Gas Chromatography(GC)

Liquid Chromatography(LC)
Mass Spectrometry(MS)
Ume-e-SalmaLiaqat
Uw-17-BIS-phD-003

Bioscience

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 Overview
 What is “chromatography”
 Gas Chromatography
 Theory of operation
 Instruments
 Applications
 Liquid Chromatography
 Comparison between LC & GC
 Mass Spectrometry

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 Chromatography

“Chromatography is the most important technique

of separation of chemical substances, and is
based on the physical interaction of a solute with
two different phases.”

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 Types of Chromatography

• Gas Chromatography
• Liquid Chromatography

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 Gas Chromatography
Definition
• GC is a common type of chromatography used in analytical
chemistry for separating and analyzing compounds that can
be vaporized without decomposition.

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 Gas Chromatography

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 Components
• Carrier gas: moves through the column, carrying the sample
along (mobile phase)
• Sample Injection: A small amount of sample is injected into
the column:
• Oven: Sample is heated to a sufficient temperature to instantly
vaporise the sample, which is then swept into the column by
the gas.
• Column: series of loops of inert solid, in gas liquid
chromatography the column is coated with porous solid coated
with liquid hydrocarbon with a high boiling point, the liquid
acts as the stationary phase.
• Detector: Flame ionisation
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 How does it work?
• Gas chromatography separates chemicals based on the ease
with which they evaporate into a gas.

• The chemical mixture being analysed is injected and carried

through the instrument by a carrier gas (commonly nitrogen).

• There is a column in the oven that makes it possible to

separate the chemicals based on how quickly they travel
through it.

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 Injection of the sample
• A very small quantity of the sample being analysed is
injected into the machine via the injection port using
a syringe.
.

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 The column & oven
• The column is normally made of stainless steel and is between 1 and 4
meters long. It has an internal diameter of just 4mm.
• The column is coiled up so that it will fit inside the temperature controlled
oven.
• Inside the column, it is packed with a very porous rock (solid which
contains many minute channels or open spaces).
• The temperature of the oven gets up to 250°C.

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 Separation

• A few things may happen to a particular molecule once

injected into the column:
• A compound with a boiling point higher than the temperature
of the column will condense at the start of the column.
• Some molecules may dissolve in the liquid stationary phase.
Some compounds will be more soluble in the liquid than
others.

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 Retention Time
• Retention time is the time taken for a substance to
emerge from the column (when a peak is seen on the
recorder)

• This time is measured from the time at which the sample

is injected to the point at which the display shows a
maximum peak height for that compound.

• Different compounds have different retention times

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 Detector

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 Interpreting the data
• The output will be recorded as a series of peaks - each one
representing a compound in the mixture passing through the
detector.

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 Calibration

• A gas chromatography test can be calibrated by

running at least 3 standards of different known
concentrations.

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 Advantages & Disadvantages of Gas
chromatography
• Advantages:
o It is the most sensitive of the chromatographic techniques
o Capable of being able to detect 10^-12g of a compound
o Suitable for volatile organics such as petrol.
• Disadvantages:
o Limited to compounds that can be readily vaporised
without decomposing.
o The sample is destroyed when it goes through the
detector, and so cannot be analysed any other way.

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 What Gas Chromatography Can
Analyse.
• Drug analysis

• Toxicology

• Organic compounds

• Analysis of body fluids for the presence of illegal substances

• Testing of fibber and blood from a crime scene

• Detect residue from explosives

• Urine samples that are routinely taken from athletes.

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 Continue….

• Alcohols in blood
• Aromatics (benzene, toluene, ethylbenzene, xylene)
• Flavors and Fragrances
• Permanent gases (H2, N2, O2, Ar, CO2, CO, CH4)
• Hydrocarbons
• Pesticides, Herbicides, PCBs, and Dioxins
• Solvents

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 Applications of GC

• Qualitative analysis: Gas chromatography can be

used for qualitative analysis.

• Quantitative analysis: Gas chromatography can be

used for quantitative analysis because the higher
the concentration, the more the flame will flare
up.

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 How does it compare to other
techniques?
• Gas chromatography is the most sensitive of all the
chromatographic techniques.

• It is limited to such compounds that have a relative

molecular masses less than 300.

• High performance liquid chromatography, can

separate compounds with a relative molecular masses
of over 1000.

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 Safety Tips
1. Perform periodic visual inspections
2. Follow the manufacturer's instructions when
installing columns
3. Turn off and allow heated areas such as the oven.
4. To avoid electrical shock, turn off the instrument .
5. Perform a radioactive leak test
6. Measure hydrogen gas and air separately when
determining gas flow rates.
7. Use only helium or nitrogen gas, never hydrogen,
21 to condition a chemical trap
 Liquid Chromatography

Definition
• Liquid chromatography (LC) is an analytical
chromatographic technique that is useful for
separating ions or molecules that are dissolved
in a solvent.

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 LIQUID CHROMATOGRAPHY

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 Liquid Chromatography

• Liquid Chromatography (LC) is a chromatographic technique

in which the mobile phase is a liquid.

• LC is a much older technique than GC, but was overshadowed

by the rapid development of GC in the 1950’s and 1960’s.

• LC is currently the dominate type of chromatography and is

even replacing GC in its more traditional applications.

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 Types of Liquid Chromatography

• Techniques in LC are classified according to the method of

solute separation;

• Adsorption chromatography
• Affinity chromatography
• Partition chromatography
• Size-exclusion chromatography
• Ion-exchange chromatography

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 Research Trends
GC VS LC

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 GC VS LC
• Advantages of LC compared to GC:
• LC can be applied to the separation of any compound that is
soluble in a liquid phase.
• LC more useful in the separation of biological compounds,
synthetic or natural polymers, and inorganic compounds.
• Liquid mobile phase allows LC to be used at lower
temperatures than required by GC.
• LC better suited than GC for separating compounds that may
be thermally labile.
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 GC VS LC
• Disadvantage of LC compared to GC

• LC is subject to greater peak or band-broadening.

• Much larger diffusion coefficients of solutes in gases vs.
liquids

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 Application of LC

1. Biochemical Screening for Genetic Disorders

2. Therapeutic Drug Monitoring and Toxicology
3. Vitamins and Related Metabolites
4. Steroid Hormones

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 Mass Spectrometry
Analytical method to measure the molecular
or atomic weight of samples

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Different Elements Can Be Uniquely Identified By Their
Masses

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 MS Principles
Different compounds can be uniquely identified by
their masses

Butorphanol L-dopa Ethanol

N -CH2-
OH COOH
HO -CH2CH-NH2 CH3CH2OH

HO
HO

MW = 327.1 MW = 197.2 MW = 46.1

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 Mass Spectrometry

• For small organic molecules the MW can be determined to

within 5 ppm or 0.0005%.

• For large biomolecules the MW can be determined within 0.01%

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 MS Principles
• Find a way to “charge” an atom or molecule (ionization)

• Place charged atom or molecule in a magnetic field or

subject it to an electric field and measure its speed or radius
of curvature relative to its mass-to-charge ratio (mass
analyzer)

• Detect ions using microchannel plate or electron multiplier

tube

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 Mass Spectrometer Schematic
Rough pumps
High Vacuum System Rotary pumps
Turbo pumps
Diffusion pumps

Ion Mass Data

Inlet Detector
Source Filter System

Vapor MALDI TOF Microch plate PC’s

HPLC ESI Quadrupole Electron Mult. UNIX
GC FAB Ion Trap Mac
Solids probe EI/CI Mag. Sector
FTMS

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 Mass Spec Principles

Sample

+
_

Ionizer Mass Analyzer Detector

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 Typical Mass Spectrum

• Characterized by sharp, narrow peaks

• X-axis position indicates the m/z ratio of a given ion
• Height of peak indicates the relative abundance of a
given ion (not reliable for quantitation)
• Peak intensity indicates the ion’s ability to desorb or
“fly” (some fly better than others).

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 Application of MS
1. Proteomics
2. Metabolomics
3. Environmental analysis
4. Pharmaceutical analysis
5. Forensic analysis

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 Safety Tips
of Ms
1. Turn off the gas source any time you turn off.
2. Check frequently for leaks, using certified leak-
checking equipment.
3. Remove ignition sources from your lab whenever
possible (including open flames, sources of static
electricity, or devices that spark).
4. Never allow gases to vent from high pressure
directly into the lab itself.

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