GAS CHROMATOGRAPHY

Introduction

 Gas chromatography – It is a process of separating component(s)

from the given crude drug by using a gaseous mobile phase.
 It involves a sample being vaporized and injected onto the head of
the chromatographic column. The sample is transported through the
column by the flow of inert, gaseous mobile phase. The column itself
contains a liquid stationary phase which is adsorbed onto the surface
of an inert solid.
 Two major types
• Gas-solid chromatography
(stationary phase: solid)
• Gas-liquid chromatography
(stationary phase: immobilized liquid)
Advantages of Gas Chromatography

 The technique has strong separation power and even complex

mixture can be resolved into constituents

 The sensitivity of the method is quite high

 It gives good precision and accuracy

 The analysis is completed in a short time

 The cost of instrument is relatively low and its life is generally long

 The technique is relatively suitable for routine analysis

Components of Gas chromatography

 Carrier gas
- He (common), N2, H2, Argon
 Sample injection port
- micro syringe
 Columns
2-50 m coiled stainless steel/glass/Teflon
 Detectors
-Flame ionization (FID)
-Thermal conductivity (TCD)
-Electron capture (ECD)
-Nitrogen-phosphorus
-Flame photometric (FPD)
-Photo-ionization (PID)
schematic diagram of a gas chromatograph
Carrier gas

 The carrier gas must be chemically inert.

 Commonly used gases include nitrogen, helium, argon, and carbon

dioxide.

 The choice of carrier gas is often dependant upon the type of

detector which is used.

 The carrier gas system also contains a molecular sieve to remove

water and other impurities.
- P inlet 10-50 psig
-F=25-150 mL/min packed column
-F=1-25 mL/min open tubular column
Sample injection- Direct Injection

1)Direct injection :into

heated port (>T oven)
using micro syringe

- (i) 1-20 uL
packed column

-(ii) 10-3 uL
capillary column
 Sample injection- rotary sample valve with
sample loop

 Split injection: routine method

- 0.1-1 % sample to column
- remainder to waste
 Split less injection: all sample to column
- best for quantitative analysis
- only for trace analysis, low [sample]
On-column injection:
-for samples that decompose above boiling
Point ( no heated injection port)
-column at low temperature to condense
sample in narrow band
-heating of column starts chromatography
 Gas Chromatography - Columns

 There are two general types of column, packed and capillary (also known
as open tubular).
 Packed columns contain a finely divided, inert, solid support material
( diatomaceous earth) coated with liquid stationary phase. Most packed
columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm.
 Capillary columns have an internal diameter of a few tenths of a
millimeter. They can be one of two types; wall-coated open
tubular (WCOT) or support-coated open tubular (SCOT).
- Wall-coated columns consist of a capillary tube whose walls are
coated with liquid stationary phase. In support-coated columns, the inner
wall of the capillary is lined with a thin layer of support material such as
diatomaceous earth, onto which the stationary phase has been adsorbed.
- SCOT columns are generally less efficient than WCOT columns.
Both types of capillary column are more efficient than packed columns.
Gas Chromatography –
Common Stationary phases
G C - DETECTORS

 There are many detectors which can be used in gas chromatography.

 Different detectors will give different types of selectivity.
 Detectors can be grouped into concentration dependant
detectors and mass flow dependant detectors.
 The signal from a concentration dependant detector is related to the
concentration of solute in the detector, and does not usually destroy
the sample Dilution of with make-up gas will lower the detectors
response.
 Mass flow dependant detectors usually destroy the sample, and the
signal is related to the rate at which solute molecules enter the
detector. The response of a mass flow dependant detector is
unaffected by make-up gas
G C – IDEAL DETECTORS

 Sensitive (10-8-10-15 g solute/s)

 Operate at high T (0-400 °C)
 Stable and reproducible
 Linear response
 Wide dynamic range
 Fast response
 Simple (reliable)
 Nondestructive
 Uniform response to all analytes
Flame Ionization Detector (FID)

 It operates by the principle that by

change in conductivity of the flame as
the compound is burnt. The change in
conductivity of the flame does not arise
by simple ionization of the compound ,
it is partial or complete stripping of the
compound to give charged hydrogen-
deficient polymers or aggregates of
carbon of low ionization potential.

 Rugged; Sensitive (10-13 g/s) ; Wide dynamic range

(107) ;Signal depends on # C atoms in organic
analyte - mass sensitive; not concentration sensitive;
Weakly sensitive to carbonyl, amine, alcohol,
amine groups; Not sensitive to non-combustibles -
H2O, CO2, SO2, Nox; Destructive
Thermal Conductivity Detector (TCD)

 It is based upon the alteration of the

thermal conductivity of the carrier gas
in the presence of an organic
compound. The platinum wires are
heated electrically and assume
equilibrium conditions of temperature
and resistance when carrier gas alone
passes over them. They are mounted in
a whetstone bridge arrangement and
when a compound emerges, the
thermal conductivity of the gas
surrounding wire alters, and hence the
temperature and resistance of the wire
change with a concomitant out of
balance signal which is amplified and
recorded.
 Rugged ;Wide dynamic range
(105);Nondestructive; Insensitive
(10-8 g/s) - non-uniform
Electron Capture Detector (ECD)

 The ECD ionizes the carrier

gas by means of a radioactive
source. The potential across
two electrodes is adjusted to
collect all the ions and a steady
saturation current, is therefore,
recorded.

 Electrons from b-source ionize carrier

molecules capture electrons and
decrease current ; Simple and reliable ;
Sensitive (10-15 g/s) to electronegative
groups (halogens, peroxides) ;Largely
non-destructive ; Insensitive to amines,
alcohols and hydrocarbons ; Limited
dynamic range (102)
 Summary of common GC detectors

Detector Type Support gases Selectivity Detectability Dynamic range

Flame ionization (FID) Mass flow Hydrogen and air Most organic cpds. 100 pg 107

Thermal conductivity
Concentration Reference Universal 1 ng 107
(TCD)

Halides, nitrates,
nitriles, peroxides,
Electron capture (ECD) Concentration Make-up 50 fg 105
anhydrides,
organometallics

Nitrogen-phosphorus Mass flow Hydrogen and air Nitrogen, phosphorus 10 pg 106

Sulphur, phosphorus,
Flame photometric Hydrogen and air tin, boron, arsenic,
Mass flow 100 pg 103
(FPD) possibly oxygen germanium, selenium,
chromium

Aliphatics, aromatics,
ketones, esters,
aldehydes, amines,
Photo-ionization (PID) Concentration Make-up 2 pg 107
heterocyclics,
organosulphurs, some
organometallics
Summary of common GC detectors

 The effluent from the column is mixed with hydrogen and air, and
ignited.
 Organic compounds burning in the flame produce ions and electrons
which can conduct electricity through the flame.
 A large electrical potential is applied at the burner tip, and a
collector electrode is located above the flame. The current resulting
from the pyrolysis of any organic compounds is measured.
 FIDs are mass sensitive rather than concentration sensitive; this gives
the advantage that changes in mobile phase flow rate do not affect
the detector's response.
 The FID is a useful general detector for the analysis of organic
compounds; it has high sensitivity, a large linear response range, and
low noise. It is also robust and easy to use, but unfortunately, it
destroys the sample
Temperature Programming

 As column temperature raised, vapor pressure analyte increases,

eluted faster

 Raise column temperature during separation – temperature

programming - separates species with wide range of polarities or
vapor pressures
Evaluation

 HETP- It is the distance on the column in which equilibrium is

attained between the solute in the gas phase and the solute in liquid
phase. Larger the number of theretical plates/ smaller the HETP, the
more efficient the column is for separation.
HETP = Length of column/n ; Where n = number of theretical plates= 16 * x2/y2
 Retention Time: Time in minute from the point of injection to the
peak maximum.
 Retention Volume: (1) VR = tR ×F (retained) (2) VM = tM ×F (non-
retained)
 average volumetric flow rate (mL/min) F can be estimated by
measuring flow rate exiting the column using soap bubble meter
(some gases dissolving in soap solution)
 But measured VR and VM depend on
- pressure inside column
Applications of Gas Chromatography

 Qualitative Analysis – by comparing the retention time or volume of

the sample to the standard / by collecting the individual components
as they emerge from the chromatograph and subsequently identifying
these compounds by other method
 Quantitative Analysis- area under a single component elution peak is
proportional to the quantity of the detected component/response
factor of the detectors.

Volatile Oils, official monograph gives chromatography profile for

some drugs. E.g. to aid distinction between anise oil from star anise
and that from Pimpinelle anisum
Separation of fatty acids derived from fixed oils
Applications of Gas Chromatography

 Miscellaneous-analysis of foods like carbohydrates, proteins, lipids,

vitamins, steroids, drug and pesticides residues, trace elements

 Pollutants like formaldehyde, carbon monoxide, benzen, DDT etc

 Dairy product analysis- rancidity

 Separation and identification of volatile materials, plastics, natural

and synthetic polymers, paints, and microbiological samples

 Inorganic compound analysis