1. Dr. Ashraf M. M. Mahmoud, Associate professor
Gas Chromatography
Dr. Ashraf M. M. Mahmoud, Associate professor

2. Common Techniques of Chromatography
Gas Chromatography (GC)
High-Performance Liquid Chromatography (HPLC)
Thin Layer Chromatography (TLC)

3. Principles of separation in Chromatography
time
Response

4. Principles Behind the Instrument
Instrumentation of GC
Principles Behind the Instrument

5. Mechanisms of Partition to Stationary Phase

6. Gas Chromatography (GC)
What is the principle ?
The technique is similar to HPLC except that the mobile liquid phase is replaced by a moving gas phase, and the injected sample should be volatile (native or derivatized).
What is the Applications of GC ?
What is the GC Instrumentation ?

7. Instrumentation of GC Oven Flow meter Gas supply Pressure regulator
controller
Septum
Detector
Oven
Column
Injector
GC Chart
Recorder 

8. Gas
supply
Pressure
regulator
Flow
controller
Flow meter
Gas
supply
Pressure
regulator
Flow
controller
Septum
Detector
Oven
Column
Injector
Recorder  GC
chart
Gas Supply
The mobile phase (carrier gas) should be chemically inert, dry and free from O2 (helium, argon, nitrogen and hydrogen).
The carrier gas should be of high purity; impurities in the carrier gas such as water vapour, air and trace gaseous hydrocarbons can cause reactions with sample and cause column deterioration and affect detector performance.
The gas supply is associated with pressure regulator and flow controller.

9. Sample Injection System
Flow meter
Gas
supply
Pressure
regulator
Flow
controller
Septum
Detector
Oven
Column
Injector
Recorder  GC
chart
Flow meter
Septum
Detector
Oven
Column
Recorder 
Carrier
gas
Injector
Sample Injection System
Preparation of the Sample :
Samples GC must be volatile.
Samples which are non volatile are converted into a volatile derivative.
The most commonly method is the silylation (reaction of trimethylsilyl,
- Si(CH3)3 with an active hydrogen atoms in the analyte (carboxylic acids, amines, imines, alcohols, phenols, and thiols).
Inorganic metals (aluminum, beryllium, and chromium) can be analyzed by GC via formation of stable, volatile metal chelates with trifluoroacetylacetone (TFA) and hexafluoroacetylacetone (HFA).

10. Sample Injection System
Septum
Injector
block
Flow meter
Septum
Detector
Oven
Column
Recorder
Carrier
gas
Injector 
Sample Injection System
Injection of the Sample :
The injector block; is heated to a temperature that is at least 50 C above the sample component with the highest boiling point; this to ensure rapid vaporization of the entire sample.
The injection system is a self-sealing silicone septum.
The sample is injected by a microliter syringe. The needle of the syringe is inserted through and the sample injected smoothly into a heated metal block at the head of the column.

11. GC Column Oven The GC column on which the actual
Flow meter
Septum
Detector
Oven
Column
Recorder
Carrier
gas
Injector 
GC Column
The GC column on which the actual
separation of sample components
is effected.
Most GC columns are made from high-purity fused silica capillary, the inner wall of the capillary coated with the stationary phase.
GC columns vary in length from less than 2 m to 50 m or more.
In order to fit into the column oven, they are usually formed as coils.
The control of column’s temperature is critical to attain a good separation in GC, thus the column is located inside a thermostated oven to control the temperature.

13. Choice of GC Columns

14. Column Configuration Packed Columns Capillary Columns
2 to 4 mm I.D. and 1 to 4 meters long.
Packed with a suitable adsorbent.
Mostly used for gas analysis.
Peak broadening due to zone (eddy) diffusion resulting from multitude of pathways a molecule can pass through column.
100 mm to 500 mm I.D. and 10 m to 100 m long
Stationary phase is coated on the internal wall of the column as a film 0.2 mm to 1 mm thick
Sharper peaks – no eddy diffusion.
Up to 500,000 theoretical plates – excellent separations.
Most popular type of column in use.

15. GC Detector The function:
Flow meter
Gas
supply
Pressure
regulator
Flow
controller
Septum
Detector
Oven
Column
Injector
Recorder  GC
chart
GC Detector
The function:
Monitoring the carrier gas as it emerges from the column..
The requirements of ideal GC detector:
The same as HPLC detectors.
Types of GC detectors:
Thermal conductivity detector (TCD)
Electron capture detector (ECD)
Flame ionization detector (FID)
Nitrogen phosphorous detectors (NPD)

16. GC Detectors Thermal Conductivity Detector (TCD):
Measures the decrease in the thermal conductivity of the carrier gas resulted from the non-conductive analyte molecules.
Electron Capture Detector (ECD):
Measures the decrease in the detector current which occurs when
an analyte molecule that tend to capture electrons appears in the
gas stream.
Flame Ionization Detector (FID):
Measures the increase in the electrical signal as a result of
ionization of the analyte molecules by the action of the detector flame.
Nitrogen Phosphorous Detectors (NPD):
Measures the increase in the current resulted from combustion of nitrogen- and phosphorous-containing compounds in the detector flame.

17. GC Detectors Characteristics of GC Detectors : Type Selectivity
Sensitivity (ng)
Temp. limit (C)
TCD
All organic compounds
5-20
450
ECD
Halogens, nitrates, and conjugated carbonyles
350
FID
Compounds with C-H bonds
0.1-10
400
NPD
Compounds containing nitrogen and phosphorous
300

18. GC Recorder Sample injected Peaks correspond to individual components
Flow meter
Gas
supply
Pressure
regulator
Flow
controller
Septum
Detector
Oven
Column
Injector
Recorder  GC
chart
GC Recorder
Sample injected
Peaks correspond to
individual components

19. Quantitative Analysis
Applications of GC ?
Peaks correspond to
individual components
Qualitative Analysis
Quantitative Analysis
Separation of Mixture Components:
Authentic
Identification of Compounds:
Unknown
Retention time comparsion
Pyrolysis gas chromatography
It is used for the identification of non-volatile materials (plastics, natural and synthetic polymers, and some microbiological materials.
It is based on the fingerprint chromatogram for the sample, which results from its thermal dissociation and fragmentation.

20. Quantitative Analysis
Concentration
Peak hight
Calibration curve
Quantitative Analysis
External Standard Method 10
100 ng/mL 10
75 ng/mL 10
50 ng/mL 10
25 ng/mL 10
Unknown 10
10 ng/mL 10
5 ng/mL

21. Quantitative Analysis
Concentration
Peak hight ratio
Calibration curve
Quantitative Analysis
Internal Standard Method 10
100 ng/mL 10
75 ng/mL 10
50 ng/mL 10
25 ng/mL 10
Unknown
Internal
Standard
Compound 10
10 ng/mL 10
5 ng/mL

22. Aspects of GC Applications:
Food Analysis
Analysis of foods is concerned with confirming the presence
and determination the quantities of the analytes (lipids,
proteins, carbohydrates, preservatives, flavours, colorants,
and also vitamins, steroids, and pesticide residues).
Drug Analysis
GC is widely applied to identification of the active components, possible impurities as well as the metabolites.

23. Environmental Analysis
The environmental contaminants; e.g. dichlorodiphenyltrichloro-
ethane (DDT) and the polychlorinated biphenyls (PCBs) are present
in the environment at very low concentrations and are found among
many of other compounds.
GC, with its high sensitivity and high separating power, is mostly
used in the analysis of environmental samples.
Forensic Analysis
In forensic cases, very little sample is available, and the
concentration of the sample components may be very low.
GC is a useful due to its high sensitivity and separation efficiency.

25. Peak Base: An interpolation of the base line between the
extremities of the peak. .
Peak Area: The area enclosed by the peak and the peak
base.
Peak Height: The distance from the peak maximum to the
peak base.
Peak Width (tW): The magnitude of the peak base
intercepted by the tangents to the inflection points of the
peak.
Retention time (tR): The time between sample injection
and the appearance of a solute peak at the detector.
Dead time (t0): The time for mobile phase to pass through a column.
Adjusted Retention Time (tR') is the time solute spent in
the stationary phase and equals to tR' = [ tR- t0].

26. tR' = [ tR- t0]. Adjusted Retention Time (tR')
The Capacity Factor (k') is used to describe the migration rates of solutes on columns, It is defined as:
For solute A, kA' = (tR,A – t0)/t0 ; = t'R,A/t0
The Selectivity Factor () of a column for the two solutes A and B in a mixture is defined as
= k'B/k'A
Chromatoaraohic column efficiency is determined by
1- Plate height (Height Equivarent to Theoretical Plate (HETP)= L/N
2- Number of Theoretical Plates(N) =16 (tR/tW)2
L = length of column
Resolution of the column (Rs) = (tR,B – tR,A)/0.5 (tw,A+tW,B)
Rs = √N/ x [α-1]/ α x [k'B/(k'B+1)]
Column efficiency Selectivity Capacity

27. Resolution and Selectivity

28. Can One Have Too Much Resolution
RS = 2.5

29. Separating Efficiency – Peak Asymmetry

30. Good Luck
Ashraf M. Mahmoud