Water As A Reagent

I. Introduction:
The quality of the analytical results produced by the laboratory depends on
a. The purity of chemicals used as analytical reagents.
b. The availability and quality of the reference materials used to calibrate assays.
The solutes and solvents used in analytical work are reagent grade chemicals,
among which water is a solvent of primary importance. Preparation of most regents and
solutions used ion the clinical laboratory requires pure water. Now the term Pure Water
is replaced by Reagent Grade Water followed by designation of type I, type II & type
III, which defines the specification of the water and is independent of method of
purification.
II. Grades of Water:
The specifications / guidelines proposed by NCCLS (National Committee of
Clinical Laboratory Standards) for the three grades of water are as follows
Type I

Type II

Type III

Microbial Content

< 10

< 1000

NS

pH

NS

NS

5-8

Resistivity M/cm

> 10

>2

> 1.0

< 0.05

< 0.1

< 1.0

Particulate Matter Water passed through 0.2 filter

NS

NS

Organic

NS

NS

Silicate mg/L

Activated Carbon

*Efficacy of deionizer is determined by the resistivity of product water. A single

deionizer is capable of producing a resistivity of excess of 1 M/cm, when connected in
series and mixed, deionizer produces water with a resistance > 10 M/cm.

III. Methods used for purification of Water:

1.Distillation Process:
Process:
Vaporizing & condensing liquid to purity or concentrate a substance or to separate
volatile from non volatile substances

Advantages:
Removes dissolved coionized solids particulate matter pyrogens or toxins, micro
organisms and to some extent dissolved ionized gases or substances with low boiling
point.
2.Deionization:
Process:
Removal of ions to produce mineral free deionized water by the use of mixed bed
residue charged with hydrogen and hydroxyl ions.
Advantage: Removes effectively dissolved ionized solids gases.
Disadvantages: Fails to remove organic matter, particulate matter, organisms, pyrogens
and toxins.
3. Reverse Osmosis:
Process: Water is forced through a semi permeable membrane that acts as a molecular
filter.
Advantage: (Similar to distillation) Removes dissolved ionized solids organics
particulate matter microorganisms.
Disadvantages: Does not remove dissolved ionized gases.
4.Carbon Adsorption: Removes only dissolved organics. Activated carbon removes
dissolved chlorine gas alone.

Organics: Best is ozone treatment UV oxidation (185nm) carbon distillation reverse

osmosis.
5.Filtration Techniques:
In normal filtration techniques water is passed through semi permeable membrane
of pore size more than 6.22 m. In Ultra filtration the pore size 0.22 m optimally (0.2
m). In the nano filtration < 0.1 m diameter pore sized membrane is used.
Advantages: Filtration removes particulate matter, micro organism.
Ultra Filtration: Removes particulate matter, micro organism and pyrogen and
endotoxin
Nanofiltration: Removes particulate matter, micro organism, dissolved solid organic
matter.
Disadvantages: None of them removed dissolved ionized gases.
6. Chemical Oxidation: Effective in removing microorganism, pyrogens ,endotoxins.
UV Oxidation & Sterilization:
UV oxidation (batch process systems) use 185nm UV light is effective in
removing dissolved organism, microorganisms.
UV sterilization use 254 nm UV light is effective actively as bacteriocidal/
bacteriostatic method. Needs filter again to remove the organisms.
7.Ozone:
Advantages: Effectively removes organic matter. Instantaneously lyses bacteria.
Disadvantages: should be coupled with a deionizer.
But ozone is incompatible with deionizer; hence it should be removed by UV
treatment before deionizing.
Choice of purification methods:
Depends on the
1. Quality of feed water (Source)

2. Quality of the end point required.

In general a filtration process followed by double distillation and deionizers is
employed to get grade I water.
IV. Uses of varies grades of Water:
Type I Water:
Used in methods requiring minimal interference and maximal precision and
accuracy. Includes metal, enzymes, electrolytes measurement and preparation of
calibrators and solutes of reference material.
Type II Water:
General laboratory testing not requiring grade I Water.
Type III Water:
Glassware washing.
Urinanalysis.
Storage and Handling of Water:
Type I water:
Cannot be stored. Should be immediately used because Resistivity decreases.
Ionic and organic contaminants will be leached from the container.

Microbial

contamination will occur.

Type II water:
Can be stored for sometime. Following instruments are to be followed if storage is
carried out. Storage distribution systems should be made of materials that minimize
bacterial

contaminant. When

water

is

removed

from

storage

to

secondary

vessel. Secondary vessel and water has to be replaced everyday.

Care should be taken to avoid touching the inside lid or dipping a pipette directly
into the primary vessel. Unused portions of water must not be returned to be primary
container.

V. Quality control and impurity testing:

Water must be monitored at regular intervals to monitor the performance of water
purification systems.
No single test can measure water purity.
It includes a battery of tests which includes.
Microbial Monitoring:
Weekly testing should be performed.
For collecting an appropriate sample the spigot should be fully opened for 1 minute, 1ml
(for bacteriological samples) to 100 ml (Standard Plate Count) may be collected.
Sample should be immediately processed.
Should not be kept for more than 1 hour at room temperature or 6 hrs in refrigerator.
Vortexing can be done ensure uniform mixing of the organism.
b. Resistivity :
Used to assess the ionic content of purified water. Higher the ion concentration,
lower the resistivity. Ion exchange tanks should be equipped with an inline resistivity
lamp which goes off when the resistivity goes off below 2 M/cm, at which point the
capacity of tank is exhausted. For grade I water, an inline resistivity meter which reads
till 18 M/cm should be used. Should measure a minimum of 10 M/cm for grade I
water. For grade II & III offline meters can be used.
pH:
Not routinely required. Can be used when a doubtful pH problem arises.

Pyrogens :
Some immunoassays are interfered by pyrogens. In such cases, it can be routinely
monitored.
Silica:
Interferences with trace metal and electrolyte analyses, enzyme determinations.
Can be measured by spectrometrical analysis.
Organic Contaminants:
Purification system is not maintained properly can act as a source of organic
contaminants.
Hence instead of using those non specific (Permanganate)/cost (HPLC)
techniques for assessing organic constituents, it is better to maintain the system
optimally or couple it with UV Oxidation techniques.
Comparison of Water Purification Process.
Purification
process

Major Class of Contaminants

Dissolved
Dissolved
Dissolved Particulate
ionized Solids ionized Gases Organics Matter

Distillation

Micro
Pyrogen
Organism /Endotoxin

+
+

+
-

+
-

+
-

+
-

Reverse
Osmosis

Carbon
Absorption

Chemical
Oxidation

+
-

+
+

+
+
+
-

+
+
+
+

+
+
-

Deionizer

Filtration
Ultra Filtration
Nano Filtration
UV Oxidation
UV
Sterilization