
 Temperature Profile
20

Temperature profile in
degrees Celsius for a
natural gas-air flame
 Nebulizer
21

¨ sucks up the liquid sample (=

aspiration)
¨ creates a fine aerosol (fine
spray) for introduction into
flame
¨ mixes aerosol, fuel and
oxidant thoroughly, creates a
heterogenous mixture
¨ the smaller the size of the
droplets produced, the higher
the element sensitivity
 a) Concentric tubes.
b) Cross flow.
c) Fritted disk.
d) Babington.

22
 Methods of sample introduction in AAS

Method Type of sample

Pneumatic nebulization Solution or slurry
Ultrasonic nebulization Solution
Electrothermal Solid, liquid or solution
vaporization
Hydride generation Solution of certain
elements
Direct insertion Solid, powder
Laser ablation Solid, metal
Spark or arc ablation Conducting solid
Glow-discharge sputtering Conducting solid
23
 Burners
24

¨ The burner head is where all of the chemical

reactions take place
¨ The burner head consists of an inlet tube; fuel and
air inlets; a nebulizer; mixing cell; and the flame (the
reaction and sample cell)
¨ There are 2 types of burners in flame spectroscopy:
i. Turbulent flow
(total consumption burner)
ii. Laminar flow
(premix burner)
25
 Turbulent Flow Burner
26

q Nebulizer & burner are combined into a single unit.

q Sample is drawn up the capillary & nebulized.
q Sample flow rate: 1 to 3 mL/min.
27

Advantage
1. introduce relatively large & representative sample
into the flame.

Disadvantages
1. A relatively short path length through flame.
2. Problems with clogging of the tip.
3. Burners noisy from electronic and auditory stand
point.
 Laminar Flow Burner
28

q Sample is nebulized by the flow of oxidant which flow

through a capillary tip.
q Resulting aerosol then mixed with fuel & flow through
a series of baffles.
q Only finest droplets went through the baffels.
q Bigger sample droplets is collected at the bottom of
mixing chamber then drained to a waste container.
q Aerosol, oxidant & fuel are burned in a slotted burner
that provides a flame of 5 – 10 cm in length
29
 Advantages
1. Provide quiet flame.
2. Provide longer path length that enhance the sensitivity
& reproducibility.
Disadvantages
1. Lower rate of sample introduction.
2. Possibility of selective evaporation of mixed solvents in
the mixing chamber could create analytical
uncertainties.
3. Mixing chamber contains a potentially explosive
mixture that can flash back if the flow rates are too
low.
30
3.3 Instrumentation for Flame AAS
 AAS Instrumentation
32

Single Beam AAS Instrument:

The modulated power source can be replaced by a chopper

 Double Beam AAS Instrument:

33
 ¨ Radiation from HCL is split into 2 beams;
1. One passes through the flame.
2. The other around the flame.
¨ A half-silvered mirror returns both beams to a single
path then pass through the monochromator then
detector
¨ Monochromator is placed between sample and
detector

34
 3.3.1 Line Sources
35

¨ Radiation source in AAS is a line source which

provide narrow emission bands
¨ Common radiation source used in AAS
1. Hollow cathode lamp (HCL)
2. Electrodeless discharge lamp (EDL)
 Hollow Cathode Lamps (HCL)
36

q Light from this lamp exactly light required for the analysis,
even no monochromator is used.
q Hollow cathode lamp MUST contain the element to be
determined.
 How does HCL works?
37

q When lamp is on, atoms are supplied with energy

that causes electrons of the atoms elevate to the
excited states.
q Upon electrons returning to ground state, wavelength
of the photon emitted are useful for the analysis.
q The photon emitted will supply the exact amount of
energy needed for the analyzed metal to undergoes
excitation.
 Excitation mechanism in HCL
38

q When lamp is on, atoms are supplied with energy that causes
electrons of the atoms elevate to the excited states
q Upon electrons returning to ground state, wavelength of the
photon emitted are useful for the analysis
q The photon emitted will supply the exact amount of energy
needed for the analyzed metal to undergoes excitation.
q When atoms return to ground state, line spectrum of that
specific atom emitted
q This light is directed at the flame where unexcited atoms of the
same element absorb the radiation and raised to the excited
state
q Absorbance is measured and related to the concentration
 Ar + e- →      Ar+ + 2e-

Reactions in the HCL:

-ionization of filler gas: Ar + e- → Ar+ + 2 e-
-sputtering of cathode atoms: M(s) + Ar+ → M(g) + Ar
-excitation of metal atoms: M(g) + Ar+ → M*(g) + Ar
-light emission: M*(g) → M(g) + hν

39
 Cathode Lamp Process
40

Excited fill gas atoms sputter metal atoms from the cathode and
excite them via a kinetic energy transfer
 Electrodeless Discharge Lamp (EDL)
41

¨Constructed of a metal or salt of interest sealed in a quartz tube

filled with a noble gas (Ne or Ar) at low pressure (1 – 5 torr)
¨The noble gas is ionized and accelerated by a strong radio-

frequency (RF) or microwave field and excite the metal or salt of

interest
¨When power is applied to the driver, an RF field is created

¨The coupled energy will vaporize and excite the atoms inside the
bulb, causing them to emit their characteristic spectrum
¨EDL can provide radiant intensities usually one to two orders of
magnitude greater than HCL
42
 3.3.2 Source Modulation
43
 q Why source modulation is employed in AAS?
1. To eliminate interference caused by emission of the
radiated flame from analyte atoms and flame gas
species.
2. To distinguish between the component of radiation
arising from the source and the component of
radiation arising from the flame background.
q Source modulator: Light chopper
(circular rotating metal disk)

44
 q The function of light chopper is to eliminate the effects of
radiation from the flame.
q Light is “chopped” with a rotating half-mirror so that
detector could received two alternating signals. One from
the radiation source and one from the flame.
q At one moment (opaque), only light emitted by flame is
read by the detector since the light from the radiation
source is cut off.
q Next moment (transparent), light from both the flame
emission and radiation source is read. Transmission from the
source light is measured since the source light is allowed to
pass.
q Absorbance of the sample is determined by measuring the
difference in radiant power between flame emission signal
and signal from the radiation source.
45
 FAAS
46
 Process occurring
during
atomization

47
 3.3.3 Interferences
48

¨ Interference: when the presence of substance

changes the signal from the analyte of interest when
analytes concentration remain the same
¨ Interferences in AAS:
a) Spectral interference
b) Chemical interference
c) Ionization interference
 Spectral interferences
49

¨ Spectral interferences: any physical process that effects the

light intensity at the analytical wavelength
¨ An absorption or emission line that arises from an interfering
species that overlaps the analyte absorption line or lies close
enough to the analyte absorption line that resolution by the
monochromator is impossible
¨ Example: A vanadium line at 3082.11Å interferes in an
analysis based upon the aluminum absorption line at 3082.15
Å.
 ¨Sources:
a) Scattering by combustion or particulate products
- Arise from the combustion products or particulate
matters from the atomization scatters the radiation from
the source
- Presence of combustion products that exhibit broadband
absorption or particulate products that scatter radiation
b) Scattering by sample matrix interference
- Arise when the emission or absorption of an interfering
species overlaps or lies so close to the analyte absorption

50
 - Example: Determination of barium in alkaline earth
mixture. The wavelength of Ba line used for atomic
absorption analysis appears in the center of a broad
absorption band for CaOH.
- The effect can be eliminated by substituting nitrous oxide
for air as the oxidant which yields a higher temperature
that decomposed the CaOH and eliminates the absorption
band

51
 q How to solve the problems?
a) Tune the monochromator to a different spectral line for
the element of interest so that there is no overlap.
- Example: A vanadium line at 3082.11Å interferes in an
analysis based upon the aluminum absorption line at
3082.15 Å. This type of interference can be avoid by
employing the aluminum line at 3092.7 Å instead
b) Utilizes a continuum source (e.g. deuterium lamp) and a
chopper. The continuous source will give broadband
absorption assumed to be due to scattering or absorption
by the sample matrix.
- The absorbance of the beam from the deuterium lamp is then
subtracted from the analyte beam (HCL) and thus a
background correction is obtained.
52
 c) Zeeman background correction.
- When an atomic vapor is exposed to a strong magnetic
field (~10 kG), a splitting of electronic energy levels of
the atoms takes place that leads to formation of several
absorption lines for each electronic transition. These lines
are separated from one another by about 0.01 nm, with
the sum of the absorbance for the lines exactly equal to
the original line from which they were formed
- Plane polarized light will allow the observation of
different bands of absorption

53
 Chemical interferences
54

¨ Chemical interferences: alteration in analyte

absorbance caused by chemical processes during
atomization
¨ Sufficient energy must be available to dissociate the
molecular form of the analyte to free atoms.
¨ If the sample contains a component which forms a
thermally stable compound with the analyte that is
not completely decomposed by the energy
available in the flame, achemical interference will
exist resulting in low result
 ¨Sources:
a) Formation of low volatility (refractory elements that
form stable compounds that are not completely
atomised):
- eg: a decrease in Ca absorbance is observed with the
increasing concentrations of phosphates,PO43- and
sulphate,SO42- due to formation of calcium phosphate
and calcium sulphate that lowers the absorbance
-Example:
Ca2+ + SO2-4 → CaSO4 (s) (non-volatile salt)
Ca2+ + PO3-4 → Ca2P2O7 (s) (non-volatile salt)

55
56

- Overcome by adding lanthanum chloride (LaCl3) to

samples, standards and blank. La3+ ions are a
releasing agent for Ca.
Ca3(PO4)2 + 2LaCl3 →3CaCl2 + 2LaPO4
- Overcome by adding protective agent such as EDTA
EDTA + Ca →  Calcium-EDTA chelate→ Ca vapor
- Overcome by using higher-temperature flame:
- Example: nitrous oxide-acetylene flame
 b) Dissociation equilibrium:
- Formation of compounds between the metal and other
components in flame (O, Cl)
- eg: MCl ↔ M + Cl
NaCl ↔ Na + Cl

- A shift in the equilibrium towards the metal oxide will

result in a reduction in absorption of radiation by the
metal analyte.

57
 Ionization interference
58

¨ Some ions will be produced in hot flames (nitrous oxide –

acetylene flame)
¨ If additional energy is applied, the ground state atom can
be excited or an electron may be totally removede from
the atom, creating an ion which could reduce the number
of ground state atoms available for the light absorption
¨ Ions have a different spectrum from atoms
¨ Most serious for Mg, Ca, Na, Li, K, Sr
¨ Results in non-linear calibration curve
¨ Overcome by adding easily ionised element (Na, K, Cs),
creating a large number of free electrons in the flame &
suppressing of the analyte
 3.3.4 Sample preparation &
59
Quantitative analysis
Sample Preparation Methods:
¨ Some samples such as liquids and homogeneous

solids need no sample preparation

¨ Sometimes need to extract the analyte from the

sample solvent
Solid sample preparation:
Microwave
a) Wet digestion
b) Dry ashing Hot plate
 a) Wet Digestion (microwave)
60

¨ Microwave digestion is the best method converting liq. or

solid sample into solution
¨ The sample is digested with a combination of different
acids under high temperature and pressure
¨ The advantages include:
- sample contained within the digestion vessel
- highly efficient
- almost any sample can be digested
q Volatile element are retained in the reaction vessel
 a) Wet digestion (hot plate)
61

¨ Hot plate digestion uses glass or Teflon beakers for

the sample preparation. Usually re-dissolve steps
are needed
¨ Certain geological samples require tough
digestions. Use acids such as HF, HNO3 and HClO4
or Lithium metaborate fusion at 1200 °C. Uses
graphite crucible to heat the sample to molten state
¨ Organics can also be digested this way
¨ Problem: Loss of volatile elements
 b) Dry ashing
62

¨ For analysis of non-volatile elemnets

¨ Mainly used for organic samples
¨ Sample is dried and burnt at 500-600 °C to
remove carbonaceous matter
¨ Adv: Matrix effects are very much reduced
 Quantitative Analysis
63

¨ The concentration of analyte can be determined by:

a) Calibration curve
b) Standard addition method
¨ Quantitative analysis still follows Beer’s Lambert
Law and the concentration of unknown are
determined by the same way using the
formula( A = εbc)
¨ Width of the flame is consider as the beam
pathlength
 Calibration Curve
64

¨ A general method for determining the concentration

of a substance in an unknown sample by comparing
the unknown to a set of standard of known
concentration
¨ Plot is a linear of radiation absorbed vs concentration
of the analyte
¨ Analysis should never be based on the measurement
of a single standard with assumption that Beer’s law is
being followed
 Standard calibration curve
65

How to measure the

concentration of
unknown?
Practically, you have
measure the absorbance
of your unknown. Once
you know the absorbance
value, you can just read
the corresponding
concentration from the
graph .
 Standard Addition Method
66

¤ used to overcome matrix effect.

¤ involves adding one or more increments of a
standard solution to sample aliquots of the
same size.
¤ each solution is diluted to a fixed volume before
measuring its absorbance.
 How to produce standard addition
curve?
67

1. Add same quantity of unknown sample (spike) to a

series of volumetric flasks.
2. Add gradual amounts of standard solution to each
volumetric flask. For example 0 mL, 5 mL , 10mL , 15mL.
3. Dilute each flask to calibration mark, mix and measure the
absorbance.
68

Standard Addition Methods

Single-­‐point  standard   Multiple  additions  

addition  method   method  
 For single-point standard addition,
69

Absorbance of A1 = εbVxCx
diluted sample Eq. 1
Vt

Absorbance of
diluted sample A2 = εbVxCx + εbVsCs Eq. 2
+ std Vt Vt
70

For single-point standard addition,

Dividing the 2nd equation by the first & then rearrange it
will give.

Cx = A1 Cs Vs
(A2 – A1 ) Vx
 Example (Standard Addition)
71

A 2.00-mL urine specimen was treated with reagent

to generate a color with phosphate which then was
diluted to 100mL. Second 2.00mL sample was added
exactly 5.00mL of a phosphate solution containing
0.03 mg/mL phosphate then was treated in the same
way as the original sample. The absorbance of the
first solution was 0.428, while the second one was
0.538. Calculate the concentration of phosphate in
milligrams per millimeter of the specimen.
 Solution:
Cx = A1 Cs Vs
(A2 – A1 ) Vx

Cx = (0.428) (0.03 mg PO43-/mL) (5.00mL)

(0.538 – 0.428)(2.00mL sample)

= 0.292 mg PO43- / mL sample

72
 • The difference between the volume of the standard
added at the origin and the value of the volume at
the intersection of the straight line with the x-axis or x-
intercept (Vs)0 is the volume of standard reagent
equivalent to the amount of analyte in the sample.
• Since,
kVscs kVxcx
A= +
Vt Vt
by solving the equation for cx,

(Vs )0cs
cx = −
73
Vs
 If Beer’s law is obeyed,
A = εbVstdCstd + εbVxCx
Vt Vt
= kVstdCstd + kVxCx
A = (kCstd)Vstd + kCxVx

k  is  a  constant  equal  to  εb/Vt  

74
 Cx can be obtained from the ratio of these two
quantities m and C

C = kVxCx
m kCstd

Cx = bCstd
mVx

75
 Example 1 (Plotting Standard Addition
76
Graph)
The chromium in an aqueous sample was determined by
pipetting 10.0 mL of the unknown into each of five 50.0 mL
volumetric flasks. Various volumes of a standard containing
12.2 ppm Cr were added to the flasks, followed by diluting
with distilled water.

1. Plot the standard addition graph.

2. Calculate the concentration of Cr in the sample.
 Solution:
1. Plot the standard addition graph (Absorbance versus volume of standard
solution).
Absorbance
0.6  

0.5  
y  =  0.0088x  +  0.2022  
R²  =  0.99993  
0.4  

0.3  

0.2  

0.1  

0  
-­‐40   -­‐30   -­‐20   -­‐10   0   10   20   30   40   50  

-­‐0.1   Volume of standard solution, mL

77
 2. Calculate the concentration of Cr in the sample (Cx).

Cx = bCstd
mVx
= (0.202 x 12.2 ppm) = 30.81 ppm
(0.008 mL-1 x 10 mL)

78
 Example 2 (Plotting Standard Addition
79
Graph)
In the determination of silicon, Si in jet-engine oils using
AAS, the standard addition method was used and the
following data was obtained.

Plot the standard addition graph and determine the

amount of Si present in the oil sample.
 Solution:
Graph Absorbance versus concentration of standard solution
Absorbance
0.2  
y  =  0.0042x  +  0.0283  
R²  =  0.97497  
0.15  

0.1  

0.05  
x- intercept = -7

0  
-­‐20   -­‐10   0   10   20   30   40  
Concentration of standard solution, ppb
-­‐0.05  

Amount of Si in jet engine oils = | x-intercept |

x-intercept ⇒ when y = 0
0.004x + 0.028 = 0
80 x = -7
= |-7.0| = 7.00 ppb
 3.3.5 Detection Limits & Accuracy
81

¨ Detection limits for FAAS vary enormously: from 1-5

ppb (eg: Ca, Cd, Cr, Cu) to more than 1000 ppb
(eg: P). Some elements (B, C, Br) cannot be
measured at all

¨ Accuracy of AAS:
- high sensitivity for most elements
- flame atomisation: concentrations at the ppm level
3.4 Flame Emission Spectroscopy
 Atomic Emission
83

¨ Electromagnetic radiation emitted when an excited

atom relaxes to lower levels by giving up the excess
energy in the form of photons

En
hv
hv

hv
E1
 Introduction
84

¨ Basic Schematic
Atomizer

Detector

¨ Scanning instruments can detect multiple elements

¨ Many lines detected so sometimes it is a quantitatively
difficult method
¨ Source can be flame, but more commonly plasma
because it is much hotter
85

¨ Each element emit its own characteristics line spectrum

¨ Quantitative analysis can be performed here by
observing what λ are emitted & comparing these
with various standard
¨ Detector permits qualitative as well as quantitative

analysis
¨ Wavelength of emitted radiation indicates what
element is present and the radiation intensity indicates
how much of the element is present
86

¨ Intensity of the emitted light increase with concentration

¨ Relationship between intensity and concentration is
usually linear

I
I = kc
Unknown concentration can be
detected by comparison with one
or a series of standards in the
same manner for the molecular
techniques
c
 Principles common to AES & AAS
87

¨ AES- it measures the radiation emitted by the excited

atoms that is related to concentration
¨ AAS- it measures the radiation absorbed by the
unexcited atoms that are determined
¨ AES - depends upon the number of excited atoms, &
greatly influenced by temperature variations
¨ AAS - depends upon the number of unexcited atoms,
the absorption intensity is not directly affected by the
temperature of the flame
88
 Relationship between AAS & AES
89

¨ Both methods use atomization of a sample and therefore

determine the concentrations of elements
¨ AAS: absorption of radiation of a defined wavelength is
passed through a sample and the absorption of the
radiation is determined. The absorption is defined by the
electronic transition for a given element and is specific for
a given element. The concentration is proportional to the
absorbed radiation
¨ AES: the element is excited. A rapid relaxation is
accompanied by emission of UV or visible radiation is
used to identify the element. The intensity of the emitted
photon is proportional to element concentration
 Excitation sources
90

¨ Flame (3000 K)
¨ Plasma (5000-10000 K)
¨ Electric arc and spark (5000 K)
¨ Lasers (10000 K)
 Excitation source - Flame
91

¨ AES using flame as excitation source is only suitable

for light elements, eg: NA, K and Ca
¨ Normal flames are not hot enough to excite heavy
metals and emit light
 3.4.1 Instruments
92

1. Nebulizer
2. Burner
3. Monochromator
4. Detector
5. Readout device / computer
P Wavelength Signal Processor
Source Selector Detector
Readout

Sample
 Flame AES
93

Slit Slit

Emitted
Detector

Lens
Flame Filterer
 q Sample is sprayed by the nebulizer into the burner
Carried into the flame
q Atomized & excited
q The emission from the excited atoms passes into the
monochromator where the selected wavelength is
passed through for measurement
q Intensity of the emitted wavelength is measured by
the detection system & indicated on the readout/
computer

94
 Disadvantages of FAES
95

¨ Spectral & chemical interferences as in AAS

¨ Limited linear range due to self absorption (photons
emitted by excited atoms partly absorbed by
ground state atoms in flame)
¨ Flame instability
¨ High emission background
 3.4.2 Interferences
96

INTERFERENCES

Chemical
Spectral
interference
interference

NOTE: same interference which occur in AAS

 3.4.3 FAAS vs FAES (comparison)
97

Flame Atomic Flame Atomic

Absorption Emission
Process measured Absorption (light Emission (light
absorbed by unexcited emitted by excited
atoms in flame) atoms in a flame)
Use of flame Atomization Atomization &
excitation
Instrumentation Light source No light source
Beer’s Law Applicable Not applicable
Data obtained A vs c I vs c
 Excitation source - Plasma
98

¨ Plasma – highly ionized, electrically neutral gaseous

mixture of cations and electrons that approaches
temperature ∼ 10, 000 K
¨ There are three types of plasma sources:
a) Inductively coupled plasma (ICP)
b) Direct current plasma (DCP)
c) Microwave induced plasma (MIP)
q ICP is the most common plasma source
 Inductively coupled plasma (ICP)
99

¨ Constructed of three concentric quartz tube

¨ RF current passes through the water-cooled Cu
coil, which induces a magnetic field
¨ A spark generates argon ions which are held
in the magnetic field and collide with other
argon atoms to produce more ions
¨ Argon in outer tube swirls to keep plasma
above the tube
¨ The heat is produced due to the formation of
argon ions
 Plasma Appearance:
a) Excitation region
¨ The bright, white, donut shaped region at the
top of the torch. Excites & ionises atoms
¨ Temp: 8000-10 000 K

b) Observation region
q The flame shaped region above the torch
with temperatures ∼ 1000 – 8000 K
q The spectrum consists of emission lines from

the analyte along with many lines from ions in

the torch

100
 Sample introduction:
a) Liquid sample
¨ Nebulizer similar to FAAS

¨ Sample nebulized in a stream of argon with a flow rate

of 0.3 – 1.5 L/min
¨ Sample aerosol enters the plasma at the base through the

central tube
b) Solid sample
q Sample atomized by electrothermal atomization and
carried into the plasma by a flow of argon gas

101
 Advantages of ICP-AES over FAES
102

¨ Temperature is two to three times higher than in a

flame or furnace, which results in higher atomization
and excitation efficiencies
¨ There is little chemical interference
¨ Atomization in the inert (argon) atmosphere minimizes
oxidation of the analyte
¨ Short optical path length minimizes the probability of
self-absorption by argon atoms in the plasma
¨ Linear calibration curves can cover up to five orders
of magnitude
 ¨ Much lower detection limit because:
- Higher temperature with the plasma will increase the
population of excited state atoms
- The plasma environment is relatively chemically inert
due to the higher population of electrons which will
minimize the interference of ionization

103
 Chapter 4
AN INTRODUCTION TO CHROMATOGRPHIC
SEPARATION
 Lesson Outcomes:

Identify and explain the different types of chromatographic

analysis
Explain the principles of gas chromatography, high
performance liquid chromatography, ion exchange and size
exclusion and etc
Interpret analytical information from chromatographic data
Recognize problems arising from resultant spectrum and
chromatograms and suggest probable solutions

2
 4.1 A General Description of
Chromatography

3
  Is a technique used to separate and identify the
components of a mixture
 Works by allowing the molecules present in the
mixture to distribute themselves between a mobile
and a stationary phase
mobile phase = solvent or gas
stationary phase = column packing material

4
 How separation occur?
 Chromatography used to separate and identify the
components of complex mixtures
 Works by allowing the molecules in the mixture to distribute
themselves between a stationary and a mobile phase
 Components that are strongly retained by the stationary
phase move slowly with the flow of mobile phase
 In contrast, components that are weakly held by the
stationary phase travel rapidly (fast)
 As a consequence of these differences in mobility, sample
components separate into discrete bands that can be
analyzed qualitatively and/or quantitatively
5
 Classification of chromatographic methods
1. Based on physical means
- The way stationary and mobile phases are brought
into contact.

2. Based on the types of mobile phase

- Either gas, liquid or supercritical fluid.

3. Based on the kinds of equilibria involved in the in

solute transfer between the phases
-Interaction of analyte between stationary and
mobile phases.
6
 Chromatographic Methods based on
physical means
Column Chromatography Planar Chromatography
 stationary phase is held  stationary phase is
in narrow tube; mobile supported on a flat plate or
phase moves by in the interstices of a paper;
pressure or gravity mobile phase moves
 Example: through capillary action or
Gas chromatography gravity
(GC), Supercritical-fluid  Example:
chromatography (SFC) Thin-layer chromatography
(TLC), Paper
chromatography (PC)
7
 Column Chromatography Planar Chromatography
8
 Chromatography based on types of mobile
phase
Column chromatography can further be differentiated
based on the types of mobile phase;
Gas
eg: gas Chromatography

Liquid
eg: liquid chromatography

Supercritical fluid
eg: supercritical fluid chromatography
9
 Gas Chromatography Liquid Chromatography

10
Supercritical Fluid Chromatography
 Classification of Chromatographic Methods

Chromatography

Partition Adsorption Ion-exchange Size-exclusion

Liquid- Gas- Liquid-solid Gas-solid Liquid-solid Liquid-solid

liquid liquid

11
 Types of chromatography on the basis
of interaction of the analyte with
stationary phase
 Adsorption – for polar non-ionic compounds
 Size Exclusion – stationary phase is a porous matrix sieving
 Ion Exchange – for ionic compounds
- Anion – analyte is anion; bonded phase has positive charge
- Cation – analyte is cation; bonded phase has negative charge
 Partition – based on the relative solubility of analyte in
mobile and stationary phases
- Normal – stationary phase polar, the mobile phase nonpolar
- Reverse – stationary phase nonpolar, the mobile phase polar
12
 Chromatography based on interaction of the
analyte with stationary phase

4. Size Exclusion – separate molecules by size;

sieving- stationary phase is a porous matrix

13
 Adsorption Chromatography
 Adsorption - of solute on surface of stationary
phase; for polar non-ionic compounds
 Components of the mixture selectively adsorb (stick)
on the surface of a finely divided solid stationary
phase.
 As mobile phase (gas/liquid) carries the mixture
through the stationary phase, the components of the
mixture stick to its surface with varying degrees of
strength and thus separate.
 Stationary phase: solid
Mobile phase: gas/liquid
14
 TLC Plates

15
 Partition Chromatography
 Partitioning is a distribution (by dissolving) of the
components between 2 immiscible phases.
Separations of the components will be based on
relative solubility of the components in the mobile
and stationary phase
 Partition - based on the relative solubility of
analyte in mobile and stationary phases
a. Normal phase – stationary phase polar, the
mobile phase nonpolar
b. Reverse phase– stationary phase nonpolar, the
mobile phase polar

16
  Example of partitioning using polar stationary
phase.
i. Polar components will retain longer than the
non-polar components
ii. Non-polar components will move quickly
through stationary phase and will elute first
before the polar components and vice-versa
 The stationary phase actually consists of a thin
film adsorbed (stuck) on or chemically bonded to
the surface of a finely divided solid particles
 If the mobile phase is gas, the volatility (vapor
pressure) and solubility in stationary phase plays
an important role
17
18
 Ion Exchange Chromatography
 Method for separating mixture of ions
 Ion Exchange - attraction of ions of opposite charges; for
ionic compounds
i. Anion - analyte is anion; bonded phase has positive
charge
ii. Cation – analyte is cation; bonded phase has negative
charge
 Sample is aqueous solution of inorganic ions or
organic ions
 Stationary phase are small polymer resin “beads”
usually packed in a glass tube.
i. These beads have ionic bonding sites on their
surfaces which selectively exchange ions with certain
mobile phase compositions as the mobile phase
19 penetrates through it.
  Ions that bond to the charged site on the resin
bead are separated from organic or inorganic
ions aqueous solution.
 The process is repeated several times by
changing of the mobile phase composition
 The process begin with initially running the
analysis using a mobile phase with all the ions in
the mixture.
 The mobile phase is then change for several
times in a stepwise fashion so that one kind of
ion at a time is removed.
 The process is repeated until complete
separation achieved.
20
21
22
 Size Exclusion Chromatography
 Size Exclusion – separate molecules by size;
sieving- stationary phase is a porous matrix
 Also called gel permeation chromatography.
 Separation technique of dissolved species is based
on the size of the components.
 Stationary phase: porous polymer resin particles
(molecular sieves).
 The components to be separated enter the pores
of these particles and are slowed down from
progressing through this stationary phase.

23
  Separation depends on the sizes of the pores
relative to the sizes of the molecules to be
separated.
 Small particles are retarded to a greater extent
than large particles (some of which may not
enter the pores at all) and separation occurs.
 Particles with size bigger than the pore size will
be eluted first from the column

24
25
 Terminologies in Chromatography
 Elution: A process in which species are washed
through a chromatographic column by addition of
fresh solvent
 Mobile phase: Is one that moves over or through
an immobilized phase that is fixed in place in a
column or on the surface of flat plate
 Stationary phase: A solid or liquid that is fixed in
place. A mobile phase then passes over or
through the stationary phase
 Retention time: Time required for the sample to
travel from the injection port through the column
26
to the detector
 4.2 Migration Rates of Solutes

27
 4.2.1 Distribution Constant
 An analyte is in equilibrium between 2 phases;
Amobile ↔ Astationary
 The equilibrium constant, K (partition constant) is
the molar concentration of analyte in the stationary
phase divided by the molar concentration of the
analyte in the mobile phase
[ A]stationary
K
[ A]mobile
4.2.2 Retention Factor
 Retention factor, k’ (capacity factor) is used to
describe the migration rate of an analyte on a
column tR  tM tM = Time taken for the mobile

k' A 
phase to pass through the
column
28
tM tR retention time
 4.2.3 Retention Time
 Retention time (tR) is the time for the analyte to pass
through the column of the time between the sample
injection and an anlyte peak reaching a detector at
the end of the column

29
 4.2.4 Dead Time
 Dead time (tM)is the time a non-retained compound
spends in the mobile phase (the amount of time the
non-retained compound spend in the column.
 tM can be expressed as the time required for an
average molecule of the mobile phase to pass
through the column

4.2.5 Adjusted Retention Time

 The adjusted retention time (tR’) is the time a
compound spends in the stationary phase
 tR’ is the difference between the retention time (tR)
and the dead time (tM) for a compound
30 tR '  tR  tM
 4.2.6 Selectivity Factor
 Selectivity factor α) is the ratio of the capacity
factors of two peaks which describes the separation
of two species (A and B on the column)
 The selectivity is always greater than one (α>1)
 If the selectivity equals to one (α=1), the
compounds cannot be seperated
 The higher the selectivity, the more separation
between two compound or peaks
k' B

k' A
*Note that species A elutes faster than species B
31
 4.3 The Efficiency of
Chromatographic Column

32
 Description of Column Efficiency
 The efficiency is related to the number of
compounds that can be separated by the column
 The efficiency is expressed as the number of
theoretical plates (N, unitless) or as the height
equivalent to a theoretical plate (HETP, milimeters)
 The efficiency increases as the height of
theoretical plate decreases, thus more
compounds can be separated by the column and
vice versa

33
 The Theoretical Plate Model of
Chromatography
 The plate model proposes that the chromatographic
column contains a large number of separate layer
(theoretical plates)
 The analyte moves down the column by transfer of
equlibrated mobile phase from one plate to the next
The column

H
Theoretical plates
*Note that the plates do not really exist, they’re just figment
of imagination in order to understand the separation
34
process in the column
 Where,
L H = height of the plate
H L= length of the column
N N= number of theoretical plate

 The number of theoretical plates that a real column

can be found by examining a chromatographic peak
after elution 2
 tR  Where,
N  16  W = width of the peak
W 
or 2
5.5tR Where,
N 2 w = w1/2 is the peak width at
w1 2 half-height
35
 4.4 Column Resolution

36
 4.4.1 The Effect of Retention and Selectivity
Factors on Resolution
 Resolution is the measure of how well species
have been separated
2[( tR )B  ( tR ) A ]
WA  WB
Where,
ΔZ = separation btwn peak A and B
WA and WB = widths at the base of peaks A and B

37
  Acceptable resolution is Rs=1.0 and the best
resolution between two peaks requires Rs>1.5
 To relate with the number of plates (N), the
selectivity factor (σ) and the retention factors (k’) of
the two solutes;
N    1  k ' B 
R   
4    1  k ' B 

Simplified: Rs = √ N

38
 Rs values of less than 1.0 are considered unresolved
peaks

39
 4.4.2The Effect of Resolution on
Retention Time
 Relationship btw the resolution of a column and
retention time
16 R H    (1  k ' B)3
2 2

(tR) B  S
 
    1  (k ' B ) 2

Where,
Ʋ = velocity of the mobile phase

Simplified: tR = Rs2

40
 Example:
17.63 min

16.40 min
Length of column: 30 cm
Peak widths:
1.30 min A = 1.11 min
B = 1.21 min

Calculate:
i) column resolution, Rs
ii) the average number of plates, N
iii) the average plate height, H
iV) length of column to achieve Rs 1.5
41
 Answer:
i) 2[(tR )B  ( tR )A ]
Rs  = 1.06
WA  WB
2
ii)  tR 
N  16  = 3.44 x 103
W 

iii) L = 8.7 x 10-3 cm

H
N

iv) (Rs)1 = √N1 = 60 cm

(Rs)2 √N2
42
 Variables Affecting Column
Efficiency

1. Mobile phase flow rate

2. Particle size
3. Diameter of column
4. Film thickness

43
 Effect of Mobile Phase Flow

 From both the plots for LC and GC, we can see that
both show a minimum in H at low linear flow rates
 H increases as the mobile phase flow rate
increases
44
 Effect of Particle Size

Effect of particle size on plate height for a packed GC column

 The numbers to the right is the packing particle

diameters
 The smaller the particle size, the more pressure is
needed to push the mobile phase through the
column
45  H increases as the particle size increases
 Effect of Diameter of The Column
 For packed column, the most important variable
that affect column efficiency is the diameter of the
particles that making up the packing
 The increase in column diameter will increase the
plate height

46
 Effect of Film Thickness
 With thick films makes the analyte molecule travel
slower. As a result, thick films will increase in plate
height

47
 4.5 Applications of Chromatography

48
 Qualitative and Quantitative
Analysis
25.6 min
 Retention time tell as about
compound identity =
qualitative
 Peak Area or height tell us
how much of compound is
there = quantitative

49
 Qualitative Analysis
 Based on retention time
- Provided the sample produce the peak at the
same retention time as a standard under identical
conditions

50
 Same compound
due to the same tR

51
 Quantitative Analysis
1. Analysis based on Peak Height
 The height of chromatographic peak is obtained
by connecting the base lines on either side of
the peak by a straight line and measuring the
perpendicular distance from this line to the
peak.
 Suitable if the peak is very narrow and have
uniform shapes or when peak area values are
not available.

52
2. Analysis based on Peak Area
 Peak areas are usually the preferred method of
quantitation since peak areas are independent of
broadening effects.
 Most modern chromatographic instruments are
equipped with computer or digital electronic integrator
that permit precise estimation of peak areas.

A = peak height (h) x w1/2

w1/2 = width at ½ height

53
 3. Calibration method (also known as external method)
 Involve preparation of series of standard solutions
that approximate the composition of the unknown.
 The peak heights or areas are plotted as a function
of concentration.
 The concentration of the component(s) to be
analysed is determined by comparing the
response(s) (peak(s)) obtained with the standard
solutions.

54
 4. Internal Standard Method
 Internal standards are widely used in
chromatography because the small quantity of
sample solution injected into the chromatograph is
not very reproducible in some experiments.
 By knowing the concentration of the standard
compound and its relative response, the
concentration of analyte can be determined.
 Internal standards are also desirable when sample
loss can occur during sample preparation steps prior
to analysis.

55
  Calibration involves plotting the ratio of the analyte
signal to the internal-standard signal (Area ratio or
height ratio) as a function of the analyte
concentration of the standards
 Signal from analyte is compared with signal from the
internal standard to find out how much analyte is
present

Area Ratio = Peak Area/Height Analyte

Peak Area/Height internal standard

Area or
height ratio
Concentration of analyte in sample

56 Concentration of analyte in standard solution

 5. Analysis based on Peak Area using Response Factor
Method
 A measure of relative mass spectral respond of an
analyte compare to each internal standard
 The response factor for compound X can be
calculated using the following equation:

Response factor = Peak Area of Compound X

Concentration of Compound X

57
  Example:
An injection containing benzene at a concentration of
2000 µg/mL is made and results in a peak area of
100,000. An injection of the sample with the unknown
concentration of benzene has a peak area of 57,000.
Calculate the concentration of benzene present in the
sample.
Answer:
Step 1: Calculate the Response Factor
Response Factor for benzene = 100000 = 50
2000
Step 2:
Concentration of benzene in sample = 57000 = 1140 µg/mL
58 50
 6. Area Normalization Method
 In the normalization method, the areas of all
eluted peaks is normalized
 The percentage content of one or more
components of the substance to be examined is
calculated by determining the area of the
peak(s) as a percentage of the total area of all
the peaks, excluding those due to solvents or
any added reagents and those below the
disregard limit
 Content of a component in the sample, C:

Ci = Ai x 100
AT
Ai = Area of component peak in the chromatogram
AT = Total area of the peaks in the chromatogram
59
  Example:
A mixture contains only three compounds, X, Y and Z. An
injection of 1 µL of the mixture resulted a
chromatogram with peak areas of 232, 650 and 984
arbitrary units. Calculate the percentage of each
compound.
Answer :
By assuming the response factor of the three
compounds is identical, the total peak area and the
percentage of each compound can be calculated.
Sample calculation: Compound Peak Composition
232 x 100 = 12.43% Area (%)
X 232 12.43
1866 Y 650 34.83
Z 984 52.73
60 TOTAL 1866 100
 Tailing and Fronting
 A common cause of tailing and fronting is a
distribution constant that varies with
concentration.
 Fronting also arises when the amount of sample
introduced onto a column is too large.

61
  Band broadening can be reduced by using:
1. Smaller particle
2. Narrower columns
3. Lower temperature (GC)
4. Thinner liquid stationary phase

62
CHAPTER 5
GAS CHROMATOGRAPHY (GC)
LESSON OUTCOMES
 Explain the principles of gas chromatography
 Draw and label the schematic diagram of the
components in GC
 Able to explain the functions of each component in
GC
 Discuss the difference between each component in
terms of parts and functions
 Interpret analytical information from spectral and
chromatographic data for GC
 Recognize problems arising from resultant
chromatograms and suggest probable solutions
2
5.1 SCOPE OF GC

3
INTRODUCTION
 GC is an technique for separating components
based on their volatilities (based on differences of
boiling point)
 In GC, the components of a vaporized sample are
separated as a result of being partitioned between
a mobile gaseous phase and a liquid or a solid
stationary phase held in the column
 Gas is called “carrier gas”
 Typical carrier gas: helium or nitrogen
 Pressure from a compressed gas cylinder
containing the carrier gas is sufficient to create the
flow through the column 4
There are two types of GC
 Gas-liquid chromatography (GLC)

mobile phase – gas

stationary phase - liquid

 Gas-solid chromatography (GSC)

mobile phase – gas
stationary phase - solid

5
PRINCIPLES
 A sample is being injected at the inlet/injector and
vaporized into the chromatographic column
 The sample is transported through the column by the
flow of inert gaseous mobile phase
 As the sample passes through the column, they are
separated and detected electronically by detector

6
5.2 INSTRUMENTATION

7
INSTRUMENTATION

A. Carrier gas
B. Injector
C. Column
Thermostated
D. Detector
oven
E. Display system -printer/monitor
8
9
5.2.1 CARRIER GAS (MOBILE PHASE)
 Function of carrier gas: to transport the analyte
through the column
 Does not interact with analyte
 Carrier gas flow quantified by linear velocity
(cm/sec) or volumetric flow rate (mL/min)
 Carrier gas contains a molecular sieve to remove
water and impurities
Gas Advantages Disadvantages
Nitrogen Cheap, readily available Long run times
Helium Good compromise, safe Expensive
Hydrogen Shorter run times, cheap Explosive
10
5.2.2 SAMPLE INJECTION SYSTEM
 The injector is a hollow, heated, glass-lined
cylinder where the sample is introduced into GC
 The results of injection should be:
- reproducible
- representative of sample
- no efficiency losses
 For optimum column efficiency, the sample
shouldn't be too large & must be injected as
quick as possible (slow injection of large
sample can causes band broadening & low
resolution)
11
 The temperature of the sample port is usually about 50
°C higher than the boiling point of the least volatile
component (warmer than oven temp.)
- to promote rapid vaporization of the injected sample.
- to prevent sample condensation in the detector.
 Sample introduction usually……
1. In the form of neat liquid or solution.

2. Introduced in a small volumes.

a. 1 μL - 20 μL for packed column.

b. 1 x 10-3 μL for capillary column

12
INJECTION MODE
1. Split Mode:
Introduces only small amount of sample into the
column
Used for concentrated sample

Produces narrow and sharp peaks

2. Splitless Mode:
Most of the sample introduced into the column

Used for low concentration samples

Wider peaks are obtained than for split injections

13
14
5.2.3 COLUMN
 There are 2 types of column:
i- Packed column
ii- Capillary column (open tubular)

Packed column: Capillary Column:

1-5m in length, 2-4mm i.d 10-100m in length, very small i.d
15
PACKED COLUMN

 Less commonly used

 Made of glass or steel
 Length: 1 to 5 m Cross-sectional view of
packed column
 Internal diameter: 2 to 4 mm
 These column is densely packed with uniform,
finely divided solid support, coated with thin
layer (0.05 to1μm) of liquid stationary phase.
 Accommodate larger samples

16
 Carrier gas flow between 10 – 40 mL/min
 Not well adapted for trace analysis
 Contain an inert & stable porous support on
which the stationary phase can be
impregnated(coated) or bound.
 Advantages:
1. Large sample size

2. Ease & convenience of use

17
CAPILLARY COLUMN (OPEN TUBULAR)
 Widely used in GC analysis
 Length: 10 – 100 m
 Coiled around a light weight of metallic support
 Types of capillary column
1. FSOT (Fused Silica Wall Coated) - i.d. 0.1 - 0.3
mm
2. WCOT (Wall Coated) - i.d. 0.25 – 0.75 mm
3. SCOT (Support Coated) - i.d. 0.5 mm
 Advantages:
1. High resolution
2. Short analysis time
3. High sensitivity
18
COLUMN TEMPERATURE
 The optimum column temperature is dependent
upon the boiling point of the sample
 Minimal temperatures give good resolution but
increase elution times
 A temperature that is roughly ≥ the average boiling
point of the sample results in a reasonable elution
period

19
ISOTHERMAL RUN
 Constant column temperature throughout the
analysis
 Eg: This chromatogram shows the effect of
isothermal temperature program at 60°C
 Results:
- increase in tR of all compounds
- the height of the later eluting peaks are reduced &
the widths increased because they are more
affected by the lower temp. program used

20
Temperature program: 60°C isothermal
Head pressure: 12 psi
Split ratio: 1/50

21
TEMPERATURE PROGRAMMING
 Temperature programming is used to accelerate
the elution rate of the late peaks (otherwise take a
very long time to elute)
 Column temperature is increased either by
continuously or in steps as the analysis proceeds
 If the sample contains a combination of low b.p
components & high b.p components, setting a high
column temp. will cause the lower b.p component
to be eluted fast & vice versa

22
 Therefore, with temp. programming, we can set the
temp. low at the beginning to separate low b.p
component & high temp. to elute high b.p components
at reasonable rate
 Eg: This chromatogram shows an ideal temp. program
for separation of 6 aromatic compounds
 Results:
- the compounds elute in order of increasing b.p
(compounds with higher b.p are more retained)

23
Temperature program: 50°C/min-100°C/min
Head pressure: 12 psi
Split ratio: 1/50

24
25
5.2.4 DETECTORS
 Some detectors are universal.
 They are sensitive to almost every compound that
elutes from the column
 They are sensitive to a particular type of compound.
Give response that is dependent on the concentration
of analyte in the carrier gas
 Characteristics of ideal detector
1. High reliability & ease to use
2. Similarity response toward all solutes or
alternatively a high predictable & selective
response toward one or more classes of solute
3. Detector should be nondestructive
26
4. Adequate sensitivity
5. Good stability and reproducibility
6. A linear response to solutes that extends over several
orders of magnitude
7. A temperature range from room temperature to at
least 400⁰C
8. A short response time that is independent of flow rate

27
 Several types of detectors;

1. Flame Ionization Detector (FID)

2. Thermal Conductivity Detector (TCD)
3. Electron Captured Detector (ECD)

28
Detector Principle of Principle class of
operation compound detected

FID Ionization of solute Organics molecule that

molecules in a can be ionized
flame
TCD Thermal Any samples
conductivity
ECD Current Compounds containing
electronegative
elements

29
FLAME IONIZATION DETECTOR (FID)
Principle of operations:
 Effluent from the column is is mixed
with H2 and air, then ignited
 Organic compounds burning in the
flame creates ions & electrons which
can conduct electricity through flame
 The burner, held at ground potential
acts as one of the electrodes.
 The second electrode called as a
collector, is kept at a positive voltage
& collects the current that is
generated.
 Signal amplified by electrometer that
generate measurable voltage. 30
Advantages Disadvantages
 Rugged  Weakly sensitive to
 Sensitive (10-13 g/s) carbonyl, amine,
 Wide dynamic range
alcohol & amine
(107) groups
 Not sensitive to non-
 Signal depends on
number of C atoms in combustibles analyte
organic analyte - such as H2O, CO2,
mass sensitive not SO2, NOx
concentration  Destructive method.
sensitive

31
THERMAL CONDUCTIVITY DETECTOR (TCD)
Principle of operation:
 Consists of an electrically heated wire
 Operating principles relies on the
thermal conductivity of the gaseous
mixture.
 When the solutes elutes from the
column there is a change in the
composition of the mobile phase &
thermal conductivity
 Resulting in a deviation from thermal
equilibrium, causing a variation in the
resistance
 This variation is proportional to the
concentration of the analyte
32
Advantages Disadvantages
 Simple  Relatively low
 Large linear dynamic sensitivity
range
 Responds to both
organic and inorganic
species
 Nondestructive;
permits collection of
solutes after
detection

33
ELECTRON CAPTURE DETECTOR (ECD)

Principle of operation:
 Sample elute from a column is passed over a radioactive β emitter,
usually nickel-63
 An electron from the emitter causes ionization of carrier gas (often
N2) and the production of a burst of electrons
 In the absence of organic species, a constant standing of current
 In the presence of organic molecules containing electronegative
functional groups that tend to capture electrons, the current
34
decreases markedly
Advantages Disadvantages
 Selectively responds to  Insensitive to functional
halogen-containing groups such as amines,
organic compounds alcohols and
such as pesticides and hydrocarbons
polychlorinated
biphenyls
 Highly sensitive
towards halogens,
peroxides, quinones
and nitro groups
35
5.3 STATIONARY PHASE
 Desirable properties for the immobilized liquid
stationary phase:
- Low volatility (ideally the boiling point of the liquid at
least 1000C higher than the maximum operating
temperature for the column)
- Thermal stability
- Chemical inertness.
- Solvent characteristics such as k and α values for
the solutes to be resolved fall within a suitable range.

36
 Separation principles
- Use the principle of “like dissolve like” where like refers to
the polarity of the analyte and the immobilized liquid
stationary phase
 Polarity of organic functional group in increasing
order
- Aliphatic hydrocarbons<olefins<aromatic
hydrocarbons<halides<ethers< esters/
aldehydes/ketones<alcohols/amines< amides<carboxylic
acids<water
 Polarity of the stationary phase should match that of
sample components
- When the match is good, the order of elution is
determined by the boiling point of the eluents 37
POLARITY OF STATIONARY PHASE
Water
Polar Carboxylic acids
Amides
Alcohol/amines
Esters/aldehydes/ketones
Ethers
Halides
Aromatic hydrocarbons
Olefins
Non-polar
Aliphatic hydrocarbons
38
 Non-polar Polar
ALIPHATIC HYDROCARBONS < ESTERS/ALDEHYDES/KETONES < ALCOHOLS/AMINES
< WATER

Pentane, Hexane Acetone, 3-pentanone Propanol, Butanol

Heptane, Octane Methyl ethyl ketone Pentanol
39
  Many liquid statationary phase are based on
polysiloxanes or polyethylene glycol (PEG)

H H H H

HO C C O C C OH Polyethylene glycol (PEG)

Use for separating polar species
H H H H
n

R R R
Polydimethyl siloxane, the R
R Si O Si O Si R
groups are all CH3. (Non-
R R R polar)
n

40
FACTORS AFFECTING GC SEPARATIONS
 Volatility of compound: Low b.p (volatile) components
will travel faster through the column than the high b.p
components
 Polarity of compounds: Polar compounds will move
more slowly, especially if the column is polar
 Column temperature: Raising the column temperature
speeds up all the compound in a mixture
 Flow rate of the gas through the column: Speeding up
the carrier gas flow increases the speed with which all
compounds move the column
 Length of the column: The longer the column, the longer
the elution time (sometimes employed for better
separation) 41
CHAPTER 6
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)
 2

Lesson Outcomes:
• Explain the principles of liquid chromatography
• Draw and label the schematic diagram of the
components in HPLC
• Able to explain the functions of each component in
HPLC
• Discuss the difference between each component in
terms of parts and functions
• Interpret analytical information from chromatographic
data for HPLC
• Recognize problems arising from resultant
chromatograms and suggest probable solutions
3
 4

Introduction
• HPLC utilizes a liquid mobile phase to separate
the components of a mixture
• Analytes are first dissolved in a solvent, & then
forced through the column under high pressure
• HPLC is very versatile because of its ability to
easily separate a wide variety of chemical
mixtures which include non volatile & thermally
unstable compounds (eg: organic, inorganic,
biological samples, synthetic or natural polymers
& thermal labile compound)
 5

Advantages of HPLC:
• Greater sensitivity – various detectors
• Reusable column – many analyses
• Ideal for ionic species and large molecules
• Sample recovery – fraction collectors

HPLC applications - any compound with solubility

in a liquid that can be used as a mobile phase
• Analytical or preparative (highly purified compound in
small or large quantities)
• Applications include analysis of sugars, pesticide
residues, organic acids, lipids, amino acids, toxins &
vitamin)
 6

Principles
• Begins by injecting the solute (analytes) onto the
top of the column
• Separations occurs as the analytes and mobile
phase are pumped through the column
• Each component elutes from the column as peak
on the recorder
• The response of detector to each component is
displayed and known as a chromatogram
 7

Guard
column

Analytical
column
Solvent reservoir

Data system and

display

Schematic Diagram of HPLC

8
 9

Instrumentation

• Mobile phase
resevoir, filtering
• Pump
• Injector
• Columns:
analytical & guard
column
• Detector
• Data system
 10

6.2.1 Mobile phase

Mobile phase reservoir, filtering:
• The common type of reservoir is a glass bottle with
special caps, Teflon tubing and filters to connect
the pump inlet and to the purge gas (He) used to
remove gas (degasser eg: sparger)
Mobile Phase:
• Readily available in pure form
• Low viscosity
• Compatibility with the detection method
• Able to dissolve the sample components
• No chemical reaction with the components
 11

Mobile phase Elution:

1. Isocratic elution 2. Gradient elution
A separation that employs A separation in which the
solvent/s of a constant composition of solvent is
composition changed continously/steps
 12

Degasser
• To removed dissolved gases and dust from the
liquid
• Dissolved gases can lead to irreproducible flow
rates and band spreading
• Both bubbles and dust also will interfere with
the performance of the detector
• The presents of particles will damage the
pumping and injection systems and/or clog the
column
 13

How to degas the solvent?

1.Ultrasound (sonicator)
2.Sparging (rapid bubbling of helium through the
solvent)

Sparging:
•Definition: A process where the dissolved gases
are swept out of the solution by fine bubbles of an
inert gas of low solubility.
•The system also contain means of filtering dust
and particulate matter
 14

Effect of dissolved gases to the HPLC system

if they are not removed from mobile phase
solvent:
• Air bubbles interfere with the performance of
detector by causing band spreading.
• Will generate peaks when pass through the
detector.
• Will interfere with pump function
 15

Why must the solvent be dust and

particulate free?
•the presents of this particles will damage the
pumping and injection systems.
•the dust and particulate will clog the column.

Method to remove dust and particulate

from mobile phase solvents:
•Filtration through a membrane
 16

6.2.2 Pump
• Function: is to force the mobile phase to flow
through the packing (deliver mobile phase
through the system)
• The pump is installed in order to maintain stable
flow and to avoid pulsation
• Types of pumps
1.Reciprocating pumps
2.Displacement pumps
3.Pneumatic pumps
 17

6.2.3 Injectors
• Function: to put sample in the mobile phase
1.Syringe injection through a self-sealing
elastomeric septum:
ØEarliest & simple mean of sample introduction
ØMicro syringes designed to withstand pressure up to
1500 psi are used
2.Sampling loop:
ØMost widely used method
ØNormal sample size is 5 to 500µL
ØMicrosample injection valves also available (sampling
volumes: 0.5 to 5 µL)
ØPermit introduction of sample at pressure up to 7000
psi.
 18

Sampling Loop
 19

3. Auto samplers:
Ø can inject a large number of samples
Ø Small vials with a septum placed in a tray
Ø Needle penetrates septum and inject the
sample
Ø Valve introduces sample into the column
 20

Autosampler
 21

6.2.4 Column
1. Analytical column:
• Straight, stainless steel calibrated tube
• Column length: 10 to 30cm
• Column i.d: 4 to10mm
• Column packing particle size: 5 to 10µm
2. Guard Column
• Function: to increase the lifetime of a analytical
column
• Prevents deactivation of the analytical column by
adsorption and minimize band broadening
• Prevents clogging of the analytical column while
extending its lifetime
• Guard column should have same internal diameter
(i.d) as analytical column
 22

• The composition of guard

column packing closely similar
to analytical column
• The particle size are larger in
order to minimize pressure
drop experienced by the
analytical column.
• Length of the column: 2 to
10cm.
• Serves to saturate the mobile
phase with stationary phase so
that losses of this solvent from
the analytical columns are
minimized
• Can be change periodically
 23

Column Packing
1. Pellicular Packing
Pellicular packings are used largely for
guard column and not for analytical
column
2. Porous Particle Packing
 24

1. Pellicular packing:
• Consist of spherical, nonporous, glass or
polymer beads with typical diameters of 30-40
µm
• A thin, porous layer of silica, alumina,
polystyrene-divinyl benzene synthetic resin or
ion-exchange resin is deposited on the surface
of these beads
• The beads may be treated chemically to give an
organic surface layer
 25

2. Porous particle packing:

• Consists of porous micro particles having
diameter ranging from 3-10µm
• The particles are composed of silica, alumina,
synthetic resin polystyrene-divinyl benzene or
ion exchange resin
• Silica particle is prepared by agglomerating
submicron silica particles under conditions
that lead to larger particles having highly
uniform diameters
• The resulting particles are often coated with
thin organic film, which are chemically or
physically bonded to the surface.
 26

Porous Particle Packing

 27

6.2.5 Detectors
• Two basic types of detector
1. Bulk property detectors
Respond to a mobile phase bulk property, such
as refractive index, dielectric constant, or
density, which is modulated by the presence of
solutes.
2. Solute property detectors
Respond to some property of solutes, such as
UV absorbance, fluorescence, or diffusion
current, that is not possessed by the mobile
phase
 28

• Detectors being chosen depends on the nature

of the sample
• Most widely used detector in HPLC are based
on absorption of UV or visible radiation
• Types of detectors….
a. UV (Absorbance)
b. Refractive index
c. Conductivity
d. Fluorescence
e. Mass-spectrometric (LC/MS)
 29

Principle class of
Principle of
Detector compound
operation
detected
Absorption of UV absorbing
UV electromagnetic species (organic
radiation chromophore)
Measures change in
Refractometer universal
refractive index
 30

Types of HPLC
(based on nature of stationary
phase & separation process)

Ion Size
Partition Exclusion
Exchange
Normal Phase
Gel Filtration

Reversed Phase Anionic

Gel Permeation
Cationic
31
 32

Partition Chromatography
• Most widely used in liquid chromatography
• Applications are to non-ionic, non-polar and
polar compounds of low to moderate molecular
weight (usually <3000)
• There are 2 types
1. Liquid-liquid – liquid stationary phase is
retained on the surface of packing by physical
adsorption.
2. Bonded phase – the stationary phase is
bonded chemically to the support group
 33

• Earlier technique was liquid-liquid type

• Currently, bonded phase method has been
widely used due to disadvantages of liquid-
liquid system
1. Loss of stationary phase by dissolution in the
mobile phase, requires recoating of the support
particles for the column
2. Stationary phase solubility problems prohibit
the use of packing for gradient elution
• Partition chromatography divided into two
modes which based on the relative polarities of
the mobile and stationary phase: normal-
phase and reversed-phase
 34

Normal phase:
• Stationary phase : Polar (eg: silica gel)
• Mobile phase : Less polar (eg. ethyl ether,
chloroform, n-hexane, THF)
• The least polar compound elutes first because in
a relative sense, it is the most soluble in the
mobile phase
• Polar compounds are retained on the polar
surface of the column packing longer
In normal-phase chromatography, the least polar
components is eluted first; increasing the
polarity of the mobile phase will decrease the
elution time
 35

Reversed phase:
• Covers 90% of all LC application
• Stationary phase : Non Polar (hydrophobic in
nature), R is C8 or C18 hydrocarbon
• Mobile phase : Polar (eg. Methanol or
acetonitrile with water)
• The most polar component appear first
• The more non polar the material is, the longer it
will be retained
In reversed-phase chromatography, the most
polar component elutes first; increasing the
mobile phase polarity, increases the elution
time
 36

6.4 Ion Exchange Chromatography

• A process by which ions held on a porous
insoluble solid are exchange for ion in solution
that is brought in contact with the solid.
• The retention of the compound in the
stationary phase depends on its charge density.
• Applications depend on the ion exchange resin
use for the analysis. Either
a. Cationic resin
b. Anionic resin
 37

• Ion exchange resins are useful as stationary

phase for LC and used to separate charged
particles
• Synthetic ion exchange resin are high
molecular weight polymers that contain large
numbers of an ionic functional group per
molecule
38
 39

a) Cation Exchange Resin

• Cation-exchange resin contain acidic groups. It
retains positively charged cations because the
stationary phase displays a negatively charged
functional group
xRSO3-H+ + M x+ →  (RSO3-­‐)xM  x+  +  xH+  
                                         solid                                                  solu4on                        solid                                      solu4on  

• M x+ represents a cation
• R represents that part of a resin molecule that
contains a sulfunic acid group
 40

a) Anion Exchange Resin

• Anion-exchange resin contain basic groups. It
retains anions using positively charged
functional
xRN(CH3)3+OH- + A- →  xRN(CH3)3+Ax- +  xOH-­‐  
                                         solid                                                      solu4on                        solid                                                            solu4on  

• Ax- represents anion

• R represents that part of a resin molecule that
contains a basic group
 41

6.5 Size Exclusion Chromatography

• Applicable to high molecular weight species
• Divided into gel filtration and gel permeation.
• Size exclusion separations are different from
other chromatographic procedures since there
are no chemical or physical interactions
involved between analytes and stationary
phase. Such interaction will lead to impaired
column efficiency
• The packings consist of small (~10um) silica or
polymer particles containing a network of
uniform pores into which solutes and solvent
can diffuse
 42

Schematic of a size exclusion column. The larger particles will elute

first because they are too big to fit inside the pores. The smallest
particles will elute last because they fit very well inside the pores.
 43

• Advantages:
1. Short and well defined separation time
2.Narrow bands, lead to good sensitivity
3.Freedom from sample loss because solutes do not
interact with stationary phase
4.Absence of column deactivation brought about by
interaction of solute with the packing
• Limitations:
1. There is no retention occurs for species with MW
beyond exclusion limit
2.All species having greater molecular weight than the
exclusion limit are so large that they are not retained
and elute together to give a peak
 44

Types of Size Exclusion

A. Gel Filtration B. Gel Permeation

• Size exclusion • Size exclusion
chromatography with chromatography with
hydrophilic packing are hydrophobic packing and
used with aqueous are used with non-polar
mobile phase organic mobile phase
• Separation of polar • Separation of non-polar
species species
 45

Applications:
• Separation of high molecular weight, natural
products molecules from low molecular weight
species
• Separation of homologs and oligomers
• Rapid determination of molecular weight
distribution of larger polymers or natural
products