The document discusses advanced chromatographic techniques and their applications in aquatic environmental studies, detailing the history and development of chromatography since its invention by Michael S. Tswett in 1906. It highlights various types of chromatography, such as liquid chromatography (LC), gas chromatography (GC), and their combinations with mass spectrometry (MS), emphasizing their role in analyzing environmental pollutants like polycyclic aromatic hydrocarbons (PAHs). The document also presents a case study involving the analysis of PAHs from soil samples collected at an oil spill site using gas chromatography-mass spectrometry (GC-MS).

1. Advanced chromatographic techniques and special
focus on Applications in Aquatic environmental
studies
By:
B. Bhaskar
Fisheries

2. Introduction
• word chromatography originated from Greek and means color
writing, (Chroma color and Graphein writing).
• Chromatography is a powerful separation technique that was first
invented by botanist Michael S. Tswett in 1906 in Warsaw.
• Tswett successfully separated chlorophyll, xanthophyll, and many
other colored species by percolating vegetable extracts through a
column of calcium carbonate.
• These pigments had different adsorptions on the calcium carbonate
column, and that yielded colored bands at various positions on the
column.
• Tswett called the colored bands a chromatogram and the method
chromatography.
• In Tswett’s method, the calcium carbonate column was referred to
as the stationary phase, while the solution of pigments and
solvents was called the mobile phase since they moved through
the column.

3. Cont...
• In the 1930’s, thin layer chromatography and ion exchange chromatography were
introduced as new chromatographic separation techniques.
• In 1941, paper chromatography and partition chromatography were introduced by
Archer Martin and Richard Synge.
• Martin and Synge also introduced gas chromatography and jointly won the Noble
Prize in Chemistry 1952 for their invention of partition chromatography.
• Liquid chromatography (LC) is an analytical technique in which species in the liquid
state are separated based on their interaction with a stationary phase and then
introduced into a detector, such as a mass spectrometer or UV-Vis
spectrophotometer.
• The mobile phase is typically an aqueous solution of organic solvents such as
methanol, acetonitrile, propanol or hexane, while the stationary phase is bare or
alkylated silica particles or polymer beads such as polysaccharide or polystyrene,
housed in a stainless steel column.
• LC operates in different modes such as normal phase (NP), reversed-phase (RP),
and hydrophilic interaction liquid chromatography (HILIC). In NP mode, the
stationary phase is more polar than the mobile phase.
• Polycyclic aromatic hydrocarbons (PAHs) are among these pollutants that represent
significant threats toward aquatic organisms because of their carcinogenic and
mutagenic properties and their persistence in the environment.

4. Mass spectrometry (MS)
• Mass spectrometry (MS) is a powerful analytical technique in which ions are
generated from either inorganic or organic compounds by a suitable
ionization technique and subsequently separated and detected in a high
vacuum region by electric and magnetic fields.
• Mass spectrometry occupies an outstanding position among analytical
methods due to its great sensitivity, detection limits, speed and a wide
range of applications.
• To give a full list of mass spectrometry applications is impossible here
because of the enormous number of applications.
• Mass spectrometry is used in many key applications such as elemental and
isotopic analysis, organic and bio-organic analysis, structure elucidation,
characterization of ionic species and chemical reactions, mass spectral
imaging, and miniaturization.
• The technique is also easily coupled to separation techniques.
• The combination of chromatography and mass spectrometry has introduced
formidable hyphenated analytical techniques that offer the separating
power of chromatography and the detection capabilities of mass
spectrometry.
• Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas
Chromatography-Mass Spectrometry (GC-MS) are the most common
examples of these techniques.

5. Ultraviolet-Visible (UV-Vis) Detectors
• Spectrophotometric UV-Vis detectors are widely used in modern
chromatography.
• Many species absorb UV or visible radiation and this absorption
characteristic is exploited in their detection.
• UV-Vis detectors operate under the absorption principle which is governed
by the Lambert-Beer law, which states that: A = ε b C where A is the
absorbance of an analyte, ε is molar absorptivity; b is cell length (usually is
measured in cm), and C is the concentration of the analyte (usually in molar
concentration units).
• UV-Vis detectors can be selectively tuned to the analyte’s maximum
absorption wavelength.
• UV-Vis detectors can only detect analytes that absorb in the UV or visible
range of the spectrum.
• The sensitivity of UV-Vis detectors depends on the specific analyte and
wavelength.
• To achieve the maximum sensitivity, the various components of the mobile
phase should be transparent at the wavelength of measurement.
• UV-Vis detectors are less sensitive than mass spectrometer detectors.

6. Conductivity Detectors
• Conductivity detectors are employed extensively in ion chromatography.
• They measure the ability of a solution containing salt to conduct electricity
between two electrodes, which is defined as conductance.
• Solution conductance is directly proportional to the salt concentration and
the mobility of the individual anions and cations.
• Conductivity increases as the ionic character of molecule increases and/or
the size of ions decrease.
• Conductivity detectors are classified as general detectors because they
respond to all or most of the ions that pass through the detector cell.
Significant improvement in the conductivity detectors was made after the
introduction of “suppressed conductivity detection”.
• In the suppressed conductivity detection the eluent is treated before
detection to make the eluent ions less detectable and the sample ions more
detectable. This makes the background signal less conducting and the
sample signal more conducting

7. Common mobile phases, stationary phases, and detection techniques
used for the separation of ILs using RP-HPLC

8. Gas Chromatography
• Gas Chromatography
• Gas chromatography (GC) is a type of
chromatography that is used to separate and
analyse volatile compounds. In this technique, the
sample is vaporized and then injected into the
chromatograph. The sample then passes through a
column that is packed with a stationary phase,
which separates the components of the sample
based on their affinity for the stationary phase.
• GC is widely used in the analysis of organic
compounds, including hydrocarbons, fatty acids,
and amino acids

9. Gas chromatography-mass spectrometry (GC-MS)
• GC-MS is a very versatile analytical tool in which substances in the gaseous state are
separated based on volatility and interaction with a stationary phase, and then introduced
into a mass spectrometer.
• The mobile phase usually is helium, nitrogen, or hydrogen gasses, while the stationary phase
is a viscous liquid such as squalene, polyethylene glycol, or polymethyl siloxane, or an
adsorbent solid such as silica, molecular sieves, alumina, and porous polymers.
• The stationary phase is housed in a polyimidecoated fused silica or metal column.
• The complexity of environmental samples requires a method that is capable of separating
individual components from a mixture before detection in order to identify and quantify
them efficiently and accurately.
• Chromatographic techniques combined with mass spectrometry, especially LC-MS and GC-
MS, are intensively used in the environmental sciences to investigate the presence of organic
contaminants.
• GC-MS is widely used for the analysis of volatile or semi-volatile compounds sampled from
solid, liquid, or gaseous sample.
• GC-MS enables the identification of many environmental organic pollutants, such as
pesticides, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs),
dioxin, and volatile organic compounds (VOCs).
• Identification and quantification of polycyclic aromatic hydrocarbons (pahs) in the
kalamazoo river oil spill by gas chromatography-mass spectrometry (GC-MS).

10. Case study for collection of samples from Oil spilled
area for GC-MS (Ref: Al Isawi, Wisam Abdulabbas Flayyih, 2016)
• Eg: River, which spans the oil spill affected area.
• Two soil samples can be collected from sites upstream to the spill,
one sample was obtained from the rupture site, and three samples
were collected from downstream sites.
• Each soil sample was divided into two layers: an upper layer (0-6
inches depth) and a lower layer (6-12 inches depth).
• The soil samples were collected using a split-spoon sampler
• After collection from sampling sites, soil samples were stored in
glass jars and transported to the laboratory to be kept in the
refrigerator.

11. Sample Preparation for GC-MS:
(Ref: Al Isawi, Wisam Abdulabbas Flayyih, 2016)
• After collection, samples were kept in capped glass jars in a
refrigerator.
• To prepare the samples for GC-MS analysis, 10 g of each collected soil
sample was placed in a 60 ˚C drying oven overnight.
• Then, 10 mL of H2O and 1 mL of hydrochloric acid (HCl) were mixed
with each sample using a stir bar, and further incubated in the oven
overnight at 60 ˚C.
• Finally, PAHs were extracted from the sample with dichloromethane
(CH2Cl2) prior to GC-MS analysis.

12. GC-MS Analysis
Apparatus: GC-MS analyses were carried out on an HP 6890 Series GC system
(HewlettPackard, Palo Alto, CA, USA) connected to an HP 5973 Mass Selective Detector
operated in the 70 eV electron impact mode under standard conditions.
• A 30 m long, 250 μm I.D and 0.25 μm film thickness ZB-5MS (Phenyl Methyl Siloxane)
Phenomenex column (Phenomenex, Torrance, CA, USA) was used with helium as a carrier
gas at a linear velocity of 1.3 mL/min.
• The injector temperature was set at 250 ˚C.
• The sample solutions were injected in the splitless mode and the injection volume was 2 μL.
• The mass spectrometer source temperature was set at 200 ˚C. The mass range was set at m/z
35-600.
• The column temperature was programmed to hold at 40 ˚C for 2 min., then the temperature
was changed at 10 ˚C/min, and then hold at 320 ˚C for 20 min.
Chemicals: A standard solution of 16 PAHs (shown in Figure 4.4) at 2000 μg/mL in
dichloromethane was purchased from Sigma-Aldrich (CRM47930, Sait Loius, MO, USA) and
used as a calibrant. GC-MS grade dichloromethane was supplied by Fisher Scientific
(Hampton, NH, USA) and used as a solvent for sample extraction and GC-MS analyses.
• The standard solution of 16 PAHs (as shown in Figure 4.4) in dichloromethane was used as a
calibrant to investigate the presence of PAHs qualitatively and quantitively at the six
sampling sites. A GC-MS chromatogram of 20 μg/mL of the PAHs standard in
dichloromethane.

13. Cont...
• results also show that the total PAH concentration in the upper layer (0-6 inches
depth) of soil is higher than that of the lower layer (6-12 inches depth).
• Five years after the oil spill accident, apparently the PAHs did not migrate
downstream from the accident site, where they first accumulated, and is only
slowly migrating through the sediment layers.
• PAH deposition at the accident site could be attributed to the fact that PAHs are
poorly soluble in water.
• Hence, they tend to deposit in the sediments and soils.
• Several studies have reported that PAHs concentrate in sediments and soils
because of their lipophilic properties

14. Cont... (Ref: Al Isawi, Wisam Abdulabbas Flayyih, 2016)
• The results indicate that the accident site (6 miles from the study area start point)
has the highest total PAHs concentration of 32,059 ng/g dry among the six
locations investigated in this research.
• The concentration of total PAHs in the lower layer of the accident site sample is
about half that of the upper layer.
• The results suggest that the PAHs did not penetrate too deeply into the soil, since
as you go deeper into the ground, the concentration of PAHs decreases.
• The concentration of total PAHs in the lower layer of the accident site sample is
about half that of the upper layer

15. Liquid Chromatography
• Liquid chromatography (LC) is a type of
chromatography that is used to separate and
analyse non-volatile compounds. In this technique,
the sample is dissolved in a liquid solvent and then
injected into the chromatograph.
• The sample then passes through a column that is
packed with a stationary phase, which separates the
components of the sample based on their affinity
for the stationary phase.
• LC is widely used in the analysis of a wide range of
compounds, including pharmaceuticals, natural
products, and food additives.

16. LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (LC-MS)
• Separation and identification of biodegradation products of
the ionic liquid 1-butyl-3- methylimidazolium chloride
(bmimcl) by liquid chromatography-mass spectrometry (lc-
ms).

17. High-Performance Liquid Chromatography
• High-performance liquid chromatography (HPLC) is
a type of liquid chromatography that is used to
separate and analyze compounds at high pressure.
• In HPLC, the sample is dissolved in a liquid solvent
and then injected into the chromatograph.
• The sample then passes through a column that is
packed with a stationary phase, which separates the
components of the sample based on their affinity
for the stationary phase.
• HPLC is widely used in the analysis of a wide range
of compounds, including pharmaceuticals, natural
products, and food additives

18. • Reversed-Phase High-Performance Liquid
Chromatography (RP-HPLC): Biphenyl Column
• Ion-Pairing Reversed-Phase High-Performance
Liquid Chromatography (IP-RP-HPLC): Post Column
Addition
• Hydrophilic Interaction Liquid Chromatography
(HILIC):

19. Ion-Exchange Chromatography
• Ion-exchange chromatography is a type of chromatography that is used to
separate charged particles based on their ionic properties.
• In this technique, the sample is passed through a column that is packed
with a stationary phase that contains charged groups.
• Ion-exchange chromatography is widely used in the purification of proteins,
nucleic acids, and other biomolecules.
• It is also used in the analysis of inorganic compounds and the separation of
organic acids.
• Size-Exclusion Chromatography
• Size-exclusion chromatography is a type of chromatography that is used to
separate molecules based on their size.
• In this technique, the sample is passed through a column that is packed
with a stationary phase that contains porous beads.
• The size of the molecules determines whether they can enter the pores of
the beads or not.
• The larger molecules cannot enter the pores and therefore elute from the
column earlier than the smaller molecules.

20. Paper Chromatography
• Paper chromatography is a type of chromatography
that is similar to TLC, but uses a piece of filter paper
as the stationary phase.
• In this technique, the sample is spotted onto the
filter paper and then placed in a chamber that
contains a mobile phase.
• Paper chromatography is a simple and inexpensive
technique that is widely used in the analysis of
organic compounds, including amino acids, sugars,
and lipids.

21. Thin-Layer Chromatography
• Thin-layer chromatography (TLC) is a type of chromatography
that is used to separate and identify compounds based on
their differential migration on a thin layer of a stationary
phase.
• In this technique, the sample is spotted onto a thin layer of
stationary phase that is coated on a plate.
• The plate is then placed in a chamber that contains a mobile
phase, which moves up the plate via capillary action.
• The components of the sample will move at different rates
on the plate based on their affinity for the stationary phase
and the mobile phase.
• TLC is widely used in the analysis of organic compounds,
including drugs, pesticides, and natural products.
• It is also used in forensic science and in the identification of
unknown compounds

23. Schematic Diagram of Gas Chromatography
, Yusra Obeidat, 2021

24. Applications of Chromatography Techniques
• Applications of Chromatography Techniques
• Chromatography techniques have a wide range of applications in various fields, including −
• Pharmaceutical Industry: Chromatography techniques are widely used in the
pharmaceutical industry for the analysis and purification of drugs. HPLC is the most
commonly used technique for the analysis of drugs, while affinity chromatography is used
for the purification of proteins and other biomolecules.
• Food Industry: Chromatography techniques are used in the food industry for the analysis
and purification of food additives, flavours, and fragrances.
• Environmental Science: Chromatography techniques are used in environmental science for
the analysis of pollutants and contaminants in air, water, and soil. GC is commonly used for
the analysis of volatile organic compounds.
• Forensic Science: Chromatography techniques are used in forensic science for the analysis of
trace amounts of drugs, explosives, and other volatile compounds.
• Biotechnology: Affinity chromatography is commonly used for the purification of proteins
and other biomolecules, while size-exclusion chromatography is used for the analysis of
protein aggregates and the determination of molecular weight.
• Petrochemical Industry: GC is commonly used for the analysis of volatile organic compounds
in petroleum products, while HPLC is used for the analysis of non-volatile organic
compounds.
• Clinical Diagnostics: Chromatography techniques are used in clinical diagnostics for the
analysis of biomolecules in blood and other bodily fluids.
• Cosmetics Industry: Chromatography techniques are used in the cosmetics industry for the
analysis and purification of fragrances, flavours, and other additives.

25. References
• https://www.tutorialspoint.com/chromatographic-techniques-types-and-
applications
• https://www.researchgate.net/figure/Schematic-Diagram-of-Gas-
Chromatography-153_fig2_349679009
• https://www.google.com/search?sxsrf=AB5stBgex21IePUu5_BEFDC5D6KddN4T
EQ:1689943336137&q=gas+chromatography&tbm=isch&sa=X&sqi=2&ved=2ah
UKEwjdrfbB6Z-
AAxXKyzgGHcdTClkQ0pQJegQIEhAB&biw=1600&bih=757&dpr=1#imgrc=MF5Zk
j9T-yesuM
• Environmental Applications of Chromatography-Mass Spectrometry Wisam
Abdulabbas Flayyih Al Isawi, Western Michigan University
• Al Isawi, Wisam Abdulabbas Flayyih, "Environmental Applications of
Chromatography-Mass Spectrometry" (2016). Master's Theses. 683.
https://scholarworks.wmich.edu/masters_theses/683
• Well, H.; Williams, T. I. Nature Publishing Group 1950, 4232, 1000-1001.
• Chatwal, G. R.; Arora, M. Analytical Chromatography, Revised and Enlarged ed.;
Mrs. Meena Pandey for Himalaya Publishing House: Mumbai, 2006; pp 1-6.