FFS CO 2 AALYTICAL CHEMISTRY (1+1)

Introduction-Analytical properties & Classification of analytical

methods

Analytical chemistry deals with qualitative and quantitative analyses. The former shows

the nature of elements or ions present in a given substance. The latter determines the

quantity of individual elements or ions resent in a given substance. The latter determines

the quantity of individual elements or compounds present in a given substance. The

results of quantitative analysis are usually expressed in percentage or often expressed in

percentage of their respective oxides.

A variety of methods, viz., chemical physical and physico-chemical can be used

for qualitative analysis.

In chemical methods of qualitative analysis, the element or ion to be detected is

converted into some new compound which has specific properties on the basis of which

its formation can be elucidated.

A distinction is made between macro, micro, semimicro and ultramicro methods

of qualitative analysis depend on the amounts of substances used for the analytical

reactions.

In macro analysis relatively large amounts of substance (0.5 – 1.0 g) or solution

(20-50 ml) are used. The reactions are carried out in ordinary test tubes, beakers or

flasks. Precipitates are separated from solutions by filtration through filter papers.

The amount of substances used in micro analysis is usually about 1/100 th of the

amount involved in macro analysis work. Semi micro analysis occupies an intermediate

position between the macro and micro-methods. Semi micro analysis has a number of

advantages over macro analysis and gives equally reliable results with careful working.
 Ultramicro analysis is used for investigation of less than 1 mg of substance. The

various analytical operations are performed under the microscope.

Based on the nature of analysis, quantitative analytical methods are grouped as

chemical analysis (inclusive of volumetric, gravimetric and gas analysis),

physicochemical analysis (inclusive of colorimatric, nephalometric, electrochemical,

chromatographic etc.) and physical methods (inclusive of quantitative sepectroscopic

analysis, luminescence analysis etc.).

Chemical analysis are classified as macro, (25 mg) semi-micro (10-15 mg) micro

(3-5 mg) submicro (1-3 mg) and ultramicro (1 mg ) in accordance with the quantity of

sample needed for analysis.

Based on the concentration of the element or constituent in the sample to be

analysed, the methods are also grouped as major (1-10 Per cent ), minor (Less than 1 per

cent ), trace (1-100 ppm) and ultra trace (1 ppm).

The mere common quantitative analytical methods include (i) volumetric analysis,

(ii) gravimetric analysis and (iii) instrumental methods of analysis.

Law of Mass action and Concept of pH and pOH-dissociation of water

Acids/bases/buffer solutions

Soil pH

pH is defined as the negative logarithm of hydrogen ion concentration or simply the

log of the reciprocal of the hydrogen ion concentration (Sorenson, 1906).
pH = -log (H+) = log 1 / (H+)

The acidity or alkalinity of a so1ution can be expressed on the scale of acidity

and alkalinity. The scale of acidity or alkalinity is called pH scale. The unit of this scale is
called pH value. This scale runs from 0 to 14. The neutral point in this scale is at pH 7.
All the values above pH 7 represent alkalinity and below 7 denote acidity. The degree of
alkalinity increases as values go above pH 7 and the degree of acidity increases as the
pH decreases below 7.

Classification

pH values Classification
<6.5 Acidic
6.5-7.5 Neutral
>7.5 Alkaline

Sorenson (1969) suggested that H ions concentration be generally expressed as

the numerical value of the negative power to which 10 must be raised in order to express
the required concentration and this value be designated by the symbol pH. Thus,
technically pH is the negative logarithm of the H ion concentration or the logarithm to the
base ten of the reciprocal of hydrogen ion concentration
i.e. H+ = 10 - pH
log [H+] = -pH log 10
- pH = log [H+] / log 10
pH = log [H+] / log 10
Since log 10 =1, therefore pH = -log [H+] OR pH = log 1/ [H+]

Potentiometry and conductometry

Determination of soil pH
The pH is defined as the negative logarithm of hydrogen ion concentration or
simply the log of the reciprocal of the hydrogen ion concentration (Sorenson, 1906).
pH = -log (H+) = log 1/(H+)
Principle
A glass electrode in contact with H+ ions of the solution acquires an electric
potential which depends on the concentration of H+ ions. This is measured
potentiometrically against some reference electrode which is usually a calomel
electrode. The potential difference between glass electrode and calomel electrode is
expressed in pH units.
Two electrodes are used in the determination of pH. One is reference electrode
which provides a standard voltage. The reference electrode is usually a saturated
calomel electrode which has two layers (1) Saturated solution of KCl and (2) mixture
of solid HgCl2 and Hg. The outer tube is usually 5-15 cm long, 0.5-1 cm in diameter.
The mixture of solid HgCl2 + Hg paste is contained in an inner tube that is connected
to the saturated KCl solution in the outer tube by means of small opening. The
resistance of this type of electrode is 2000-3000 ohms.
The outer electrode is glass electrode that consists of a tube enclosing a lead
wire made of Ag coated with AgCl. This wire is again enclosed in a wax insulation. To
the tube at the bottom is attached a glass bulb made of a special kind of glass which
is sensitive to H ions. The thickness of the glass membrane varies from 0.03 to 0.1
mm and has a resistance of 50 to 500 mega ohms.
When these two electrodes are dipped in solution, the saturated solution of KCl
comes out of reference electrode through the small holes and forms an invisible ionic
bridge between electrodes through which current passes. The H+ ions are absorbed
by glass electrode and depending on the amount of H+ ions present in the solution, an
electric potential develops between electrodes. This potential difference is measured
in terms of pH by suitable galvanometer.
Apparatus
1. pH meter
2. 100 mL beakers
3. Glass rod
Reagents
1. Buffer solution (pH 4.0, 7.0 and 9.2)
Procedure
Standardization of pH meter

Switch on the instrument and allow it to warm for 10 minutes

Keep the pH selector switch on zero position

Set the temperature compensation control to the solution temperature

Adjust the zero adjustment knob so that the pointer in the meter reads
exactly zero, when the electrodes are immersed in distilled water.

Lift the electrodes from distilled water and wipe it dry using filter paper
and dip them in standard buffer solution of known pH (4.0, 7.0 and 9.2)

Change the function switch to particular pH range (0-7 or 7-14) and
adjust the standardization knob till the pointer reads the correct pH value
of the buffer solution. Do not disturb the zero knob adjustment.

pH measurement

Weigh 20 g of air dry soil passed through 2 mm sieve and transfer to a clean
100 mL beaker

Add 50 mL of distilled water

Using glass rod, stir the content intermittently and allow it to stand for half an
hour.

Wash the electrodes carefully with a jet of distilled water and wipe it dry with
a piece of filter paper.

Stir the soil suspension again just before taking the reading.

Immerse the electrodes into the beaker containing soil water suspension and
change the function switch to the particular pH range.

Record the meter reading.
Determination of Soil E.C
The electrical conductivity (E.C) measurement gives the total amount of
soluble salts present in the soil and is expressed as millimhos/cm or dSm-1

Principle
As the amount of the soluble salts in a solution increases the electrical
conductivity also increases. This electrical conductivity is measured in terms of
the resistance offered to the flow of current using a conductivity bridge.
It is known that solutions offer some resistance to the passage of electric
current through them, depending upon the concentration of salts present. Hence
EC is measured in terms of Electrical resistance between parallel electrodes
immersed in the soil suspension of water. In such a system, the solution
between the electrodes becomes the electrical conductor to which the physical
laws relating to resistance are applicable. The electrical resistance “R” is directly
proportional to the distance “L” between the electrodes and inversely
proportional to the cross sectional area “A” of the conductor.
Hence R = L/ A or R= r x L/ A
Where r = proportionality constant known as electrical resistivity
If L = 1 cm and A = 1 cm2 then R = r.
Where r is called specific resistivity. Hence specific resistance is the
resistance of a conductor 1 cm in length and 1 cm2 in area.
Higher the salt content, higher the passage of current and lesser the
resistance to the flow of the current. Hence the reciprocal of specific resistivity is
called as specific conductivity. Therefore specific conductivity is defined as the
conductivity of a solution enclosed in a cell whose electrodes are exactly 1 cm
and possess a surface area of 1 cm2. The resistance is expressed as ohms/cm
and the conductivity is expressed in reciprocal ohms or mhos per cm. It is not
possible to make a conductivity bridge having electrodes 1 sq.cm. in area and
place exactly 1 cm apart. Hence, the factor called the cell constant is determined
for the given cell. Modern conductivity meters are calibrated to read directly the
electrical conductance with given cell.
Apparatus
1. Conductivity bridge
2. 100 mL beaker
3. Glass rod

Reagents
1. 0.01 N KCl solution
2. Saturated CaSO4 solution
Procedure
• Switch on the conductivity bridge and wait for 10 minutes
• Check the instrument, with saturated CaSO4 solution and 0.01N KCl solutions
 • The E.C of CaSO4 and KCl solutions should be 2.2 dSm-1 and 1.41 dSm-1 respectively
• Use the same soil water suspension used for measuring pH for the determination
of E.C.
• Stir the contents and allow the soil to settle for 15 minutes
• Wash the electrodes carefully and immerse them into soil solution
• Adjust the temperature correction
• The readings on the scale at this position indicate the electrical conductivity
• Multiply this by the cell constant (noted on the cell itself) to get specific
conductivity.

Lectures 10&11 Theory Of Indicators

Indicators are chemicals used in volumetric analysis to indicate the end point of a
reaction. They are classified as
a) Self indicators b) Internal indicators and c) External indicators d. Mixed indicators

Self indicators
Any one of the two solutions involved in the titration will produce a colour
change at the end point eg. titration involving KMnO4.
Internal indicators
These indicators are directly added to the solution being titrated. These are acted
upon only at the end of the reaction.
eg. Phenolphthalein, methyl red etc.
External indicators
These are not directly added. If they are added directly they will be acted upon
immediately by the reagents involved. In this case a few drops of the solution from the
titration flask are taken out from time to time and tested outside with the indicator
solution for completion of the reaction. eg. Potassium ferricyanide in the estimation of
Fe by titration with K2Cr2O7.
Mixed indicators
In some titrations involving weak acids Vs weak bases, the end point cannot be
easily detected by using a single indicator. In such cases one indicator is mixed with
another indicator or an indicator is mixed with an organic dye. Either one or both of
them may be sensitive to pH. such mixed indicators enable us to detect the end point
more accurately even within a narrow range of pH.
eg. Methyl green and Phenolphthalein = pH 8.4 - 8.8
red-violet at pH < 8.4, grey pale blue at pH 8.4 - 8.8 and pink above pH 8.8.
Universal indicators
Mixture of two or more indicators. These are not employed in quantitative
analysis. These are employed to determine the approximate pH range of a solution.
They are available in two ranges viz., narrow where the colour change is in the pH range
of 0.2 to 0.4 units and wide where the colour changes in 2pH units say 6-8, 8-10.
pH / Test papers
These are filter papers impregnated with indicators. They serve a similar purpose
as that of universal indicators. They are also in two ranges namely narrow and wide.
Indicators may also be neutralisation indicators (Acid - base) eg. Methyl Red,
Redox indicators eg. Diphenylamine, Metal ion indicators eg. Murexide, Eriochrome
black-T, Adsorption indicators eg. Potassium chromate in chloride estimation.
Theory of Indicators
Ostwalds (1984) theory:
Also referred to as ionic theory of indicators. According to this theory neutralisation
indicators, are weak organic acids or bases in which undissociated molecules differ in
colour from their ions".
eg. Phenolphthalein is a weak acid which does not dissociate in acid medium and
hence no colour with acid solution. In alkali medium the molecules dissociate and
combine with alkali to produce a pink coloured salt.
H ph H = ph
NaOH Na+ + OH-
ph + Na+ ph - Na (Na phenolphthate)
ph - Na ph Na+ (pink colour)
Similarly methyl orange which is yellow unionised condition produces red colour
when ionised. The undissociated molecules of basic indicators can be denoted by IndOH
and their cation by ind+. They dissociate as
Ind OH Ind+ OH
Addition of acid or alkali to the solution shifts the dissociation equilibrium to the
right or left and the solution acquires colour of undissociated Ind OH molecule or Ind+
ions respectively.
Chromophore theory:
According to this theory the colour change of indicators is not due to ionisation
but due to change in the structure of indicators. The colour change of indicators is due to
some intra molecular rearrangement, which changes the structure of the indicators.
 The colour of organic compounds is due to the presence of chromophores in their
molecules. These chromophores may be radicals (atomic groups) or group of double
bonds. These include
i. Nitro groups O = N = O which can be converted into O = N - NH - group
ii. Azo group: - N + N - which may be converted into N - NH - group
iii. Quinoid group transformed from benzenoid.
iv. Carbonyl group C =O
v. Double bonds close to each other
The colour of organic compounds is also due to the presence of auxochromes.
These cannot themselves produce colours but when present along with chromophores
they augment the action of the latter and deepen the colour produced by them.
Examples of auxochromes - OH2 - NH2, OCH3 - N (CH3)2 etc.
According to this chromophore theory the colour change in indicators is the
result of Tautomerism which is a reversible inter conversion of isomeric forms. Due to
this intramolecular regrouping when the structure changes with the formation or
disappearance of groups which influence colour namely chromophores and auxochromes
the colour of indicator also changes.
eg. Paranitrophenol.
The formation of quinoid nucleus under alkaline condition is the cause for colour
change of paranitrophenol under alkaline condition. When it is acidified the equilibrium
is shifted in the opposite direction and the colour changes from yellow to colourless.
The isomers, involved in tautomerism are called as tautomers.
Choice of indicator
Two quantitative methods are available for choice of indicators
1. Calculation of indicator error
2. Plotting of titration curves

1. Calculation of indicator error

The colour of indicator changes over a narrow range of pH and this range depends
only on the properties of the given indicator and does not depend upon the reacting acid
or base. Due to this the colour change of an indicator generally takes place not at the
equivalence point but with deviation. Such deviation is called as indicator error. It is the
error caused by the difference between the titration exponent of a given indicator and the
pH at equivalence point. Several methods are available for calculating indicator error.

2. Plotting of titration curves

1. Strong acid Vs. Strong base
The salt formed does not hydrolyse. Hence the pH at equivalence is 7.0. In such
cases the indicator with a titration exponent (pT) of 7.0 is suitable. The titration
exponent (pT) is the pH at which the titration in the presence of a given indicator is
ended.
Region of abrupt change is between 4.00 and 10.0. Hence, indicators with pT of
4.0 - 10 can be sued i.e. methyl orange - phenolphthalein.
The indicator used must have a titration exponent within the region of abrupt
change in the titration curve.

2. Weak acid Vs Strong base

The salt formed is alkaline. The pH at equivalence is > 7.0. Equivalence point
lies in the alkaline range. Break of pH in the curve is at pH 7.73 0 10.0 Phenolphthalein
is the best indicator.

3. Weak base Vs Strong acid

The salt formed is acidic. Equivalence point is in acid range (pH 5.12). The
region of abrupt change is 4.0 and 6.2. Hence, suitable indicators are methyl red and
methyl orange.

4. Weak acid Vs Weak base

There is no pH break. This means that titration cannot be performed accurately
with any of the known indicators. Hence, in titration by neutralisation method one of
the reacting substances must be a strong electrolyte.
Volumetric analysis:

Volumetric analysis enables us to ascertain the given substance quantitatively by

a careful measurement of the volume of a solution of known concentration required to

react with the solution to be analysed. The substance is allowed to react with a reagent of

known strength and from the volume of the reagent used for the complete interaction of

the substance, the amount of the substance present is determined.

Volumetric methods of analysis are largely employed in the technical work, since

the speed of working and the accuracy are combined with economy of reagents and the

possibility of estimating one or more substances in the presence of others without

effecting any separation. Unlike gravimetric methods, here the final measurement is

made by volume measurement .

The method involves:

1. The preparation of one or more standard solutions of accurately known

concentration

2. The use of accurately graduated vessels, by which volumes can be rapidly

measured.

3. Some means of recognizing the end point, ie., the completion of reaction.

Advantages:

i). Economy of time: tedious process of weighing is not walways

required. Volumetric methods are more rapid.

ii). Minute quantities of substances which would be very difficult to

weigh accurately, can be worked out easily.

Titrimetry:

Titrimetry is based on the fact that the product of volume and concentration of a

one reacting substance is equal to the product of volume and concentration of the second

reacting substance involved in the titration, which can be shown in the reaction equations:

V1N1 – V2N2

Where

V1 - Volume if the first reacting substance

N1 - Concentration of first reacting substance

V2 - Volume of the second reacting substance

N2 - Concentration of the second reacting substance.

Titrimetry involves determining the quantity of reagent which exactly

corresponds to the above reaction equation and is chemically quivalent ot the substances

being determined.

Acidimetry and alkalimetry:

These are the methods where the interaction between hydrogen (H)+ and hydroxyl

(OH)- ions occur. If an acid is added to the base (or vice versa), the pH of the solution

will change during the course of titration and so by using a pH sensitive indicator like

methyl orange, methyl red or phenolphthalein the end point can be determined.

2 NaOH + (COOH) → 2 COONa

2 Na2 CO3 + 2HCl → 2 Nacl + H2o + co2↑

Oxidation – reduction or redox titrations:

These involves the change in the oxidation states of the reaction substances. The

commonly used, oxidising agents include potassium permanganate and potassium

dichromate, while the reducing agents are ferrous ammonium sulphate and oxalic acid.

Based on the reaction substances involved, redox titrations are grouped into four

categories as follows:

i). Permanganate titration (permanganametry)

ii). Dichromate titrations (Dichrometry).

iii). Iodine titration (Iodimetry)

iv). Copper reduction titrations

Precipitation titrations:

The reacting constituents precipitate during the course of titration. When that

substance is completely precipitated, the additional drop of the titrant will react with the

indicator making the end point. Since the setitrations involve the silver compounds they

are also called as argentimetric or argentimetric titrations.

AgNO3 + NaCl → AgCl +NaNO3

AgNO3 + KCNS → AgCNS + KNO3

Complex formation or complexometric titrations:

The reacting constituents form undissociable stable complex thereby the

concentration of a particular ion in the solution gradually decreases. When all the ions

have been completely complexed any additional drop of the titrant will react with the

indicator marking the end point.

Common terminologies used in volumetric analysis:

Standard solution: A solution of known concentration or strength is called as standard

solution. It contains a weight of the solute in a known volume of the solution.

Methods for the expression of Concentration

1. Formality or formal concentration

The ‘formality’, F, of a solution expresses the number of gram-formula weights of

solute present in one litre of solution. If follows that this term also gives the number of

milliformula weighs of solute in 1 ml. of the solution.

2. Molarity or Molar concentration:

The ‘Molarity’ M, of a solution defines the number of gram-molecular weights

(or moles) of a species in 1 litre of solution or the number of millimolecular weights in 1

ml of solution.

The molar concentration and the formal concentration of a given solution will

frequently differ. Thus the solution that results when 1 gram-formula weight of oxalic

acid (90.04g. H2C2O4) is dissolved in sufficient water to give 1 litre is by Because this

solute undergoes dissociation to the extent of approximately 22 per cent, however, the

species concentration of prepared by dissolving 1.00 gram-formula weight of sodium

chloride (58.44 g.) in sufficient water to give 1.0 litre of solution will be 1.00 F with

respect to NaCI, by definition ; the concentration of both Na+ and CI- will be 1.00 M.

The molar concentration of the species NaCL will be Zero. This is the general situation

for solutions of strong electrolytes.

From these examples it is clear that quantitative information is needed with regard to the

composition of the solute in solution before its molar concentration can be specified. On

the other hand, formal concentration may be calculated from the specifications for

preparation of solutions and the formula weight of the solute.

3. ormality or ormal concentration:

 The ‘normality’ N, of a solution expresses the number milliequivalents of

dissolved solute contained in 1 mil litre (Thus, a 0, 2N solution of silver nitrate contains

0.20 milliequivalent of this substance in each milliliter of the solution).

4. Percentage concentration:

Chemists frequently denote the concentration of solutions in term of percentages

and this may be done in several ways:

Three of the common methods are as follows:

Weight per cent Weight of solute x 100

Weight of solution

Volume per cent Volume of solute x 100

Volume of solution

Weight – Volume Weight of solute, x 100

Per cent Volume of solution, ml

Weight = Volume per cent is frequently used to indicate concentration of dilute

aqueous solutions of solid reagents ; thus a, a 5 per cent silver nitrate solution is prepared

by dissolving 5 g per cent silver nitrate solution is prepared by dissolving 5 g. of silver

nitrate in water an diluting to 100 ml

5. parts per millions:

In expressing the concentrations of very dilute solutions, percentage

compositions become awkward to use because of the number of zeros needed to place the

decimal point. Under these circumstances the concentration is more conveniently

expressed in parts permillion (ppm.) . This term is defined by the equation

ppm Weight of solute, x 1,000,000

Volume of solution, ml
 Thus an aqueous solution containing 0.003 per cent nickel by weight contains 3

ppm nickel.

Titer:

The titer of a solution is the weight of a substance that is chemically equivalent to

1 ml of that solution. Thus, a silver nitrate solution having a titer of 1.00 mg of chloride

would contain just enough silver nitrate in each milliliter to react completely with that

weight of chloride ion. The concentration of a reagent to be used for he routine analysis

of many samples is advantageously expressed in terms of its titer.

6. Milliequivalent:

A Milliequivalent represents 1/1000 of an equivalent weight. The equivalent

weight of an element or radical is the weight in grams of the element or radical required

to displace or to unite with one atomic weight of H or to combine with one atomic weight

of CI or of any other univalent element. The equivalent weight of H is 1.008 and that of

CI is 35.46. since their respective atomic weights are 1.008 and 35.46. The equivalent

weight of K is 39.1 because it takes one atomic weight of K to combine with one atomic

weight of CI to from KCI. The equivalent weight of the ammonium radical NH4 to

combine with 35.46 gm CI which is one atomic weight of CL. The equivalent weight of

Ca is 20 because it takes two atomic weights of CI for one atomic weight of Ca which is

40 to make up the compound CaCI2.

Example:

100 germs of soil, when treated with NH4CH3Coo1 gave up the following cations:

8.064 mg of H; 60 mg of Ca; 8 mg of Mg and 78.2 mg of K. In terms of milliequivalents

per 10 grams of soil, each cation may be calculated as follows:

8.064 1.008 = 8 milliequivalents for H; 60 (40 2) = 3 m.e. for Ca; 8 (24 2) = 0.67 for mg;

78.2 39.1 = 2 for k. The total cation exchange capacity for the particular soil will thus be

8+3+0.67*2=13.67 milliequivalents/100 grams of soil.

ormal Solution : A solution (normal) contains one gram equivalent of the substance in

one litre of the solution. The Strength of such a solution is represented by 1 N or simply

N.

ormality : It is the number of gram equivalent of the substance dissolved in one litre of

the solution.

Normality x gram equivalent = Weight of substance

Weight in one litre

If any two of these three are known, the third can be calculated.

Molar/solution:

A Molar solution of a substance contains one gram molecular weight or one mole

of a substance in one litre of the solution. The strength of such a solution is represented

by 1 M or simply M.

Molarity : The molarity (M) of a solution is the number of gram molecular weights or

moles of solute in one litre of the solution.

Thus a 3 M solution of sulphuric acid contains 3 moles of H2SO4 (3 x 98g)

In one litre of the solution.

Molar Solution :

A molar solution of N substance containg one gram molecular weight of a

substance dissolved in 1000g of a solvent. The strength is represented by 1 M

Molarity :

The molarity (m) of solution is the number of moles of solute dissolved in 1000 g

of solvent. Thus a 3 m aqueous solution of sulphuric acid contains 3 moles of H2SO4 (3 X

98 g) in 1000 g of water.

Formality :

The formality (F) of a solution is the number of gram formula weights of the

solute in one litre of the solution. Thus 1 F Na2CO3 Solution contains 106 g of sodium

carbonate in one litre of the solution.

Percentage of solution:

A known weight of any substance dissolved in 100ml. Two per cent boric acid

solution means 2 gram of boric acid dissolved in 100 ml.

Strength in terms of g per litre. N/10 HCl means 3.65 gram of HCl present in a
litre of the solution.

Colorimetric analysis

Colorimetry is quantitative analysis in which the quantity of a coloured

constituent is determined by measuring the relative amount of absorption of light passing

through a solution of the constituent.

Comparison is made directly or indirectly, by either visual or electrical means,

between the intensity of light passing through the solution and the intensity of light

passing through the solution and the intensity of light of the same wave length passing

through a solution (or series of solutions) of the same substance at known concentrations.

The energy of electromagnetic radiations is inversely related to the wave length.

Visible range
 Colour Wave length, nm Colour Wave length, nm

Violet 400 - 435 Green 500 - 560

Blue 435 - 480 Yellow-green 560 - 580
Green blue 480 - 490 Yellow 580 - 595
Blue green 490 - 500 Orange 595 - 610
Red 610 - 750

The energy of these electromagnetic radiations decreases from gamma to radio

frequency waves.

Laws of absorbance

Lambert's Law
When a monochromatic light passes through an absorbing medium, its intensity

decreases exponentially as the length of the medium increases.

Beer's Law
The intensity of ray of monochromatic light passing through and absorbing

medium decreases exponentially as the concentration of the absorbing medium increases.

If a beam of monochromatic light of intensity Io falls on a homogeneous layer of

some substance, part of the light (of intensity Ir) is reflected from the substance, part (Ia)

is absorbed, and part (It) is transmitted through the layer, so that

Io=Ir + Ia + It (1)

In case of aqueous solutions Ir is small in comparison with Ia It and may be

disregarded. Then,

Io =Ia + It (2)

Since the absorption of light by the solvent (wate,r alcohol, ether etc.) is much

less the absorbance is mainly rleated to concentration (c) and the thickness of the layer of

solution (h). The relationship can be represented as

Io
Log ------- = Σhc = A (3)
It
 Where Σ is the extinction coefficient and A, absorbance. According to Beer and

Lambert's law, "the optical density of a solution is proportional to the product of the

concentration of the absorbing (coloured) substance and the thickness of the layer of

solutiion. From equation (3) we have

Io
------- = 10 Σhc
It
and hence

It=Io x 10 ΣhcIn the colorimetric analysis the basic principles involved are

Lambert's law and Beer's Law.

Apparatus
Instruments for the measurement of selective absorption of radiation by solution

are known as Colorimeters.

Simpler Visual and Photoelectric devices for the visible region.

Absorptiometers include the class of colorimeters, but can be applied to other

spectral region as well.

Spectrophotometers much narrower band of wave lengths is employed, as

produced by a monochromator.

The absorptiometers are also classified as single - or dual - beam instruments.

The double beam type may have two matched detectors, or the radiation may be flashed

alternatively over the two paths to a single receptor.

The construction of the instrument includes following common components.

Some of the simpler instruments may omit one or more items, and the sequence in which

the radiation passes may also vary.

i) Source of radiation : Incandescent lamp / H2 or D2 arc lamp / Day light.

ii) Intensity control : Iris diagram / variable slit / Rheostat in lamp circuit.

iii) Wave length control : Color filter / mono chromator

iv) Sample holder : Test tube / cuvette.

v) Receptor : Photographic plate / phototube or photo multiplier / photocell / eye

vi) Indicator: Galvanometer / potentiometer / pen recorder / oscilloscope

Operating Instructions

Operating instructions for the Klett - Summer - Son Photoelectric colorimeter

1. Before turning the colorimeter lamp on, be sure that a filter is in place in the

space provided for it.

2. Switch on the instrument

3. Flip the short circuit switch on the side of the instrument to the ON position.

4. Place the clean colorimeter tube containing distilled water in the instrument.

5. Turn the scale to zero by means of the large knob on the front of the

instrument.

6. Turn the zero adjustment knob, until the pointer is brought to zero on the

scale. Allow few minutes for the lamp to warm up, and again not move as the

zero adjustment knob is turned, the short circuit switch is probably in the

wrong position.

7. Remove the distilled water tube and place the tube containing the standard

solution in the instrument. Turn the scale knob until the pointer returns to

zero. The reading on the scale is the reading of the standard solution.

8. Repeat the step 8, using unknown solution.

9. Put the switch to off position and disconnect the power.

Emission and absorption spectroscopy

Flame photometry
(Estimation of Potassium and Sodium)

Principle
Certain elements when excited in flame emit radiation. The excitation causes
one of the outer electrons of neutral atoms to jump to an outer orbit of higher energy
level or the atoms may be excited sufficiently to loose an electron completely. When
excited atoms return to lower energy levels, light of characteristic wavelength is
emitted. The flame photometer measures this radiation intensity, which is proportional
to the concentration in a solution.
Preparation of standard solution
Potassium
1.907 g of AR grade KCl is dissolved in one litre of distilled water. This gives
1000 ppm of K. 100 mL of 1000 ppm solution diluted to one litre will give 100 ppm K
solution. From this various standards are prepared ranging from 10 to 100 ppm.
Sodium
2.54 g of AR grade NaCl is dissolved in one litre of distilled water. This gives
1000 ppm of K. 100 mL of 1000 ppm solution diluted to one litre will give 100 ppm K
solution. From this various standards are prepared ranging from 10 to 100 ppm.

Instrumentation

1. Sample solution sprayed or aspirated as fine mist into flame.

Conversion of sample solution into an aerosol by atomiser (scent spray) principle.
No chemical change in the sample in this stage. [NB atomiser does not convert
anything into atoms].
2. Heat of the flame vaporizes sample constituents. Still no chemical change.
 3. By heat of the flame + action of the reducing gas (fuel), molecules & ions of the
sample species are decomposed and reduced to give ATOMS.
eg Na+ + e- --> Na
4. Heat of the flame causes excitation of some atoms into higher electronic states.
5. Excited atoms revert to ground state by emission of light energy, hν, of
characteristic wavelength; measured by detector.

The light emitted from glowing gases is characteristic of the elements in that gas, and
the brightness of each band of light is directly proportional to the quantity of that
element in the glowing gas. The flames of salts viz., LiCl, NaCl and KCl, on burning
chlorine emits outside of the visible range, so only the spectra of the metals are seen.
Lithium is the smallest atom and emits bright red light; sodium emits yellow light; and
potassium emits lilac light.

Emission Na ||
of K ||
___________________________________

400 500 600 700 800

λ (nm)

Operating the instrument

Attach the lead from the indane gas cylinder to the burner.

Fix the atomizer in its place and introduce it into distilled water kept in a vial.

Start the compressor and adjust the air pressure to 10 psi.

Open the gas and light the burner through the window. Adjust the flow of gas to
give a central bluish cone.

With distilled water set zero by using the zero adjustment knob. Then introduce
100 ppm K solution and adjust to read 100 on the scale. Again introduce distilled
water and adjust to zero. Repeat this process till the spot of light adjusts to zero
and hundred, without zero adjustment.

Now introduce the various standard solutions, record the readings and draw the
standard curve.

Take the sample in a small vial and introduce through the atomizer. Record the
reading of the galvanometer. Calculate the Na and K using the standard curve.

Distilled water should be used in between two unknown solutions and also before
switching off the instrument.

ATOMIC ABSORPTIO SPECTROPHOTOMETRY

(Estimation of Fe, Zn, Mn, and Cu)

Principle
Chelating agents are useful tools for assessing readily available micronutrients in
soils. They combine with free metal ions in solution to form soluble complexes. DTPA
offers a most favorable combination of stability constants for simultaneous complexing
of zinc, copper, manganese and iron. To avoid excessive dissolution of calcium carbonate
that can release occluded micronutrients (not available to plants), the extractant is
buffered in slightly alkaline pH range and in particular by including soluble Ca 2+. TEA is
used for buffering because it burns clearly during atomization. At selected pH of 7.3, ¾ th
of TEA is protonated and is present as HTEA+. When the extractant is added to soils,
additional Ca2+ and some Mg2+ enter the solution, largely because the protonated TEA
2+
exchanges with Ca and Mg2+ from soils exchange sites. This increases the
concentration of ionic Ca2+ by two to three fold and suppresses dissolution of calcium
carbonate in calcareous soils with2:1 solution to soil ratio. The extracted micronutrients
are estimated using Atomic Absorption Spectrophotometer (AAS).
Reagents
DTPA extractant: (1) Dissolve 1.96 g of DTPA, (2) 15 mL of Triethanolamine
and (3) 1.47 g of Calcium chloride and make up the volume to one litre with double
distilled water. Adjust the pH to 7.3 by using dilute HCl.
Instrumentation
Atomic absorption spectroscopy offers the researches unique advantages over
older methods of analysis and has helped them to expand the knowledge about the
chemistry of essential plant nutrients, particularly micronutrients, as well as the pollutant
elements which prove hazardous not only for crop plants but also for animal and human
health. The ease of operation of the instrument, straight forward standardization in many
determinations, lower detection limits and good precision of analysis make the atomic
absorption spectroscopy still more useful.
Principle
A portion of the sample is converted into atomic vapours usually by a flame, and
irradiated by the light from a source whose emission lines are those of the metal being
analysed. The absorption of the light by the atoms of the element in the vaporized sample
is related to the concentration of the desired metal in it and is measured at a specific
wavelength which is characteristic of the element. The concentration of the metal in the
solution is measured by comparison with absorbance measurements on standards of
known composition.
Instrument Features
Atomic Absorption Spectrophotometer (AAS) has the following basic components
i) A light source that emits resonance line radiation
ii) A sampling cell, usually a flame, into which the sample is fed as an aerosol
iii) A monochromator that is used to isolate the absorbing resonance line from other
nonabsorbing lines.
iv) A detector that measures the amount of absorption
v) Electronic amplification of the absorption signal and
i) A readout-system that normally is a strip chart recorder, a digital display, a meter
or a printer.

I. Light source
The narrow line emission radiation which is absorbed by the dissociated atoms is
typically generated by a hollow cathode lamp. This line should be of sufficient spectral
purity and intensity to achieve a linear calibration graph with low noise level from the
AA spectrometer. The lamp, filled with a rare gas, generally argon or neon at low
pressure, has its cathode made of or lined with the element or elements of interest.
Hollow cathode lamps emit only the spectrum of the cathode element together with that
of the fill gas. The anode is merely a straight metal wire. The front face of the lamp is
made of quartz or any of several types of glass depending on the wavelength range it
must transmit.
Optimum lamp currents vary among elements and designs ranging from 5 to 50 ma at
100-300 V. Hollow cathode lamps are stable and reliable and are typically guaranteed for
5,000 ma-hr of operation. For a lamp operated at 10 ma, this would provide 500 hours of
guaranteed performance.

II. Flame atomization system:

The important parts of the flame atomization system are:
a) ebulizer: The main functions of the nebulizer are to draw up solution, regulate
uptake rate, and convert solution into aerosol and to provide the main pathway for the
oxidant gas.
b) Adjustable impact bead: It helps aerosol formation. Larger droplets formed by the
nebulizer are broken up into smaller droplets. The position of the impact bead provides
control over sensitivity and noise. It can also be set to reduce chemical and atomization
interferences. The standard impact bead is made of glass. High concentrations of
dissolved solids in analytical solutions can etch or erode it. Hydrofluoric acid will
chemically attack it and it should therefore be complexed with boric acid or a borate salt.
c) Mixing paddlers: The mixing paddles are static in the spray chamber. The double
reversal of the gas in two sets of paddlers ensures thorough mixing of acetylene and
oxidant.
d)Flame and burner :The only purpose of a burner and flame is to get the element of
interest into the atomic state. Burners used for AAS are all of the premix, laminar flow
type. The sample aerosol is aspirated and mixed with the gases before combustion.
During combustion, the element is reduced to the atomic state after which it can absorb
radiation. Flames in use are produced by two types of gas mixtures: air-acetylene and
nitrousoxide-acetylene. Air-acetylene is the cooler flame (2100-2400oC) and is used for
those elements that do not form refractory compounds and have relatively low ionization
potentials. Nitrous oxide - acetylene is a hotter flame (2600-2800oC) and must be used
for elements forming refractory compounds (e.g. SiO2, Al2O3, TiO2 and Cr2O3). It can
also be used for many of the other elements, giving better detection limits and better
freedom from chemical interferences, provided ionization does not seriously deplete the
atomic population.

III. Monochromator
If a linear relationship between absorbance and concentration is to be obtained, it is
obviously essential to isolate completely the line to be measured from all other lines. The
slit width used should be the maximum which will permit this condition to be achieved so
as to give a maximum signal to noise ratio at the detector. The light from the source unit,
diverging from the entrance slit, is rendered parallel and sent to the dispersing device.
The dispersed light is then focused on the exit slit of the monochromator, through which
it passes into the detector section.

IV. Detector recording: A flame into which a sample has been aspirated not only can
absorb radiation but also can emit radiation; therefore, many AAS units may be used for
flame emission spectrometers. All sources are pulsed or modulated either electronically
or mechanically so that the detector and electronics can distinguish the source emission
from the flame emission. In AAS, the electronics are then tuned to accept only the
modulated signal and to ignore the signal produced by the flame.
The intensity of the radiation is measured with and without the sample to obtain a
reading in either percent absorption or absorbance (optical density) and the concentration
of the element of interest is determined by comparing that reading to one obtained for a
standard or series of standards.
Types of Atomic Absorption spectrophotometer
There are four basic types of AAS instruments, viz., 1) Single beam, 2) Double
beam, 3) Dual-channel double-beam and 4) Double beam sequential multi element.
Procedure

Weigh 10 g of air dry soil

Transfer it to a 100 mL polythene shaking bottle

Add 20 mL of DTPA extractant and shake the contents for 2 hours in a mechanical
shaker.

Filter through Whatman No. 1 or 3 tilter paper

Feed extract into the atomic absorption spectrophotometer

Measure the available Cu, Fe, Mn and Zn by using the wavelengths of 324.75,
248.33, 279.48 and 213.86 nm respectively.
Critical limits for available micronutrients:
Fe Zn Mn Cu
Calcareous: 6.3 µg g-1 Loam :1.2 µg g-1 2.0 µg g-1 1.2 µg g-1
Non Calcareous: 3.7 µg g-1 Clay : 2.0 µg g-1

Ion Chromatography- Principles and measurement methods

Chromatography is a technique used to separate the components of a chemical
mixture. Ion chromatography is a modern method of analysis for the determination of
ions on the basis of ion exchange i.e. anions being separated on an anion exchange
column and cations on a cation exchange column. Modern ion chromatography actually
began with a paper published in 1975 by Small, Stevens and Bauman. The electrical
conductivity of a solution varies with the concentration of ions present in it, and this
principle forms the basis of separation of all ionic species in solutions. The instrument
used for separation of ions on the basis of differences in electrical conductivity of a
solution is commonly known as ion chromatograph.
Basic Components of an Ion Chromatograph
Essentially, an ion chromatograph consists of following basic components
i. Eluent pump and reservoir : It is required to pump the eluent through the
analytical column, guard column and suppresser column
ii. Sample injection valve : It is used to inject the sample solution for analysis
iii. Guard column : It protects the analytical column by removing suspended
particulates and other poisonous substances from the eluent stream
iv. Analytical column : It is required separation of ionic species
v. Suppressor column : It converts the highly conducting eluent to a form with a low
conductance.
vi. vi. Conductivity cell : It measures the electrical conductivity of the ions
separated by analytical column moving down the suppressor column and
vii. Recorder : It records the signal received from the conductivity cell in the form of
a peak
Ion chromatographs (IC) can be divided into two major groups
 (i) (i) those that operate on the principle of elucent suppression and
(ii) (ii) those with no suppressor column.
Eluent Suppressed Ion Chromatography
In the eluent-suppressed IC, the ion species are resolved by conventional elution
chromatography followed by passage through a suppressor column, wherein the eluent
coming from the separating column is neutralized. Thus, only the ionic species of interest
leave the bottom of the suppressor column in a background of H2CO3, which exhibits a
low conductivity, in the case of anion or water in case of cation, and they are monitored
subsequently by the conductivity cell.
Anions
In the determination of anions, the IC is equipped with an analytical column packed with
an anion exchange resin in the HCO3- form whereas the suppressor column contains a
strong acid high capacity cation exchange resin in the H+ form. The eluent used is
normally a mixture of NaHCO3 and Na2CO3. Although other dilute mixtures of NaHCO3
+ NaOH, Na2CO3 + NaOH are also used. The anions are separated and converted to their
strong acids in a background of H2CO3, which has low conductivity. The presence of
strong acids in H2CO3 is measured by a conductivity cell and reported as peak on a
recorder. The peak height/area is directly proportional to the concentration of ion in
solution. From the calibration graphs prepared for peak height vs concentration of ions in
standard solution the concentration of ionic species in the sample solution are calculated.

the suppressed ion chromatography anions are separated on an anion exchange column
that contains an anion exchange resin of low exchange capacity. A dilute solution of a
base such as sodium carbonate (2.7 mM) and sodium bicarbonate (0.3 mM) is used as an
eluent. Immediately following the anion exchange column is a cation exchange
suppressor unit that converts the eluent to carbonic acid.

Na+HCO3- + Memb-SO3-H+→ H2CO3 + Memb-SO3- + Na+

The counter ion of the sample anion e.g. Na+ is converted to H+. A conductivity detector
is used and the background conductance from the carbonic acid is low. The suppressor
column contains a membrane unit made of a sulfonated polymer (Memb-SO3-H+).
 The eluent and sample ions exchange with H+ ions in the membrane as follows:
Eluent
Na+HCO3- + Memb-SO3-H+ →H2CO3+Memb-SO3-Na+
Sample-ion
Na+Cl= + Memb-SO3-H+ → H+Cl- + Memb-SO3-Na+
A dilute H2SO4 solution flows continuously around the outside of the membrane
of suppresser unit. This solution replaces sodium ions in the membrane with H+ ions from
H2SO4, thus regenerating the membrane.

H2SO4 == 2H+ + SO4”

Memb-SO3-Na+ + H+ == Memb-SO3-H+ + Na+

This system gives an excellent separations for a variety of anions e.g. F-, Cl-, NO2-, PO4”,
Br-, NO3- and SO4”.
Cations
A similar system can provide good separation of a number of cations. For cation
separations, the analytical column contains a cation exchange material in the form of H+
form and the suppressor unit contains anion exchange resin in the OH- form. The eluent
cation in this case is mostly H+ ion through 22mN H2SO4 or 5mM HCl and is converted
by the suppressor unit to H2O. The alkali metals (Li, Na, K, Rb, Cs) are separated and
converted to their hydroxides and conductivity of the metal hydroxides is measured by a
conductivity cell and reported as peak by the detector. The eluent and sample ions
exchange with OH- ions in the suppressor column as follows
Eluent
H+ Cl- + R-OH- == Resin-Cl- +H2O

Sample ion
M+A- + Resin-OH- = Resin-A + M+OH-
Single column ion chromatography
In this system, a low capacity exchange column and low conductivity eluent are
used without the need for a suppressor column but this system is about 20 times less
sensitive than eluent suppressed system.
 Separation of ionic species is a function of the length of the separator column,
eluent composition and concentration and flow rate. Table1 shows the retention times of
different cations and anions by the IC system. The separation of these ions is based on
their relative affinity for the separator column resin. The retention time of an ion
increases as its affinity for the resin increases.
Table 1. Retention times for selected ions

Anion Retention Cation Retention

time (min) time (min)

F- 1.4 Li 3.1
Cl- 1.9 Na 3.8
NO2- 2.3 NH4 4.3
PO43- 3.4 K 5.5
-
NO3 4.8 Mg 8.2
SO2- 8.2 Ca 10.0