COURSE CODE: ESST3003

COURSE TITLE: Environmental Monitoring and Assessment

TITLE OF ASSIGNMENT: Chromatographic and spectrophotometric techniques in

monitoring the aquatic environment.

Chromatographic and spectrophotometric methods are used in environmental monitoring

to determine the presence of certain water quality paraments and its concentrations. There are
different chromatographic and spectrophotometric techniques used in environmental analysis.
Spectroscopic techniques include visible, ultraviolet, fluorescence, atomic absorption, and
infrared spectroscopy. (Hofmann. 2010). Chromatography allows for identification of
components in a sample by separation of analytes individually. It generally consists of two
phases: stationary, that contains a solid or liquid absorbed to a solid surface and mobile or
moving phase, that consists of a liquid or gas component. Chromatographic techniques include
high pressure liquid chromatography, thin-layer, gas, and column chromatography. Generally,
detectors are required for gas and liquid chromatography to identify the mixture's components as
these methods cannot identify the components of a substance on its own. Therefore, flame
ionization, thermal conductivity detectors and mass spectrometry is often used. (Coskun. 2016).
Chromatography and spectrophotometry are often used for testing water quality as it requires
low sample volumes and gives an accurate and precise analysis of the parameter being tested.
Different chromatographic methods are used to test for a range of water quality
parameters. Column chromatography can be used to test for anions such as nitrates (NO2-, NO3-),
sulphates (SO42-), phosphates (H2PO4-) and chlorides (Cl-). For example, to identify and analyze
nitrates in water samples, ion chromatography, a type of column chromatography is used. The
water samples must first be specifically prepared before they are subjected to chromatography.
Samples are filtered using a membrane or diluted using deionized water. It can be stored in
plastic or glass containers and kept at a temperature of 4◦C. (Amin et al. 2008). After preparation
the sample is now subjected to anion separation. The sample is injected into the ion
chromatogram and passes through the analyte column (exchange resin) known as the stationary
phase. The exchange resin converts the nitrates to acids, which will then be detected. Here the
nitrate and nitrite ions are separated by passing through a cation resin column with positive
charged organic polymers such as the ammonia ion. The mobile phase involves a suppressing
column that reduces the conductivity of nitrite and nitrate ions. The system produces a
conductance vs time graph where the concentration can be determined. (Zhang. 2007). These
concentrations are compared to the standards in the water pollution rules which are less than or
equal to 10 mg/L for nitrates, less than or equal to 250 mg/L for sulphates, less than or equal to
0.5 mg/L for phosphates and 120 mg/L to 230 mg/L for long term chlorides.
High Performance Liquid Chromatography or HPLC is another chromatographic
technique used to analyze water quality parameters. It involves the same phases of
chromatography which is the stationary and mobile phase. It can be used to detected pesticides
stated in 2019 Water Pollution Rules such as diazinon, endosulfan, chlordane and endrin. For
example, if we want to test for these four pesticides in a water body, the sample must be prepared
before it is analyzed. A stock solution of diazinon, endosulfan, chlordane and endrin are prepared
by dissolving it in acetonitrile and preparing dilutions of varying concentrations. A solvent is
added which usually consists of water and alcohol, to make a polar solution for operation by
reverse phase chromatography and acts as the mobile phase. The water sample is then injected
into the chromatogram in which the pump would then carry the solvent and the sample into the
column, usually consisting of silica or alumina beads where separation takes place. The
stationary phase consists of glass beads surrounded by a thin layer of silica or alumina packed in
a column in which the water sample passes through, and the pesticides present in the sample
interacts with the column. Each pesticide has varying rates of being attracted to the surface of the
column molecules (adsorption). The stronger the interaction or adsorption, the longer the
retention time. The detector at the end of the chromatogram, usually an ultraviolet detector, then
detects a signal for the time when the pesticides are eluted from the column and produces a
chromatogram for the peak vs time. (Barkovich 2020). The standard pesticide concentrations and
the peak areas are used to produce a calibration curve. Using this curve allows for the
determination of concentrations of the pesticides in the water sample which can be compared to
the standard pesticide values to analyze if concentrations have exceeded the standards or if it is
in within the limits for protection of aquatic systems. (Rajput et al. 2018).
Gas chromatography (GC) is mainly used to monitor the concentrations of volatile
organic compounds that are present in water/groundwater bodies. This includes organic
pollutants stated in the water pollution rules such as: benzene, ethylbenzene, toluene, cyanide,
and tributyltins (TBT's). To determine the concentration of these organic chemicals in a water
sample, standard solutions of each organic chemical is prepared by dissolving each chemical in
methanol. Since these organic compounds are volatile, they are stored in sealed glass vials and
kept in a cold environment to prevent any loss due to vaporization. Collecting and preparing
water samples for GC analysis must also be stored in sealed glass vials and kept in a cold
environment, ideally at 4°C. The internal standards are used to prepare the samples by spiking
them with each organic standard solution. (Aeppli et al. 2008). The small amount of sample is
then injected into the sample port that will vaporize the spiked water sample. The carrier gas
which is inert and oxygen free such as nitrogen or argon, and then carries the vaporized
substances to the column. This carrier gas acts as the mobile phase and once it arrives at the
column of the gas chromatogram which is the stationary phase, the vapor interacts with the liquid
coated silica. This column, can be capillary or packed, containing silica which allows the organic
compounds to dissolve at different degrees in the liquid. Organic compounds that are less soluble
will separate faster than those with a high solubility. At the end of the column, there is a detector
which will measure the components of the sample as they elute from the water sample and the
nitrogen/helium carrier gas. The detector commonly used in gas chromatography is mass
spectrometry which would measure the masses of each organic compound through the separation
process. When the samples leave the column, it enters the mass spectrometer through an inlet
line which allows the sample to be ionized and fragmented using electrons. This is analyzed by
the spectrometer according to mass to charge ratio which will produce a chromatogram of
retention times and peak area. The chromatograms of the standard concentrations and samples
can then be used to determine the sample concentration using the formula:
Concentration = (sample peak area/standard peak area) x standard concentration.
The concentrations of the samples can then be compared to the standard concentration according
to the water pollution rules (Thet and Woo. 2020). These standards for protection of aquatic life
and systems are less than 370 (µg/L) for benzene, less than 2 (µg/L) for toluene, less than 4
(µg/L) for cyanide and less than 0.072 (µg/L) for tributyltins (TBT's).
Spectrophotometry is another method used to monitor the aquatic environment by
measuring and identifying water quality parameters of unknown concentrations present in a
water sample. It involves the use of wavelengths to measure the intensity of light in a substance
which produces an absorbance value. The concentration of the water parameter is related to
absorbance by the Beer-Lambert’s law which states that there is a linear relationship (A = ϵ l c).
(Vo. 2020). This enables us to assess the overall health of an aquatic system by identifying any
toxic contaminants and if any of these parameters have exceeded their standards, according to
the water pollution rules. There are varying spectrophotometric methods that can be used to
quantify and identify parameters for example, visible (UV), atomic absorption, and infrared
spectroscopy.
Ultra-Violet visible spectroscopy also known as ultraviolet visible spectroscopy can be
used to measure water quality parameters such as chemical oxygen demand (COD), nitrates,
nitrites, total phosphates, and turbidity. To analyze total phosphate concentrations in a water
sample by UV-VIS spectrophotometry, reagents are used for example, potassium di-hydrogen
phosphate and ammonium molybdate are prepared by dissolving and diluting them. These were
used to prepare standard solutions of varying concentrations which is measured at 830nm to
produce a standard phosphate calibration curve using absorbances. After collecting the water
samples, it is then filtered to remove any sediments that is available. Aliquots of the water is
taken and is added into a cuvette together with ammonium molybdate and hydrazine sulphate.
These reagents would react with the phosphates present in the water sample to produce a blue
color which is measured by the spectrometer to give an absorbance value. A reference or blank
sample that does not contain any reagent is also added to the spectrometer that allows the
instrument to be calibrated and reduce errors. (Ganesh et al. 2012). The spectrophotometer
measures the absorbance value by passing light from a source into the monochromator, which
contains a prism, that refracts light through the cuvette. The amount of light absorbed by the
sample is detected which produces the absorbance value. These values can be used to determine
the phosphate concentration in the water sample using the calibration curve. (Raja and Barron.
2021). These concentrations are then compared to the standard phosphate concentration for
protection of aquatic life which is less than 0.5 mg/L, according to the water pollution rules.
Atomic absorption spectroscopy (AAS) is one spectroscopic method used to monitor the
aquatic environment. It can detect heavy metals such as lead, zinc, cadmium, and iron in water
sources. To analyze a water sample for heavy metal by AAS, the water sample should be
collected in clean plastic bottles washed with deionized water and nitric acid to reduce
contamination. Lead should not be collected in glass bottles as the glass contributes to
contamination. After collection the samples are preserved using nitric acid which lowers the pH,
to avoid chemical and biological reactions from occurring. It is then stored at 4oC. Before the
sample is subjected to analysis it must first be prepared by adding nitric acid, and slowly boiling
the sample to allow for evaporation. This process is known as digestion, which destroys the
sample matrix and reduces interference. Standard solutions of varying concentrations of each
heavy metal are also prepared to allow for instrument calibration and determination of sample
concentration. (Sharma and Tyagi. 2013). The sample is then inserted into the spectrophotometer
which is subjected to atomization. A flame atomizer allows the samples to be vaporized and
ionized from a radiation source. The heat from the flame atomizer breaks down the sample into
individual atoms. The radiating source passes light through the sample in which some of it is
absorbed and passes through the monochromator before it is detected. The spectrometer
separates the different wavelengths of lights and can have two beam types: single or double
beam. A single beam spectrometer passes radiation through the sample while double beam
passes radiation through a reference and the sample, which is more accurate as it reduces errors.
The obtained absorbance values for samples are used to determine the concentration of lead from
standards. This is done by obtaining the equation of the line from the standard concentration
calibration curve, which is y = mx + c where y is the absorbance and x is the standard
concentration. The sample absorbance values are inserted into this equation and rearranged to
determine the concentration of the sample(x). (Raja and Barron. 2021). This is compared to
standard heavy metal concentrations which is less than 65µg/L for protection of aquatic life and
systems, according to the water pollution rules.
Infrared spectrophotometry can be used to detect organic compounds in a water system
such as benzene, ethylbenzene, and toluene. It is suitable for detecting these chemicals as the
infrared system identifies the different functional groups present in an organic compound. It uses
infrared radiation to excite the organic molecules, resulting in vibrations at varying frequencies.
One type of infrared spectroscopy that is used to detect organic molecules in water samples is
known as Fourier Transform Infrared Spectrometer (FTIR). When it emits radiation through the
sample, the detector picks up the vibration signals producing a spectrum of the energy absorbed
in the form of peaks, in which each peak corresponds to a frequency number in the spectrum.
(Gowen et al. 2012). To analyze the concentration of organic compounds, present in the sample,
the intensity of the peak is used as it indicates how concentrated the substance is. This can then
be related to the Beer Lambert’s equation, A = ϵ l c where A is the absorbance, ϵ is the
absorptivity, l is the path length and c is the concentration. The absorptivity provides a measure
of the intensity of the peak for the functional group of an organic molecule at a specified
frequency. The peak intensity can be compared to the standard concentrations in which the peaks
will be more intense than that of samples. (Libre texts. 2021).
Chromatography and spectroscopy assists in the monitoring of the aquatic environment
by identifying and quantifying different water parameters and pollutants present in an aquatic
system. There are different chromatographic methods that can assist in monitoring of the aquatic
environment: column, liquid, and gas chromatography. Spectrophotometric methods include
visible, atomic absorption and infrared spectrophotometry. These methods determine the
concentrations of pollutants which can be compared to the standards of the 2019 Water Pollution
Rules, to determine if these contaminants are within limits for protection of aquatic life, or if the
concentrations are exceeded.

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