Water analysis trace pollutant extraction techniques for chromatographic and High Performance Liquid Chromatography (HPLC)

1. Presented By:
Engr. Abdul Latif Sajrani
M.S (Environmental Science)
EnvironmentalSampling
&
Analytical Techniques
SubjectName:
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3. 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
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4.  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.
4

5. 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)

6. Chromatography
6

7. Chromatography
The 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

9. 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

10. Definition of chromatography
 Tswett (1906) stated that " chromatography is a method
in which the components of a mixture are separated on
adsorbent column in a flowing system”.
 IUPAC definition (International Union of pure and
applied Chemistry) (1993):
Chromatography is a physical method of separation in
which the components to be separated are distributed
between two phases, one of which is stationary while the
other moves in a definite direction.
The stationary phase may be a solid, or a liquid supported
on a solid or gel, the mobile phase may be either a gas or a
liquid.
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11. Principles of Chromatography
 Chromatography is a physical process.
 Any Chromatography system is composed of three
Components :
 Stationary phase
 Mobile phase
 Mixture to be separated
 We can only control stationary and mobile phase as
mixtures are the problem we have to deal with.
 Chromatography is a dynamic process in which the
mobile phase moves in definite direction. 11

12. 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.
12

13. Gas Chromatography (GC)
13

14. 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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15. 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

16. 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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18. 18

19. High Performance Liquid
Chromatography (HPLC)
19

20.  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.
20
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

22. 22
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 soluble
in mobile phase
GC
•Sample must be volatile
HPLC
GC
•Separates both polar and
non polar compounds
•PAH - inorganic ions
•Samples are nonpolar
and polar
Comparison of HPLC and GC

24. 24
Comparison of HPLC and GC

25. 25
Sample Thermal Lability Sample Molecular Weight
HPLC
•Analysis can take place
at or below room
temperature
GC
•Sample must be able
to survive high
temperature injection
port and column
HPLC
GC
•No theoretical upper limit
•In practicality, solubility is
limit.
•Typically < 500 amu
Comparison of HPLC and GC

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Sample Preparation Sample Size
HPLC
•Sample must be filtered
•Sample should be in
same solvent as mobile
phase
GC
•Solvent must be volatile
and generally lower
boiling than analytes
HPLC
GC
•Sample size based upon
column i.d.
•Typically 1 - 5 L
Comparison of HPLC and GC

27. 27
Separation Mechanism Detectors
HPLC
•Both stationary phase
and mobile phase take
part
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 organic
compounds
Comparison of HPLC and GC

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Carbohydrates
1. 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
2
3
4
5
mAU
time
6
How can We Analyze the Sample?

29. Separations
29
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

30. Separations
30
Injector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph

31. Separations
31
Injector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time

32. Separations
32
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

33. Separations
33
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

34. Separations
34
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

35. Separations
35
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

36. Separations
36
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

37. Separations
37
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

38. Separations
38
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

39. Separations
39
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

40. Separations
40
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

41. Separations
41
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

42. Separations
42
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

43. Separations
43
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

44. Separations
44
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

45. Separations
45
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

46. The Chromatogram
46
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.

47. HPLC Analysis Parameters
47
Mobile Phases
Flow Rate
Composition
Injection Volume
Column
Oven Temperature
Wavelength
Time Constant

48. Modes of High Performance Liquid
Chromatography
48
Types of Compounds Mode Stationary
Phase
Mobile Phase
Neutrals
Weak Acids
Weak Bases
Reversed
Phase
C18, C8, C4
cyano, amino
Water/Organic
Modifiers
Ionics, Bases, Acids Ion
Pair
C-18, C-8 Water/Organic
Ion-Pair Reagent
Compounds not
soluble in water
Normal
Phase
Silica, Amino,
Cyano, Diol
Organics
Ionics Inorganic Ions Ion
Exchange
Anion or Cation
Exchange
Resin
Aqueous/Buffer
Counter Ion
High Molecular Weight
Compounds
Polymers
Size
Exclusion
Polystyrene
Silica
Gel Filtration-
Aqueous
Gel Permeation-
Organic

49. HPLC Applications
49
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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References
Reeve, R.N. (2002) Introduction to environmental analysis. Wiley.
http://en.wikipedia.org/wiki/Mikhail_Tsvet
http://192.215.107.101/ebn/942/tech/techfocus/1071main.html
http://www.chem.usu.edu/~sbialk/Classes/565/opamps/opamps.html
Skoog, Holler, and Neiman. Principles of Instrumental Analysis. 5th ed. Orlando:
Harcourt Brace & Co., 1998.
http://weather.nmsu.edu
http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm
http://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.html
http://weather.nmsu.edu/Teaching_Material/SOIL698/Student_Material/HPLCHP1090/
HPLCINJ.HTM
http://test-
equipment.globalspec.com/LearnMore/Labware_Scientific_Instruments/Analytical_Instru
ments/Chromatographs/HPLC_Columns
http://www.chemistry.adelaide.edu.au/external/soc-rel/content/lc-col.htm

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