The document provides information about gas chromatography (GC). It begins with definitions, stating that GC separates compounds based on their volatility between a stationary and mobile gas phase. The basic principle is that a sample is vaporized and injected into a column, where components are separated as they are eluted by an inert gas and detected. Key components of a GC system are described, including the carrier gas, injector, column placed in an oven, and various detectors. Common detectors mentioned are the flame ionization detector and electron capture detector. The document also discusses the process of derivatization used to modify compounds to make them more suitable for GC analysis by increasing volatility or detectability.

1. Gas Chromatography (GC)
▶ Introduction
▶ Definition
▶ Principle
▶ GC Instrumentation
▶ Detectors
▶ Derivatisation

2. Definition:
Gas Chromatography (GC) is an analytical procedure employed
for separating compounds based primarily on their volatilities by
equillibrium between stationary and mobile(gas )phase
Depending upon type of stationary phase use it is classified as
▶Gas / Liquid (partition) GLC :gas chromatography in which the stationary phase
is a liquid
▶Gas / Solid (adsorbent) GSC: gas chromatography in which the stationary phase
is a solid
GC used for separation and analysis of volatile, gaseous substances.

3. Basic Principle of GC –
Sample vaporized by injection into a heated system, eluted through a
column by inert gaseous mobile phase and detected.
Principle of gas chromatography:
The sample is dissolved in solvent
The sample solution injected(at sample injection unit) into the instrument
enters a gas stream which transports the sample( vaporise form of
sample)into a separation tube known as the "column." (Helium or nitrogen is
used as the so-called carrier gas.)
The various components are separated inside the column.
The detector measures the quantity of the components that exit the
column.
To measure a sample with an unknown concentration, a standard
sample with a known concentration is injected into the instrument.
The standard sample peak retention time (appearance time) and area are
compared to the test sample to calculate the concentration.

5. Instrumentation of GC
GC Instrumentation: Block Diagram

6. GC Instrumentation: Block Diagram

7. Component of GC
▶ Carrier Gas:
▶ Flowcontroller
▶ sampleInjector system :
▶ Column:
▶ Oven:
▶ Detectors
▶ Amplification and recordersystem

8. Carrier Gas:
It is use to carry sample through the column
▶ Helium (commonlly use)
▶ Others: N2, H2, Ar and Air.
▶ Safety, availability, non-flammability, cost and efficiency are factors for gas selection.
▶ Purity of 99.995 % and higher is considered for selection as well.
▶ Pinlet = 10-50 psi

9. Flow controller :
carrier gases are available in pressurized tanks and pressure regulators, gages
and flow meters are used to control flow rate of the gas
2 type of flow regulators :
i) Soap bubble meters
ii) Rotameters

10. Soap bubble meters Rotameters
Rubber bulb containing soap solution is
converted into soap bubble by carrier gas
pressure & travel inupword direction
which measure flow ate of carrier gas
A glass tube consist of float , carrier gas
pressure moves folat in upword direction
which measure flow ate of carrier gas

11. Sample Injection system
▶ Transfers the analyte into the column.
▶ It provides the means to introduce a sample into a continuous flow of
carrier gas.
▶ Injectors are usually heated to ensure analyte’s transfer to a gas phase.
▶ Volatile liquid or gaseous sample is injected through a septum.
▶ Vapor is swept through column.
▶ Types:
1)Split injection method
2)Splitless injection method
3)On – Column
4) Automatic sampler
5) Headspace sampling

12. Split vent is
closed

13. Usually consists of heated liner (a glass sleeve, prior to the column
(200–300 °C).
▶ A sample is introduced into a heated small chamber via a syringe through a septum –
the heat facilitates volatilization of the sample and sample matrix.
▶The carrier gas then either sweeps the entirety (splitless mode) or a portion (split mode)
of the sample into the column.
▶In split mode, a part of the sample/carrier gas mixture in the injection
chamber is spliting the vaporised sample into 2 parts small fraction enter in column
and remaining is exhausted through the split vent/waste
•In splitless mode the split valve opens after a pre-set amount of time
Splitless - all the sample is introduced (but only for
limited time period)
• These method is suitable for higher boiling point comp.
Split/ Splitless injection method

14. Headspace Sampling
•In which analysis of volatile components of mixture
•The components are directly analysed without extraction from samples
•The volatile component with high vapor pressure above liquid are in form of gase
distributed in the headspace of a seales samle vial
•The injection of gases samle into Gc column done by gas syringe or specially
designed valve

15. Direct on column injection system
It is suitable for thermo- labile and
very high boiling point compound
sample is introduced directly on to the column
Automatic sampler
▶It consist of tray which holds a large no. of sample vials all are rotated into
position under syringe as needed
▶The column and inlet can then be heated, releasing the sample into the gas phase.
▶ This ensures the lowest possible temperature for chromatography and keeps
samples from decomposing above their boiling point.
▶ Analytes are injected directly on the column.
▶ This technique is suitable for thermally unstable compounds.

16. columns
▶ Gas chromatography columns are of two designs:
1)packed columns and
2) capillary columns.
▶Packed columns are typically a glass or stainless steel coil (typically 1-5 m total length and 5
mm inner diameter) that is filled with the stationary phase, or a packing coated with the
stationary phase.
▶ Capillary columns are a thin fused-silica (purified silicate glass) capillary (typically 10-
100 m in length and 250 mm inner diameter) that has the stationary phase coated on the
inner surface.
▶Capillary columns provide much higher separation efficiency than packed columns but are
more easily overloaded by too much sample.

17. Columns: Separate the analytes. 2-50 m coiled stainless steel/glass/Teflon.
▶ The main chemical attribute regarded when choosing a column is the
polarity of the mixture, but functional groups can play a large part in column
selection.
▶ The polarity of the sample must closely match the polarity of the column stationary
phase to increase resolution and separation while reducing run time.
▶ The separation and run time also depends on the film thickness (of the stationary
phase), the column diameter and the column length.
Packed
1. Solid particles either porous or non-porous coated with thin (1 μm) film of liquid
2. 3 - 6 mm ID; 1 - 5 m length
▶ Capillary (open tubular) silica columns
1. 0.1 - 0.5 mm I.D. (internal diameter); 15 - 100 m length
2. Inner wall modified with thin (0.1-5 μm) film of liquid (stationary phase)
3. Easy to install
4. Well defined stationary phase.

18. Packed
• liquid coated silica particles (<100-300 mm diameter) in glass tube
• best for large scale but slow and inefficient.
▶ Capillary/Open Tubular
• wall-coated (WCOT) <1 mm thick liquid coating on inside of silica tube
•support-coated (SCOT) 30 mm thick coating of liquid, coated support on inside of silica tube
• best for speed and efficiency but only small samples.
▶ The polarity of the solute is crucial for the choice of stationary phase compound, which in an
optimal case would have a similar polarity as the solute.
▶Common stationary phase compounds in open tubular columns are cyanopropylphenyl dimethyl
polysiloxane, carbowax polyethylene glycol, biscyanopropyl cyanopropylphenyl polysiloxane
and diphenyl dimethyl polysiloxane.
▶ For packed columns more options are available. Solid stationary phase adsorbents are SiO2
(silica gel), Al2O2 (alumina), charcoal and Na/ Ca Al Silicates.
Stationary phase

20. Oven:
▶ 0-400 °C ~ average boiling point of sample.
▶ Accurate to <1 °C.
▶ The column(s) in a GC is/are contained in an oven, the temperature of
which is precisely controlled electronically.
▶ The rate at which a sample passes through the column is
directly proportional to the temperature of the column.
▶ The higher the column temperature, the faster the sample moves through
the column.
▶ However, the faster a sample moves through the column, the less it interacts with the
stationary phase and the less the analytes are separated.
▶ A method which holds the column at the same temperature for the entire analysis is
called "isothermal".
▶Most methods, however, increase the column temperature during the analysis, the initial
temperature, rate of temperature increase (the temperature "ramp"), and final
temperature manipulations are called the temperature program.

21. Selecting Temperature Conditions:
▶ Temperature of injector: ensures evaporation of sample, but do
not decompose it (200 – 300 °C).
▶ Temperature of the column (GC oven).
▶ Effect of injection.
▶ For the split injection– no specific requirements.
▶ For the splitless and on column injection – solvent trapping technique
▶ Oven temperature - optimized to improve the separation.
▶ Temperature of the detector: has to be high enough to
prevent
condensation of analytes on the detector.

22. Detector
▶ The detector is placed at the exit of the column.
▶ It is employed to detect and provide a quantitative measurement of the various
constituents of the sample as they emerge from the column in combination
with the carrier gas.
Detector use in GC are :
1. Kathorometer or Thermal Conductivity Detector (TCD)
2. Flame Ionisation Detector (FID)
3. Electron Capture Detector (ECD)

23. Types of detectors
Non specific
Thermal
conductivity
Atomic emission
photoionization
Destructive FID,
NPD,PID, CD
Non destructive
TCD,ECD
Specific Flame
ionization
Nitrogen-Phosphorous
Flame photometric
Helium/argon ionization
Electron capture
chemiluminiscence
23

24. Flame Ionisation Detectors:
•The FID was invented by scientist Harley and Pretorious
and separately by McWilliams and Dewer.
•It makes use of an oven, wherein a flame is produced by
burning hydrogen gas in presence of oxygen or air.
•Effluent from the column is directed into a air/hydrogen
flame.
•A definite potential difference is maintained between the
two electrodes with the help of a series of batteries.
•Amplifier and recorder record chromatograms.

25. Working
• A portion of eluate coming from the column is directed
into the furnace through the wire loop.
• Solvent evaporates and organic compounds pyrolyses and
forms ions.
• These ions are attracted towards
the respective electrodes.
• This changes the potential difference between the
electrodes and hence the current in the circuit.
• As electrical resistance of flame is high and resulting current
is small, an electrometer is employed.

26. • It works on the principle of wheatstone’s bridge.
• Out of four resistances in the circuit, the magnitude of three
resistances remains constant.
• But that of fourth resistance varies as per change in the
temperature.
• This change is potential difference in the capacity of the
solute and the carrier gas to absorb heat (thermal
conductivity differences).
• The change in the temperature changes the
resistance and hence the current in circuit.
Thermal conductivity detector

27. Electron Capture Detector ECD
▶ It uses a radioactive beta particle (electron) source to measure the degree of
electron capture. ECD is used for the detection of molecules containing
electronegative / withdrawing elements and functional groups like halogens,
carbonyl, nitriles, nitro groups, and organo - metalics.
Working
•beam of electrons is produced by the beta emitter
•When carrier gas passes over the emitter, the gas ionises producing electrons
•In absence of compound, ionization of carrier gas produces a constant standing
current
•When solute is eluted out from the column, it captures electron towards it. Hence
current decreases
•This decrease gives idea about the concentration of a solute in the sample

28. DERIVATIZATION OF
HPLC AND GC

29. DERIVATIZATION :-
Derivatization is the process of chemically modifying a compound to
produce a new compound which has properties that are suitable for
analysis using a GC and HPLC
The chemical structure of the compound remains the same and
just modifies the specific functional group of reacting compounds to
derivative of deviating chemical and physical properties in order to
make them detectable and analyzable
Derivatization is needed in GC, HPLC , UV- Visible Spectroscopy
etc.

30. WHAT DERIVATIZATION ACCOMPLISH ?
• Increase volatility
 eliminates the presence of polar OH, NH & SH groups
 Derivatization targets O, S, N & P functional groups .
• Increases detectability.
• Increases stability.
• To reduce adsorption of polar samples on active surfaces of column
walls and solid support.

31. TYPES OF DERIVATIZATION : -
I. Silylation.
II. Alkylation.
III. Acylation.
IV. Chiral derivatization.

32. 1. SILYLATION :-
Most prevalent method, readily volatizes the sample.
 Mechanism:
 This process produces silyl derivatives which are more volatile, more thermally stable.
 Replaces active hydrogen with Tri methyl Silyl Groups.
 Silylation occurs the nucleophilic attack(SN2). The better the leaving group, the better the silylation.
 Solvents: (functional groups)
alcohol > Phenol > Carboxyl > Amine > Amide > Hydroxyl
 Advantages : -
• Ability to silylate a wide variety of compounds.
• Large number of silylating reagent available.
• Easily prepared.
 Disadvantages :-
• Silylation reagent are moisture sensitive.
• Must use aprotic organic solvents.

33. 2. ALKYLATION : -
Alkylation reduces molecular polarity by replacing active hydrogen with an alkyl group. These
reagent are used to modify compounds with acidic hydrogen. Such as carboxylic acidic and
phenols. These reagents makes esters, ethers, alkyl amines and alkyl amides. The principle
reaction employed for preparation of these derivatives is nucleophilic displacement.
 Advantages :-
• Wide range of alkylation reagents available.
• Reaction condition may vary from strongly acidic to strongly basic.
• Some reaction can be done in aqueous solutions.
• Alkylation derivatives are generally stable.
 Disadvantage :-
• Limited to amines and acidic hydroxyls.
• Reaction conditions are frequently severe.
• Reagent are often toxic.

34. 3. ACYLATION : -
Acylation reduces the polarity of amino, hydroxyl, and thiol groups. In comparison to silylation
reagent, the acylating reagents target highly polar, multifunctional compounds, such as
carbohydrates and amino acids.
 Advantages : -
• Addition of halogenated carbons increased detectability by ECD.
• Derivatives are hydrolytically stable.
• Increases sensitivity by adding molecular weight.
• Acylation can be used as a first step to activate carboxylic acids prior to esterification
 Disadvantages :-
• Acylation derivatives can be difficult to prepare.
• Reaction product (acid by–product) often need to be removed before analysis.
• Acylation reagent are moisture sensitive.
• Reagents are hazardous and odorous.

35. 4. Chiral derivatization : -
These reagents target one specific functional group and produce individual diasteriomers
of each of the enantiomers. There are two ways to separating enantiomers by
chromatography:
I. Separation on an optically active stationary phase.
II. Preparation of diastereomeric derivatives that can be separated on a non stationary
phase.
 REAGENTS : -
A. TPC (N-trifluroacetyl-L-prolyl chloride)
Used for optically active amines, most notable amphetamines.
B. MCF [(-)methylchloroformate]
Used for optically active alcohols.
- If an optically pure reagent is used to prepare diasteriomeric derivatives, then only two
derivatives are formed. The enantiomeric ratio is reflected in the relative peak size.

36. SPICIAL TYPES OF HPLC DERIVATIZATION : -
 For UV-Vis spectrophotometric detection.
 For flourimetric detection.
 For chiral analysis.
 According to when and where the derivatization is done
o Pre-column derivatization.
o Post-column derivatization.

37. PRE-COLUMN DERIVATIZATION :-
• Performed before the analytical separation is attained.
• Sample is derivatiszed manually or automatically and injected into the HPLC column.
• Separation of components occurs after derivatization
 Advantages :-
• Fewer equipment and reaction chemical restriction.
• Can be performed manually or automatically.
• No time restrictions on the kinetics of derivatization of reactions .
 Disadvantages :-
• Introduction of contaminants.
• Loss of analyte through adsorption.
• Sample degradation and incomplete reaction.
• Poorer precision due to increased complexity.

38. POST-COLUMN DERIVATIZATION :-
• Performed after analytical separation of compound but prior to detection.
• Addition pump is used for addition of derivatizing agent to the elute sample from
column.
 Advantages :-
 Minimal artifact formation.
 Complete reaction is not essential as long as it is responsible and the
chromatography of analyte remains unaffected.
 Disadvantages :-
 Band broadening.
 Added complexity for method development and routine use.

40. Thank
you….