Chromatographic and High Performance Liquid Chromatography (HPLC)
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Transcript of Chromatographic and High Performance Liquid Chromatography (HPLC)
Presented By:Engr. Abdul Latif Sajrani
M.S (Environmental Science)
Environmental Sampling&
Analytical Techniques Subject Name:
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Contents Page No.
Water Analysis……………………………………......................04
Organic Trace Pollutants………………………………………...05
History of Chromatography……………………………………..07
Definition of Chromatography………………………………....10
Principles of Chromatography…………………………………...11
Paper Chromatographic Technique. ……………………………..12
Gas Chromatography (GC)………………………………………14
Principle of Gas Chromatography (GC)…………………………15
High Performance Liquid Chromatography (HPLC)……………19
The differences between HPLC & GC…………………………..23
The separation process…………………………………………..29
The Chromatogram……………………………………………..46
HPLC Applications………………………………………….…..49
References……………………………………………………….50 3
A chemical analysis of a water solution in which specific ions
and their concentrations are determined and recorded.
The character of the water solution then can be described in
terms of the individual ion concentrations and the total dissolved
solids, in units of ppm or mg/liter.
A complete analysis will include measurement
of pH, hardness, and bacteriological testing.
For limits of these criteria recommended for good quality
domestic water, suggested by U.S. Environmental Protection
Agency (EPA), consult EPA 822-R-94-001, May 1994 or CSU.
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Include
Naturally occurring compounds from decomposition of
OM
Anthropogenic pollutants
Degradation and inter-reaction products of pollutants
Substances derived from sewage treatment
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Typical analysis:
Individual compounds or groups of compounds
Total analysis of all organic components
Field screening for specific pollutants prior to lab analysis
Qualitative identification of trade products in spills and discharges
Organic Trace Pollutants (OTP)
Chromatography
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ChromatographyThe word “CHORMATOGRAPHY” was suggested by a Russain
Scientist, Michael Tswett in 1906.
M. Tswett was the first to use the term "chromatography" derived
from two Greek words "Chroma" meaning color and "graphein"
meaning to write.
The technique of paper chromatography was introduced into
biological research by Martin and Synge in 1941.
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1901 - invented chromatography
1903 - Mikhail Tswett separated plant pigments
using paper chromatography
liquid-solid chromatography
1930’s - Schuftan & Eucken use vapor as the
mobile phase
gas solid chromatography
1941 - paper chromatography was introduced into
biological research by Martin and Synge.
History of Chromatography
Invention of Chromatography
Mikhail Tswett invented
chromatography in 1901
during his research on
plant pigments.
He used the technique to
separate various plant
pigments such as
chlorophylls, xanthophylls
and carotenoids. Mikhail Tswett
Russian Botanist(1872-1919) 9
Definition of chromatography Tswett (1906) stated that " chromatography is a method
in which the components of a mixture are separated onadsorbent column in a flowing system”.
IUPAC definition (International Union of pure andapplied Chemistry) (1993):
Chromatography is a physical method of separation inwhich the components to be separated are distributedbetween two phases, one of which is stationary while theother moves in a definite direction.
The stationary phase may be a solid, or a liquid supportedon a solid or gel, the mobile phase may be either a gas or aliquid.
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Principles of Chromatography
Chromatography is a physical process.
Any Chromatography system is composed of threeComponents :
Stationary phase
Mobile phase
Mixture to be separated
We can only control stationary and mobile phase asmixtures are the problem we have to deal with.
Chromatography is a dynamic process in which themobile phase moves in definite direction.
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Flow sheet for the use of Paper Chromatographic Technique
Pour the solvent system into the petriplates
Apply the sample in the centre of the filter paper
Place the filter paper between the plates
Run the Chromatogram till the end of paper
Visualization of spots
Calculate the Rf Value
Air Dry
Rf Value:
Define as the ratio of the
distance traveled by a given
compound as compound to
the distance traveled by the
solvent.
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Gas Chromatography (GC)
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Gas Chromatography (GC)
Gas chromatography is a chromatographic technique
that can be used to separate volatile organic compounds.
It consists of
a flowing mobile phase
an injection port
a separation column (the stationary phase)
an oven
a detector.
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Principle Gas Chromatography The organic compounds are separated due to
differences in their partitioning behavior between the
mobile gas phase and the stationary phase in the
column.
Mobile phases are generally inert gases such as helium,
argon, or nitrogen.
The injection port consists of a rubber septum through
which a syringe needle is inserted to inject the sample.
The injection port is maintained at a higher temperature than
the boiling point of the least volatile component in the sample
mixture.
Cont…15
Since the partitioning behavior is dependent on
temperature, the separation column is usually
contained in a thermostat-controlled oven.
Separating components with a wide range of boiling
points is accomplished by starting at a low oven
temperature and increasing the temperature over time
to elute the high-boiling point components.
Principle GC
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High Performance Liquid
Chromatography (HPLC)
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The differences between High Performance Liquid Chromatography and Gas Chromatography.
The components of the high performance liquid chromatograph (HPLC).
The separation process.
The chromatogram.
The most common modes of HPLC.
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In This Section, We Will Discuss
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I need a quantitative
separation of
carbohydrates in some
of our products
as soon as possible.
I’ll need a separation
technique.
I’ll get
on it!
You’ve Got a Problem to Solve
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I have two separation techniques in my lab,
High Performance Liquid Chromatography
and Gas Chromatography. Which should I use?
Separation Techniques
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Sample Volatility Sample Polarity
HPLC
•No volatility requirement
•Sample must be solublein mobile phase
GC
•Sample must be volatile
HPLC
GC
•Separates both polar andnon polar compounds
•PAH - inorganic ions
•Samples are nonpolarand polar
Comparison of HPLC and GC
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Comparison of HPLC and GC
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Sample Thermal Lability Sample Molecular Weight
HPLC
•Analysis can take placeat or below roomtemperature
GC
•Sample must be ableto survive high temperature injectionport and column
HPLC
GC
•No theoretical upper limit
•In practicality, solubility islimit.
•Typically < 500 amu
Comparison of HPLC and GC
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Sample Preparation Sample Size
HPLC
•Sample must be filtered
•Sample should be insame solvent as mobilephase
GC
•Solvent must be volatileand generally lower boiling than analytes
HPLC
GC
•Sample size based uponcolumn i.d.
•Typically 1 - 5 L
Comparison of HPLC and GC
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Separation Mechanism Detectors
HPLC
•Both stationary phaseand mobile phase takepart
GC
•Mobile phase is a sample carrier only
HPLC
GC
•Most common UV-Vis•Wide range of non-
destructive detectors•3-dimensional detectors•Sensitivity to fg (detector
dependent)
•Most common FID,universal to organiccompounds
Comparison of HPLC and GC
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Carbohydrates1. fructose
2. Glucose
3. Saccharose
4. Palatinose
5. Trehalulose
6. isomaltose
Zorbax NH2 (4.6 x 250 mm)
70/30 Acetonitrile/Water
1 mL/min Detect=Refractive Index
1
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4
5
mAU
time
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How can We Analyze the Sample?
Separations
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Separation in based upon differential
migration between the stationary and
mobile phases.
Stationary Phase - the phase which
remains fixed in the column, e.g. C18,
Silica
Mobile Phase - carries the sample
through the stationary phase as it
moves through the column.
Injector
Detector
Column
Solvents
Mixer
Pumps
High Performance Liquid Chromatograph
Waste
Separations
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Injector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
Separations
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Injector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time
Separations
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Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Separations
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Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Separations
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Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Separations
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Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Separations
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Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Separations
37
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Separations
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Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Separations
39
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Separations
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Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Separations
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Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Separations
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Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Separations
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Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Separations
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Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
Separations
45
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
The Chromatogram
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Injection
to
tR
mAU
time
tR
to - elution time of unretained peak
tR- retention time - determines sample identity
Area or height is proportional
to the quantity of analyte.
HPLC Analysis Parameters
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Mobile Phases
Flow RateComposition
Injection Volume
Column Oven Temperature
WavelengthTime Constant
Modes of High Performance Liquid Chromatography
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Types of Compounds Mode StationaryPhase
Mobile Phase
NeutralsWeak AcidsWeak Bases
ReversedPhase
C18, C8, C4cyano, amino
Water/OrganicModifiers
Ionics, Bases, Acids Ion Pair
C-18, C-8 Water/Organic Ion-Pair Reagent
Compounds notsoluble in water
NormalPhase
Silica, Amino,Cyano, Diol
Organics
Ionics Inorganic Ions Ion Exchange
Anion or CationExchange Resin
Aqueous/Buffer Counter Ion
High Molecular WeightCompoundsPolymers
Size Exclusion
Polystyrene Silica
Gel Filtration-AqueousGel Permeation-Organic
HPLC Applications
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Chemical
Environmental
Pharmaceuticals
Consumer Products
Clinical
polystyrenes
dyes
phthalates
tetracyclines
corticosteroids
antidepressants
barbiturates
amino acids
vitamins
homocysteine
Bioscience
proteins
peptides
nucleotides
lipids
antioxidants
sugars
polyaromatic hydrocarbons
Inorganic ions
herbicides
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ReferencesReeve, R.N. (2002) Introduction to environmental analysis. Wiley.http://en.wikipedia.org/wiki/Mikhail_Tsvethttp://192.215.107.101/ebn/942/tech/techfocus/1071main.htmlhttp://www.chem.usu.edu/~sbialk/Classes/565/opamps/opamps.htmlSkoog, Holler, and Neiman. Principles of Instrumental Analysis. 5th ed. Orlando: Harcourt Brace & Co., 1998. http://weather.nmsu.eduhttp://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htmhttp://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.htmlhttp://weather.nmsu.edu/Teaching_Material/SOIL698/Student_Material/HPLCHP1090/HPLCINJ.HTMhttp://test-equipment.globalspec.com/LearnMore/Labware_Scientific_Instruments/Analytical_Instruments/Chromatographs/HPLC_Columnshttp://www.chemistry.adelaide.edu.au/external/soc-rel/content/lc-col.htm
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