Submitted by:Sharath. H.N

M Pharma 1st year

Bacteriological and chemical analysis of Water

Contents

• Introduction to Analysis of water• Bacteriological Analysis of Water• Chemical analysis of water• Solid phase extraction

• One of the things which make Earth a unique planet in this universe is continuous availability of water, a vital

• requisite for the existence of life. Water is also the essential prerequisite of agriculture and industrial production, the source

• of food needed for the survival of life. Thus, life on earth is entirely and exclusively dependent on water.

• Water-related diseases continue as a major health problem globally. An estimated, four billion cases of diarrhea yearly represented 5.7% of the worldwide disease in the year 2000 (WHO, 2002).

• So Physical , chemical and bacteriological analysis of water was of great need for beneficity of people.

WHO Definition

Defines safe drinking water as water that “does not represent any significant risk to health over the lifetime of consumption, including different sensitivities that may occur between life stages.”

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Categories of Contaminants

Chemicals

Microbiological

Physical

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Categories of Contaminants

Chemicals· Organic· Inorganic· pH

Microbiological· Bacteria· Virus· Protozoa· Helminths

Physical· Turbidity· Colour· Odor· Taste

Water Sampling-Microbiological sampling-Indicator organisms for pathogen presence

Physical sampling-turbidity, conductivity, total dissolved solids etc

Chemical sampling- pH, dissolved oxygen, phosphates, chemical oxygen demand, biological oxygen demand, mineral impurities (iron, manganese, chloride, lead, sodium etc)

MICROBIOLOGY OF WATER

Pathogens

• 4 types of pathogens– Bacteria– Virus– Protozoa– Helminths

bacteriaa on the tip of a pin

micro-organism that cause disease

FACTORS AFFECTING NUMBER AND TYPE OF BACTERIA IN WATER

•Type of water:Surface or deepMineral springs

•Presence of organic matter•Temperature•Light•pH•Dissolved oxygen•Rainfall•Season•Storage•Filtration

Microbiological Testing Methods

• 3 methods to determine presence of bacteria in water:– Presence-Absence (P-A)– Most Probable Number (MPN)– Membrane Filtration

Presence-Absence (P-A)

• Simplest method• Add water sample to a bottle containing broth and let it sit

for 24-48 hours• Color will change if indicator organism is present• Does not show numbers of bacteria!• If the sample is positive, the water should be re-tested using

membrane filtration to determine the number of bacteria• Not recommended by WHO for analysis of surface water

and untreated community water supplies• Not recommended for testing the efficiency of household

water treatment technologies (e.g. biosand filter)

Presence-Absence

Positive for E. coli

Negative

Positive for Coliforms

Most Probable Number (MPN)• Tells the number of bacteria that are most likely to be

in the water sample

• Add water sample or diluted sample to 5 or 10 or test tubes (or larger tray 50 – 96 tubes)

• Incubate for 24-48 hours

• Gas production and/or cloudiness will be visible if the indicator organism is present

• Using a table provided, report the number of positive tubes as number of colonies per 100 mL of sample.

• Typically used for wastewater or turbid samples

Most Probable Number (MPN)# Positive

TubesMPN Index

(CFU/100mL)0 <1.11 1.12 2.23 3.64 5.15 6.96 9.27 12.08 16.19 23.010 >23.0

Sample Table for 10 tube test

Most Probable Number (MPN)

• Characteristics:– Quantitative results– Simple to understand and use– Relatively inexpensive– Can be used with turbid water– More labor-intensive than P-A– Requires some training– Requires incubator & other equipment

Membrane Filtration• Most accurate method to count bacteria• Filter 100 mL of a water sample

– Add broth to a Petri dish which provides nutrients for the indicator organism to grow

– Filter the water using the filtration equipment– Transfer the filter paper to the Petri dish – Incubate for 24-48 hours depending on the broth

• If the indicator organism is present, colonies will appear on the filter paper and can be counted

• Results are reported as the number of colonies per 100 mL of water sample (CFU/100mL)– CFU = colony forming units

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Membrane Filter Technology

• A membrane is a thin material that has pores (holes) of a specific size

• Membranes trap larger particles that won’t fit through the pores of the membrane, letting water and other smaller substances through to the other side

Membrane Filtration EquipmentField Kits

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Membrane Filtration Equipment

Incubator

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Membrane Filtration EquipmentNalgene Testing Kit

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Membrane Filtration• Advantages:

– Able to count the number of bacteria– Most accurate test method– Ability to test many samples at once– Internationally recognized method– Rapid– Easy & Economical.– Gives direct result. – Useful in rural areas.– Samples can be tested in the field

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Membrane Filtration• Limitations:

– More labour intensive than MPN, P-A– Requires more training– Requires additional equipment– Cost of consumables can be high in many countries

– Turbid water interferes with bacterial growth.

– Noncoliforms interferes with counting of coliforms.

– Toxic substances in the water may be absorbed by filter and interferes with bacterial growth.

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Chemical testing of water

• Chemical Testing Methods– Test strips– Colour disc comparators– Colorimeters & photometers– Digital meters– Arsenic specific kits

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Chemical Tests

• There are many different chemicals that can be found in our drinking water

• Difficult and expensive to test for all chemicals so we need to select a few that are a priority in the local area– Iron, Manganese– Arsenic, Fluoride– Chlorine– Total Dissolved Solids

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Chemical Test Methods

• Test (reagent) strips• Colour disc comparators• Colorimeter and photometer• Digital meters

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Test (Reagent) Strips• Designed to react with

specific chemicals– pH, chlorine, hardness, etc.

• Compare colour on stick to colour chart

Advantages:– Inexpensive– Easy and simple– Provides rough estimate

Limitations:– Requires visual interpretation of

colour– Low accuracy +/- 10 %

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Colorimeters & PhotometersPhotometer (Wagtech)

• Uses light source to measure chemical concentration

• Test a range of chemicals

Advantages:– More accurate

Limitations:– More expensive– Power source necessary– Proper training required

Colorimeter (HACH)

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Arsenic Test Kits• Designed specifically for

arsenicAdvantages:

– Fairly accurate – range 2 to 100 ug/L

– Portable– Relatively easy to use

Limitations:– Requires visual interpretation

of colour– Expensive

Solid phase extractionSolid phase extraction (SPE) is an increasingly useful sample

preparation technique. With SPE, many of the problems

associated with liquid/liquid extraction can be prevented, such as

incomplete phase separations, less-than-quantitative recoveries,

use of expensive, breakable specialty glassware, and disposal of

large quantities of organic solvents.

SPE is more efficient than liquid/liquid extraction, yields

quantitative extractions that are easy to perform, is rapid, and can

be automated. Solvent use and lab time are reduced.

SPE is used most often to prepare liquid samples and extract semivolatile or nonvolatile analytes, but also can be used with solids that are pre-extracted into solvents.

SPE products are excellent for sample extraction, concentration, and cleanup. They are available in a wide variety of chemistries, adsorbents, and sizes.

Selecting the most suitable product for each application and sample is important.

Based on types of seperations solid phase extraction was classified into 4 types

•Reversed Phase•Normal Phase•Ion Exchange•Anion exchange•Cation exchange

Reversed Phase SPE

Reversed phase separations involve a polar or moderately polar sample matrix and a nonpolar stationary phase.

The analyte of interest is typically mid- to nonpolar. Several SPE materials, such as the alkyl- or aryl-bonded silicas (LC-18, ENVI-18, LC-8, ENVI-8, LC-4, and LC-Ph) are in the reversed phase category.

Here, the hydrophilic silanol groups at the surface of the raw silica packing (typically 60Å pore size, 40μm particle size) have been chemically modified with hydrophobic alkyl or aryl functional groups by reaction with the corresponding silanes.

Normal Phase SPE

Normal phase SPE procedures typically involve a polar analyte,a mid- to nonpolar matrix (e.g. acetone, chlorinated solvents, andhexane), and a polar stationary phase Polar-functionalized bondedsilicas (e.g. LC-CN, LC-NH2, and LC-Diol), and polar adsorptionmedia (LC-Si, LC-Florisil, ENVI-Florisil, and LC-Alumina) typicallyare used under normal phase conditions.

Retention of an analyte under normal phase conditions is primarily due to interactions between polar functional groups of the analyte and polargroups on the sorbent surface.

Ion Exchange SPEIon exchange SPE can be used for compounds that are charged when in a

solution (usually aqueous, but sometimes organic).

Anionic (negatively charged) compounds can be isolated on LC-SAX or

LC-NH2 bonded silica cartridges. Cationic (positively charged) compounds

are isolated by using LC-SCX or LC-WCX bonded silica cartridges.

The primary retention mechanism of the compound is based mainly on the

electrostatic attraction of the charged functional group on the compound to

the charged group that is bonded to the silica surface.

Anion Exchange SPE

The LC-SAX material is comprised of an aliphatic quaternary amine group that is bonded to the silica surface.

A quaternary amine is a strong base and exists as a positively-charged cation that exchanges or attracts anionic species in the contacting solution — thus the term strong anion exchanger (SAX

Cation Exchange SPE

The LC-SCX material contains silica with aliphatic sulfonic acid groups that are bonded to the surface.

The sulfonic acid group is strongly acidic (pKa <1), and attracts or exchanges cationic species in a contacting solution – thus the term strong cation exchanger (SCX).

References:•International Journal of Innovative research in Science, Engineering and Technology•WHO Guidelines•SUPELCO Pharma bulletein•Internet source

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