4/15/2016

GAS CHROMATOGRAPHY

Dr. Eddy Owaga

Overview
1. Introduction
2. Principle of GC
3. GC Instrumentation - carrier gas, sample injection port,
column, detectors, data processor
4. Advantages and disadvantages of GC
5. Applications of GC

1
 4/15/2016

 The father of modern

gas chromatography is
Nobel Prize winner
John Porter Martin,
who also developed the
first liquid-gas
chromatograph. (1950)

GC-MS
Quadri-
TOF

2
 4/15/2016

INTRODUCTION-GC
Separation of gaseous & volatile substances
 Simple & efficient in regard to separation
GC consists of GSC (gas solid chromatography)
GLC (gas liquid chromatography)
Gas → Mobile phase
Solid / Liquid → Solid phase
GSC not used because of limited no. of Solid phase

(i) Gas - Solid Chromatography (GSC)

 The stationary phase, in this case, is a solid like silica or

alumina.

 It is the affinity of solutes towards adsorption onto the

stationary phase which determines, in part, the retention
time.

 The mobile phase is a suitable carrier gas.

 Most useful for the separation and analysis of gases like

CH4, CO2, CO, ... etc.

 The use of GSC in practice is considered marginal when

compared to gas liquid chromatography.

3
 4/15/2016

(ii) Gas - Liquid Chromatography (GLC)

• The stationary phase is a liquid with very low volatility

while the mobile phase is a suitable carrier gas.

• GLC is the most widely used technique for separation

of volatile species.

• The presence of a wide variety of stationary phases

with contrasting selectivities and easy column
preparation add to the assets of GLC or simply GC.

Overview
1. Introduction
2. Principle of GC
3. GC Instrumentation - carrier gas, sample injection port,
column, detectors, data processor
4. Advantages and disadvantages of GC
5. Applications of GC

4
 4/15/2016

How a Gas Chromatography Machine Works

– First, a vaporized sample is injected onto the

chromatographic column.

– Second, the sample moves through the column

through the flow of inert gas.

– Third, the components are recorded as a

sequence of peaks as they leave the column.

Chromatographic Separation

– Deals with both the stationary phase and the

mobile phase.
• Mobile – inert gas used as carrier.

• Stationary – liquid coated on a solid or a solid

within a column.

10

5
 4/15/2016

• Chromatographic Separation:
– In the mobile phase, components of the sample are
uniquely drawn to the stationary phase and thus, enter
this phase at different times.

– The parts of the sample are separated within the column.

– Compounds used at the stationary phase reach the

detector at unique times and produce a series of peaks
along a time sequence.

11

Chromatographic Separation contd…:

– The peaks can then be read and analyzed by a forensic

scientist to determine the exact components of the
mixture.

– Retention time is determined by each component

reaching the detector at a characteristic time.

• The number of components in a sample is determined by the

number of peaks.
• The amount of a given component in a sample is determined by the
area under the peaks.
• The identity of components can be determined by the given
retention times.
12

6
 4/15/2016

Overview
1. Introduction
2. Principle of GC
3. GC Instrumentation - carrier gas, sample injection port,
column, detectors, data processor
4. Advantages and disadvantages of GC
5. Applications of GC

13

14

7
 4/15/2016

Schematic layout of GC instrumentation

To Waste or Flow Meter

Two-Stage
Regulator Syringe Detector
Injector

Flow Controller

Carrier Gas Cylinder Column

15

Schematic layout of GC instrumentation

16

8
 4/15/2016

17

Schematic layout of GC instrumentation

18

9
 4/15/2016

Schematic layout of GC instrumentation

19

Overview
1. Introduction
2. Principle of GC
3. GC Instrumentation - carrier gas, sample injection port,
column, detectors, data processor
4. Advantages and disadvantages of GC
5. Applications of GC

20

10
 4/15/2016

CARRIER GAS

» Hydrogen ( H2 ): better thermal conductivity

Disadvantage: it reacts with unsaturated compounds &

inflammable

» Helium ( He): excellent thermal conductivity; it is expensive

» Nitrogen ( N2): reduced sensitivity; it is inexpensive

21

REQUIREMENTS OF INERT GAS

 Inertness
 Suitable for the detector
 High purity
 Easily available
 Cheap
 Should not cause the risk of fire
 Should give best column performance

22

11
 4/15/2016

Flow regulators & Flow meters

 deliver the gas with uniform pressure/flow rate

 flow meters:- Rota meter & Soap bubble flow meter

Rota meter

 placed before column inlet: it has a glass tube with a float held
on to a spring. Level of the float is determined by the flow rate
of carrier gas

23

Rota meter

24

12
 4/15/2016

• -The carrier gas pressure ranges from 10-50 psi. Higher pressures
potentially increase compression possibility while very low
pressures result in large band broadening due to diffusion.

• -Depending on the column dimensions, flow rates from 1-150

mL/min are reported.

• -Conventional analytical columns (1/8”) usually use flow rates in the

range from 20-50 mL/min while capillary columns use flow rates
from 1-5 mL/min depending on the dimensions and nature of
column.
25

Overview
1. Introduction
2. Principle of GC
3. GC Instrumentation - carrier gas, sample injection port,
column, detectors, data processor
4. Advantages and disadvantages of GC
5. Applications of GC

26

13
 4/15/2016

Injectors
INJECTORS

 The sample is introduced into the injector through a self-

sealing silicone rubber septum.

 The carrier gas flows through the injector carrying vaporized

solutes.

 The temperature of the injector should be adjusted so that

flash vaporization of all solutes occurs.

 If the temperature of the injector is not high enough (at least

50 degrees above highest boiling component), band
broadening will take place.
27

Syringe

Septum

Carrier
Gas

Vaporization
Chamber

To
Column

28

14
 4/15/2016

29

Types of injectors

30

15
 4/15/2016

Types of injectors

31

32

16
 4/15/2016

33

34

17
 4/15/2016

Sample preparation

1. The prerequisite in GC separation is that all solutes being separated

must be: (a) fairly volatile, and (b) thermally stable.
(c) Usually, the solute should be dissolved in a non-aqueous matrix (H2O
changes column behavior).

2. Lack of volatility prevents the direct use of GC for many solute. One way
to overcome this difficulty is to derivatize the solutes into more volatile forms.

OH

Cl O
2,4-dichlorophenoxyacetic acid
O
(A cancer suspect agent).
Cl

Silylation
35

Derivatization of a solute can be used for any of the following

reasons

(a) To increase the volatility of the solute.

(b) To increase the thermal stability of solute

(c) To improve the response for the solute on certain detectors (e.g.,
incorporating halogen atoms into a solute so that it can be detected using
an electron capture detector).

(d) To improve the separation of the solute from other sample

components (i.e., changing the structure of a solute will also
affect its retention on the column)

36

18
 4/15/2016

• Most derivatization reactions can be classified into

one of three group:
(a) Silylation

(b) Alkylation

(c) Acylation

Most of these reactions are performed using minimal

amount of sample and reagents (i.e., 0.1~2.0 mL) are typical
carried out at room temperature. Some, however, do require
heating to moderate temperatures (60 ~ 100 OC).

37

Silylation

(a) This is the most common type of derivation techniques used in GC.

(b) It involves replacing an active hydrogen on the solute (i.e. R-OH,

RCOOH, R-NH2, etc.) with an alkylsilyl group (usually –SiMe3). The
result of this reaction is that the solute is converted into a less
polar, more volatile and more thermally stable form.

(c) The most common reagent used in silylation is trimethylchlorosilane

(TMS). Examples of its use are shown below:
Si Si
R OH + Cl Me3 R O Me3 + HCl

OH Si Me3
Si
Cl O O + Cl Me3 Cl O O

Cl Cl

The resulting Product of this reaction is usually just referred to as a TMS-

derivative. 38

19
 4/15/2016

Overview
1. Introduction
2. Principle of GC
3. GC Instrumentation - carrier gas, sample injection port,
column, detectors, data processor
4. Advantages and disadvantages of GC
5. Applications of GC

39

COLUMNS
• Important part of GC
• Made up of glass or stainless steel
• Glass column- inert , highly fragile
COLUMNS can be classified
(i) Depending on its use
1. Analytical column
1-1.5 meters length & 3-6 mm d.m
2. Preparative column
3-6 meters length, 6-9mm d.m
40

20
 4/15/2016

41

(ii) Depending on its nature

1.Packed column: columns are available in a packed manner

Stationery Phase for GLC: polyethylene glycol, esters, amides,

hydrocarbons, polysiloxanes…

2.Open tubular or Capillary column or Golay column

• Long capillary tubing 30-90 M in length

• Uniform & narrow d.m of 0.025 - 0.075 cm

• Made up of stainless steel & form of a coil

• Disadvantage: more sample cannot loaded

42

21
 4/15/2016

3. SCOT columns (Support coated open tubular column)

 Improved version of Golay / Capillary columns, have

small sample capacity

 Made by depositing a micron size porous layer of

supporting material on the inner wall of the capillary
column

 Then coated with a thin film of liquid phase

43

Columns

• Packed

• Capillary

44

22
 4/15/2016

Wall-coated open tubular (WCOT) <1 mm thick liquid coating on inside of silica tube
Support-coated open tubular (SCOT) 30 mm thick coating of liquid coated support on inside of silica
tube] 45

• Packed - As suggested by the term, it is filled with a coated inert solid

support such as fire brick, alumina, and graphite with a specific mesh size.
The coatings are called phases and for best results are chemically bonded
to the support. Chemical bonding provides for longer column life and less
bleeding (major source of background noise) contributing to lower
sensitivity. Column dimensions 1/8” - 1/4” ID x up to about 6’ using glass
or stainless steel.
• Advantages - higher capacity (higher conc).
• Disadvantages: low resolution and low S/N.

• Capillary - Here the phase (film) is coated on the inside diameter of the
capillary wall with film thickness range of 0.1 to 5μ where the ticker film
provides for better resolution but also allows for more bleed. Typical
dimensions .25mm - .53mm ID x up to 60m made of fused silica coated
with polyamide.
• Advantages: high resolution and better S/N.
• Disadvantages: low capacity and cost.

46

23
 4/15/2016

packed capillary

47

48

24
 4/15/2016

Capillary columns advantages compared to

packed columns
1. higher resolution
2. shorter analysis times
3. greater sensitivity

Capillary columns disadvantage compared to

packed columns
1. smaller sample capacity
2. Need better experience
49

Liquid Stationary Phases

• In general, the polarity of the stationary phase should match
that of the sample constituents ("like" dissolves "like").

• Most stationary phases are based on polydimethylsiloxane or

polyethylene glycol (PEG) backbones:

50

25
 4/15/2016

51

Equilibration of the column

 Before introduction of the sample

 Column is attached to instrument & desired flow

rate by flow regulators

 Set desired temp.

 Conditioning is achieved by passing carrier gas

for 24 hours

52

26
 4/15/2016

Temperature Control Devices

• Preheaters: convert sample into its vapour form, present
along with injecting devices
• Thermostatically controlled oven: temperature
maintenance in a column is highly essential for efficient
separation.
Two types of operations
 Isothermal programming:-
 Linear programming:- this method is efficient for
separation of complex mixtures

53

Instrumentation - Oven
Temperature Control
• Isothermal • Gradient

240

200
Temp (deg C)

160

120

80

40

0
0 10 20 30 40 50 60
Time (min)

54

27
 4/15/2016

Isothermal - Keep oven at one temp through the run. Not very
useful. Possibly useful for series of very similar compounds
differing by boiling points such as alcohols ( MeOH, EtOH, n-PrOH,
i-PrOH, BuOH, i-BuOH).
BP 64.6 78.3 97.2 82.4 117.6 99.5

Gradient - temp profile: 40 deg hold for 10 min then 10deg/min to

240 deg and hold there for 20 min.
• Advantages: 1- resolution and 2- analysis time.

55

Overview
1. Introduction
2. Principle of GC
3. GC Instrumentation - carrier gas, sample injection port,
column, detectors, data processor
4. Advantages and disadvantages of GC
5. Applications of GC

56

28
 4/15/2016

Detection Systems
Characteristics of the Ideal Detector:
The ideal detector for gas chromatography has the following
characteristics:

1. Adequate sensitivity

2. Good stability and reproducibility.

3. A linear response to solutes that extends over several orders

of magnitude.

4. A temperature range from room temperature to at least

400oC.

57

Characteristics of the Ideal Detector contd..

5. A short response time that is independent of flow rate.

6. High reliability and ease of use.

7. Similarity in response toward all solutes or a highly

selective response toward one or more classes of solutes.

8. Nondestructive of sample.

58

29
 4/15/2016

59

1.Thermal Conductivity Detector

(Katharometer, Hot Wire Detector)

Measures the changes of thermal conductivity due to

the sample (mg). Sample can be recovered.

60

30
 4/15/2016

Thermal Conductivity Basics

When the carrier gas is contaminated
The TCD is a nondestructive, by sample , the cooling effect of
concentration sensing detector. A the gas changes. The difference in
heated filament is cooled by the flow cooling is used to generate the
of carrier gas. detector signal.

Flow
Flow

61

Thermal Conductivity Detector

• When a separated compound elutes from the column , the

thermal conductivity of the mixture of carrier gas and
compound gas is lowered. The filament in the sample column
becomes hotter than the control column.

• The imbalance between control and sample filament

temperature is measured by a simple gadget and a signal is
recorded

62

31
 4/15/2016

Measures heat loss from a hot filament –

filament heated to const T
• when only carrier gas flows heat loss to metal block is
constant, filament T remains constant.
• when an analyte species flows past the filament generally
thermal conductivity goes
down, T of filament will rise. (resistance of the filament will
rise).

63

Relative Thermal Conductivity

Relative Thermal
Compound
Conductivity
Carbon Tetrachloride 0.05
Benzene 0.11
Hexane 0.12
Argon 0.12
Methanol 0.13
Nitrogen 0.17
Helium 1.00
Hydrogen 1.28

64

32
 4/15/2016

Advantages of Thermal Conductivity Detector

 Linearity is good
 Applicable to most compounds
 Non destructive
 Simple & inexpensive

Disadvantages
 Low sensitivity
 Affected by fluctuations in temperature and flow
rate
 Biological samples cannot be analyzed
65

(ii) Flame Ionization Detector

 Is a destructive detector
 The effluent from the column is mixed with H & air, and
ignited.
 Organic compounds burning in the flame produce ions and
electrons, which can conduct electricity through the flame.
 A large electrical potential is applied at the burner tip
 The ions collected on collector or electrode and were
recorded on recorder due to electric current.

66

33
 4/15/2016

FIDs are mass sensitive rather than conc. sensitive

ADVANTAGES:

• µg quantities of the solute can be detected

• Stable

• Responds to most of the organic compounds

• Linearity is excellent

• DA: destroy the sample

67

Flame Ionization Detector

68

34
 4/15/2016

69

(iii) ELECTRON CAPTURE DETECTOR

• The detector consists of a cavity that contains two

electrodes and a radiation source that emits  - radiation
(e.g.63Ni, 3H)

• The collision between electrons and the carrier gas

(methane plus an inert gas) produces a plasma containing
electrons and positive ions.

70

35
 4/15/2016

• If a compound is present that contains electronegative atoms,

those electrons are captured and negative ions are formed, and
rate of electron collection decreases

• The detector selective for compounds with atoms of high

electron affinity.
• This detector is frequently used in the analysis of chlorinated
compounds
• e.g. – pesticides, polychlorinated biphenyls

71

72

36
 4/15/2016

73

74

37
 4/15/2016

Interfacing GC with other Methods

• Chromatographic methods (including GC) use retention times
as markers for qualitative analysis.

• This characteristic does not absolutely confirm the existence

of a specific analyte as many analytes may have very similar
stationary phases.

• GC, as other chromatographic techniques, can confirm the

absence of a solute rather than its existence.

• When GC is coupled with structural detection methods, it

serves as a powerful tool for identifying the components of
complex mixtures.

• A popular combination is GC/MS.

75

76

38
 4/15/2016

Mass Spectrometry
Analytical method to measure the molecular
or atomic weight of samples

77

Different elements can be uniquely identified by their

masses

78

39
 4/15/2016

MS Principles
Different compounds can be uniquely
identified by their masses
Butorphanol L-dopa Ethanol
N -CH2-
OH COOH
CH3CH2OH
HO -CH2CH-NH2

HO
HO

MW = 327.1 MW = 197.2 MW = 46.1

79

80

40
 4/15/2016

Overview
1. Introduction
2. Principle of GC
3. GC Instrumentation - carrier gas, sample injection port,
column, detectors, data processor
4. Advantages and disadvantages of GC
5. Applications of GC

81

Chromatographic Analysis

– The number of components in a sample is determined by

the number of peaks.

– The amount of a given component in a sample is

determined by the area under the peaks.

– The identity of components can be determined by the

given retention times.

82

41
 4/15/2016

Semi-Quantitative Analysis of Fatty Acids

C18
10

C16 8

6
Response
Detector

C14 4

0.5 1.0 1.5 2.0 2.5 3.0

Sample Concentration (mg/ml)
Retention Time

C 
T h e c o n t e n t % o f C1 4 f a t t y a c i d s =  
C + C  + C 


= t h e c o n t e n t % o f C1 4 f a t t y a c i d s
83

Tentative Identification of Unknown Compounds

Mixture of known compounds

Response

Octane Decane
1.6 min = RT
Hexane

GC Retention Time on Carbowax-20 (min)

Response

Unknown compound may be Hexane

1.6 min = RT

Retention Time on Carbowax-20 (min)

84

42
 4/15/2016

Overview
1. Introduction
2. Principle of GC
3. GC Instrumentation - carrier gas, sample injection port,
column, detectors, data processor
4. Advantages and disadvantages of GC
5. Applications of GC

85

ADVANTAGES OF G.C

• Very high resolution power, complex mixtures can be resolved

into its components by this method.

• Very high sensitivity with TCD, detect down to 100 ppm

• It is a micro method, small sample size is required

• Fast analysis is possible, gas as moving phase- rapid

equilibrium

• Relatively good precision & accuracy

• Qualitative & quantitative analysis is possible

86

43
 4/15/2016

Overview
1. Introduction
2. Principle of GC
3. GC Instrumentation - carrier gas, sample injection port,
column, detectors, data processor
4. Advantages and disadvantages of GC
5. Applications of GC

87

Applications of GC
-pesticides analysis
-free fatty acids analysis

88

44
 4/15/2016

In summary…
1. Introduction
2. Principle of GC
3. GC Instrumentation - carrier gas, sample injection port,
column, detectors, data processor
4. Advantages and disadvantages of GC
5. Applications of GC

Questions?
89

45