Gas chromatography (GC) is a common type of chromatography that separates compounds by vaporizing them and passing them through a column with a carrier gas. It can be used to test purity, separate mixtures, and identify unknown compounds. Key components include an inlet to introduce the sample, a column to separate components, and a detector. The carrier gas moves the vaporized sample through the column where components interact differently with the stationary phase and elute at different retention times.

1. Gas chromatography
Gas chromatography (GC) is a common type
of chromatography used in analytic chemistry
for separating and analyzing compounds that can
be vaporized without decomposition.
GC include testing the purity of a particular substance,
or separating the different components of a mixture.
GC help in identifying a compound.
In preparative chromatography, GC can be used to
prepare pure compounds from a mixture.

3. Filters/Traps
Air
Hydrogen
Gas
Carrier
Column
Gas Chromatography
• gas
system
• inlet
• column
• detector
• data
system
Data system
Syringe/Sampler
Inlets
Detectors
Regulators
H
RESET

4. Schematic Diagram of Gas Chromatography

5. Schematic Diagram of Gas Chromatography

6. Basic Block Daigram!

7. In gas chromatography, the moving phase (or "mobile phase") is a
carrier gas, usually an inert gas such as helium or an unreactive gas
such as nitrogen.
The stationary phase is a microscopic layer of liquid or polymer on
an inert solid support, inside a piece of glass or metal tubing called
a column. The instrument used to perform gas chromatography is
called a gas chromatograph (or "aerograph", "gas separator").
The gaseous compounds being analyzed interact with the walls of
the column, which is coated with different stationary phases. This
causes each compound to elute at a different time, known as
the retention time of the compound. The comparison of retention
times is what gives GC its analytical usefulness.

8. • Gas chromatography is in principle similar to column
chromatography (as well as other forms of
chromatography, such as HPLC, TLC), but has several
notable differences.
• Firstly, the process of separating the compounds in a
mixture is carried out between a liquid stationary phase
and a gas moving phase, whereas in column
chromatography the stationary phase is a solid and the
moving phase is a liquid. (Hence the full name of the
procedure is "Gas-liquid chromatography", referring to the
mobile and stationary phases, respectively.)
• Secondly, the column through which the gas phase passes
is located in an oven where the temperature of the gas can
be controlled, whereas column chromatography (typically)
has no such temperature control.
• Thirdly, the concentration of a compound in the gas phase
is solely a function of the vapor pressure of the gas.

9. Gas chromatography is also similar to fractional
distillation, since both processes separate the
components of a mixture primarily based on boiling
point (or vapor pressure) differences. However,
fractional distillation is typically used to separate
components of a mixture on a large scale, whereas GC
can be used on a much smaller scale (i.e. micro scale).
Gas chromatography is also sometimes known
as vapor-phase chromatography (VPC), or gas-liquid
partition chromatography (GLPC).

10. GC analysis
A gas chromatograph is a chemical analysis instrument for
separating chemicals in a complex sample. A gas chromatograph
uses a flow-through narrow tube known as the column, through
which different chemical constituents of a sample pass in a gas
stream (carrier gas,mobile phase) at different rates depending
on their various chemical and physical properties and their
interaction with a specific column filling, called the stationary
phase.
As the chemicals exit the end of the column, they are detected
and identified electronically. The function of the stationary
phase in the column is to separate different components,
causing each one to exit the column at a different time
(retention time). Other parameters that can be used to alter the
order or time of retention are the carrier gas flow rate, and the
temperature.

11. In a GC analysis, a known volume of gaseous or liquid analyte is injected
into the "entrance" (head) of the column, usually using a micro syringe (or,
solid phase microextraction fibers, or a gas source switching system).
As the carrier gas sweeps the analyte molecules through the column, this
motion is inhibited by the adsorption of the analyte molecules either onto
the column walls or onto packing materials in the column. The rate at
which the molecules progress along the column depends on the strength
of adsorption, which in turn depends on the type of molecule and on the
stationary phase materials.
Since each type of molecule has a different rate of progression, the
various components of the analyte mixture are separated as they progress
along the column and reach the end of the column at different times
(retention time).
A detector is used to monitor the outlet stream from the column; thus,
the time at which each component reaches the outlet and the amount of
that component can be determined. Generally, substances are identified
(qualitatively) by the order in which they emerge (elute) from the column
and by the retention time of the analyte in the column.

12. Columns
• Packed columns are 1.5 – 10 m in length and have an internal diameter of
2 – 4 mm. The tubing is usually made of stainless steel or glass and
contains a packing of finely divided, inert, solid support material
(e.g. diatomaceous earth) that is coated with a liquid or solid stationary
phase. The nature of the coating material determines what type of
materials will be most strongly adsorbed. Thus numerous columns are
available that are designed to separate specific types of compounds.
• Capillary columns have a very small internal diameter, on the order of a
few tenths of millimeters, and lengths between 25–60 meters are
common. The inner column walls are coated with the active materials
(WCOT columns), some columns are quasi solid filled with many parallel
micropores (PLOT columns). Most capillary columns are made of fused-
silica(FSOT columns) with a polyimide outer coating. These columns are
flexible, so a very long column can be wound into a small coil.

13. Detectors
A number of detectors are used in gas
chromatography. The most common are
the flame ionization detector (FID) and
the thermal conductivity detector(TCD). Both
are sensitive to a wide range of components,
and both work over a wide range of
concentrations

14. Methods
• The method is the collection of conditions in which the GC operates
for a given analysis. Method development is the process of
determining what conditions are adequate and/or ideal for the
analysis required.
• Conditions which can be varied to accommodate a required analysis
include inlet temperature, detector temperature, column
temperature and temperature program, carrier gas and carrier gas
flow rates, the column's stationary phase, diameter and length,
inlet type and flow rates, sample size and injection technique.
Depending on the detector(s) (see below) installed on the GC, there
may be a number of detector conditions that can also be varied.
Some GCs also include valves which can change the route of sample
and carrier flow. The timing of the opening and closing of these
valves can be important to method development.

15. Carrier gas selection and flow rates
Typical carrier gases
include helium, nitrogen, argon, hydrogen and
air. Which gas to use is usually determined by
the detector being used.

16. Stationary compound selection
The polarity of the solute is crucial for the
choice of stationary compound, which in an
optimal case would have a similar polarity
than the solute. Common stationary phases in
open tubular columns are cyanopropylphenyl
dimethyl polysiloxane, carbowax
polyethyleneglycol, biscyanopropyl
cyanopropylphenyl polysiloxane and diphenyl
dimethyl polysiloxane. For packed columns
there are more options available.

17. Instrumentation
Injection Port - Sample introduction
Manual - Direct Injection
Automated - Autosampler
- Purge and Trap

18. Temperature Control
Instrumentation - Oven

19. Columns
• Packed
• Capillary

20. Polarity
Non-polar
Polar
+
-

21. GAS LIQUID CHROMATOGRAPHY
Principles
Partition of molecules between gas (mobile
phase) and liquid (stationary phase).

22. THE CHROMATOGRAPHIC PROCESS - PARTITIONING
(gas or liquid)
MOBILE PHASE
STATIONARY PHASE
Sample
out
Sample
in
(solid or heavy liquid coated onto a solid or support system)

23. Most Common Stationary Phases
1. Separation of mixture of polar compounds
Carbowax 20M (polyethylene glycol)
2. Separation of mixtures of non-polar compounds
OV101 or SE-30 (polymer of methylsilicone)
3. Methylester of fatty acids
DEGS (diethylene glycol succinate)

24. DETECTORS
Flame Ionization Detector (Nanogram - ng)
High temperature of hydrogen flame (H2 +O2 + N2)
ionizes compounds eluted from column into flame.
The ions collected on collector or electrode and were
recorded on recorder due to electric current.

25. Schematic Diagram of Flame Ionization Detector
Collector
Jet
Flame
Detector electronics
 - 220 volts
Column
Chassis ground
Signal output

26. Thermal Conductivity Detector
Measures the changes of thermal conductivity due
to the sample (mg). Sample can be recovered.

27. Thermal Conductivity Detector
Principal: The thermal balance of a heated filament
Electrical power is converted to heat in a resistant
filament and the temperature will climb until heat
power loss form the filament equals the electrical
power input.
The filament may loose heat by radiation to a cooler
surface and by conduction to the molecules coming
into contact with it.

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

29. When a compound elutes, the thermal
conductivity of the gaseous mixture of carrier gas
and compound gas is lowered, and the filament in
the sample column becomes hotter than the
other control column.
Its resistance increased, and this imbalance
between control and sample filament resistances
is measured by a simple gadget and a signal is
recorded
Thermal Conductivity Detector

30. • Responds to all compounds
• Adequate sensitivity for many compounds
• Good linear range of signal
• Simple construction
• Signal quite stable provided carrier gas glow rate,
block temperature, and filament power are controlled
• Nondestructive detection
Thermal Conductivity Detector

31. Electron Capture Detector
For pesticide analysis (picogram).
Accept electrons of carrier gas.

32. Exhaust
Chimney
Igniter
Hydrogen
Inlet
Column
Effluent
Polarizing Electrode
Collector Electrode
Schematic Diagram of Flame Ionization Detector

33. Electron Capture Detector

34. Electron Capture Detector

35. Gas Chromatography Application

36. SEMI- QUANTITATIVE ANALYSIS OF FATTY ACIDS
C
C
C
Detector Response
RetentionTime
14
16
18
Peak Area (cm )
Sample Concentration (mg/ml)
2
4
6
8
10
0.5 1.0 1.5 2.0 2.5 3.0
2
The content % of C fatty acids =
C
C + C + C






= the content % of C fatty acids
14
14

37. TENTATIVE IDENTIFICATION OF UNKNOWN COMPOUNDS
Response
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane
Decane
1.6 min = RT
Response
Unknown compound may be Hexane
1.6 min = RT
Retention Time on Carbowax-20 (min)

38. Response
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Response
GC Retention Time on SE-30
Hexane
RT= 4.0 min on SE-30
Retention Times

39. GLC ADVANTAGES
1. Very good separation
2. Time (analysis is short)
3. Small sample is needed - ml
4. Good detection system
5. Quantitatively analyzed

40. DISADVANTAGES OF GAS CHROMATOGRAPHY
Material has to be volatilized at 250C without decomposition.
R C OH CH3OH H2SO4
O
R C O CH3
O
CH2 O C R
CH O C R
CH2 O C R
O
O
O
CH3OH
O
R C O CH3
CH3ONa
Fatty Acids Methylester
Reflux
+ 3
Volatile in Gas
Chromatography
Volatile in Gas
Chromatography
+ +

41. GC-MS
Gas Chromatography-Mass Spectrometry
An Hybrid technique which couples the powerful
separation potential of gas chromatography with the
specific characterization ability of mass spectroscopy.
Supporting & Servicing Excellence

42. • GC History
• What is GC
• Key Components
• Separation Process
• GC Theory
• Carrier Gas
• Injectors
• Columns
Overview

43. What is GC?
• GC is a Separation Technique
• Sample is usually a complex mixture we
require to separate into constituent
components.
• Why: usually to quantify some or all
components e.g. Pharmaceuticals,
Environmental pollutants, etc
• Occasionally as a qualitative tool

44. What is the sample?
• Usually a mixture of several components
• Sample usually introduced as a liquid
• Components of interest (analytes) usually in
low concentrations (<1% to ppb levels)
• Samples dissolved in volatile solvent

45. Comaparison: HPLC & GC
HPLC
• non-volatile samples
• thermally unstable compounds
• macromolecules
• inorganic and ionic samples
• More complex interface to Mass
Spec .
GC
•volatile & thermally stable
•rapid analysis
•good resolution
•easily interfaced to Mass Spec

46. Key components of GC
• Hardware to introduce the sample
• Technique to separate the sample into components
• Hardware to detect the individual components.
• Data Processing to process this information.

47. Separation Process
• Sample is introduced into system via hot, vaporising injector.
• Typically 1ul injected
• Flow of “Carrier Gas” moves vaporised sample (i.e. gas) onto
column
• Column is coated with wax type material with varying affinity
for components of interest
• Components are separated in the column based on this
affinity.
• Individual analytes are detected as they emerge from the end
of the column through the Detector.

48. Example Chromatogram (Capillary)
1 2 3 4 5
Minutes
-87
0
250
500
750
mVolts
0.541
0.754
1.113
1.474
2.038
2.853
3.210
4.463
5.320
5.562
c:starexampleslevel4.run File:
Channel:
Last recalc:
c:starexampleslevel4.run
A = TCD Results
25/07/1993 18:35
WI:2
WI:4
Time 
Inject Point
Detector
Response

49. •
1
2
4
6
3
5
7
8
10
9
11
12
13
14
15
16
17 18
1. -HCH
2. -HCH
3. -HCH
4. Heptachlor
5. -HCH
6. Aldrin
7. Heptachlor epoxide
8. Endosulfan I
9. 4,4’-DDE
10. Dieldrin
11. Endrin
12. 4,4’-DDD
13. Endosulfan II
14. 4,4’-DDT
15. Endrin aldehyde
16. Endosulfan sulfate
17. Methoxychlor
18. Endrin ketone
Analysis of Halogenated Pesticides
2ppb in Water

50. Chromatogram

51. GC Step by Step
• Carrier Gas
• Injector
• Column
– Capillary
– Stationary Phase
• Detectors
– Mass Spectrometer

52. Carrier Gas
 Inert
 Helium
 Choice dictated by detector, cost, availability
 Pressure regulated for constant inlet pressure
 Flow controlled for constant flow rate
 Chromatographic grade gases (high purity)

53. Column Types
Packed Columns
Length: <2m
Diameter: 1/8” & ¼” OD
Capillary Columns
Length: 10m to 100m
Diameter: 180um,
250um, 320um &
530um I.d

54. • Capillary Column Flow
– 250 um 1 ml/min
– 320 um 1.5 ml/min
– 530um up to 2.0 ml/min
Typical column flow rates

55. Purpose of Injection
• Deposit the sample into the column in the narrowest band
possible
• The shorter the band at the beginning of the chromatographic
process - tall narrow peaks
• Gives maximum resolution and sensitivity
• Therefore type of injection method and operating conditions
is critical in obtaining precise and accurate results

56. Splitless injector Design
“Unique”Dual Split Vent design
•Improved Precision
•More Efficient Sweep
Graphite/Viton Seal
•Reduced Sample Contact
Shortened Capillary Guide
•Minimal Cold Spots
•Minimal Upswept Volume
Large Internal
Volume
•Minimum
Solvent Tailing

57. Cross Section of PTV Injector
Modern
Temperature
Programmable
Injector (Varian
1079)
Programmable
Temperature
Vapourising
Injector

58. Split & Splitless Injection
• Most common method of Injection into Capillary
Columns
• Most commonly misunderstood also!
• Same injector hardware is used for both techniques
• Electronically controlled Solenoid changes Gas Flow
to determine Injector function.

59. Split Injection
• Mechanism by which a portion of the injected solution is discarded.
• Only a small portion (1/1000 - 1/20) of sample goes through the column
• Used for concentrated samples (>0.1%)
• Can be performed isothermally
• Fast injection speed
• Injector and septa contamination not usually noticed

60. Splitless Injection
• Most of the sample goes through the column (85-100%)
• Used for dilute samples (<0.1%)
• Injection speed slow
• Should not be performed isothermally
• Solvent focusing is important
• Controlled by solenoid valve
• Requires careful optimisation

61. On Column Injection
• All of the sample is transferred to the column
• Needle is inserted directly into column or into insert
directly above column
o Trace analysis
o Thermally labile compounds e.g Pesticides, Drugs
o Wide boiling point range
o High molecular weight

62. Large Volume Injection
• To enhance sensitivity in Envoirnmental applications.
• Uses 100µL syringe: Inject up to 70 µl
• Very slow injection with injector temperature a few degrees below solvent
boiling point, split open, flow at about 150 mls/ min
• Solvent vents out of split vent, thus concentrating the analytes
• Close split
• Fast temperature ramp to top column temperature +20°C
• Column programming as per sample requirements

63. Columns

64. Material of Construction
• Metal (1957)
• Glass (1959)
• Fused Silica (1979)
• Aluminium Clad (1984)
• Inert Metal (1990)

65. Capillary Column Characteristics
• Length (10M - 50M)
• Internal Diameter (0.1mm - 0.53mm)
• Liquid Stationary Phase
• Film Thickness (0.1um - 5um)
• Polarity (Non-polar - Polar)

66. Stationary Phases
Choice of phase determines selectivity
Hundred of phases available
Many phases give same separation
Same phase may have multiple brand names
Stationary phase selection for capillary columns much simpler
Like dissolves like
Use polar phases for polar components
Use non-polar phases for non-polar components

67. Column Bleed
 Bleed increases with film thickness
 Polar columns have higher bleed
 Bleed is excessive when column is damaged or degraded
 Avoid strong acids or bases
 Adhere to manufacturer’s recommended temperature limits
 Avoid leaks

68. Choosing a Column
• Internal Diameter
• Film Thickness
• Length
• Phase

69. Internal Diameter, Smaller ID’s
• Good resolution of early eluting compounds
• Longer analysis times
• Limited dynamic range

70. ID Effects - larger ID’s
• Have less resolution of early eluting compounds
• Shorter analysis times
• Sufficient resolution for complex mixtures
• Greater dynamic range

71. Film Thickness
 Amount of stationary phase coating
 Affects retention and capacity
 Thicker films increase retention and capacity
 Thin films are useful for high boilers
 Standard capillary columns typically 0.25µm
 0.53mm ID (Megabore) typically 1.0 - 1.5µm

72. The maximum amount that can be injected without significant peak
distortion
 Column capacity increases with :-
 film thickness
 temperature
 internal diameter
 stationary phase selectivity
 If exceeded, results in :-
 peak broadening
 asymmetry
 leading
Column Capacity

73. Length effects - isothermal analysis
• Retention more dependant on length
• Doubling column length doubles analysis times
• Resolution a function of Square Root of Length
• Gain 41% in resolution
• Is it worth the extra time and expense?-

74. Length effects - programmed analysis
• Retention more dependant on temperature
• Marginally increases analysis times
• Run conditions should be optimised

75. Summary - Effect of ID, Film
Thickness, and Length
ID
• Choice based on
capacity and resolution
• Use 0.25mm for MSDs
• Use 0.32mm for
split/splitless & DI
• Use 0.53mm for DI &
• purge & trap
Film Thickness
• Thick film for low
boilers
• Thin film for high
boilers
• Thicker films for larger
ID's
Length
 Gain in resolution is
not double
 Isothermal: tR  L
 Programmed: tR is
more dependent on
temperature

76. Basic GCMS

77. • Basic Mass Spectrometry Theory
• Types of Ionisation
- Electronic Ionisation
- Chemical Ionisation
• Interpretation of Mass Spectra
• Ion Trap Theory
• Components of the Ion Trap
Overview

78. Ion Trap Mass Spectrometry

79. Basic Mass Spec.Theory
• Mass Spec. is a Microanalytical Technique used to obtain information
regarding structure and Molecular weight of an analyte
• Destructive method ie sample consumed during analysis
• In all cases some form of energy is transferred to analyte to cause
ionisation
• In principle each Mass Spectrum is unique and can be used as a
“fingerprint” to characterise the sample
• GC/MS is a combination technique that combines the separation ability of
the GC with the Detection qualities of Mass Spec.

80. Basic GCMS Theory(1)
• Sample injected onto column via injector
• GC then separates sample molecules
• Effluent from GC passes through transfer line into the
Ion Trap/Ion source
• Molecules then undergo electron /chemical
ionisation
• Ions are then analysed according to their mass to
charge ratio
• Ions are detected by electron multiplier which
produces a signal proportional to ions detected

81. Basic GCMS Theory(2)
• Electron multiplier passes the ion current
signal to system electronics
• Signal is amplified
• Result is digitised
• Results can be further processed and
displayed

82. Types of Ionisation
• Electron impact ionisation
• Chemical Ionisation

83. Definition of Terms
Molecular
ion
The ion obtained by the loss of an electron from
the molecule
Base peak
The most intense peak in the MS, assigned 100%
intensity
M+ Symbol often given to the molecular ion
Radical
cation
+ve charged species with an odd number of
electrons
Fragment
ions
Lighter cations formed by the decomposition of
the molecular ion.
These often correspond to stable carbcations.

84. Electron Ionisation(1)
• Sample of interest vaporised into mass spec
• Energy sufficient for Ionisation and Fragmentation of
analyte molecules is acquired by interaction with
electrons from a hot Filament
• 70 eV is commonly used
• Source of electrons is a thin Rhenium wire heated
electrically to a temp where it emits free electrons

85. Electron Ionisation

86. Electron Ionisation
• The physics behind mass spectrometry is that a charged particle passing
through a magnetic field is deflected along a circular path on a radius that
is proportional to the mass to charge ratio, m/e.
In an electron impact mass spectrometer, a high energy beam of electrons
is used to displace an electron from the organic molecule to form a radical
cation known as the molecular ion. If the molecular ion is too unstable
then it can fragment to give other smaller ions.
The collection of ions is then focused into a beam and accelerated into the
magnetic field and deflected along circular paths according to the masses
of the ions. By adjusting the magnetic field, the ions can be focused on the
detector and recorded.

87. Chemical ionisation
• Used to confirm molecular weight
• Known as a “soft” ionisation technique
• Differs from EI in that molecules are ionised by interaction or
collision with ions of a reagent gas rather that with electrons
• Common reagent gases used are Methane , Isobutane and
Ammonia
• Reagent gas is pumped directly into ionisation chamber and
electrons from Filament ionise the reagent gas

88. Chemical Ionisation(2)
• First - electron ionization of CH4:
– CH4 + e-  CH4
+ + 2e-
• Fragmentation forms CH3
+, CH2
+, CH+
• Second - ion-molecule reactions create
stable reagent ions:
– CH4
+ + CH4  CH3 + CH5
+
– CH3
+ + CH4  H2 + C2H5
+
• CH5
+ and C2H5
+ are the dominant methane CI reagent ions

89. Chemical Ionisation(3)
• Form Pseudomolecular Ions (M+1)
– CH5+ + M  CH4 + MH+
– M+1 Ions Can Fragment Further to Produce a Complex CI
Mass Spectrum
• Form Adduct Ions
– C2H5+ + M  [M + C2H5]+ M+29 Adduct
– C3H5+ + M  [M + C3H5]+ M+41 Adduct
• Molecular Ion by Charge Transfer
– CH4+ + M  M+ + CH4
• Hydride Abstraction (M-1)
– C3H5+ + M  C3H6 + [M-H]+
» Common for saturated hydrocarbons

90. EI vs CI for Cocaine analysis
• EI Spectrum of Cocaine
• Extensive Fragmentation
• Molecular Ion is Weak at m/z 303

91. Methane CI of Cocaine
Pseudomolecular Ion and Fragment Ions

92. Proton Affinity
• Proton Affinity Governs CI Susceptibility
• The higher the affinity the more tightly bound
the proton is to the parent species
• The greater the difference in proton affinities
between the analyte and reagent gas the
more energy transferred to the protonated
molecule –more fragmentation

93. Interpretation of Mass Spectra(1)

94. Intepretation of Mass Spectra(2)
•The MS of a typical hydrocarbon, n-decane is shown above.
The molecular ion is seen as a small peak at m/z = 142.
•Notice the series ions detected that correspond to fragments that differ by 14 mass
units, formed by the cleave of bonds at successive -CH2- units

95. Interpretation of Mass Spectra(3)

96. Interpretation of Mass Spectra(4)
•The MS of benzyl alcohol is shown above.
•The molecular ion is seen at m/z = 108.
•Fragmentation via loss of 17 (-OH) gives a common fragment seen for alkyl
benzenes at m/z = 91.
•Loss of 31 (-CH2OH) from the molecular ion gives 77 corresponding to the phenyl
cation.
• Note the small peaks at 109 and 110 which correspond to the presence of small
amounts of 13C in the sample (which has about 1% natural abundance).

97. Determining Isotope Patterns in Mass Spectra
•Mass spectrometers are capable of separating and detecting individual ions even those
that only differ by a single atomic mass unit.
•As a result molecules containing different isotopes can be distinguished.
•This is most apparent when atoms such as bromine or chlorine are present (79Br : 81Br,
intensity 1:1 and 35Cl : 37Cl, intensity 3:1) where peaks at "M" and "M+2" are obtained.
•The intensity ratios in the isotope patterns are due to the natural abundance of the
isotopes.
•"M+1" peaks are seen due the the presence of 13C in the sample.

98. THIN LAYER CHROMATOGRAPHY
Stationary Phase ---------> Silica Gel
Mobile Phase ------------->Solvent (developing)

99. Isotope Patterns 2,Chloropropane

100. •Examples of haloalkanes with characteristic isotope patterns.
•The first MS is of 2-chloropropane.
•Note the isotope pattern at 78 and 80 that represent the M and M+2 in a 3:1
ratio.
•Loss of 35Cl from 78 or 37Cl from 80 gives the base peak a m/z = 43,
corresponding to the secondary propyl cation.
•Note that the peaks at m/z = 63 and 65 still contain Cl and therefore also show
the 3:1 isotope pattern.

101. 1,Bromopropane

102. • The second MS is of 1-bromopropane.
• Note the isotope pattern at 122 and 124 that
represent the M amd M+2 in a 1:1 ratio.
• Loss of 79Br from 122 or 81Br from 124 gives the
base peak a m/z = 43, corresponding to the propyl
cation.
• Note that other peaks, such as those at m/z = 107
and 109 still contain Br and therefore also show
the 1:1 isotope pattern.

103. ION TRAP THEORY

104. • Ionize analytes within the ion trap
– Use energetic electrons to ionize
• Store ions and continue to ionize until the optimum trap capacity is
reached
– Optimum ion time calculated by software
• Increase the voltage on the Ring Electrode of the ion trap to scan ions out
in order from low to high mass
– This voltage-time relationship called the EI/MS Scan Function
• Store the mass-intensity information as a mass spectrum

105. Gate
Filament
Ring Electrode
Trapped Ions
Analytes + He
Carrier Gas
Gate
Filament
Ring Electrode
Trapped Ions
Analytes + He
Carrier Gas
Filament
Trapped
Ions
CARRIER GAS
Ring electrode
Gate
Electron Ionization Happens Inside the Ion Trap

106. Mass Spectrum of Toluene

107. Mass Spectrum of Caffeine

108. Mass Spectrum of Glycerin

109. Mass Spectrum of Cholesterol

110. Mass Spectrum of Aspirin

111. The detector contains two filaments: one exposed
only to carrier gas, while the other is exposed to the
carrier gas for sample analysis.
When the gas for the sample analysis is only carrier
gas , the two filaments can be balanced.
Instead of a direct measurement of filament
temperature, the filament resistant, which is a
function of temperature, is measured.
Thermal Conductivity Detector

112. The ability of a colliding molecule to carry off heat
depending on its thermal conductivity. Hydrogen
and helium have high thermal conductivity and
therefore will be more efficient at “cooling” a
heated filament than other gases will
Thermal Conductivity Detector

113. Thermal Conductivity Detector
The TCD will respond to any substance
different from the carrier gas as long as its
concentration is sufficiently high enough.

114. Thermal Conductivity Detector

115. Thermal Conductivity Detector

116. Electron capture compound, X (highly electonegative element), tends to capture free
electrons and increase the amount to ion recombination
X (F, Cl and Br) + e  X-
Ion recombination : X- + N2
+ = X + N2
The current will decrease and this decrease constitutes the signal.
Halogens, lead, phosphorous, nitro groups, silicone and polynuclear aromatics.
Insecticides, pesticides, vinyl chloride, and fluorocarbons
Electron Capture Detector

117. Electron Capture Detector

118. HPLC

119. Picture of HPLC instrument

120. Components
• Mobile phase reservoirs
• HPLC Pump(s)
• Mixing valves
• Sample injector (manual or auto)
• Column
• Detector
• Plumming
• Mobile phase waste container

121. HPLC-UV
Mobile
Phases
A and B
HPLC
Pump
syringe
6-port
valve
Sample
loop
HPLC
column
Detector
MP waste
Jacket for controlling
column temperature

122. HPLC Separations
• Different analytes have different equilibria between
the mobile phase and stationary phase
• Equilibrium is dynamic; thus we can view it as a given
analyte molecule spending a fraction of time
dissolved in the mobile phase
• Since different solutes gave different fractions, a
separation of the analytes occur as they are pushed
through the column by the mobile phase

123. Types of HPLC
• Reverse-phase (polar mobile phase/non-polar
stationary phase/somewhat polar analytes)
• Normal Phase (non-polar mobile phase/polar
stationary phase/non-polar analytes)
• Adsorption (non-polar mobile phase/polar stationary
phase/non-polar analytes); isomer separation
• Ion-Exchange (salts/ionic stationary phase)
• Size-exclusion (aqueous/gel for large MW solutes,
>104)

124. Columns
• Length (5-15 cm); much shorter than GC column
• Diameter (4 mm down to 50mm)
• Particle size (3, 5, or 10 mm)
• Different phases bonded to silica
• Typically detection limit is decreased by decreasing
the column diameter
• Optimal linear flow rate conserved; so optimal
volumetric flow rate decreases with the square of
the radius
• 4 mm/ 1.0 mL/min; 1 mm/60 mL/min

125. Reversed phase stationary phase
• Most common; n-octyldecyl, C18
Si-O-Si-(CH2)17-CH3
CH3
CH3
Si-O-Si-(CH
2
)
17
-CH
3
CH
3
CH
3
Si-O-Si-(CH
2
)
17
-CH
3
CH
3
CH
3
Si-O-Si-(CH
2
)
17
-CH
3
CH
3
CH
3

126. P
P
C18 Phase designed to retain very polar
compounds

127. Reverse-phase mobile phases
• Water
• Methanol
• Acetonitrile
• THF
• Additives, salts, acids, bases
• Ion pairing

128. Gradients in reverse-phase
• For complex mixtures
• Polar non-polar
– Buffer A 100 % H2O
– Buffer B 100 % MeOH or acetonitrile

129. 0 5 10 15 20 25 30 35 40 45
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative
Abundance
11.36
17.23
12.57
12.74
17.68
36.21
1.21 15.13 24.95
24.53
22.46
2.54
3.01 21.73
5.43 6.14 25.20
20.41 48.55
27.31 37.18
29.53 32.43 40.11 45.43
RP-HPLC Separation of a Tryptic Digest of BSA

130. HPLC Method Development
• Isocratic, Fig 25-25 Harris
• Find the best methanol separation
• Use Table 25-25 to guide you in finding the best
acetonitrile and THF separations
• Based on separations try binary mixtures
– Methanol, 38 %
– Acetonitrile, 30 %
– THF, 22 %
– 19 % MeOH/15% acetonitrile, 15 % acetonitrile/11% THF,
19 % MeOH/11% THF
– Trinary mixture, 13:10:7
• Temperature/computer simulations

131. Gradients
• First step
– long, simple gradient
– Adjust accordingly
– Can become complex
• Do you need a gradient?
If Dt/tG > 0.25, then a gradient is appropriate
Dt = time between first and last peak
tG = time of gradient

132. RT: 0.00 - 35.03
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
8.59
10.02
12.56
3.15
2.99 13.93
3.60
14.61
2.38
5.53 16.89 17.78
7.53 20.03
1.70 22.71 32.84
29.14 31.25
25.24 27.28
NL:
2.27E8
Base Peak F: +
c ESI Full ms [
300.00-
1300.00] MS
chem361gbsaf
Dt = 22-8 = 14 min
tG = 22-4 min = 18 min
Dt/tG = 14/18 = 0.63 > 0.25

133. Normal Phase
• Bare silica
– Mobile phases, (ethyl acetate/ hexane)
• HILIC columns
– Attach polar groups to silica
– Methanol to water

134. Ion Exchange
• Ion exchange resins
– Strong cation, -SO3
-H+
– Weak cation, - COO-H+
– Strong anion, - N(CH3)3
+OH-
– Weak anion, - NH3
+OH-
• Bound to polystyrene support
• Mechanism
– RSO3
-H+ + P RSO3
-P+ + H+

135. Ion Exchange Gradients
• Mobile Phase A – H2O
• Mobile Phase B – 500 mM NaAc

136. Ion chromatography
• Separation of small ionic species
– PO4
3+, SO4
2-, BrO3-, NO2-, F-, Cl-, ect
– Mg2+, Na+, Ca2+, Li+, Ba2+, ect
– -Detected by differences in conductivity

137. Size Exclusion Chromatography
• Stationary phase is a gel
• Fractionates sample on the basis of size
• Elution volume vs. molecular weight
• Pore size of the gel defines the MW range
• Exclusion limit – (10 6), permeation limit (103)
• Ve = V0 + KVi
• Large molecules can not diffuse into the pore,
Ve = V0

138. Stationary and Mobile phases
• Gel filtration – hydrophilic packing (styrene
and divinylbenzene) and aqueous mobile
phase
• Gel permeation –hydrophobic packing
(sulfanated divinylbenzenes and
polyacrylamides) and non-polar organic
mobile phases

139. Affinity Chromatography
• A “handle” is attached to a solid support,
which is packed into a column
• This handle selectively binds to a certain
analyte or group of analytes
• Examples
– Antibodies to capture specific proteins
– avidin binds to biotin

140. ICAT reagent
• Selectively capture cysteine-containing peptides
Wall of column
avidin
biotin
linker
iodoacetamide
C
S A T W
M
P
A

141. Introduction
• HPLC is a form of liquid chromatography used to separate
compounds that are dissolved in solution. HPLC instruments
consist of a reservoir of mobile phase, a pump, an injector, a
separation column, and a detector.
• Compounds are separated by injecting a sample mixture onto
the column. The different component in the mixture pass
through the column at differentiates due to differences in
their partition behavior between the mobile phase and the
stationary phase. The mobile phase must be degassed to
eliminate the formation of air bubbles.

142. HPLC system

143. FOUR TYPES OF LIQUID
CHROMATOGRAPHY
• Partition chromatography
• Adsorption, or liquid-solid
• chromatography
• Ion exchange chromatography
• Size exclusion, or gel, chromatography

144. COMPOSITION OF A LIQUID
CHROMATOGRAPH SYSTEM
• Solvent
• Solvent Delivery System (Pump)
• Injector
• Sample
• Column
• Detectors (Diode Array)
• Waste Collector
• Recorder (Data Collection)

145. Uses of HPLC
• This technique is used for chemistry and biochemistry research analyzing
complex mixtures, purifying chemical compounds, developing processes
for synthesizing chemical compounds, isolating natural products, or
predicting physical properties. It is also used in quality control to ensure
the purity of raw materials, to control and improve process yields, to
quantify assays of final products, or to evaluate product stability and
monitor degradation.
• In addition, it is used for analyzing air and water pollutants, for
monitoring materials that may jeopardize occupational safety or health,
and for monitoring pesticide levels in the environment. Federal and state
regulatory agencies use HPLC to survey food and drug products, for
identifying confiscated narcotics or to check for adherence to label
claims.

146. HPLC Chromatograph injectors
• The function of the injector is to place the sample into the
high-pressure flow in as narrow volume as possible so that the
sample enters the column as a homogeneous, low-volume
plug. To minimize spreading of the injected volume during
transport to the column, the shortest possible length of
tubing should be used from the injector to the column.
• When an injection is started, an air actuator rotates the valve:
solvent goes directly to the column; and the injector needle is
connected to the syringe. The air pressure lifts the needle and
the vial is moved into position beneath the needle. Then, the
needle is lowered to the vial.

147. HPLC columns
• The column is one of the most
important components of the
HPLC chromatograph because
the separation of the sample
components is achieved when
those components pass
through the column. The High
performance liquid
chromatography apparatus is
made out of stainless steel
tubes with a diameter of 3 to
5mm and a length ranging
from 10 to 30cm.
• Normally, columns are filled
with silica gel because its
particle shape, surface
properties, and pore structure
help to get a good separation.
Silica is wetted by nearly every
potential mobile phase, is inert
to most compounds and has a
high surface activity which can
be modified easily with water
and other agents. Silica can be
used to separate a wide variety
of chemical compounds, and its
chromatographic behavior is
generally predictable and
reproducible.

148. Picture of an HPLC column

149. WHAT AFFECTS SYSTEM
Column Parameters
• Column Material
• Deactivation
• Stationary Phase
• Coating Material
Instrument Parameters
• Temperature
• Flow
• Signal
• Sample Sensitivity
• Detector

150. WHAT AFFECTS SYSTEM
Sample Parameters
• Concentration
• Matrix
• Solvent Effect
• Sample Effect

151. Several column types
(can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange

152. Normal phase
• In this column type, the retention is governed
by the interaction of the polar parts of the
stationary phase and solute. For retention to
occur in normal phase, the packing must be
more polar than the mobile phase with
respect to the sample

153. Reverse phase
• In this column the packing material is relatively
nonpolar and the solvent is polar with respect to the
sample. Retention is the result of the interaction of
the nonpolar components of the solutes and the
nonpolar stationary phase. Typical stationary phases
are nonpolar hydrocarbons, waxy liquids, or bonded
hydrocarbons (such as C18, C8, etc.) and the solvents
are polar aqueous-organic mixtures such as
methanol-water or acetonitrile-water.

154. Size exclusion
• In size exclusion the HPLC column is consisted
of substances which have controlled pore sizes
and is able to be filtered in an ordinarily phase
according to its molecular size. Small
molecules penetrate into the pores within the
packing while larger molecules only partially
penetrate the pores. The large molecules
elute before the smaller molecules.

155. Ion exchange
• In this column type the sample components
are separated based upon attractive ionic
forces between molecules carrying charged
groups of opposite charge to those charges on
the stationary phase. Separations are made
between a polar mobile liquid, usually water
containing salts or small amounts of alcohols,
and a stationary phase containing either acidic
or basic fixed sites.

156. Selectivity Factor
• K’ values tell us where bands elute relative to
the void volume. These values are unaffected
by such variables as flow rate and column
dimensions. The value tell us where two peaks
elute relative to each other. This is referred to
as the selectivity factor or separation factor
(now and then as the chemistry factor).

157. Types of Liquid Column Chromatography
(LCC)
• LLC (Liquid Liquid)
• LSC (Liquid Solid -
adsorption)
• SEC (Size Exclusion)
• GLC GSC
• SFC (Supercritical
Fluid)

158. Types of Detectors
• Absorbance (UV with
Filters, UV with
Monochromators)
• IR Absorbance
• Fluorescence
• Refractive-Index
• Evaporative Light
Scattering Detector
(ELSD)
• Electrochemical
• Mass-Spectrometric
• Photo-Diode Array

159. EVALUATION PARAMETERS
• EFFICIENCY
• RESOLUTION
• INERTNESS
• RETENTION INDEX
• COLUMN BLEED
• CAPACITY FACTOR