Gas Chromatography

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A simple GC system consists of:

1. Gas source (with pressure and flow regulators)

2. Injector or sample application system (sample inlet)

3. Chromatographic column (with oven for temperature

control)

4. Detector & computer or recorder

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 Principles

 A gas mobile phase flows under pressure through

a heated tube stationary phase

 The analyte is loaded onto the head of the column

via a heated injection port (evaporates)

 It condenses at the head of the column, which is at

a lower temperature

 The oven temperature is then either held constant

or programmed to raise gradually.

 Separation of mixture occurs according to the

relative lengths of time spent by compounds in
stationary phase

 Monitoring of the column effluent can be carried

out with detectors

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 Carrier gas: packed column Flow = 25-150 mL/min
 He (common), open tubular column Flow = 1-25 mL/min
 N2,
Column : 2-100 m coiled
 H2 stainless steel/glass/Teflon/fused silica,
Pinlet 10-50 psig packed vs. unpacked

 Oven: 0-400 °C ~ average boiling point

of sample

Accurate to <1 °C

 Detectors: FID, TCD, ECD, NPD,

MSD. (SINGLE OR TANDEM)

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Carrier Gas
 The purpose of carrier gas is to transport the sample
through the column to the detector.
 The selection of proper carrier gas is very important
because it effects both column and detector performance.
 Carrier gas usually used in GC are :
Helium
Hydrogen
Nitrogen
mixture of 95% Argon and 5% methane.
 The detector that is employed usually dictates the carrier to be used

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 TCD : Hydrogen and Helium most
suitable carrier gases
 FID : Nitrogen and Helium
 ECD : mixture of 95% argon and
5% methane.

 Cylinders holding carrier gases are painted with

different colours to identify carrier gas.
 Hydrogen is painted Red
 Oxygen painted Black
 Argon painted Green.
 High purity carrier gases are used for quantitative
analysis.

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Injection port
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 Injection port : Sample is
introduced into Gas
Chromatograph
 Sample introduced in the form of
solution by a microliter syringe
 A high temperature resistance
rubber septum is kept in the
injection port.
 Injection port temperature = Max.
450o C.
 Sample is vaporized as soon as it is
introduced into the injection port.
 The sample vapour along with the
carrier gas enter the column.

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 Column
 Column is the heart of the chromatographic system

 Sample mixture is separated into individual compounds depending on the

principle of partition or adsorption.
 Column : stainless steel – 2 to 18 feet length, 1/8II dia.

 Stationary phase: Adsorbent (or) liquid phase coated on solid support (GSC/GLC)

 Glass wool inserted into the tubing ends.

 Common stationary phases

 Methyl silicone , SE-30 (non polar)

 Phenyl silicones (Medium polar)
 Diethylene glycol succinate (DEGS) (polar)

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 Capillary Columns

 Goley introduced capillary columns

 Capillary columns are made from fused silica, usually coated on
the outside with polyamide to give the column flexibility.
 The wall of the column is coated with the liquid stationary phase
(0.1 -5 µm)
 Most common coating is based on organo silicone polymers.
 Length = 25 to 100m , 0.15 to 0.5 mm dia.

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 Bonded-Phase
- many bonded phases exist, but most separations can be formed with the
following phases:
CH3 Advantages:
1. Dimethylpolysiloxane
O Si

CH3 -more stable than coated liquid phases

n

2. Methyl(phenyl)polysiloxane
CH3 C6H5 -- can be placed on support with thinner
and more uniform thickness than liquid
O Si O Si phases
CH3 C6H5
n m
3. Polyethylene glycol (Carbowax 20M)
H H

HO C C O H

H H n

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GC Oven

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 GC Oven
 GC oven incorporate a fan-ensures uniform heat distribution throughout the
oven.

 Temperature conditions :
 isothermal
 Gradual increase in temperature

 Oven programming rates can range from 1o C/min to 40oC/min.

 Eg:60oC (1 min)/5oC/min to 100oC (5 min)/10oC/min to 200oC (5 min).

 Materials of different volatilities can be separated in a reasonable time

and also injection of the sample can be carried out at low temperature,
where it will be trapped at the head of the column and then the
temperature can be raised until it elutes.

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 Column Oven
 Isothermal Analysis
 Temperature Programming Analysis
 Column Temperature Eg.1

􀂄 Separations/Resolutions are good, but the analysis time is too long.

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 Column Temperature Eg.2

 Short analysis time, but separations/resolutions are not good.

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Column Temperature Eg.3

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 Start from low temp and then raise the temp to a higher one in
a certain rate of increase.

 e.g.: 50 C (2 min) →20 C/min → 200C (5 min)

 80 C (5 min) → 10C/min → 150C (3 min) → 5C/min → 180C
(15 min)

 The lower the rate of temp increase, the better the resolution is.

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 Theory of Gas Chromatography
 Adsorption: the molecules of analyte reside on the
stationary phase for sometime and then desorbed and
eluted out along with the mobile phase.
 Solid adsorbent is used as a stationary phase.
 Partition : the molecules of analyte get distributed
between the mobile and stationary phases.
 Stationary phase : high molecular weight liquids.
 If the con. Of the analyte is more in stationary phase, the
elution takes more time.
 If concentration of analyte is less in stationary phase, the
elution takes less time.

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 Partition coefficient (β)
 GC separation = distillation separation.

 β = Weight of the solute per gram of the stationary phase

Weight of the solute per cc of the carrier gas

 β is characteristic of each one of the components

 In addition to partition, other physical interactions like –

hydrogen bonding, wander Waal’s forces

 Nature of analyte, stationary and mobile phases.

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Sampling: Derivatization Techniques
 i) To permit analysis of compounds not directly
amenable to analysis due to inadequate volatility or
stability
 ii) To improve the analysis by better chromatographic
behavior or detectability.
 Replacement of N-H, O-H and S-H groups by
alkylation, acylation, silylation increases the volatility.
 Closely related compounds, optical isomers can be
separated after derivatization.

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 Silylation
Typical reagents
 i) Hex methyl disilazane (HMDS)
 (CH3)3SiNHSi(CH3)3

 ii) Trimethyl chlorosilane (TMCS)

 (CH3)3SiCl
 Iii) Dimethyl chlorosilane (DMCS)
 (CH3)2SiCl
 iv) Tetramethyl disilazane (DMCS)
 (CH3)2HSiNHSi(CH3)2H
 The reagent is used to replace an active protons to improve
thermal stability or to reduce tendency to form hydrogen bonds.
 Ex: sugars, thiols, steroids, amino acids

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 Acetylation
 To replace active hydrogen
 Alcohols, amino acids, 1 or 2 amines
 Amino acids are usually esterified to methyl or butyl
esters prior to acetylation.
 Typical reagents : acetic anhydride & Trifluoroacetic
anhydride dissolved in pyridine & DMF.
 Trifluoroacetyl derivatives.

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 Detector

 1. Detector is to indicate the presence and measure the

amount of component eluted out from the column.

 2. High detector temperature should be set to prevent

condensation of sample

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 Type of Detectors

1. Flame Ionization Detector (FID)

2. Thermal Conductivity Detector (TCD)
3. Flame Thermionic Detector (FTD )
4. Flame Photometric Detector (FPD)
5. Electron Capture Detector (ECD)
6. Mass Spectrometer Detector (MSD)
TCD, FID, MSD are general detectors.

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 Type of Analysis
 FID ::-Detects any compounds that can be oxdised in
hydrogen/air flame
 ECD:-Selective to electronegative moieties
e.g.halogens
 TCD:-Detects any component including N2 and O2
except the gas used for the carrier gas.

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 FID
 Most organic compounds can be detected

These compounds are oxidized in

hydrogen / air flame

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 Flame Ionization Detector
 For organic compounds analysis

 Hydrogen and air are needed to create the flame

 Sample is brought to hydrogen flame and converted into ions.

Current will be generated.

 The current is proportional to the amount of the organic compound

present
 Advantages

1. High Sensitivity to organic compounds

2. Little or no response to water, CO2, the common carrier gas

impurities hence zero signal to when no sample is present

3. Stable baseline which is not mostly affected by fluctuations

in carrier gas temp, flow rate and pressure.

4. Good linearity over a wide range of sample conc. Range

5. Presence of halogens in the compound decreases sensitivity

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 TCD
 Any components including N2 and O2 except the gas used for
carrier gas can be detected by TCD
 Wheatstone bridge is used as principle of detection.

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Advantages over FID
 Since all compounds, organic and inorganic, have a thermal
conductivity different from helium, all compounds can be detected
by this detector

 The TCD is often called a universal detector because it responds

to all compounds.

 Non destructive type of detector hence can be used for trapping

separated components and preparative analysis

 Low cost and versatile

 The TCD is not as sensitive as other detectors but it is non-specific

and non-destructive

 The TCD is also used in the analysis of gases (argon, oxygen,

nitrogen, carbon dioxide) because it responds to all these pure
substances unlike the FID which cannot detect compounds which
do not contain carbon-hydrogen bonds.

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 Electron Capture Detector (ECD)
􀂄 Very selective and sensitive to electrophilic compounds
(e.g. halogens, phosphorous and nitro group)

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 ECD
􀂄 Detects electrophilic compounds
 e.g.: halogenated compounds
 􀂄 Good for low concentration of these kind of
compounds
 􀂄 Contains 63Ni radioactive

􀂄 Examples of analyses:
 analysis of PCBs (polychlorinated biphenyls)
 analysis of PBBs(polybrominated biphenyls)
 analysis of PBDEs (polybrominated diphenyl ethers)
 analysis of organ chlorine pesticides (DDT, BHC, etc.)

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ECD Application

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 Headspace (HS)
Extraction/Injection

Figure 1. TurboMatrix Automated Headspace Sampler (right) with the Clarus 500 Gas Chromatograph (left).
 Static Head Space
 Analysis of the vapour in equilibrium in a sample

 keep in a closed vial at constant temperature.

 Temperature range: from 40ºC to 190ºC.

 Solid or liquid samples are introduced in a sealed vial with an inert gas

(at controlled pressure) in the headspace.

 The system is kept at constant temperature during a fixed time until the

volatile compounds reach the equilibrium between the matrix and the
head space.

 A fixed volume of the head space is transferred to the GC injector

through the proper heated line.

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WHEN DO WE USE HEAD SPACE?

 When direct injection is not possible.

 When results are better than direct injection (depends
on compound/matrix).
 Samples that need a previous extraction (alcohol in
blood, water pollutants, etc.).
 When analyzing only volatile compounds (food,
beverages, perfumes, Residual solvent analysis in
drugs…).

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Advantage
 The non volatile compounds are not introduced in the
GC System.
 Better sensitivity.
 Sample preparation is not needed.

Disadvantage
 Quantification is done only in the vapor phase.
 Quantification problems (internal standard).
 Special equipment.

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 Strengths Limitations

 Capable of quantitative accuracy  Only thermally stable and

 Greater separation power than volatile compounds can be
HPLC when used with capillary analysed
columns  Sample may require
 Used to determine compounds
derivatisation to convert it to a
volatile form
which lack chromophores.
 Quantitative sample
 The mobile phase does not vary introduction is more difficult
and does not disposal –cheap than because of the small volume of
HPLC solvents sample injected
 Aqueous solutions and salts
cannot be injected into the
instrument
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 Applications
 Characterization of some unformulated drugs with regard to
detection of process impurities
 Limit tests for solvent residues and other volatile impurities in
drug substances
 Sometimes used for quantification of drugs in formulations,
particularly if the drugs lacks a chromophore
 Characterization of some raw materials used in synthesis of
drug molecules
 Characterization of volatile oils (excipients)
 Cough mixtures, tonics, fatty acids in fixed oils
 Measurement of drugs and their metabolites in biological
fluids

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 Considerations for
Residual Solvent Analysis
USP Method 467

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 The Role of Solvents?
• They may be critical to the synthetic process:
–Enhance yields
–Improve crystallization
–Increase solubility
• The list of regulated solvents will most likely grow
–Improved toxicological testing
–New, unknown toxic affects

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<467> Organic Volatile Impurities
• Solvents classified by risk assessment
–Class 1: Solvents to be avoided
–Class 2: Solvents to be limited
–Class 3: Solvents with low toxic potential
• Drug formulations containing these solvents
must be tested
• Only the solvents used or produced in the
manufacturing and/or purification process must
be evaluated

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<467> Methodology Overview

 Drug product is dissolved in solution

–Water Soluble Articles: Water
–Water Insoluble Articles: DMF,
 Headspace injection
 GC analysis with FID detection
–Procedure A: G43 (ZB-624) Phase
–Procedure B: G16 (ZB-WAXplus) Phase

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 GC Conditions
Column : Zebron ZB-624
Dimensions : 30 meters x 0.32 mm x
1.80 μm
Injection : Split 5:1 1 µL @ 140 °C
Oven Profile : 40°C for 20min to 240°C at
10°C/min for 20min
Carrier Gas : Constant Flow Helium, 35
cm/sec
Detection : Flame Ionization @ 250 °C

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 Class 1 Solvents
 Testing required
 Solvent levels must meet Concentration Limit

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 Class 1 Solvents As Impurities

 Class 1 solvents are highly regulated

 Concentration Limit requires very low level
 These levels may be present in Class 2 or 3
solvents as impurities
 Example: Toluene possible impurity –Benzene
 Cross contamination possible

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Class 1: ZB 624 30m x 0.32mm x 1.80 μm

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 Class 2 Solvents
 Solvents are assigned a permitted daily exposure (PDE) limit
 Each solvent is assigned a Concentration Limit allowable in
any component of the drug product

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Class 2: ZB -624 30m x 0.32mm x 1.80 μm

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 Class 3 Solvents

 If only Class 3 solvents are present…

 Solvent level is determined by <731> Loss on Drying
 The PDE limit, unless otherwise specified, is 50mg/day
or 5,000 ppm
 If solvent level is above the PDE limit, it must be
identified and quantified

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 Class 3:
ZB 624 30m x 0.32mm x 1.80 μm

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Limitations of the Method <467>

 Long analysis time >30min

 Poor resolution of some compounds
 Long wait equilibration time for headspace samples
 Poor detection of some compounds
 No definitive identification of contaminates
 –FID does not give information about the peak

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 Column Phases

What is the ideal phase?

 Depends on the goal of the separation…what
are the target analytes

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 G43 Phase
 Phase Structure

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G16 Phase

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Phenyl methyl Polysiloxane

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Selectivity Xylene Isomers

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 Detector

The USP Method specifies FID

• Benefits:
–Responds to a wide range of compounds
–Large dynamic range
–Inexpensive
–Stable and easy to use
•Negatives:
–No information about the analyte
–Poor response for highly chlorinated compounds

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  Oral liquid Pharmaceutical Drug Products:
 APIs+ flavoring agents + dyes, and preservatives.
 Benzoate salts are commonly used as preservatives in
OLDPs for their bacteriostatic and fungistatic properties.
 Benzoate salts have been reported to form residual levels of free
benzene under heat and acidic conditions in beverages and soft drinks[1-
3].

 Benzene is a potential carcinogen that can cause or increase the risk of

leukemia and benzene exposure is also associated with other types of
blood cancers, and pre-cancers of the blood.
 ICH Guideline has set up a limit of 2ppm for residual benzene in
pharmaceutical drug products

[1] Benzene residues in soft drinks, FDA (1990) Dec. 7 memo, related (1991) Jan memo.
[2] Benzene in Soft drinks, FDA statement, (2006) April 13, News 01355.
[3] Letter Regarding Benzene Levels in Soft Drinks, FDA, (2006) March 21.

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