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

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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

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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

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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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