BIOANALYTICAL

TECHNIQUES FOR
INORGANIC ANALYSIS

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The metal present in food and drinking water consumable by
human beings and of toxicological importance have received
considerable attention in recent years. The part played by
metals ions in various form in health and disease have
attracted attention of biomedical scientists. There is strong
evidence showing the relationship between natural
environment and various diseases.

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 IMPORTANCE OF WATER ANALYSIS

 The type and amount of metals and other materials found

in water to determine its usefulness. Ground water, lake,
river and other water resources can differ greatly in mineral
content, turbidity, pathogenic content, pH, alkalinity and
many other properties used as indicator for water quality.
 Because variation of aforesaid parameters from their
standard values, may lead to serious health hazards.
 It has also been reported that there is relationship
between hardness of drinking water and incidence of
Cardiovascular diseases. Also investigations carried out in
different countries reveals that death rates are higher in areas
which are supplied with soft water with low Magnesium
contents.
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Intake and excretion of water are controlled through
regulatory mechanisms that are also dependent on
electrolytes.
 Imbalances of fluid distribution lead to fluid volume
deficit or fluid volume excess with related health effects.
The quality of drinking water is regulated in most
countries under recommendations and legal requirements
for developing countries framed by World Health
Organization (WHO) recommended in 1984 and onwards
Drinking water is most important material for human
consumption and as such must be free of bacterial,
pathogenic organisms, toxic metals and toxic organic
compounds.

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Food taken by human beings in various form is
important for the regular supply of energy to body to
regulate most biological systems. Studies by various
scholar have proved that inorganic elements are also
involved in many physiological and metabolic functions of
the human body and for maintaining normal health.
 Due to environmental pollution it is becoming evident
that food may also contain toxic elements due to
biocycling for which the body has a tolerance level and
beyond this level toxicity may adversely affect the
biochemical systems.

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 Milk
Vegetables and fruit
Juices
Soft drink
Meat products
Oil and fats
Spices
Grains
And others

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 Minerals in milk
 Depends on feed, season, breed, stage of
lactation, infections
 Mineral content constant at 0.7%
 Milk contains much more potassium than
blood plasma
 Some minerals in milk present at levels
beyond solubility – thus in colloidal form (e.g.
Ca, Mg, phosphate, citrate)
 Colloidal minerals removed by
 Precipitation with curd when milk is coagulated
with rennin
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 4. Metal uptake in canned foods
 Canned foods take up minerals from container
 Tin & iron from the tin plate
 Tin and lead from the solder
 In acid foods canned in the absence of O2, tin
forms the anode of the tin-iron couple → tin
goes slowly in solution → protect product for 2
years or longer
 Rapid deterioration
 When iron forms the anode → in presence of
oxidizing agents dissolution of tin is accelerated →
after all tin is in solution, H2 forms → can swells →
and leak out
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How to diagnosis metals in human body

Biological Samples
Body Fluids: Blood, CSF
(serum, erythrocytes)
Excretory by Products
(Urine, sweet, feaces)

Body Tissue
(Biopsy material, hair& nails)

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 SAMPLE PREPARATION FOR
INORGANIC ANALYSIS
 Wet Digestion Methods
1) Acid Digestion—Wet Ashing
2) Microwave Digestion
3) Pressure Ashing
 Dry Ashing
 Ultrasonic Sample Preparation
 Advance extraction methods

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 DRY ASHING
 Dry Ashing involves heating of the sample
with high organic matter in a silica or
porcelain crucible in a muffle furnace under
normal atmosphere at 400-800°C.
 This allows organic matter to be destroyed.
 After decomposition, the residue is dissolved
in acid and transferred to a volumetric flask
prior to analysis .
 However, the method may also lead to the
loss of volatile elements, e.g .Hg, Pb, Cd, Ca,
As, Sb, and Cr.
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 Dry Ashing: Disadvantages
 losses due to volatilization
 resistance to ashing by some materials
 difficult dissolution of ashed materials
 high risk of contamination
 It has largely been replaced by wet ashing

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 Sample Preparation Steps Prior to
Analysis of Metals
Sample Preparation Instruments

Extraction, Atomic Absorption (AA);

concentration, Graphite Furnace Atomic
speciation Absorption (GFAA);
Inductively Coupled Plasma
(ICP); ICP/MS
Extraction, UV-VIS molecular absorption
derivatization, spectrophotometry

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 Major functions of sample
preparation are:
 To degrade and solubilize the matrix, to release all
metals for analysis.
 To extract metals from the sample matrix into a
solvent more suited to the analytical method to be
used.
 To concentrate metals present at very low levels to
bring them into a concentration range suitable for
analysis.
 To separate a single analyte or group of analytes
from other species that might interfere in the
analysis.
 To dilute the matrix sufficiently so that the effect of
the matrix on the analysis will be constant and
measurable.
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 Reagents Commonly Used in Sample
Dissolution or Digestion
Reagent Sample Type
Water Soluble salts

Dilute acids Dry-ashed sample residues, easily

oxidized metals and alloys, salts
Concentrated acid Less readily oxidized metals and
(e.g., HNO3) alloys, steels, metal oxides
Concentrated acid with Metals, alloys, soils, particulates
added oxidizing agent from air, refractory minerals,
vegetable matter
Hydrofluoric acid Silicates and other rock samples
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1 January 2020 16
 ‘Digestion’ and ‘extraction’.
 Digestion infers the complete destruction
of the sample matrix (organic matter )
whereas
 Extraction considers the liberation from
the matrix of the analyte of interest
(without the requirement for complete
destruction of the matrix).
 In the latter case, it may still be possible
to identify ‘organic’ parts of the matrix by
using appropriate techniques.
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Extraction and acid digestion

Acid mixtures used for

digestion. The least
aggressive mixture
that digests the sample
thoroughly should be used.

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 Microwave Digestion
 Two types of microwave heating
systems commercially available,
 Such as:
 a closed-vessel system
 open-focused

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 Schematic of a pressurized
microwave digestion system.

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 Microwave Assisted Closed Vessel
Digestion - advantages
 Closed Vessel / sealed containers,
fabricated of high-temperature polymers
 chance of airborne dust contamination is
eliminated.
 The sealed, pressurized containers reduce
evaporation, so that less acid digestion
solution is required, reducing blank values.

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 Microwave Assisted Closed Vessel
Digestion - procedure
 In a typical application, 0.2 to 2 g of sample is dried,
weighed, and loaded into an extraction vessel. A
certain amount of select solvents is also added. Then
parameters such as temperature, pressure, and
extraction time are set according to the instructions
from the microwave manufacturer. A preextraction
heating step (typically, 1 to 2 minutes) is needed to
bring the system to the preset values. Subsequently,
the samples are extracted for about 1-2 to 20 minutes.
After the extraction, the vessels are cooled, and this
normally takes less than 20 minutes. Finally, the
extract is filtered, concentrated, and analyzed.
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 Sound waves are mechanical vibrations in a solid, liquid or
gas and are intrinsically different from electromagnetic
waves. While the latter (radio waves; infrared, visible or
ultraviolet light; X-rays; gamma rays) can pass through
vacuum, sound waves must travel in matter, as they
involve expansion and compression cycles traveling
through a medium. Expansion pulls molecules apart,
whereas compression pushes them together

‘‘cavitation’’. The whole process

takes place within about 400 ms

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-Ultrasound energy causes its chemical effects through the
phenomenon of cavitation, which consists of the production of
microbubbles in a liquid when a large negative pressure is applied
to it.
-When extraction applications of ultrasound are concerned,
particle fragmentation can enhance the ability of the extractant to
leach metals.

-The leaching is facilitated by the generation of reactive species,

e.g. radicals, as a result of the ultrasonic irradiation. Thus, when
water is sonicated H and OH radicals are formed, which in turn
result in the formation of oxygen gas and hydrogen peroxide.
Metals may be encapsulated within cell walls that must be broken
down by the combined effects of dilute acid attack and
ultrasonication in order to bring metals into the liquid extractant
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The collapse of bubbles created by the
sonication of solutions results in the generation
of extremely high local temperature and
pressure gradients, which may be regarded as
localized ‘‘hot spots.’’
Suslick et al. estimated the temperature of these hot
spots to be about 5000C ^ similar to the surface of the
sun. The pressure is roughly 1000 atm, equivalent to
that at the Marian Trench the deepest point in the
ocean. The size of the bubbles is very small relative to
the total liquid volume, so the heat that they produce
is rapidly dissipated with no appreciable change in
environmental
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 Speciation and fractionation
Studies
Speciation is defined as 'the process of
identifying and quantifying the different defined
species, forms or phases present in a material' or
'the description of the amounts and types of these
species, forms or phases present' .
In some cases, it is possible to identify, by using
single or sequential extractions, operationally
defined determinations which identify 'groups' of
metals without clear identification. The reasons
why speciation is important is that metals and
metalloids can be present in many forms, some of
which are toxic.
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Speciation Studies
One approach to investigate the speciation of metals and
metalloids in environmental samples has been the linking of
chromatographic separation with quantitation by atomic
spectroscopy. In this situation, the use of a suitable
chromatographic technique, e.g. gas or liquid
chromatography, is being used to separate a metal complex
prior to detection of the metal by an atomic spectroscopic
technique, e.g. inductively coupled plasma–mass
spectrometry. However, while chromatography is capable of
highly reproducible separations, it can only accept liquid
samples. In this situation, the use of specific methods of
extraction are required to remove the extract, without
altering its chemical form (speciation) from the sample
matrix.
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 Stainless steel is not used for sampling of, liquid
samples, blood and scalp hair For Cr and Ni
analysis

Glass should not be used for collection of, juices,

drinks, milk, serum for Al and Pb Measurement.

plastics with a Cd-based softener are to be

avoided when sampling for Cd measurement

Zn-doped stoppers should not be used when Zn is

to be measured
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