CHROMATOGRAPHY

Preparative vs Resolution vs Speed

 Gas Chromatography
Achieve separation by using suitable;

 Columns with proper stationary phase, diameter of

column, stationary phase loading, column length.

 Injection modes/ oven temperatures to optimize the of

loading and separation of the sample mixture on the
column.

 Temperature (or pressure) programs for the column

 Detector that is suitable for the analyte(s)of interest

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 Instrumentation

Instruments;
 Carrier Gas
 Flow regulators and meters
 Sample injection system
 Columns & ovens
 Detectors

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Schematic Diagram of Gas
Chromatograph

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 H2 inlet
(detector) N2 inlet
Air inlet
(make-up gas)
(detector)

He inlet
(carrier gas)
 Process Flow Schematic

Detector (flame
Sample injection ionization
detector or FID)
Carrier gas
Air
(nitrogen or
helium) Hydrogen

Long Column (30 m)

 Carrier Gas (mobile phase)
Requirements:
 It should be inert and available at low cost
High purity
Easily available
Less risk of explosion or fire hazards
Pressure:
-Inlet  10 to 50 psi
-packed column  25 to 150 mL/min.
- capillary column  1 to 25 mL/min.
 Sample injection port
 Calibrated micro syringes are used to inject liquid
sample
 Purge: volatile components are removed from
sample by gentle heating
 Rubber or silicone diaphragm(septum)
 Sample port T: over 50°C than column
 Packed C: sample sizes,1 to 20 μL
 Capillary C: 10 to 30 μL
splitter is used to deliver a fraction of
injection (1:50 to 1:500)
 Avoid over loading
 Slow injection & oversized samples cause band
spreading & poor resolution
Split Injection:
 Injection is split, with only a fraction of
the sample (usually 1% - 20%) actually
making it to the column

 The most common method of injecting

samples onto small diameter, open-tubular
columns.
Even for injections 20 μL, only a
fraction (adjustable) makes it on
to the column

 Not for analytes with a wide range of

boiling points
some may be swept out the split
vent before they are volatilized
Splitless Injection:
 Sample - vaporized in the injector and ALL of
the sample is swept onto the column by the
carrier gas

 Relatively small samples (≤10 μL)

 Sample spends a large amount of time in the

injector

 Best for trace (1 -100 ppm range)

concentrations of high boiling point analytes in
low boiling point solvents
extra time in the injector helps
volatilize the analytes.
 On-Column Injection:
 used widely in packed-column GC, less
in capillary GC

 sample - deposited directly on the

column

 Good for thermally unstable

compounds

 Good for quantitative analysis at low

concentrations

 entire sample reaches the detector

Smaller injections (Capillary GC)
 Column Configurations
 Two types of columns are used in gas
chromatography, packed and open tubular
or capillary.

 Packed column length from less than 2 m to

5m

 Capillary columns from few m to 100 m

 They are constructed of stainless steel, glass,

fused silica, or Teflon.
 Column
Types of columns
1.packed columns
2. Open tubular or capillary.

Packed column-3m Capillary column- 30m

PLOT
 Column ovens
 Column temperature is very important in GC

 The column is ordinarily housed in a thermostatic oven.

 they are usually formed as coils having diameters of 10 to 30

cm.

 The optimum column temperature depends upon the boiling

point of the sample and the degree of separation required.

 Roughly, a temperature equal to or slightly above the average

boiling point of a sample results in a reasonable elution time
(2 to 30 min).
Length vs. resolution
Diameter vs. resolution
Stationary phase thickness vs.
resolution
 Sample capacity vs. resolution
Sample capacity: the amount of sample that can be
injected onto a column without overloading. Often
expressed as grams of sample per gram of packing.

Overloading is defined as the point at which the sample

mass injected makes the column efficiency decrease by
10% from its normal value; sometimes called sample
loading.

packed (preparative, larger capacity, low resolution)

capillary (analytical, smaller capacity, high resolution)

 Capillary column

Pack column
 Column Temperature
Temperature too high, causes co-elution: poor resolution but faster separation
Temperature too low, longer elution times: compromise temperature or program
 The Stationary Phase

requirements are:

Low vapor pressure

Thermal stability
Low viscosity (for fast mass transfer)
High selectivity for compounds of interest
 Detectors
Detect the difference between a pure carrier gas & eluted compound

 Ideal detector:
 High sensitivity to even small concentration
 linearity, ie, less response to low concentration &proportional
response to high concentration
 Large linear dynamic range
 Compatible with the column, carrier gas, solvent, etc.
 Useful at a range of temperatures
 Good stability and reproducibility
 Rapid response time
 Easy to use
 Stable, Predictable response
 Inexpensive
 operation from RT to 400 oC
 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 Phosphoroes Detector(NPD)
6. Photo Ionization Detector(PID)
7. Flame Photometric detector(FPD)
8. Electrolytic conductivity detector (Hall detector)
9. Absolute Mass Detector(AMD)
10.Thermionic Detector(TD)
 Flame Ionization Detector (FID)
Most widely used, Air-H2 flame
Number of ions depends on
number of reduced (methylene)
carbons in molecule
The positive ions will be attracted to the
cylindrical cathode.
Negative ions and electrons will be attracted to
the jet anode.
Organic compounds  Produces ions and
electrons  pyrolyzed(temp of flame)  burner
tip and electrode
Ions &electrons move to ward the collector
less sensitive for non-hydrocarbon groups
 Insensitive to noncombustible gases(CO2, SO2,
NO2 and H2O)
Insensitive to functional group Adv: High sensitivity, low noise ,wide
(carbonyl, alcohol, halogen and amine) linear range, easy to use, fast response
D.Adv: Destroy the sample
 Thermal Conductivity Detector (TCD)
Element (platinum, gold or tungsten wire) is
electrically heated at constant power
– Temperature depends on thermal
conductivity (He & H)of surrounding gas.
Hydrogen and helium have higher thermal
conductivity and carrier gas provide best Thermal conductivity detector
sensitivity cell

Six times greater than the Organic compounds

Poorer sensitivity than FID, but more universal
Advantages: simplicity, large range,
inexpensive,linearity is excelent.
organic & inorganic species
DA: low sensitivity ng/mL
Arrangement of the twin detectors
 Electron Capture Detectors (ECD)
The sample elute from a column is passed over a
radioactive β-emitter(nickel-63)
 Selectively to halogen-containing organic sample
like pesticides and, polychlorinated biphenyls
Ni-63: radioactive β-emitter-- electron --ionization
of carrier gas (N2)
High electronegative group (halogen, peroxide,
quinones and nitro group) in the sample capture
the electron
Highly selective and sensitive, nondestructive
Insensitive to amines, alcohols and Hydrocarbons
AD: High sensitivity, analyse the polyhalogenated
organic compounds
Small linear range
 Thermionic detector (nitrogen phosphorus
detector)
 N or P containing organic compounds
 phosphorus atom is approximately ten times
greater than to a nitrogen atom and 104 to 106 larger
than a carbon atom.
 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.
 Column effluant + H2 +air(hot gas)  electrically
hearted Rb2SiO4 (rubidium silicate) bead at 180 V 
plasma (600 – 800°C )  ions  to determine
compounds
useful for detecting and determining the many
phosphorus-containing pesticides.
 Photo Ionization Detector (PID)
 UV light
(10.2 eV H2 or 11.7 eV Ar lamp)
photoionization of molecular 
current to flow between based
electrodes

 Most sensitive for Aromatic and S, P

easily photoionized molecules

 Linear range is high

 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
Additional peaks are obtained…..impurities
are present….compound is not pure
 Quantitative analysis:
Direct comparison method:
comparing the area of the peak, peak height, width of peak.

Calibration curves:
standards of varying concentration are used determine peak areas

Internal standard method:

A known concentration of the internal standard is added separately
to the standard solution
The peak area ratio of sample and internal standard….
unknown concentration is easily determined
 Elemental analysis

 Determination of C,H ,O ,S and N .

 Determination of mixture of drugs

 Isolation and identification of drugs

 Isolation and identification of mixture of

components(amino acids ,plant extracts ,volatile
oils)
 Column Bleed
At high temperature stationary phase may vaporize into the carrier
gas, resulting in column “bleed:
 GC - Derivatization
Why is chemical derivatization needed?

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.
 GC - Derivatization
Chemical 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

Derivatizing Reagents: Common derivatization methods can be

classified into 4 groups depending on the type of reaction
applied:
 Silylation
 Acylation
 Alkylation
 Esterification
 Multidimensional GC
The use of two or more columns to resolve a sample.
• Normal column chromatography frequently fails to
resolve all components in a complex mixture.
 Chromatographic analysis
Best - single column single analysis
Second best - two separate assays using
different columns or conditions
Last resort - multi dimensional GC - when
Sample is limited
Time is limited
Equipment is limited
 Approaches
Enrichment
• Used to increase amounts of trace components
 Approaches
Heart cutting

 Grabs an unresolved portion of a sample for improved separation

 Approaches
Back flushing

 Reverse column flow to drive off highly retained components

 Chiral Separations
 Chromatography with a chiral (optically active) stationary phase
is one of the few ways to separate enantiomers.
Eg: -Cyclodextrin a cyclic sugar made of seven glucose molecules.
Programmed temperature (120 oC – 200 oC) chiral separation on a 0.25 mm x 25 m open tubular column with a
stationary phase containing 10 wt% fully methylated -Cyclodextrin chemically bonded to dimethyl polysiloxane.
Helium is the most common carrier gas and is compatible with most detectors.
For a flame ionization detector, N2 gives a lower detection limit than He.
H2, He, and N2 give essentially the same optimal plate height (0.3 mm) at
significantly different flow rates.
Fastest separations can be achieved with H2 as carrier gas, and H2 can be run
much faster than its optimal velocity with little penalty in resolution.
There are drawbacks to using H2. It can catalytically react with unsaturated compounds
on metal surfaces, and it cannot be used with a mass spectrometric detector, because H2
breaks down vacuum pump oil in the detector.

Separation of two polyaromatic hydrocarbons on a wall-coated open tubular column with different carrier gases.
Resolution, R, increases and analysis time decreases as we change from N2 to He to H2 carrier gas.