Analysis of Trace Pollutants - NPTel

Environmental Chemistry and Analysis Prof. M.S.Subramanian

Indian Institute of Technology Madras

MODULE 2.3

Analysis of trace pollutants in water BioConcentration 1 Accumulation in sediments 2 Biomagnification 2 Degradation 3 Gas liquid Chromatography (GC) 7 Mobile phase 8

Sample injection 8

Column and stationary phase 9

Detectors 11 Extraction procedures or sample preparation 12 Solvent extraction 13

Head space analysis 14

Purge and trap 14

Solid phase extraction (SPE) 14

Analysis of Individual Trace Pollutants 15 Analysis of DDT 16

Alumina-silver nitrate column 16

Silica gel column 17

Technique for measuring total concentration of group of Compounds

19

Phenols 19

Surfactants 20

Total hydrocarbons 20



MODULE 2.3

Analysis of trace pollutants in water This chapter gives the outline of the analysis of organic compounds and

inorganic metal ions present in water at trace levels (i.e at µg l-1). The need to

analyse these species at such a low concentration of µg l-1 studies from the fact

that they tend to bioaccumulate (especially the neutral organic compounds and

metal ions) and are found in organisms at concentration exceeding background

levels by many factors of ten. Analysis of species at such low concentration

needs a preconcentration step so that they are amenable for instrumental

analysis.

BioConcentration:

Bioconcentration is a widely recognised phenomenon whereby

organisms accumulate particular component chemicals in their tissues. The

uptake of these chemicals occurs via a variety of mechanisms depending on the

organism and the chemical. Micro organisms, aquatic plants, fish, and other

animals living in water are capable of accumulating soluble metals and organic

chemicals. Organic compoundswhich do not contain polar groups such as OH or

NH2 , or which are not ionic, will have low solubility in water. Among the same

group of compounds the solubility generally decreases with increasing molecular

mass.As the solubility in water decreases, the solubility in organic solvent

increases.

Since fatty tissues are organophilic, the lower solubility organic

compounds tend to accumulate in these tissues, particularly that is found in

organs in closest contact with aqueous fluids e.g. kidneys. The tendency to

accumulate in fatty tissues is greater, the greater the molecular mass of

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compound. The ability to accumulate in an organism is measured by the term

bioconcentration factor which is given by the relation:

concentration of a compound in an organismbioconcentration factorconcentration in surrounding water

=

Accumulation in sediments:

As discussed earlier the species which have low solubility in water tend to

adhere to any available solid; the larger the surface area greater will be its ability

to adsorb the compound. Such kind of material is found in sediments which are in

abundnce in estuaries where there are often discharges from major industries.

Organisms such as mussels and scallops which feed by filtration of sediments

ingest these organics.

Biomagnification:

The tendency for a pollutant which is present at very low concentrations in

a water body to get accumulated to an unacceptable level in a food chain is

explained in fig.1 with pesticide as an example.

As we go up the food chain it can be seen that the concentration of

pesticide from lake water which was only at 0.02 mg.kg-1 has been increased to

1600 mg kg-1 in birds where one species is dependent for survival on the

consumption of previous species.

If a pollutant is present in the first organism, then as we proceed down the

food chain there will be increase in concentration in each subsequent species.

This is illustrated in fig 1

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Lake water ................................. 0.02

Concentration of pesticide (DDT) (mg kg-1)

Plankton ................................... 5.0

Non-predatory fish ................................... 40-100 (fatty tissue)

Predatory fish

..................... 80-2500 (fatty tissue)

Birds feedingon fish

..................................... 1600 (fatty tissue)

Fig.1 A typical Food chain

(Redrawn by permission of John Wiley & Sons Ltd, Chichester, England, 779777 from

Environmental analysis by Roger N. Reeve and John D. Barnes, p.25,1994)

Degradation:

The accumulation of the compound in the fatty tissue also depends on the

rate at which it is metabolised to produce a water soluble product which can be

excreted. The rate of metabolism of a compound depends on the structure of the

molecule and it has been found that the rate is slow especially with chlorinated

compounds.

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If we take as an example p, p'-DDT, the metabolism of this compound

occurs in two stages as shown in fig.2.

H

ccl2

cl cl clclc

ccl3

c

p,p'-DDEp-p' DDT

slow

cl c

H

cl

cooH

p,p'-DDA

(water solubility increased by the presence of cooH group)

Fast

Fig 2 Metabolism of p,p’-DDT



The first stage is rapid and normally takes only a few days for completion

but the second stage is extremely slow, often taking many months in some

species. It is because of this reason ,the first degradation product is often found

as the predominant species in environmental samples.

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The o,p'-isomer of DDT is present only as a minor component in

commercial DDT sample which however is metabolised rapidly by the reaction

shown in Fig.3.

H

cl cl clOHc

ccl3

c

o,p' DDT

Fast

ccl3

Hcl

(Water solubility increasedby the presence of OH group)

Fig 3. Metabolism of o, p’-DDT



In addition to DDT, other chlorine containing pesticides, polychlorinated

biphenyls and dioxins which are bioaccumulative compounds, there are a

number of non-bioaccumulative organic compounds which are suspected to be

carcinogens. A typical example is chloroform, which can be produced in trace

quantities during the disinfection of water by chlorination and which is thought to

be harmful at µg l -1concentrations.

Analysis of such compounds at such low levels requires pretreatment

procedure. These pretreatment procedures are necessary to remove potential

interferences and in many instances to increase the analyte concentration to

within the instrument sensitivity.

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The preconcentrations methods will be followed by chromatographic

separation of the components. As most organic compounds have significant

volatility even at room temperature, gas chromatography would be expected to

be a useful technique. The alternative of high performance liquid chromatography

is used only where there are advantages over established gas chromatographic

methods.

In the determination of groups of compounds such as phenols, and also of

classes of detergents, where the total concentration of the group of substances is

required rather than the concentration of individual compounds

nonchromatographic methods are used. Ultraviolet / visible absorption

spectrometric techniques can be used to determine the total concentration of

related classes of organic compounds.

A detailed description of gas chromatophic method is given in this chapter.

The liquid chromatographic methods, flame spectroscopic methods, ICP AES,

anodic stripping voltammetry amd chelation ion chromatography are discussed in

the next chapter.

(i) Gas liquid chromatographic methods:

The following precautions have to be taken in the storage and the subsequent

analysis of the organic trace pollutants:

a) To minimise the volalitisation of the organic sample (e.g pesticide) the sample

should be stored at 4oC, if the analysis is not performed immediately.

(b) Since the organic compounds are tend to undergo microbial degradation, it is

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better to store these compounds below 0oC in deep freezer to minimise the

microbial activity.

(c) Plastic sample containers should not be used for storing these compounds

because of the possibility of leaching of potential monomers and additives into

the samples and hence only glass containers should be used.

(d) The samples should be analysed as soon as possible after collection to

minimise the loss of the analyte by adsorption on the walls of the container.

The volume of the sample required for injection into a chromatograph is of the

order of a few microlitres and that for spectrophotometric detection a few mililitres

and this solution might have been obtained through a preconcentration step from

several litres of the original sample. Precaution has to be taken to avoid

contamination or loss of material at these low concentration during the

subsequent analysis.

Gas liquid Chromatography (GC):

Gas chromatography(GC) is a separation technique where volatile, thermally

stable solutes migrate through the column containing a stationary phase (which

is a liquid adsorbed on or chemically bonded to a solid) at rates dependent on

their distribution ratios. These are inversely proportional to their volatilities, which

in turn are determined by their partial vapour pressures and hence their boiling

points. Solutes are therefore generally eluted in order of increasing boiling

point,except where there are specific interactions with the stationary phase.

Usually an elevated temperature in the range of 50-350oC is normally employed

to ensure that the solutes have adequate volatility. Gas liquid chromatography

has high separation efficiencies and can be interfaced with highly specific

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sensitive detectors. The main components of a gas liquid chromatograph is

shown in fig.4.

In gas liquid chromatography, the mobile phase is a gas and the stationary

phase is a liquid adsorbed on or chemically bonded to a solid. In the liquid

stationary phase the solutes can dissolve and the sorption process is called

partition. Specific interactions of the solute in the stationary phase may alter the

order of elution from that of increasing boiling points. Gas liquid chromatography

has high separation efficiencies and can be interfaced with highly specific and

sensitive detectors.The main components of a gas chromatograph is shown in

fig.4.

Computer or recorder

Column oven

Detectoroven

column

Exit

Carrier gas

Injectoroven

Siliconerubberseptum

Injectorport

Detector

Fig 4 Components of gas liquid chromotograph

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Mobile phase:

The mobile phase is known as the carrier-gas because its sole purpose is to

transport solutes through the column, thus not contributing to chromatographic

selectivity. Commonly used carrier gases are nitrogen for packed columns and

hydrogen or helium for capillary columns.

Sample injection:

Small volumes of liquids or solutions (0.1-10 µl) are generally injected into a

heated injection port, through which the carrier gas continuously flows, from a

calibrated microsyringe used to pierce a self-sealing silicone-rubber septum. Split

and splitless modes of injection are used. In the latter the whole injection passes

straight to the column whereas in the former the sample is split in the ratios of

50:1 and upto 500:1 with a large part vented waste. This avoids overloading the

column and is especially an important facility where capillary column are used.

Column and stationary phase:

The column is the central component of a gas chromatograph since it is

where the separation occurs. There are two types of GC columns and they are

capillary (open tubular) and packed columns. Capillary columns consist of long

narrow bore, high-purity quartz tabing with a thin layer of liquid or stationary

phase coated or chemically bonded to the surface of inner wall. Very long lengths

(up to 100 m) can be used resulting in high efficiencies for the separation of

complex mixtures. Capillary columns are available with a range of internal

diameters and thicknesses of the stationary phase. The narrower the bore and

the thinner the coating, the greater the efficiency, but the lower the sample

capacity. Packed columns are much shorter than the capillary columns, rarely

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exceeding 2m in length. The stainless steel or glass tubing has an internal

diameter of 2 or 3mm and is filled with a granular material that acts a support for

a thin coating of a liquid stationary phase for GLC . Alternatively the liquid

stationary phase can be bound to the solid support through a chemical reaction.

Solid supports are inert, porous silaceous materials with a large surface area.

The particle sizes vary between 0.125 mm to 0.25 mm. The smaller the particle

size and the thinner the coating of stationary phase, the lesser is the band

spread.

Given below are the three types of columns with decreasing separation

efficiency (fig.5)

• Narrow-bore capillary columns:

Typical dimensions 30 - 60m length, 0.2 mm i.d, flow rate

0.4 ml min-1 of He

• Wide -bore capillary columns:

Typical dimensions 15 - 30m length, 0.53mm i.d., flow rate

2.5ml min-1 of He

• Packed columns:

Typical dimensions 2m length, 2mm i.d., flow rate

20ml min-1of He

Fig 5. Typical dimensions of capillary and packed columns

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Many of the organic compounds of environmental interest are of high

relative molecular mass and have low volatility. High oven temperatures are

necessary for these and consequently silicone polymers are often the favored

stationary phases. Best separation efficiencies are achieved when the stationary

phase has a similar polarity to the components of analyte. Fuel oils are separated

on non-polar columns (e.g .dimethylsilicone), pesticides and chlorinated solvents

are often separated on medium-polarity columns

(e.g. diphenyl / dimethylsilicone), whereas 2, 3, 7, 8- tetrachlorodibenzo-p-dioxin

can be separated from its isomers using highly polar columns (e.g.

cyanopropylsilicone).

In order to confirm whether peak observed in a chromatogram is due to a

single component rather than two, the chromatogram should be produced on two

columns with different polarities. Under such circumstance, it would be unlikely

that the peaks would remain unresolved on both column. Capillary columns

because of their greater separation efficiency decreases the probability

unresolved peaks.

Detectors:

Depending upon the characteristics of the analyte and the circumstances of

its determination different detectors are used in GC. The one with the most

comprehensive response is based on the change in thermal conductivity in the

mobile phase as a component is eluted. Principally this results from the mass

difference between the molecules eluted and those of the carrier gas. The

thermal conductivity detector is robust and reliable, but has only moderate

sensitivity and a limited dynamic range. The preferred option for general purpose

GC use, is flame ionisation detector (FID). The eluate stream is mixed with air

and hydrogen allowing combustion of most components. Ionic species produced

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in the combustion facilitate the passage of an electronic current between two

electrodes in contact with flame. The magnitude of current reflects the number of

ions and thus the amount of the eluted compound present. There are some

selective detectors which can detect only certain compounds, yielding a simple

chromatogram. The ideal situation for the determination is to have a detector that

can detect only this type of analyte. One such, is the electron capture detector

(ECD).

High energy electrons (b-particles) from a radioactive source such as 63 Ni or 3H, are used to ionise the carrier gas stream. As a result, in the absence

of an eluted component, a steady electronic current will flow between the

electrodes. An eluting component which is electronegative will 'capture' electrons

and reduce the current flowing, leading to a peak in the chromatogram. ECDs

are very sensitive detectors and have been extensively employed in the

determination of chlorinated pesticides. Another selective detector mentioned is

flame photometric detectors (FPD). It selectively monitors radiation emitted by

the species such as S2 and HPO produced in the combustion of sulphur or

phosphorous containing compounds.

Nowadays the interfacing of GC with dedicated mass spectrometers is

quite common. Not only the mass spectrometer can function as aid to peak

identification but also be adjusted for selective response to a single type of ion

originating in one component of the mixture.

Extraction procedures or sample preparation:

Although gas chromatography is primarily a technique for analysing

mixtures of volatile solutes, there are many compounds and materials that are

either nonvolatile, have volatile components in a non-volatile matrix or thermally

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labile. Direct injection of the sample would deposit nonvolatile solids on the

column which would cause blockage and shorter the column lifetime. In some

instances, samples may contain solutes at such low levels that they must be

preconcentrated prior to analysis.

Special extraction methods have been developed for handling such

samples and they are

(a) solvent extraction

(b) head space analysis

(c) purge and trap technique

(d) solid phase extraction

(a) Solvent extraction:

Extraction of a sample component into a small volume of organic solvent

can be highly selective and may serve to both concentrate and enhance

technique sensitivity. The water sample is shaken with an immiscible organic

solvent in which the analyte species are soluble. The organic layer is separated,

and after drying, is injected into the chromatograph. The extractions can be made

selective by adjusting the pH of the aqueous layer. For extracting selectively

acidic components the sample solution should be made acidic. Similarly for

extractive selectively basic components, the sample solution should be made

basic.

While choosing the solvent for extraction, the compatibility with the

detector also should be taken into account. For example, chlorinated solvents

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should not be used for extraction if electron-capture detector is used. For that

hexane or light petroleum will be suitable.

(b) Head space analysis: This involves chromatographing the vapours

derived from a sample by warming it in a partially filled vial sealed with septum

cap. After equilibration under controlled conditions, the proportions of volatile

sample components in the head space above the sample are representative of

those in the bulk sample. The head space vapours, which are under slight

positive pressure, are sampled by a modified automated injection system or gas

syringe, and injected on to the column. The procedure is useful for mixtures of

volatile and nonvolatile components, such as residual monomers in polymers,

alcohol or solvents in blood samples, and flavours and perfumes in manufactured

products, as it simplifies the chromatograms and protects the column from

contamination by non volatile substances.

(c)Purge and trap:

This is a procedure where volatile solutes can be collected on a solid

sorbent in a preconcentration step using a purge gas stream. The organics are

collected in a short tube of absorbent material such as activated charcoal, or a

porous polymer (e.g. Tenax). After the collection period, the tube is flash heated

to release the organics into the chromatograph.

(d) Solid phase extraction (SPE):

It is a relatively new techique and is used as a primary means of

sample pretreatment. The removal of interfering matrix components in general

and the preconcentration of trace and ultratrace levels of analytes are other

important uses of SPE. It is versatile, rapid and requires only small volumes of

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solvents, or none at all in the case of solid phase micro extraction. SPE has been

applied in the determination of pesticides and herbicides in polluted surface

water and soils, polycyclic aromatic hydrocarbons (PAHs) in drinking water,

polluted industrial and urban atmospheres, and drugs in biological fluids.

Most of the SPE is carried out using a small bed of sorbent

(25-500 mg) contained in a cartridge made from a polypropylene syringe barrel,

the sorbent being retained in position by polyethylene fritted disks. A typical

cartridge procedure consists of four distinct steps. They are

(1) Solvent conditioning which involves wetting the surface of the sorbent and to

create the same pH and solvent composition as those of the sample. (2) sample

loading in which the sample solution is passed through the cartridge with the

object of either retaining the analytes of interest whilst the matrix components

pass through or retaining the matrix components whilst the analytes pass

through.(3) Rinsing is necessary to remove all the components which are not

retained by the sorbent. (4) Elution is the final step to recover retained analytes.

A further development is the use of extraction discs where the

absorbent material is held with in a filter disc. The sample is passed through the

disc by suction and the solutes get extracted into the disc. By using a suitable

solvent the extracted components are eluted.

Analysis of Individual Trace Pollutants

In the analysis of individual components in a mixture which are present at

low concentrations, simple extraction of the analyte and injection of the extract

into the chromatograph is not sufficient. Further treatment of the sample is

necessary and it involves (1)extraction (2) clean-up to remove interfering

components (3) concentration of the extract.

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To illustrate the method the analytical scheme for the commercial

pesticide DDT is discussed in detail as an example.

Analysis of DDT:

DDT is now considered as a contaminant. Commercially available DDT

insecticide is not a single compound, the major active component (p-p'-DDT) only

being 70 - 80% of the total content. In addition it also contains (15-20%) o,p'-DDT

one of the minor components, p, p'-DDD (similar in structure to p,p'-DDT with a

CHCl2 side chain rather than CCl3) (1-4%) is, in fact more toxic to insects than

p,p'-DDT. Hence three peaks are expected in a chromatograph.

If environmental samples are considered a number of decomposition and

metabolic products will also be present. In fact for many samples, the highest

concentration component is not p, p' DDT but its primary metabolic product,

DDE. Thus after considering the environmental DDT samples, a total of atleast 5

peaks are expected including the peaks for the two decomposition products p,p'

DDE(aerobic decomposition) and p,p' DDD (anaerobic decomposition). In

addition the interfering components in the sample such as other pesticides and

polychlorinated biphenyls may be present.

For the analysis of individual DDT components, in such a sample the

following pretreatment is given before injecting the sample into GLC.

1. The organic components are extracted into hexane (A 40 fold increase in

concentration is achieved by extracting 2litre of the sample into 50ml solvent).

2. The hexane extract is dried using 5g sodium sulphate.

3. The sample is further concentrated to 1ml by partial evaporation on a steam

bath followed by bubling nitrogen.

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The dried sample is placed on alumina-silver nitrate column.

(a) Alumina-silver nitrate column

In this column polar components in the extract are retained. The silver

nitrate helps to retain compounds containing unsaturated carbon-carbon bonds.

Hence non polar materials including DDT components are eluted using 30ml of

hexane. The hexane extract is again concentrated to 1ml and placed on the top

of silica gel column.

(b) Silica gel column

This is a less polar column and therefore can be used to separate

potential non-polar interferences from the sample. By passing hexane (10ml)

through the column, polychlorinated biphenyls (PCBS) are eluted out, but the

DDT Components are retained in the column. DDT is then eluted with a more

polar solvent mixture (12ml of 10% diethyl ether in hexane).

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0 30 40 50

p,p'-DDE

o,p'-DDT

p,p'-DDT

Time (min)

25m x 0.32mm id methyl silicone capillary column with a temperature gradient to 220oC.

Fig 6 Chromatographic separation of DDT components.



The eluates are again concentrated to 1ml before injection into the

chromatograph. A typical chromatogram is shown in fig 6. The detection limit for

each component is approximately 10ngl-1.

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Technique for measuring total concentration of group of compounds:

This technique includes the measurement of total phenols, surfactants

(total, anionic, cationic and non-ionic surfactants), and total hydrocarbons. Visible

spectrophotometry is often used for phenols and surfactant analysis after the

formation of derivatives. IR absorption technique is used for the total

hydrocarbon concentration.

Phenols:

Phenol analysis is important in water pollution studies because of the

objectionable taste of chlorinated phenols in chlorinated drinking water containing

as little as 1 ppb. The effluents from coke-oven and coal distillation plants are the

major source for phenol pollution. The determination first involves the distillation

of phenols and other phenolic compounds from waste water. Then the distilled

phenol is allowed to react with 4-aminoantipyrene at pH10 in the presence of

K3Fe(CN)6. A red dye is formed which is extractable into chloroform. The

absorbance is measured at 460nm.

N + OH NOH 3C OH

The method is quite sensitive and has a detection limit of about 1ppb of phenol.

N 3C N

NH 2 H 3 C H N O3C

Red dye

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Surfactants:

Similarly anionic surfactants can be determined by spectrophotometry

using methylene blue method. Under basic conditions a salt is formed between

methylene blue and the surfactant and this salt can be extracted into chloroform.

The absorbance of the extract can be measured at 652 nm and the concentration

can be determined by comparison with a calibration graph.

Total hydrocarbons:

For the determination of total hydrocarbon content, the hydrocarbons are

extracted from acidified water using a non-hydrocarbon solvent such as

carbontetrachloride and the (IR) absorption is measured at 2920 cm-1,

corresponding to C-H stretching frequency.

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