GAS

CHROMATOGRAPHY
Sri Noegrohati
Fac. Of Pharmacy, Sanata Dharma Univ
What is Gas Chromatography
+ Gas chromatography is the process of separating volatile compounds in a complex
mixture by injecting a gaseous or liquid sample into a gaseous mobile phase which
is percolating over a stationary phase
+ Combination of different types of columns having a various composition of
stationary phase with proper detectors selective/specific to the type of analyte in
sample made GC as one of the widely accepted tools for the separation of compounds
because of its simplicity, sensitivity, and effectiveness.
+ Commonly used for analysis of various bioactive compounds in pharmaceutical and
phytochemical analysis..
 FUNDAMENTAL OF THE SEPARATION
+ The basis for gas chromatography separation is the different strengths of interaction of the
analytes (in the mobile phase) with the stationary phase  differences in the distribution of
components in the GC system
+  analytes should be easily volatilised  molecular weight < 1250 Da, and thermally
stable  don’t degrade in the GC system

Gas chromatography
Solutes KH is Henry's law constant
KH,A = CS,A /PA
Stationary phase KH Mobile phase
Liquid or Solid) b.p/ vapor p. (Inert gas)
and polarity.
 Henry's law constant
+ Vapor pressure is the pressure above a
liquid surface caused by the evaporation of
liquids/solid
+ In GC, the vaporized analyte is solubilized
in liquid stationary phase  The solubility
of gaseous analyte in a stationary phase
follows:

Vapor pressure Vapor pressure SA,S = KH,A PA.

obey Raoult Law: obey Dalton Law: + SA,S = Concentration of A in Stationary
phase (Mol/L or M)
+ PA = Partial pressure of A in mobile
phase
P0A is the vapor pressure of pure A and A + KH,A = Henry's law constant of A (M/atm)
is the mole fraction of in the mixture. 
Ptotal is total pressure in a closed system KH,A = SA,S /PA
and PA, PB is partial pressures of A,B
The Chromatographic Process
 During a GC separation, the sample is vaporized and carried by the mobile gas phase (i.e.,
the carrier gas) through the column. In the analytical column, the components of a sample
are partitioned between mobile phase and stationary phase
 The stationary phase can be either:
 a finely divided solid particles (adsorbent) (gas-solid chromatography (GSC)
 a high boiling liquid coated on supporting solid particles (packed GLC column)
or a thin film of liquid coated on the walls of the column tubing (capillary GLC
Column).
 The mobile phase (=carrier gas) is comprised of an inert gas i.e., helium, argon, or
nitrogen.
 The GC separates the mixture into its component based on their relative vapor pressure
and affinities for the stationary phase  differences in Henry's law constant, KH.
+ The separated compounds are eluted from the column ,identified and quantified in a
detector (FID,ECD, NPD, Mass Detector)
Gas chromatography application in
pharmaceutical analysis
+ Due to the very high efficiencies of separation power, the extreme sensitivity of the
detection and the precision and accuracy of the data, GC are mostly applied in quality
control, qualitative and quantitative analysis to ensure the purity of the produced
material, to check for trace contaminants, eliminate inconsistencies in pharmaceutical
products:
+ 1) Residual solvent analysis;
+ 2) Analysis of various functional groups;
+ 3) Percentage of purity of pharmaceutical compounds；
+ 4) For the analysis of drugs of abuse;
+ 5) In pharmaceutical R & D’s to determine the identity of natural products which
contains complex mixture of similar compounds;
+ 6) In the metabolomics studies.
+ Currently, there is also a growing use of the method within the pharmaceutical industry to
separate chiral compounds
 heated injection ports
High Resolution Gas Chromatography -
which rapidly volatilizes
the components in a
Mass Spectrometer, HRGC-MS
liquid sample

temperature can be controled

during the separation
 How does gas chromatography work?
1. After injection into the heated sample ports 
the chemical components of the sample mixture
are vaporized, mixed with gaseous mobile
phase
2. The inert gaseous mobile phase transports the
sample molecules into the analytical column
without reacting with the sample
3. The sample components are separated in the
heated analytical column by their different
interactions with the stationary phase and
their relative vapor pressure  different KH
4. The outlet of the column is inserted into the
detector which responds to the chemical
components eluted at unique time, tR  signal
 chromatogram by the acquisition
software
 Sample Introduction techniques:
1. Direct Injection
Split/splitless injector
+ inject the sample in the minimum possible volume
 use a calibrated microsyringe to deliver a few
microliters sample through a rubber septum and into
the vaporization chamber located within a heater
block.
+ The injector block is heated at least 50 C above
the boiling point of the least volatile solute,
which ensures a rapid vaporization of the
sample’s components
+ When a capillary column is used  significantly
smaller injection volume is required to avoid
overloading the column with sample  use split
injection mode, but, in trace analysis  splitless
injection mode,
Split/splitless injector for capillary column
 2. Indirect Injection:
Head Space
+ Headspace is the gas phase or vapor portion of a
sample in a sealed chromatography vial.
+ Headspace injection is a technique involving the
indirect determination of volatile constituents in
liquid or solid samples by analysing the associated
vapour phase that is in thermodynamic
equilibrium with the sample in a closed system 
limited to high volatility compounds while the rest
of the sample is much less or non-volatile  no need
for extraction and cleanup step  Green Chemistry
Principle of direct + Used predominantly for the determination of volatile
headspace constituents present in trace concentrations
extraction.
+.
Optimization of
the Separation
in GC
 Factors Influence the Separation
 Vapor pressure:
 Boiling temperature is temperature when vapor pressure reaching 1 atm The lower the
boiling point  more volatile  more dissolved compound out of the stationary
phase into the gas phase  the higher the vapor pressure  shorter tR
 The temperature of the column does not have to be above the boiling point because every
compound has a non-zero vapor pressure at any given temperature, even solids.
 polarity of components versus the polarity of stationary phase: If the polarity of the
compound and stationary phase are similar  “likes dissolved likes”  longer tR.
KH is temperature dependent, and also depends on the chemical nature of the
stationary phase
 Column temperature: An excessively high column temperature  all components mainly stay
in the gas phase  very short tR, Lower column temperature  components able to interact
with the stationary phase  better separation
 Carrier gas flow rate: A high flow rate  shorter tR, but a poor separation

High temperatures and high flow rates decrease the retention time, but also
deteriorate the quality of the separation
Fundamental Equation to Control the
Separation (see 1st lecture)
 Optimize Resolution by Column Efficiency
 Columns is the heart of the GC system separation of analytes
 Column Type: classified as packed or capillary columns
Column Type Packed Column Capillary Column
Modern technology. Today most GC
History First type of GC column used applications are developed using
capillary columns

Packed with silica particles Not packed with particulate material. Made
Composition onto which the stationary of chemically treated silica covered with
phase is coated. thin, uniform liquid phase films.

Efficiency, N Low High

Outside diameter 2-4 mm 0.4 mm
Column length, L 2-4 meters 15-60 meters
Advantages Lower cost, larger samples Faster, better for complex mixtures
 Capillary column
Four parameters that must be specified in a capillary column:
1. The stationary phase: will determine the final resolution obtained and will influence other
selection parameters. Changing the stationary phase is the most powerful way to alter
selectivity  in GC analysis.
2. The column length is related to the overall efficiency (N= L/H) of the column and to overall
analysis time. A longer column L  will increase N  better peak efficiency and quality of
the separation, but it will also increase analysis time.
3. The column internal diameter (ID) By decreasing the column internal diameter, lower
mobile phase velocity  column efficiency >>(and therefore resolution) and column
capacity  better separations, but column overload and peak broadening may become
an issue.
4. The sample capacity of the column depend on film thickness. A shorter run time and
higher resolution can be achieved using thin films, however these films offer lower
capacity.
 Three Principal Capillary columns types

 wall-coated open tubular column (WCOT) typically 0.25 nm thick stationary phase is
coated
 porous-layer open tubular column (PLOT), porous solid support : alumina, silica gel,
and molecular sieves
 support-coated open tubular column (SCOT) is a PLOT column that includes a liquid
stationary phase.
 Efficiency Optimization

+ Van Deemter equation (1st lecture sl. No.

32-37)
Empty
 Packed column (1-5 m):
H = A + B/u + Cu
WCOT  Open tubular column WCOT, SCOT,
PLOT (10 – 100 m):
 no Eddy diffusion  lower H 
separations are more efficient
SCOT
H = B/u + Cu
 No back pressure  length >>

SRI NOEGROHATI, FAR USD 17

 1. Stationary phases  Likes dissolves Likes
trade temperature representative
stationary phase polarity
name limit (oC) applications
low-boiling aliphatics
squalane nonpolar Squalane 150
hydrocarbons
amides, fatty acid methyl
Apezion L nonpolar Apezion L 300
esters, terpenoids
alkaloids, amino acid
slightly
polydimethyl siloxane SE-30 300–350 derivatives, drugs, pesticides,
polar
phenols, steroids
phenylmethyl alkaloids, drugs, pesticides,
moderately
polysiloxane (50% OV-17 375 polyaromatic hydrocarbons,
polar
phenyl, 50% methyl) polychlorinated biphenyls
trifluoropropylmethyl alkaloids, amino acid
polysiloxane (50% moderately derivatives, drugs,
OV-210 275
trifluoropropyl, 50% polar halogenated compounds,
methyl) ketones
cyanopropylphenylme
thyl polysiloxane 50%
polar OV-225 275 nitriles, pesticides, steroids
cyanopropyl, 50%
phenylmethyl)
Carbowax aldehydes, esters, ethers,
polyethylene glycol polar 225
20M phenols
 2. The length  N=L/H

 various lengths depending on the inner diameter.

 Longer columns  Higher N  Higher Rs but also increases analysis time 
use only for very complex samples which demand the utmost in separation
power.
3. Internal Diameter  Mobile phase velocity
4. Film Thickness  mass transfer non equilibrium (C)
 Phase Ratio 
 dc is column diameter (mm) and df is film thickness (mm)
 A column with high phase ratio represents large column volume-to-stationary-
phase volume  small k’ (nS/nM , nM>nS)  < tR
 Carrier Gas Type and Linear Velocity
 carrier gas choice and linear velocity significantly affect column separation
efficiency (N)
 since there is no Eddy diffusion  In capilary GC  lower H  Higher N
 higher Rs
 Nitrogen provides the best efficiency, but small changes in linear velocity 
large negative changes in efficiency.

Hydrogen : 40-50 cm/sec

Helium : 25-35 cm/sec
Nitrogen : 10-15 cm/sec
 Detectors
 The detector senses a physicochemical property of the analyte  provides a
response  amplified  converted into an electronic signal to produce a
chromatogram.
 Detectors that exhibit an enhanced response to certain analyte types are
known as "Selective Detectors“:
 During the last 10 years MASS SPECTROMETER has become a standard
detector that allows specific detection and lower detection limits 
 does not require the exhaustive separation of all components present in
the sample
 provides most information in qualitative identification and quantitative
analysis with only micrograms of sample.
Common Detectors

• Specific and sensitive

 Mass spect Det. Flame Ionization Det.

+ The effluent from the column enters the mass + The effuent from the column mixes with H
spectrometer’s ion source in a manner that and is burned in the presence of excess air.
eliminates the majority of the carrier gas. In the Combustion produces a flame that contains
ionization chamber the remaining molecules—a electrons and the cation CHO Applying a
mixture of carrier gas, solvent, and solutes— potential between the flame’s tip and the
undergo ionization and fragmentation. The mass collector gives a current that is proportional
spectrometer’s mass analyzer separates the ions to the concentration of cations in the flame.
by their mass-to-charge ratio and a detector counts
the ions and displays the mass spectrum.
Pharmaceutical Gas Chromatography
Mass Spectrometers
+ Via the GC/MS method, a gaseous mobile phase transports the volatile compounds
molecules through the gas chromatographer and elutes into the mass spectrometer, most
commonly via electron impact ionization.
+ In the pharmaceutical industry, GC-MS is used in R&D, production, and quality control/
quality assurance.
+ GC-MS is considered a high-level benchmark for drug substance identification
because it can perform a 100% specific test, to identify a particular substance.
+ In medicinal chemistry and pharmaceutical biotechnology, GC-MS is used in
synthesis and characterization of compounds.
+ Gas Chromatography Mass Spectrometers are used to identify impurities in active
pharmaceutical ingredients.
 METHOD DEVELOPMENT GOALS PLAN

The goal: to obtain adequate separation in reasonable time.

The procedure should be RUGGED.
STEPS:
1. Determine whether it is qualitative / quantitative analysis
2. Select a method for sample preparation (sample clean up: Solid phase extraction,
liquid-liquid extraction)
3. Choice of the right detector (sensitivity range, destructive/ non-destructive/
responsiveness to analytes).
4. Choice of column (diameter, length, particle size, thickness/type of st. phase)
5. Choice of injection technique (in GC: split/splitless/on column.
6. Method Validation
 Advantages
 GC is a simple, rapid, quantitative and easily automated technique
 GC is therefore preferred to HPLC for gases, most low-boiling samples, and
many higher boiling samples that are thermally stable under the conditions of
separation.
 GC also has available several very sensitive and/or element-specific
detectors that permit considerably lower detection limits.
 GC is widely used for the analyses of raw materials, solvents, intermediate
products and organic pollutants in air, water, and finished products.