Gas Chromatography

Gas Chromatography
an analytical separations technique useful

for separating volatile organic compounds

consists of :
Flowing mobile phase (inert gas - Ar, Ne, N)
Injection port ( rubber septum - syringe injects
sample)
kept at a higher temperature than the boiling point

Principles
Separation due to differences in partitioning

behavior
selective retardation

Key Information

organic compounds separated due to

differences in their participating behavior

between the mobile gas phase and the
stationary phase in the column
in contrast to other types of chromatography,
the mobile phase does not interact with
molecules of the analyte; its only function is
to transport the analyte through the column

Gas Chromatography
Separation column containing stationary phase
since partitioning behavior independent of
temperature - kept in thermostat - controlled oven

Detector

Schematic of a gas
Chromatograph

The Beginning
concept of GC announced in 1941 by

Martin and Synge (also did liquid partition

chromatography)
10+ years later GC used experimentally
1955, first commercial apparatus for GC
appeared on the market

Today
estimate : 200, 000 gas chromatographs are

currently used through out the world.

30+ instrument manufactures
130 different models
cost 1,500 to 40,000 dollars
improvements: computers- automatic control
open tubular columns-separate a multitude of
analytes in relatively short times

Uses of Gas Chromatography

Determination of volatile compounds (gases

& liquids)
Determination of partition coefficients and
absorption isotherms
Isolating pure components from complex
mixtures

Instrumentation

Instrumentation
flowing mobile phase
injection port
separation column
detector

GC detectors
another powerpoint

Liquid Chromatography
much slower diffusion in liquid
as compared to gas

Liquid liquid extraction

repeated extraction is basis
for LC

Retardation of solutes in liquid

onto a solid phase

Elution chromatography
Increasing polarity of

Solvents mixed

pure solvents
hexane
ether
acetone
methanol
water
acetic acid

%hexane and %

methanol
miscible
can be mixed
continuously (solvent
programming)

Types of Liquid Chromatography

Liquid-solid: adsorption on solid

which is generally polar (silica gel,

alumina, magnesium silicates) or
reverse phase (cellulose, poly amides)
Ion exchange: specific interactions
with ionic species (change relative
strengths of acid or base)

Types of Liquid Chromatography

Liquid-liquid: partition between 2

bulk phases (one immobilized) is

highly selective
Liquid exclusion: molecular sieve
separates molecules on basis of ability
to diffuse into immobile support

Retardation based on size of

molecule as it diffuses into
porous solid

High Performance Liquid

Chromatography
Once called High Pressure Liquid
Chromatography

What is HPLC?
The most widely used analytical separations technique
Utilizes a liquid mobile phase to separate components

of mixture
uses high pressure to push solvent through the column
Popularity:
sensitivity
ready adaptability to accurate quantitative determination
suitability for separating nonvolatile species or
thermally fragile ones

HPLC is.
Popularity:
widespread applicability to substances that are of prime
interest to industry, to many fields of science, and to the
public
Ideally suited for separation and identification of

amino acids, proteins, nucleic acids, hydrocarbons,

carbohydrates, pharmaceuticals, pesticides, pigments,
antibiotics, steroids, and a variety of other inorganic
substances

History lesson
Early LC carried out in glass columns
diameters: 1-5 cm

lengths: 50-500 cm
Size of solid stationary phase
diameters: 150-200 m
Flow rates still low! Separation times long!
Eureka! Decrease particle size of packing causes increase in

column efficiency!
diameters 3-10 m
This technology required sophisticated instruments
new method called HPLC

Advantages to HPLC
Higher resolution and speed of analysis
HPLC columns can be reused without repacking or

regeneration
Greater reproducibility due to close control of the
parameters affecting the efficiency of separation
Easy automation of instrument operation and data
analysis
Adaptability to large-scale, preparative procedures

Advantages to HPLC
Advantages of HPLC are result of 2 major advances:
stationary supports with very small particle sizes and
large surface areas
appliance of high pressure to solvent flow

Schematic of liquid
chromatograph

LC column

LC injector

Types of HPLC
Liquid-solid (adsorption) chromatography
Liquid-liquid (partition) chromatography
Ion-exchange chromatography
Size exclusion chromatography

Partition Chromatography
Most widely used
Bonded-phase Chromatography
Silica Stationary Phase:

OH

OH
O

Si
Siloxanes:

O
Si
O

OH

Si

O
O

OH
O

Si

Si
CH 3

Si

R
CH3

R= C 8, C18

Partition Chromatography II
Reverse Phase Chromatography
Nonpolar Stationary Phase
Polar Mobile Phase

Normal Phase Chromatography

Polar Stationary Phase
Nonpolar Mobile Phase

Column Selection
Mobile-Phase Selection

Partition Chromatography III

Research Applications
Parathion in Insecticides:
O
CH3CH2O P O
CH3CH2O

NO2

Cocaine in Fruit Flies: A Study of

Neurotransmission by Prof. Jay Hirsh, UVa

Adsorption Chromatography
Classic
Solvent Selection
Non-polar Isomeric Mixtures
Advantages/ Disadvantages
Applications

What is Ion Chromatography?

Modern methods of separating and determining ions

based on ion-exchange resins

Mid 1970s
Anion or cation mixtures readily resolved on HPLC
column
Applied to a variety of organic & biochemical systems
including drugs, their metabolites, serums, food
preservatives, vitamin mixtures, sugars,
pharmaceutical preparations

The Mobile Phases are...

Aqueous solutions
containing methanol, water-miscible organic solvents
also contain ionic species, in the form of a buffer
solvent strength & selectivity are determined by kind
and concentration of added ingredients
ions in this phase compete with analyte ions for the
active site in the packing

Properties of the Mobile Phase

Must
dissolve the sample
have a strong solvent strength leads to reasonable
retention times
interact with solutes in such a way as to lead to
selectivity

Ion-Exchange Packings
Types of packings
pellicular bead packing
large (30-40 m) nonporous, spherical, glass,
polymer bead
coated with synthetic ion-exchange resin
sample capacity of these particles is less
coating porous microparticles of silica with a thin film
of the exchanger
faster diffusion leads to enhanced efficiency

Ion-Exchange Equilibria
Exchange equilibria between ions in solution and ions on

the surface of an insoluble, high molecular-weight solid

Cation exchange resins
sulfonic acid group, carboxylic acid group

Anion exchange resins

quaternary amine group, primary amine group

CM Cellulose
Cation Exchanger

DEAE Cellulose
Anion Exchanger

Eluent Suppressor Technique

Made possible the conductometric detection of eluted

ions.
Introduction of a eluent suppressor column
immediately following the ion-exchange column.
Suppressor column
packed with a second ion-exchange resin
Cation analysis
Anion analysis

Size Exclusion
Chromatography(SEC)
Gel permeation(GPC), gel filtration(GFC)

chromatography
Technique applicable to separation of high-molecular
weight species
Rapid determination of the molecular weight or
molecular-weight distribution of larger polymers or
natural products
Solute and solvent molecules can diffuse into pores -trapped and removed from the flow of the mobile phase

SEC(continued)
Specific pore sizes.average residence time in the pores

depends on the effective size of the analyte molecules

larger molecules
smaller molecules
intermediate size molecules

SEC Column Packing

Small (~10 m) silica or polymer particles containing

a network of uniform pores

Two types (diameters of 5 ~ 10 m)
Polymer beads
silica-based particles

Advantages of Size Exclusion

Chromatography
Short & well-defined separation times
Narrow bands--> good sensitivity
Freedom from sample loss, solutes do not interact

with the stationary phase

Absence of column deactivation brought about by
interaction of solute with the packing

Disadvantages
Only limited number of bands can be accommodated

because the time scale of the chromatogram is short

Inapplicability to samples of similar size, such as
isomers.
At least 10% difference in molecular weight is required
for reasonable resolution

Instrumentation
Instruments required:

Mobile phase reservoir

Pump
Injector
Column
Detector
Data system

Schematic of liquid
chromatograph

Mobile phase reservoir

Glass/stainless steel reservoir
Removal of dissolved gases by degassers
vacuum pumping system
heating/stirring of solvents
sparging
vacuum filtration

Elution methods
Isocratic elution
single solvent of constant composition
Gradient elution
2 or more solvents of differing polarity used

Pumping System I
Provide a continuous constant flow of the

solvent through the injector

Requirements

pressure outputs up to 6000 psi

pulse-free output
flow rates ranging from .1-10 mL/min
flow control and flow reproducibility of .
5% or better
corrosion-resistant components

Pumping System II
Two types:
constant-pressure
constant-flow
Reciprocating pumps
motor-driven piston
disadvantage: pulsed flow creates noise
advantages: small internal volume (35-400 L), high
output pressures (up to 10,000 psi), ready adaptability
to gradient elution, constant flow rates

Pumping System III

Displacement pumps
syringe-like chambers activated by screw-driven
mechanism powered by a stepper motor
advantages: output is pulse free

disadvantage: limited solvent capacity (~20 mL) and

inconvenience when solvents need to be changed
Flow control and programming system
computer-controlled devices
measure flow rate

increase/decrease speed of pump motor

Sample Injection Systems

For injecting the solvent through the column
Minimize possible flow disturbances
Limiting factor in precision of liquid chromatographic measurement
Volumes must be small
.1-500 L
Sampling loops

interchangeable loops (5-500 L at pressures up to 7000 psi)

LC column

LC injector

Liquid Chromatographic Column

Smooth-bore stainless steel or heavy-walled glass

tubing
Hundreds of packed columns differing in size and
packing are available from manufacturers ($200$500)
Add columns together to increase length

Liquid Chromatographic
Columns II
Column thermostats
maintaining column temperatures constant to a few
tenths degree centigrade
column heaters control column temperatures (from
ambient to 150oC)
columns fitted with water jackets fed from a constant
temperature bath

Detector
Mostly optical
Equipped with a flow cell
Focus light beam at the center for

maximum energy transmission

Cell ensures that the separated
bands do not widen

Some Properties of Detector

Adequate sensitivity
Stability and reproducibility
Wide linear dynamic range
Short response time
Minimum volume for reducing zone broadening

More Properties of Detector

High reliability and ease of use
Similarity in response toward all analytes
Selective response toward one or more classes of

analytes
Non-destructive

Types of Detector
Refractive index
UV/Visible
Fluorescence
Conductivity
Evaporative light scattering
Electrochemical

Refractive Index I
Measure displacement of beam with respect to

photosensitive surface of dectector

Refractive Index II
Advantages
universal respond to nearly all solutes
reliable
unaffected by flow rate
low sensitive to dirt and air bubbles in the flow cell

Refractive Index III

Disadvantages
expensive
highly temperature sensitive
moderate sensitivity
cannot be used with gradient elution

UV/Visible I
Mercury lamp
= 254nm
= 250, 313, 334 and 365nm with filters
Photocell measures absorbance
Modern UV detector has filter wheels for rapidly

switching filters; used for repetitive and quantitative

analysis

UV/Visible II

UV/Visible III
Advantages
high sensitivity
small sample volume required
linearity over wide concentration ranges
can be used with gradient elution

UV/Visible IV
Disadvantage
does not work with compounds that do not absorb light
at this wavelength region

Fluorescence I
For compounds having natural

fluorescing capability
Fluorescence observed by
photoelectric detector
Mercury or Xenon source with
grating monochromator to isolate
fluorescent radiation

Fluorescence II
Advantages
extremely high sensitivity
high selectivity
Disadvantage
may not yield linear response over wide range of
concentrations

Conductivity
Measure conductivity of column

effluent
Sample indicated by change in
conductivity
Best in ion-exchange
chromatography
Cell instability

Evaporative Light Scattering I

Nebulizer converts eluent into mist
Evaporation of mobile phase leads to formation of

fine analyte particles

Particles passed through laser beam; scattered
radiation detected at right angles by silicon
photodiode
Similar response for all nonvolatile solutes
Good sensitivity

Evaporative Light Scattering II

Electrochemical I
Based on reduction or

oxidation of the eluting

compound at a suitable
electrode and measurement of
resulting current

Electrochemical II
Advantages
high sensitivity
ease of use
Disadvantages
mobile phase must be made conductive
mobile phase must be purified from oxygen, metal
contamination, halides

Data System
For better accuracy and precision
Routine analysis
pre-programmed computing integrator
Data station/computer needed for higher control levels
add automation options
complex data becomes more feasible
software safeguard prevents misuse of data system

Electrophoresischarged species
migrate in electric field
Separation based on charge or
mobility

Capillary electrophoresis
higher voltages can be used as
the heat can be dissipated

Capillary electrophoresis