PHARMACEUTICAL ANALYSIS I

CHROMATOGRAPHY IV: GAS CHROMATOGRAPHY

a. Instrumentation/Layout
b. Principles behind Gas Liquid and Gas Solid Chromatography
c. Column types
d. Stationary phase
e. Types of detectors
f. Qualitative and quantitative analysis
g. Applications and examples
h. Derivation of samples
DR. CHIN
Instrumentation/Layout
Filters/Traps
A
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Column
Data system
Syringe/Sampler
Inlets
Detectors
Regulators
H
RESET
gas system
inlet
column
detector
data system
Schematic Diagram of Gas Chromatography
Gas Chromatography
Stationary Phase:
Gas-solid chromatography : solid, underivatized support
Gas-liquid chromatography: liquid-coated support
Bonded-phase gas chromatography chemically-derivatized support
Carrier Gas
The mobile phase gas is called as carrier gas and must be
inert, pure and dry.
E.g. nitrogen, helium, argon, hydrogen and carbon dioxide.
The Oxygen is not used as carrier gas because the presence
of oxygen may induce oxidation in the stationary phase and
lead to a loss of column performance.
The carrier gas flows through the instrument from a
pressurized cylinder.
The carrier gas systems also contains a molecular sieve to
remove water and other impurities.
Carrier Gas: Flow Rate
Flow rate is controlled by 2 stage regulator on the gas
cylinder and additional controls within the instrument.
Inlet pressures usually range from 10 to 50 psi above
room pressure, which leads to flow rates of
25 to 150 mL/min with packed columns
1 to 25 mL/min with open-tubular columns
If the inlet pressure remains constant, flow rates will
be constant.
Flow rates can be established by soap-bubble flow
meter, located at the head of column
Sample Injection System
It is important because column efficiency depends on
this system.
Column efficiency requires sample of suitable size and
sample has to be introduced as a plug of vapor slow
injection of oversized samples causes band spreading
and poor resolution
Method of sample injection:
Microflash vaporizer direct injector (Commonly used)
Rotary sample valve (Reproducible results)
Solid samples: sealed into thin-walled vials that can be
inserted at the head of column and punctured or
crushed from the outside
Sample Injection
Sample is injected using a syringe
into a flowing stream of hot mobile
phase
High temperature (at least 50
o
C
above boiling point of sample)
causes vaporization of sample
Introduces a narrow plug of
sample vapor onto the column
Various designs
For packed columns, inject 1 to 5
L of sample
For capillary columns, a split valve
is used to introduce a small
fraction of sample onto column
A flash vaporization injector
Introduced as a plug of vapor
with suitable size
Slow injection or oversized
samples cause band spreading
and poor resolution
Micro syringes
Injection ports
microsyringe
Gastightsyringe
Sample Injection System
Split/Splitless Injector
Splitless Injection
(where the split vent is closed)
attempts to transfer all of the
sample to the column and is
used for trace analysis.
Split Mode
only a small portion (maybe 1-
10% of the sample moves into
the column, and the rest is sent
to waste. This is used when the
analytes are in high
concentration and would
overload the column.
Sample is injected through the
septum with a syringe.
Sample injection system
Column efficiency required that samples be of suitable
size and introduced as a plug of vapor.
Slow injection of oversized sample cause band
broadening and poor resolution.
Injector changes sample to gas and reproducibly
introduces resulting gaseous sample into column.
Types of injector system are dependent upon physical
state of sample.
For gaseous sample
Rotary Valve is preferred and that is same as in HPLC but
here sample loop is larger.
In position 1 carrier gas flows directly into column and
valve is in that position, the sample is flushed and filled
with gaseous sample.
Upon switching value to inject position 2, carrier gas
sweeps through sample loop and flushes sample into
column.
For liquid sample
A gas syringe is used.
Sample is injected with 1-.5- or 10-L graduated syringe
through septum into heated flash evaporation injector.
The sample is rapidly vaporized because injectors
temperature is maintained above B.P. of highest boiling
component in sample.
Carrier gas flushes vaporized sample from injector and
into column.
Typical liquid sample volume used with packed column
varies from 0.1 5L and injector temperature is held
at least 10C above the column temperature.
For solid sample
A solid sample is more difficult to convert to gas.
Sometimes it can be chemically converted to volatile
derivative.
E.g. Fatty acid is converted to volatile methyl ester
with BF
3
or BCl
3
in methanolic solution.
In some cases pyrolysis equipment is attached to
GC and used to vaporize solid sample.
In pyrolysis apparatus sample is placed and heated
sufficiently (1000C) to vaporize the sample or to
form volatile sample decomposition product and
volume sample swept into GC with carrier gas.
Columns
Length: <2 m to >50 m
They are made of:
Stainless steel
Glass
Fused silica
Teflon
In order to fit into an oven for thermostating, they are formed as
coils having diameters of 10 to 30 cm.
Types of analytical columns generally used in GC are:
Packed columns
Less efficiency compared to capillary columns
Capillary columns
More efficiency compared to packed columns
i. Wall coated open tubular columns
ii. Support coated open tubular columns
Columns
Packed Column
Most packed columns are 1.5 - 10m in length and have an
internal diameter of 2 - 4mm.
They have greater sample capacity compared to open tubular
columns but generates broader peaks, longer retention time
and lower resolutions.
Useful for preparative work.
Packed columns contain a finely divided, inert, solid support
material (commonly based on diatomaceous earth ) coated
with liquid stationary phase.
Most widely used support is Kieselguhr.
The support holds stationary phase in place so that as large
a surface area as possible is exposed to the carrier gas.
Usual particle size supports are 60 80 # (260 170 m) or
80 100 # (170-149m)
Capillary Columns
Capillary columns have an internal diameter of a few
tenths of a millimeter.
Increased length leads to longer separation times.
Stationary phase thickness and column diameter
increases leads to increased sample capacity and can
provide increased resolution.
Thick stationary phases bleed more and contaminate
MS detection system.
Capillary Columns
Advantages:
Highly efficient
Disadvantages:
Small sample capacity
Fragileness of columns
Mechanical problems associated with sample
introduction and
connection of the column to the detector
Difficulties in coating the column reproducibility
Short life times of poorly prepared columns
Tendencies of columns to clog
High cost
Liquid phase
Solid support
coated with liquid
phase
Porous Adsorbent
Porous Layer Open
Tubular
(PLOT)
Wall-coated Open
Tubular
(WCOT)
Support-coated Open
Tubular
(SCOT)
Capillary Columns: Open Tubular Columns
Wall Coated Open Tubular Column (WCOT)
Simply capillary tubes coated with a thin layer of stationary
phase
Made up of
Stainless steel
Aluminium
Copper
Plastic
Glass
The glass was etched with gaseous hydrochloric acid, strong
aqueous hydrochloric acid, or potassium hydrogen fluoride
to give a rough surface, which bonded to stationary phase
more tightly.
Fused Silica Open Tubular Column (FSOT)
These have much thinner walls than their glass counterparts.
The tubes are given added strength by an outside protective
polyimide coating, which is applied as the capillary tubing is
being drawn.
The resulting columns are quite flexible and can be bent into
coils having diameters of few inches.
Advantages over glass columns:
Physical strength, much lower reactivity toward sample
components and flexibility
Support Coated Open Tubular Column (SCOT)
Most widely used SCOT have inside diameters of 320
and 260 m
High resolution SCOT have inside diameters of 200 and
150 m
It needs sample splitter and high sensitive detector
Megabore columns of internal diameter 530 m
Less efficient than smaller diameter capillary columns
but more efficient than packed columns
Type of Column
FSOT WCOT SCOT Packed
Length (m) 10-100 10-100 10-100 1-6
ID (mm) 0.1-0.3, 0.53* 0.25-0.75 0.5 2-4
Efficiency (Plate/m) 2000-4000 1000-4000 600-1200 500-1000
Sample size (ng) 10-75 10-1000 10-1000 10-10
6
Relative pressure Low Low Low High
Relative speed Fast Fast Fast Slow
Flexible Yes No No No
Chemical inertness Best Poor Poorer Poorest
Comparison of Capillary Columns
Capillary Column: Stationary Phase
HO-CH
2
-CH
2
-(O-CH
2
-CH
2
)
n
-OH
polyethylene glycol
Effect of Temperature on GC
Column temperature is an important variable.
It must be controlled to a few tenths of degree
for precise work.
So, the column is housed in thermostated oven.
Optimum column temp. depends upon the b.p.
of the sample and the degree of separation
required.
A temp. equal to or a slightly above the average
b.p. of a sample results in a reasonable elution
time (2 to 30 min).
For samples with a broad boiling range,
temperature programming is employed,
whereby the column temperature is increased
either continuously or in steps as the separation
proceeds.
Optimum resolution is associated with minimal
temperature.
But lower temperature requires more time to
complete analysis, as the elution time of analytes
increase.
Types of detectors
1. Thermal Conductivity Detector(TCD)
2. Flame Ionization Detector(FID)
3. Atomic Emission Detector(AED)
4. Electron Capture Detector(ECD)
5. Nitrogen Phosphorous Detector(NPD)
6. Photo Ionization Detector(PID)
7. Flame Photometric detector(FPD)
8. Electrolytic conductivity detector (ELCD)(Hall
detector)
9. Absolute Mass Detector(AMD)
10. Infrared Detector (IRD)
Characteristics of the Ideal Detector
Adequate sensitivity
Good stability and reproducibility
A linear response over several orders of magnitude
solutes
A temperature range from room temperature to 400
o
C
A short response time that is independent of flow rate
High reliability and ease of use
Highly predictable and selective response toward one
or more classes of solutes
Nondestructive of sample
Thermal Conductivity Detector (TCD)
Principle: Change in thermal conductivity (cooling ability) of the gas
stream brought about by the presence of analyte molecules
It is also called as Katharometer
Helium and hydrogen are preferred carrier gases.
The thermal conductivities of helium and hydrogen are 6 10 times
greater than those of most organic compounds.
So, in the presence of even small amounts of organic materials, a
relatively large decrease in the thermal conductivity of the column
effluent takes place
Consequently, the detector undergoes a marked rise in temp.
The conductivities of other carrier gases are closely resemble those
of organic constituents.
Thermal Conductivity Detector
Advantages:
Simple, Universal, large
dynamic range, response to
both organic and inorganic
species, non destructive
Disadvantages:
Low sensitivity
Not recommended with
capillary columns because
only very small amounts of
samples can be introduced,
so such small amounts cant
be detected.
Thermal Conductivity Detector
A universal detector
Flame Ionization Detector (FID)
Most widely used detector.
The effluent from the column is mixed with H
2
and air and then
ignited electrically.
Most organic compounds when pyrolyzed at the temperature of
a hydrogen/air flame, produce ions and electrons that can
conduct electricity through the flame, which is measured.
No. of produced ions is proportional to the no. of reduced
carbon atoms in the flame.
It is mass-sensitive rather than concentration-sensitive because it
responds to the no. of carbon atoms entering the detector per
unit of time
It can be useful for the detection of pollutants in natural water
samples, because it is insensitive to water.
Flame Ionization Detector
For most organic compounds
Flame Ionization Detector (FID)
Advantages: High sensitivity, a
large linear response range,
low noise, rugged and easy to
use.
Disadvantage: Destructive
Less sensitive to non
hydrocarbon groups.
Insensitive to H
2
O,CO
2
,SO
2
Electron Capture Detector (ECD)
It is choice of detectors for environmental samples because this
detector selectively detects halogen containing compounds, such
as pesticides and polychlorinated biphenyls.
Working:
The effluent from the column is passed over a radioactive
emitter (Nickel63)
An electron from the emitter causes ionization of carrier gas
(N
2
) and production of a burst of electrons.
The current decreases markedly, in the presence of organic
molecules
It is highly sensitive to molecules containing electronegative
functional groups such as halogens, peroxides, quinones and nitro
groups.
Electron Capture Detector (ECD)
It is insensitive to amines,
alcohols and hydrocarbons.
Important application of this
detector is detection and
determination of chlorinated
insecticides.
Advantages: Highly selective
and sensitive, nondestructive
Disadvantage: Low linear
response range.
Atomic Emission Detector (AED)
The eluent is introduced into a microwave-energized helium
plasma that is coupled to a diode array optical emission
spectrometer.
Molecules are energized by a plasma source.
Then molecules are separated into excited atoms.
As electrons return to their stable state, they emit light, which is
element specific.
These spectra are then observed with diode array spectrometer,
capable of detecting radiations from about 170 to 780 nm
It is relatively new detector and yields elemental information
about the separated components in a mixture.
The sample is destroyed
It is specific to P and S, can measure upto ppb.
Six elements detect simultaneously .
Determine the hetero atoms(H,P,S,O),silicon , heavy metals(Pb , Hg),tin,
arsenic ,copper ,iron.
Nitrogen phosphorous detector (NPD)
The column effluent is mixed with
hydrogen, passes through the flame tip
assembly, and is ignited.
The hot gas then flows around an
electrically heated rubidium silicate bead.
Then large no. of ions from phosphorus or
nitrogen containing compounds are
produced.
It results in the production of increased
current.
This is a detector of choice for analysis of
organophosphorus pesticides and
pharmaceuticals.
Compared with the FID , the thermionic
detector is approximately 500 times more
sensitive to phosphorus-containing
compounds and 50 times more sensitive
to nitrogen bearing species.
Flame photometric detector (FPD)
It is a selective detector that is
primarily responsive to
compounds containing sulfur
and phosphorus.
It is widely used in the analysis
of air and water pollutants,
pesticides and coal
hydrogenation products.
In this detector, the eluent is
passed into a lowtemperature
hydrogen/air flame, which
converts part of phosphorus to
HPO and Sulfur to S
2
.
Suitable filters are employed to
isolate these bands, and their
intensity is recorded
photometrically.
filters
photomultiplier
H2 + air
Column effluent
Electrolytic Conductivity Detector (ELCD)
Halogens, sulfur, or nitrogen compounds mix with a
reaction gas in a reaction tube.
The products are mixed with a suitable liquid, which
produces a conductive solution, and the change in
conductivity is monitored.
Molecules are ionized by
excitation with photons from
an UV lamp.
The charged particles are
then collected, producing
current.
This detector is used for
analyses of aromatic and UV
ionized compounds.
PHOTO IONIZATION DETECTOR(PID)
Mass Selective Detector (MSD)
Molecules are bombarded with electrons, producing ion
fragments that pass into the mass filter
The ions are filtered based on their mass/charge ratio.
It gives qualitative identification of components by matching
the compounds mass spectrum with spectra included in
libraries.
Disadvantages:
Main difficulty is changing from highly pressurized gas
mixture to vacuum containing isolated components.
The sample is destroyed.
Infrared Detector (IRD)
Molecules absorb infrared energy, the frequencies of which
are characteristic of the bonds within that molecule.
This detector is a powerful tool for distinguishing isomers.
It gives qualitative identification of components by matching
the compounds IR spectrum with spectra included in
libraries.
Non destructive
Detectors for GC
Gas-Solid Chromatography (GSC)
It is based upon adsorption of gaseous substances on solid
surfaces.
Distribution coefficients are larger than those for Gas-liquid
chromatography
It is useful for the separation of air, hydrogen sulfide, carbon
disulfide, nitrogen oxides, carbon monoxide, carbon dioxide and the
rare gases.
It is performed with both packed and open tubular columns
Open tubular columns are called porous layer open tubular
columns (PLOT)
A thin layer of adsrobent is affixed to the inner walls of capillary.
Two types of adsorbents:
Molecular sieves
Porous polymers
Molecular Sieves
The sieves are classified according to the maximum
diameter of molecules that can enter the pores.
Molecules smaller than these dimensions penetrate
into the interior of the particles where adsorption
takes place.
For such molecules the surface area available is
enormous when compared with the area available to
larger molecules.
Thus molecular sieves can be used to separate small
molecules from large.
Porous Polymers
Porous polymer beads of uniform size are
manufactured from styrene cross-linked with
divinylbenzene.
The pore size of these beads is uniform and is
controlled by the amount of cross-linking.
These will be used to separate gaseous polar species
such as hydrogen sulfide, oxdies of nitrogen, water,
carbon dioxide, methanol and vinyl chloride.
Applications and Examples
Separation and analysis of volatile organic compounds
(gases and liquids)
Testing purity of compounds
Compound identification
Isolation of pure compounds (microscale work) from
complex mixtures
Determine relative amounts of components in mixture
Determination of partition coefficients and absorption
isotherms.
Qualitative analysis
Retention time data should be useful for identification
of mixtures
Comparing the retention time of the sample as well as
the standard
Checking the purity of a compound: compare the
standard and sample chromatograms
If additional peaks are obtained, impurities are present
and hence the compound is not pure.
From the percentage area of the peaks obtained, the
percentage purity can also be reported.
Quantitative analysis
Direct comparison method:
comparing the area of the peak, peak height, width
of peak.
Calibration curves:
standards of varying concentration are used to
determine peak areas .
A graph of peak area vs concentration of the drug
is plotted.
From the peak area of unknown sample, by
intrapolation, the concentration of the sample can
be determined.
Advantage:The errors, if any are minimised
Quantitative Analysis: Internal Standard
Method
In this method, a compound whose retention time is near to that
of analyte is used as internal standard.
A known concentration of the internal standard is added
separately to the standard solution and sample solution whose
concentration is not known.
The peak area ratio of sample and internal standard.unknown
concentration is easily determined.
A graph of ratio of peak areas vs concentration of the standard is
plotted.
From the peak area ratio of sample and internal standard, by
intrapolation, the concentration of the unknown solution is
determined from the graph.
The ratio of analyte peak area to internal standard peak area will
remain unaffected by slight variations in injecting volumes, pipetting
and chromatographic conditions.
Derivatisations
GC is best for separation of volatile compounds which are
thermally stable.
Not always applicable for compounds of high molecular weight
or containing polar functional groups. These groups are difficult
to analyze by GC either because they are not sufficiently
volatile, tail badly, are too strongly attracted to the stationary
phase, thermally unstable or even decomposed.
It is a technique of treatment of the sample to improve the
process of separation by column or detection by detector.
Common derivatization methods can be classified into 4
groups depending on the type of reaction applied:
Silylation
Acylation
Alkylation
Esterification
Precolumn Derivatisation
There are two types:
Precolumn derivatisation
Post column derivatisation
Precolumn derivatization prior to analysis is generally done to:
increase the volatility and decrease the polarity of compounds;
reduce thermal degradation of samples by increasing their
thermal stability;
increase detector response
Improve separation and reduce tailing
Example:
Carboxylic acids, sugars, phenols, alcohols, etc. can be converted
to less polar compounds by using reagents like BSA reagent (Bis
trimethy silyl acetamide reagent)
They can also be converted to acetyl derivative or triflouro
acetyl derivative.
Post column Derivatisation
To improve the response shown by detector .
The components may not be detected by detector
unless derivatisation is done.
Usually, this is online detection technique, where the
flow rate is neither stopped nor altered.
The components may be converted in such away
that their ionisation or affinity towards electrons is
increased.
Pretreatment of solid support:
solid support is to hold the stationary phase liquid as
a thin film.
OH
O
OH
OH
HO
CH
2
OH
1
2
3
4
5
6
+
Si
CH
3
CH
3
CH
3
5Cl
O-Si(CH
3
)
3
O
O-Si(CH
3
)
3
O-Si(CH
3
)
3
(CH
3
)
3
-Si-O
CH
2
O-Si(CH
3
)
3
1
2
3
4
5
6
5 HCl +
Derivatization of Glucose with Trimethylchlorosilane
Glucose Trimethylchlorosilane